CROSSABILITY, GE-NOME RELATIONSHIPS AND INHERITANCE STUDIES IN INTERGENERIC

HYBRIDS OF PIGEONPEA

A THESIS SUBMITTED FOR THE DEGREE OF

Doctor of Philosophy

BY

P: SATEESH KUMAR, M-sc. ( A Q

SCHOOL OF LIFE SCIENCES UNIVERSITY OF HYDERABAD

HYDERABAD - 600 134 INDIA

DECLARATION OF ORIGINALITY

T h i s t h e s i s r e p o r t s t h e o r i g i n a l work of t h e a u t h o r ,

excep t a s o the rwise s t a t e d . I t has not been submit-

t e d p r e v i o u s l y f o r a degree a t any U n i v e r s i t y .

P. Sa teesh Kumar

C E R T I F I C A T E

T h i s i s t o c e r t i f y t h a t t h e T h e s i s e n t i t l e d

"CROSSABILITY, GENOME RELATIONSHIPS AND INHERITANCE

STUDIES I N INTERGEhERIC HYBRIDS OF PIGEONPEA" i s

b a s e d o n t h e r e s u l t s o f t h e w o r k c a r r i e d o u t b y

M r . P . S a t e e s h Kumar, M . S c . ( A g . ) , f o r t h e d e g r e e

o f DOCTOR OF PHILOSOPHY u n d e r my s u p e r v i s i o n . T h i s

w o r k has n o t b e e n s u b m i t t e d t o a n y d e g r e e o r d i p l o m a

o f a n y o t h e r U n i v e r s i t y .

31 January 1985

DR. N.C. SUBRAHMANYAM (SUPERV I SOR School o f L i f e Sciences U n i v e r s i t y of Hyderabad Hyderabad-500134, I n d i a

I wish to express my gratitude to Dr. N.C. Subrahmanyam, University of Hyderabad and Dr. Donald G. Faris, ICRISAT for their encouragement and advice during the course of this investigation. I am indebted to than for the time and efforts spent on ny behalf.

I wish to express my thanks to Dr. J.P. Moss, ICRISQ. for keeping at my disposal the facilities in the groundnut cytogenetics laboratory at I W W .

I thank the Director of Research, IClUW; Program Leader (Pulses) ,. ICRIW and the Dean, School of Life Sciences, University of Xyderabad for providing the necessary research facilities.

My + M s are due to Drs. L.J. Reddy, D. Shanm, A.K. Singh, R.P.S. Pundir, K.B. Saxena and D.C. Sastri of ICFUSAT for helpful suggestions during the course of this investigation. I am also thankful to Dr. Ian Dudas for helpful discussions during the final stages of preparation of this dissertation.

Iwish to thank Drs. L.J.G. van der Maesen and P. Renwandan for the supply of Atylmia seed and for help on taxonomic details.

I would like to express my sincere =reciation to Mr. G. Shinde for all the help rendered during the course of this investigation.

My a~preciation is extended to the personnel of the Photolab, ICICISAT for their excellent processing and printing of the photographs presented in the deeaertation.

My amreciation is also extended to Messrs R. Vijaya Kurrrar, A. Nageshwar Rao M. Chenchi Reddy and to the field staff of the Pigeonpea Breeding Subprogram of IGUSAT and to the staff,of the Plant Culture Unit of University of Elyderabad for their uninhibited help during the course of field experiments.

I acknowledge with thanks the help rdered by ny frierds MeSSrs M.K. R q , D.V. Ranga Rao, A.N. Murthy, V.B. Reddy and P. Muralikrlshna.

I gratefully acknowledge the financial generosity of ICRIsA'r.

I would like to express my a~reciation to Mrs. K. Syamalanba for typing the dissertation.

Finally, I owe a personal debt to my late grand parents for their encou:agemt and involvement in my research efforts.

The present investigation w a s ini t iated with the brmd objectives of, assessment of genome relat ionship between the G&LUS and its related wild species, screening the wide crosses for haploids tbrough selective chromsome elimination, developnent of m W s to inprove crossability between species, studying the inheritance of descernible characters in the hybrids obtained and develop in VitEP regeneration techniques in pigeonpea. The present study involved crossing C. & a ~ with twelve v i e s of &ylcsia and two species of m. A t o t a l of 24 intergeneric cross conbinations including reciprocals were attenpted.

Eight (A. . . . r A. A. lanceolata, A.

-, A. =, A. and A. se&ed out of the 12 species hybridized with C. a when Cajarus w a s the female parent. From the reciprocal crosses a low rate of success was obtained in two conbinations (A. albicans x C.

and A. sericea x C. u. When fajmus was the female parent the degree of crossability varied not only with the

species in question, but also with the genotype. Che of the'- cultivars, ICP-102 failed t o cross with any of the species. The ra te of success w a s very high (1.37-19.5%) when A. lineata w a s the male parent followed by C. a x A. albicans crosses (1.09-9.6%). In the other six anbinations the success was low ranging from 0.9-3.05%. C.

cultivars which had a c o m n female parent in their pedigree did not vary mch in their abi l i ty t o cross with

species (R. cothii and R.

Post-pllinatiion homne treatments were used t o inprove the crossability in successful C&rus-&yl~~L~ crosses and also t o assess their influence in developing new hybrid con-binatiops. Effects of gibberellic acid (GA31, kinetin and their mixture on S&mw, x crossability. were tested. GIU and kinetin were used independently a t concentrations ranging from 10 ppn to 80 ppn or as a 1:l mixture keeping the net homne concentration in the same range. Following hormone treatments the rate of success w a s increased through increased pod sett ing and n&r of seeddpod. GA3 a t a concentration of 50-60 ppa was found t o be the m s t effective treatment. Higher hormone concentrations had a detrimental effect. In unsuccessful crosses homne treatments delayed bud drop by 3-4 days, permitting prolonged ovule developent. Resu l t s indicate tha t the hormone treatment helps in post-fertilization dwelopnent leading t o an increased rate of success. Results suggest that the Auetralian species of Atyhsh are more diverged fran Cajanua than the Asian & y l d a .

A l l the proqeny were subjected t o a detailed cytological andlysie. There waa no evidence of chromaom elimination in any of the m & y l Q a i B crosses.

Cytolcgical studies i n pigeonpa unequivocally established the p r s e n c e of tw nucleolar organizers per genome in S&r.u and A!&!~Q& species. Detailed meiotic s tudies in the hybriris revealed a high degree of gemme homlogy and recorrbinati~n between the a i l t iva ted and the wild species. In the hybrids eleven bivalents were ccmrcn a t metaphase-I with o c c a s i o ~ a l univalents. Univalents were m r e fresuent i n t h e hvbrids with the Australian &yl&ai (A. arandifoliaand8. latlsecala). C h i a m freauencv w a s also low i n ' these hvbrids in comrarison with the ohers: Anaphase separation was ;egular in mst 'of the hybrids again with the exception of the hybrids w i t h A. arandifolia and A. latisepala. In both these cases m u l t i ~ o l a r spindle abnormalities were ammn. Inversion loops a t pachytene and anaphase I bridges were found i n hybrids of C. s&n with A. arandifolia indicating inversion heterozygosity in t h e F, hybrids.

W r i d s between C. +an and A. were t h e most f e r t i l e while the hybrids wrth A. arandifolia and A. l m were highly s t e r i l e . Other hybrids exhibited a considerable arount of s t e r i l i t y in s p i t e of regular bivalaent formation a t metaphaseI and normal disjunction. The genotype of the f m i e parent a l so influenced the pollen f e r t i l i t y . Hybrids with the genotype, 18-7035 were consistently l e s s f e r t i l e in all c o ~ m a t 1 0 n s .

Nucleolar var iat ion in n h r , s i z e and dis tr ibut ion was recorded a f te r division-I and division-I1 i n three hybrids (C. f&dU.x A. m r C. U X A. lUEh and C. X A. $uuL&lS. I n these three hybrids three t o four brvalents were found attached t o the diplotene/diakinesis nucleolus as against one t o two bivalents i n t h e parents. Variation i n nucleolar nrrmber and dis tr ibut ion a r e interpreted to have originated from pairing and reconbination between nucleolar organizer chromosome(s) of one parental species with t h e nonnucleolar o r g h i z e r chromosme(s) of the other. Detailed analysis of these hybrids provided evidence for "crypt ic s t ruc tura l hybridityn and f o r intergenomic (allosyncetic) rearbinat ion. Nucleolar behaviour was used as a mrker t o t r a c e t h e products of recodinat ion in these hybrids. The nuc lwlar variat ion in the hybrids suggests t h a t t h e s t e r i l i t y i n t h e hybrids inspi te of regular meiosis is a consequence of duplicatirma and/or deficiencies originating from allosyndetic reambination part icular ly between t h e s t ruc tura l ly a l t e red chromsomes i n t h e hybrids.

Most of the &yJaaia characters l i k e seed strophiole, seed mottling, and pod hairiness were expressed in t h e hybrids indicating the i r daninant nature while l e a f l e t shape and twining nature shared incorrplete dominance. The F2 segregation f o r l e a f l e t &ape and pod hairiness rwealed that these t r a i t s a r e governed by one locus while twining nature, seed strophiole and seed nott l ing were governed by tuo loci . Substantial var iat ion for quantitative t r a i t s was found in the F2 generation which include8 transgressive segreganta f o r leaf l e r r g t h r leaf width, pod length, seed w e i g h t and protein content in the

hybrids, underlining the iap6rtance of Atylsda species in tk iaprovmt of fajanua. The F2 variation in these hybrids sbtantiated the cytological ,evidence for a close relationship between c. and A. -.

Techniq~es were standardized for regeneration of plants from immture ecjcryos (&ryos at least 11 &ys.oldl of C. ,@ and from wtyl&s of C. &+n, A. -, A. albuxxs and A. -. Genotypic differences were wident with respect to frequency of regeneration in both arbryo and cotyledon cultures. In cotyledcr. cultures the regeneration potential varied with the region Of ti.e cotyledonary explant with nodal halves being mre effective bi-ile in errbryo cultures an age dependent response was wident .

C O N T E N T S

LIST OF TABLES

LIST OF FIGURES

1. SCOPE OF THE INVESTIGATION

2. MATERIAL AND METHODS

3. SPECIES CROSSABILITY AND HYBRID MORPHOLOGY

3.1 . RESULTS

3.1.1 CROSSABILI'PI

3.1.2 EFFECT OF HORMONE TREATMENTS

3.1.3 HYBRID MORPHOLOGY

3.2 CiSCUSSION

4. MEIOTIC BEHAVIOUR

4.1 PARENTS

4.1.1 RESULTS

4.1.2 DISCUSSION

4.2 HYBRIDS

4.2.1 RESULTS

4.2.1.1. NUCLEOLAR DISTRIBUTION AFTER TELOPHASE-I AND I1 IN F1 HYBRIDS

4.2.1.2 CHROMOSOME BEHAVIOUR IN F2 POPULATIONS

4.2.2 DISCUSSION

4.2.2.1 F2 GENE~TION

5. INHERITANCE OF QUALITATIVE TRAITS

5.1 RESULTS

5.2 DISCUSSION

6. VARIATION FOR QUANTITATIVE TRAITS

6.1 RESULTS

6.2. DISCUSSION

Page

7. IN VITRO REGENERATION

7.1 RESULTS

7.1.1 TISSUE CULTURE

7.1.2 EMBRYO CULTURE

7.1.3 ANTHER CULTURE

7.2 ' DISCUSSION

8. GENERAL DISCUSSION AND CONCLUSION

REFERENCES

PUBLICATIONS FROM THE THESIS

LISP OF TABLES

PAGE

12 Table I. Studies on Intergeneric hybridization in pigeonpea.

Table 11. Origin and salient economic features of the species used in the study.

Table

Tab1 e

111. Seed germinability in E2 ~opllations.

IV. Constituents of basal mdia used in In . regeneration studies.

Table V. Crossability of and Rhyn&&a species with five genotypes of Caidwa dm-

Table VI. Crossability of species with C&mus a having a c o m n £ a l e background.

Table VII. Percent pod-set in intergeneric crosses of S & u s &.an and &y&iia species followed by horrrone treatments.

VIII. Expression of qualitative traits in parental species and F1 hybrids between faiarucajan and ALyhski species.

Table

Table IX. Expression of quantitative traits in pareptal species and F1 hybrids between S & b sajm and ALyLxla species.

Table X. Expression of quantitative traits in parental species acd F1 hybrids between S&nw and ALyhski species.

XI. Meta-1 configurations and pollen fertility in fajmu c&in and species.

XII. Metaphase-I configurations, Andphase1 abnodities, pcllen sterility and ovule fertility in El hybrids betwea C. s&n and different species of &yJ.g&.

Table

Table XIII. Nucleolar variation at telophase-I1 in a hybrid between C. and

Table XIV. Inheritance 1; ttern of seed strophlole in C. x ~ & & a a species nybrlds.

Table XV. Inheritance pattern of seed mottles in C. x species hybrids.

Table XVI. Inheritance pattern of pod hairiness in C. s.ajdn x A t y h i a species hybrids.

Table XVII . Inheritance pattern of twining nature i n C. mjan x At&& species hybrids.

lrPble XVIII. Inheritance pattern of lea£let shape in C. x &&ski species hybrids.

Table XIX. Variation for pod length (an) in the F2 population of hybrids between C. and &&sia species.

Table XX. Variation for 100-seed weight (g) in the F2 population of hybrids between C. and ALy&ia species.

W l e XXI. Variation for seeds per pod i n the F2 population of hybrids between C. siim and &ylgsia species.

l ab le XXII . Variation for mid-leaf width (an) in the F2 population of hybrids between C. 0 and A t y l d a species.

m l e XXIII. Variation for mid-leaf length (an) i n the F2 p p l a t i o n of hybrids between C. and Atyhaia species.

W l e XXN. Percent shoot regeneration from wtyleQn cultufes of different cul t ivars of and "qecies of A&&& on IS medium sqpleinented with 2,4-D (0.5 mg/l) and BR (2 nq/l).

Table XXV. Percent plantlet regeneration and shoot rweneration from cotyledon cultures of different f&nus culti&rs and species of &&.sia on MS Wum supplenented with 2r4-D (0.5 41) BA(2 na/l) and NA?i (1 Iq/l).

TAble XXVI. Percent plantlet r w v e r y i n d r y 0 c ~ l t u r e s of pigeonpea on MS and media s;;~plemented with 1 rg/l of 2,4-D.

PAGE

107

109

110

110

116

Fig. 1. Effect of gibberellic acid andlor kinetin on percent pod and seed set in four ~-~ crosses.

Fig. 2. Effect of gibberellic acid andlor kinetin on delaying bud abscission.

a. Caianusrojan as fanale parent.

b. Ca.ianus a as. male parent. Fig. 3. ltJigs and leaflets of parents and hybrids.

Fig. 4. Twigs and leaflets of parents and hybrids.

Fig. 5. Variation for leaf shape and ultrastructure in C. X 8. hybrids.

Fig. 6. Meiosis in C. sajm cv Pant A2 and I I E t ~ ~ in cultivars of C. &.

Fig. 7. Meiosis in Aty.&ia species.

Fig. 8. Meiosis in At&&& species.

Fig. 9. Nucleolar distribution in pollen nother cells of C Esljan and species of A t y h b .

Fig. 10. Frequency distribution of nucleoli at telo-JI in pollen mpther cells of C, cajm and & y h h species.

Fig. 11. Meiosis in hybrids between C. EBian and A. abisals.

Fig. 12. Meiosis fn hybrids between C. &an and A. serieea and C. &an and A. s&&Ua.

Fig. 13. Meiosis in hybrids between Cr &an and A. scarabaeoides and C* Enjan and A* line9ta.

PAGE

33

Fig. 14. Meiosis in hybrids betwen C. raipn a d A. arandifolia*

Fig. 15. k i o s i s in hybrids between c. & and A. lahsala.

Fig. 16. Nucleolar variation in pollen mother cells of hybrids between C. &an and A. il'blcans.

Fig. 17. Nucleolar variation in pollen mother cel ls of hybrids between C. and A. l h a b and C. 0 and A. gmnd&L~.

Fig. 18. Frequency distribution of nucleoli a t telo-11 in daughter nuclei of hybrids

' between C. sajm and .&yl&.a species.

Fig. 19. Frequency distribution of F2 segregants i n different fe r t i l i ty groups.

Fig. 20 ~romosome pairing a t pachytene in hybrids between C. 0 and species of A t y h b .

Fig. 21 Meiosis in tetraploid C. 0.

Fig. 22 Possible nucleolar variation i n pollen mther cel ls of the hybrids between C. and

species with out reconbination and follawing recabination between nucleolar organizing and non-nucleolar organizing chromomlres.

Fig. 23. YitrP regeneration of sajanu plants from cotyledons and h m t u r e d r o y s .

PAGE

75

Plants of greatest irrportance to agriculture belong to the

families Graminae and Legrmrtnosae. Leglnninosae amprises a wide

array of genera (600) and q e c i e s (1300) (Delwiche, 1978) and

occupies a unique place in the plant kingdom. Sweral legumes

particularly from the subfamily Papilionoidae are of great

m m i c importance next t o cereals (Cobley and Steele, 1976).

Grain legums or plses are the major source of dietary protein

in developing countries. The high protein content and relatively

low cost of these food legumes have earned than the t i t l e "Pcor

m's Meat" which effectively underlines their v i t a l importance

in developing nations. legmes have also played a major role in

patterning the agricultural s y s t m in the tropics. Their

nitrogen fixing abil i ty enables then to sustain high yields in

the face of mFnimrm inpts, inprove so i l f e r t i l i t y along with

their adaptability to diverse e n v i r o m t s .

Pigempea &an (L.) Millsp.1 the only cultivated

species in -, subtribe Cajaninae is an important pulse crop in

the tropics and ranks f i f t h emong the edible legumes. India has

the largest .area in the world under grain legume cultivation

where pigeonpea oocupies an area of 2.8 million ha. Pigeonpea is

m s t l y ansumed in the form of s p l i t p l s e (dhal) . Green pods

and seeds are used as vegetable in sotw parts of the tropics.

Seed protein content in pigeonpea averages about 23%. The

average yield of pllees on a national basis anpared t o cereals

baa been very low and th is gap continues to widen. The .green

revolution" has not increased the pulse yields because of the

aphasia on cereals. Apart from socio-economic co~siderations ir!

cultivating legumes on marginal lands without fertilizer and poor

plant protection measures, genetic factors have also contributed

to the slow pace of pigeonpea irrproveuent. In pigmnpea as with

m y other leg- the inbreeding behaviour resulted in severe

restriction on genetic variability and inprovement has therefore

been confined to selection and perpetuation of useful kinds of

gene action. Plant breeding history shows that diverse gene

p l s are the foundations for effective crop improvement

programnes. The primary objective in plant breeding is to widen

the genetic base of a cultivated species. If the needed

variation is limited as in the case of pigeonpa the options for

the breeders are:

1. Incorpration of alien vaiation,

2. Induction of nutations or

3. Exploitation of somclonal variation

The a~plicebility of nutation breeding in crop irrgrovment

i s limited since a.,vast majority of mutations are deleterious and

it still r a i n s a hit and trial method with l M t e d scope for

directed attenpts.

clonal variation is a recent technique which offers a

lot of potential for the future but it has not been possible to

exploit these variants on a field scale except in a few crop

(Scowcroft and Larkin, 1982) . Tbe otkr and possibly the most viable recourse of

introducing variation into a species is through transfer of

genetic material from one species to another by hybridization.

The genetic potential of wild relatives is widely damnstrated in

plant breeding and in evolutionary studies. Wild relatives have

helped to fill the voids in traditional breeding programres and

have helped to exploit the potential in several crops (Stalker,

1980).

The first reo~rded interspecific hybrid was made in 1717

between carnation and sweet William by Thomas Fairchild (see

Allard, 1960). Since then there has been extensive literature on

distant hybridization and the first a-made cereal, "Triticale"

was an outcome of intergeneric hybridization. Extensive studies

on distant hybridization have been made in crops like wheat

(Sears, 1972; Sears, 1975;) barley (see Bothmr and Hagberg,

1983), Maize (see MangelsdDrf, 1974; Harlan and de Wet, 1977) , Eblanurn (see Swaminathan and Magoon, 1961; Atskaitis and

Vinitskus, 19751, cotton (Blank & el, 1972; Meyer, 1973;

Meyer, 1974), Nicotiana (seesmith, 1968; Mann & f., 1963;

berbec, 1974) , . : f ~ t o (see Rick, 1982) , and rice (see Nayar, - 1973) .

Distant hybridization studies have also been carried out in

several important legumes such as, Phhsealus, Yigna, W,

E h m , Arachis (5nartt. 1979). Species in the genus E h a s s d u

have been a subject of wide interest. The possibility of gene

exchange between species has led to several studies on

interspecific hybridization especially between P. and

E. coccinwfi (lendel, 1866; Tschermak-Seysenegg, 1942;

Lwprecht, 19481 Rudorf, 1953; Kedar and -is, 1960; Thornas,

1964; Al-yasiri and Coyne, 1966; Rutger and Becknw, 19701

Sna~tt, 1970; m c h a n d , 1971; Marechal, 1971; Ibrahim and

m e , 19751 Elaq & a, 1980; Savwa, 1981; Shii & d r 1982;

Conti, 1983). Hybridization with other ph;rseolus species - including the wild forms has also received wide attention (erz,

1952; Elonma, 1956; m e , 1964; Smrtt, 1970; Braak and

Kogtra, 1975; Le Merchand & dl, 1976; Tan Boun Suy, 1979;

Mrang, 1979). Interspecific hybridization in Phaseolus in recent

years has been widely used in the irrprovement of Phaseolus sp.

with respect to disease resistance, insect resistance, nitrogen

fixation and several agronomic characters (F-li & dl, 1981;

Bmerot & dl, 1981; Zapata e,k al, 1982; Hunter eL al, 1982; - Alvarez, 1981; Lapinskas, 1980).

Wide hybridization has received a fair degree of attention

in the genus -. Studies on hybridization between G. max

and G. soja have been extensive (Karasawa, 1936; Ting, 1946;

William, 1948; Tang and Chen, 1959; Tang and Tai, 1962; Ahmed

& fir 1977 and 1979; Kiazuma stt dl, 1980; Malik and Singh,

1982: Gai gL a, 1982). Atmad & dl (1977 and 1979) made a

detailed cytolcqical analysis of these hybrids and found

structural differences between the two genome6 in the form of

inversions. There have &so been attempts at hybridization

between the wild species (Palmer, 19651 and in 1968 Palmr and

Hadley reparted a successful cross between G. tabacina and

E. latFfolia (E. tomentosa). Later Broue & al. (1979) and - Putiwsky and Broue (1979) obtained two intraspecific Md six

interspecific hybrids. Recently -mil and Eyrmitz (1983)

reprted a range of intra and interspecific hybrids in the

subgenus Glgche along with detailed cytological studies in the

hybrids. Attaopts at hybridization between cultivated m w

and the wild species were m t successful with the conventional

methods (Ladizinsky & & 1979; Hood and Allen, 1980). Newell

and Hymswitz (1982) were able to obtain a hybrid between E. mix

and P. tomentella with the aid of ovule culture. Apart fran

alding in understanding the species relationships, interspecific

hybridization in G l y s = h has also helped in widening the genetic

base for quality characters of P. max particularly protein

content (Ala &dl, 1979; Erikson and Beversdorf, 1981; Kiazuma

& al, 1980).

%e genus Y i d a exhibits dysploidy with x= 5, 6 or 7. Host

of the interspecific crosses m far involve species with

chrmsome nuhers x=6 (subspecies aativa, &d nigra) . Hybrids between 2n=10 subspecies (e have also been studied

(Mettin and Hanelt, 1973). Very little informtion is available

on crosses awng.' subspecies with the chromsome nrnober 2n=14.

Crosses betwea~ types with the same chromosome nuher result in

fertile or sanifertile hybrids and meiosis in the F1 hybrid$

indicated the presenceof chromoscinal structural hybridity as

evidenced by heteromrphic bivalents, trivalents, quadrivalents

etc (Mettin and Hanelt, 1973). Crosses involving forms with

different chramsome n h r s have also been attaqrted (Zohary and

Plitmann, 1979) and hybrids were obtained in numerous

ca7binations. Same of these hybrids erhibited hybrid vigour . Hybrid between a&bza (6) and llxdaka (rcf) shoved pentavalents

in the rl :cieratioii aid ir. F2, segregants st~~wetl diffrru.~t= in

chrcmeome nlmbers ranging from 11-14 (Nerson, 1970). W a t a n a b e

and Yamada (1958) reported crosses between aativa and

ancrustifolia in which the pollen fertility was less than 5% and

the F2 segregated alsrost exclusively for parental types.

Yamamto (1971, 1974 a,b) studied crosses between e, m&kaz&a, gihaa and and in the hybrids he found

poor meiotic pairing and consequently low fertility. He reported

a stable kasyotype produced by the substitution of one chromsome

of satFva in a karyotype derived from the

interspecific hybrid. Later x e (1975) studying the anylose

isoyme pattern in a Pilosa x -gm hybrid showd the

occurrence of reconbination In the hybrid progeny. Cubero (1982)

has sumnarized the interspecific hybridization studies in the

genus W. In general hybridzation studies in this genus have

shawn that the various chromsome types are not fully

reproductively isolated from one another, and hybridization

studies have also.: shown that hybridization and recurrent

reconbination between distinct karyotypes antributed to the

build up of chrolnosomdl polymorphism. Species crosses in this,

genus helped in the generation of variability for traits like

seed hardiness (Donnelly, 1980).

In the genus Yigna the closest relative of green and black

gram (Y. t.adiata and Y. mu& is Y. s&hkU. Hybridization

between these species is achiwed with relative ease (AIIRDC,

1977) . Eybridization studies betxeen Y. w, y. nuugrz,

y. LmS3ellulata and Y. mgubds have been numerous (Dana,

1966a,b,c; Al-yasiri and CoyneL1966; Biswas and Dana, 1975,

1976; Smrat 1973; <hen & & 1977; and Chowdhry,

1977; Md H a r m , 1977, 197&, 1978b; machado & pl,

1982; Chen & 1983) . Although Machado EL f. (1982) f ran

their cytological studies could not conclude whether the pairing

observed in the interspecific hybrid was autosyndetic or

allosyndetic, Chen & fir (1983) from their F2 studies concluded

that reemhination and gene exchange takes place between these

species. In !,?igna also the related species have been frequently

used for the transfer of cha.racters like disease resistance and

protein content (Singh, 1981; Gill & &, 1983; and Lukoki and

Otoule, 1981).

Several wild Arachin species have been successfully crossed

with the cultivated species A. &agaea. The cultivated species

crosses easily with A. mrrentina. The first report of this

cross came from Krapvickas and Rigoni (1952)' with subsequent

reports from Kumar & al (1957) and Raman (1959). Gregory & al

(1973) have produced hybrids between A. and wild .;I

diploids in the section &a&h. These hybrids have also been

reported by Mss (1977). Raman (1976) has reported hybrids of

a with A. y i J . l ~ ~ and A. duarmda. Intersectional

crosses sUCh BS Arachin . x -, Eredoidefi x

Ereceoidesx-,-XErectoides.

and x Erectoids are possible (Gregory et & 1973). In

addition Raman (1976) reported hybrids behveen A. and

A- -, A. hme&&di and A. d b s i and between

A- montimle and A. gl&r&a though the authenticity of m y of

these has been questioned. Inspite of ploidy differences the

wild species of Amdais have gained a lot of impbrtance with

respect to transfer of specific characters like resistance to

insects, viruses, nematodes, fungi, etc. (ICRIW, 1982).

Rybridization between species of Elaun (Mtiwm, elatius,

hknnik, f u h m and &sshhml has also received sane attention

(Zohary and Hopf, 19731 Gritton and Wiezbicka, 1975; Belova,

1979 and 1980). Success has also been repxted from crosses

involving Eislrm and U c h (Stegajlo, 1963; Sobolev eL a, 1968; Sabolev and Burgil, 1970) . The chromosome nlmber of these

hybrids was between 12 and 16 (2n) . Sobolev et al (1968)

reported mnhtmulogous chromsome pairing in the h*rids and the

chromosomes formed distinct groups.

Cultivated pigeonpea is bestowed with a wealth of related

wild species in the genus m. The genus is also

cansidered close to -. All the species of and

have the same basic chromomme nlmber as that of

(X=ll). Ther6 are about 34 species of Atylg&a, of which

18 are +Bnic to Lndia, 15 are natives of Australia and one

species is found in Africa (van der Maesen, pers. comn.) . The

taxanomic delimitation of fajmw and into distinct

genera which has been dbne on the basis of the seed stro@dole

has been a point of contention (Mac ChbL1975; van der Maesen,

1980). Many of the A Q l m h species possess ~ e ~ e r a l desirable

characters such as disease and pest r e s m c e , high protein

content, plmtoperiod insensitivity, drOUght tolerance

(Remmandan, 1980). Inspite of limited variability for certain

imp3rtant qualitative traits in C. Eirjan and the existence of a

wide wealth of species, studies on intergeneric

hybridization in pigeonpea are surprisingly few and far btween.

Naithani (1941) was the first to give details atout the

meiotic division of pigeonpea although it was M y (1933) vtso for

the first tine reported the chromsome n-r of cajanus as n-11

which was donfirmed by Akinola & al. (1972). Krishnaswamy and

Ayyangar (1935) provided information about meiotic pairing and

&aim frequencies in pigeonpea and this was followed by the

studies of Kumar & al (1945, 1966) , Bhattachax jet? (1956) and

Reddy and De (1983) all of which mentioned normal &om-

pairing and regular formation of eleven bivalents at meiosis.

The karyotype description of Naithani (1941) showed no

variation for chromsome morphology between variet'ies. This

attempt was followed by that of w i k a r and Tbakur (1956) who

gave the total conplemnt length as 75.4um.

These initial attenpts were followed by the works of

Shrivastava & al (1973) and Sinha and Kumar (1979) wlw reported

significant varietal variation for a m ratios and &orno&

lengths and by those of Sharrna and Gupta (1982) and Pundir

(1981).

There have been two publications on pachytene karyotyp

analysis (Reddy, 1981a and Dundaa ek al 1983). While Reddy

identified the pachytene chronosomes by the length and centramere

positions, Dundas & al (1983) used chronmmere patterns for

chromsore i6e~t i f ication. Only recently the C i e ~ ~ a C banding

techniaue was attempted for Riseonpea without much success

(mania and Lavania, 1982).

The satellite chrotmsome n h r s in pigeonpa have been a

subject of controversy and from the literature one can see that

thenmber varies from0 to 2. Sinha and Kcmvlr (1979) a d

Shrivastava s.t dl (1973) reported that some cultivars used in

their studies had one Satellite (SAT) chromsoxre while others

have none. The karyotypes presented by Shanm and Gupta (1982)

do not show any SAT chromsome while those of Pundir (1981)

showed t w . ArrPng the species of Atvlosia. A. lineata was used

extensively by Kunrar & dl (19581, Sikder and De (1967) and Reddy

and De (1983) in their studies on meiotic pairing. K u t m r & &.

(19581, Deodikar and Thakur (19561, Sikder and De (1967)

Shrivastava & (1973) and Pundir (1981) studied the somatic

karyotype of A. lineata and reported a close relationship between

the karyotypes of Gujaws and mineatn. Sikder and De

(1967), Reddy (1973) and Pundir (1981) studied the karyotypes of

A. lineata. A. a and A. ecarabaeoides. The karyotypes of.

these species were also presented by Reddy (1981 a,b,c) based on

pachytene chromsorne analysis. The other species for

which chrcmsome nmbers were'reported were A. (Bir

and K m i , 1973, 1977; k d i r and Singh, 1981); A. mgaa

( S a n j e and Satyananda, 1979); A. vblubllifi (Rao, 1978 and

Pundir and Singh, 1978) 1 A. f ' *% (Radir and Singh, 19781,

Atvlofiia (Rao, 1978 and Pundir and Singh 1978) and

A. caianifalia (punclir, 1981).

Attempts at --- hybridization are presented in

Table I. The first report on intergeneric hybridization in

pigeonpea dates back to 1956 when Deodikar and Thakur crossed

C. cajm with -. The hybrid was fairly fertile.

K- EL al (1958) extended the earlier work on to hybrid

cytology and found regular bivalent fonmtion in the hybrid. A

hybrid betwem C. and A. Bcarabaeoides was obtained by Roy

and (1967) who expressed doubts about the generic status of

A t y b s h . Rec?dy (1973) analysed pachytene chromosome pairing in

Ca ianusa , Btvlos ia l ineata ,A. -andA.ser icea

and their hybrids. These pachytene studies in general revealed a

high degree of chromosome homDlogy between C. and the three

species of -. Studies on the krheritance of a few

qualitative traits was also done in this study. Miya~yagarn and

Spence (1978) reported hybrids between Qjaua and A.

while further attagits (Pundir,l98l; Re&3y et al, 1980 and in

the present study) to cross Sajdnus with & failed.

Further attempts at fajmus - hybridization by Pundir

(1981) involved karyotype conprisons between the cultivated 'and

the wild species and meiotic pairing in the F1 hybrids. These

studies revealed a great degree of karyotypic similarities

between species. All the studies on ~ - & y & A a

hybridization revealed a close relationship between the species

of the two genera and regular pairing in their hybrids which

nevertheless exhibited a fair degree of sterility.

T a b l e I. Studies on i n t e r g e n e r i c h y b r i d i z a t i o n i n plgeonpea.

Year &ykxJa species used Authors

1956 A. Deodikar and Thakur

1958 A. Kumar at al

1967 A. scarabaecldea Roy and De

1973* A. w. A. and Reddy A. sar5aa

A r l yanayagam and Spence

1981 A- a l b t c a n s r A. serlcea, A. Pundi r A. scarabecldes, A. trlnervea and A. calanlfolla

1984 A. ceLldAa (sub-sp. retlculata). A. Dundas 0-a and A. auilhLh

*This work was publlshed I n 1981.

bcept at ICRfSA!T there bas been no report on the use of

AtylQah species in pigeonpa breeding progrms. At ICRISX!

their use is mstly confined to breeding high protein lines

(Reddy et al, 1979) and to a limited extent for breeding for

insect resistance, dwarfs and isolation of cytoplasmic m l e

steriles (L.J. Reddy pers. cam.). Only a few &yh~& species

have been used in this direction so far. While there has been a

rapid accurmlation of literature on distant hybridization in

other crops, the limited interest in pigeonpea in this regard

stew from the small chromosome size and the paucity of attanpts

in distant hybridizations in pigeonpea. Genome relationships

between C a j a u ~ and Atvlafiia species is still obscure.

A. &mi&Ua, which is mrphologically very similar to

except for the seed strophile, was identified.as early as 1920

(Van der Maesen, 1980) but an attempt to cross these two species

was not reported until 1981 (Pundir) . A clear .understanding of the cytcgenetic relationships

between the cultivated and wild species is a prerequisite for

the effective use of wild species in the crop's inprovement.

Distant hybridization is nostly aimd at introducing new genetic

variability or to achieve a new g a m i c constitution in such a

way that the characters of the parental species m e recmbined

effectively. These possibilities are directly related to the

degree of genetic relatedness between the parents. It has been

found that, the closer the gemme relationship between tbe

cultivated and the wild species the greater the m t of genetic

recmbmation and consequentry variability.

Apart from being a swrce of novel genes and variability,

wide hybridizations have also prove3 to be a potential means of

haploid production. For instance Kasha and Rao (1970) reported a

high frequency of haploids from crosses betweM 8. yulgazs and

B. kulhsun. These mlgxs haploids originate from fer t i l iza t ion

followed by selective elimination of bultaim chrorrosom during

early stages of &ryo devel-t (Subrahrmnym and Kasha,1973) . Selective chrctrosome e l m t i o n leading t o haploid formation is

widespread auong ktxiiem interspecific crosses (Subrahnwyam,

1977, 1979, 1980) and also from intergeneric crosses (Barclay,

1975; Shigenobu and Sakaaoto, 1981; Botkmer & &, 1984).

Barclay (1975) reported wheat haploids (polyhaploids) from

crosses between X. aestivMl and El. hulk^^^^. Selective

chromosome e l m b a t i o n is also known in hybrids between

a. h k a m and B. . . . ( G w and G u p t a , 1973).

Haploid production via chromeome elimination has become a

successful ccanrercial venture in Barley breeding (Kasha and

Reinbergst 1980) ':

. Pigeonpa being a long duration and pbtosensit ive plant can

nonmlly be grown only once in a year. This places a severe

constraint in pigeonpea breeding p r o g r m s . Developnent of

haploids in pigeonpea would have an inmeme value in shortening

the time required for breeding a cultivar. UnfortuMtelyt the

anther culture technique has mt shown promise in any of the

l a w species. From the experience in barley it a d

worth while t o screen Gaj.am5 - A t y h s h crosses for selective

chrorroale e i h L . ~ ~ i u r r as a a u r c e of haplcids.

Pundir (1981) reported that g ~ ~ e of the species

used in his study failed to cross in any direction. Natural

barriers to interspecific hybridization may be of p r e or

post-fertilization nature. The advent of the d r y 0 culture

technique has helped in overcaning such problens especially the

post-fertilization basriers. Rescue of interspecific progeny

through ' d r y o culture was done for the first time in a Liam

x L. austtiaenn cross by Liaba_- (1929). Raghavan (1977)

lists 75 species (updated by Stewart,l981) in which the &ryo

culture techniques have been standardized. New hybrid

ambinations through the use of d r y o culture include sweral

legumes (lionma, 1955; Bajaj and Bow, 1971; Braak and Kooistra,

1975; Alvarez et al, 1981; Mok & al, 1978; Ann and Hartman,

1978; Newell and Hymwitz, 1982; Sastriand Eloss, 1982; Gosal

and Bajaj, 1983; Chen et al, 1983).

In several instances it has been fcund that the &ryo

culture technique helps in rescuing the hybrid progeny only in

dination with h o m n e treatments (Islam, 1964; Kruse, 1974;

Bajaj 'k al, 1980; Alonso and K k b e r , 19801 Sastri st al, 1981 r

Sastri and MOSS, 1982; Kuwada and Mabuchi, 1976) . Honmne

treatments prolong the enbryo deve1-t on the fenale parent so

that the d r y 0 at excision is more enenable to culture.

Further, hormone t r e a w t s are also known to *rove the rate of

success in compatible crosses. In tbe light of the literature it

seaaed worth while to standardize eubryo culture technique for

pigeonpea and to study the effect of bornrme treatments in

intergener~c crosses invoiving pigeonpea.

Cbtaining species hybrids and assessment of g m

relationshfps is a first step in the exploitation of wild species

in the Lrprwement of any cultivated species. The next logical

step is the utilization of such hybrids in the breeding programne

before which it would be essential to study the inheritance

pattern and also assess the quantum of variability generated.

Studies on inheritance provide information on the ~ossible n&r

of genes governing a character and their interaction. Evaluation

of the variation in the F2 generation help in understanding the

extent of reconbination and variability. Genetic studies provide

a clear direction to the handling of segregating generations.

There have been several studies on the genetics of qualitative

and quantitative traits in pigeonpea (Deshpande and Jeswani,

1956; D'cruz and Deokar, 1970; l-kmoz and Abram, .1971; Pandey,

1972; Sharma & aL 1972; Joshi, 1973; Chaudhary and

lbmbrer1977; Dahiya and Brar, 1977; Dahiya & aL 1977;

Kapr, 1977; Malhotra and Sodhi, 1977; Re&3y eL Bl, 19791

Sidhu and d u 1980; Saxena and Shame, 1981 to n a ~ a few).

Only tm studies have so far be^ amducted on the genetics of

x &yUxia crosses (RedCty F& tt, 1980 and Pundir, 1981).

Tissue culture technique are emerging as a major

sqplanentary aid to traditional plant breeding p~ocedures in

crop inprwement prograrmres (Vasil SL al., 1982) due to their

increasing importance in clonal propagation (Hura- shige, 1974) , productim of haploids (CMh-Chhg Chu, 1982), production of

disease free plants (Gengenbach ek dl, 1977) and induction of

genetic variants (Scowcroft and Lah~nr 1982).

A basic knowledge of differentiation ie essential for the

effective use of this technique. Tissue culture techniques have

been developed for several grain legumes (Kartha & B;LI 1981) but

such studies on pigeonpea are limited (Rao and Swwr 1975;

Mehta and Ram, 1981). - In.the light of the above considerations and constraints the

present investigation was undertaken to understand the

relationship between Cajaus and & & h s h and Rhvnchosia in order

to allow easier transfer of useful t ra i t s from the wild t o the

cultivated species with the following broad objectives:

1. Assessment of crossability relationships between

Cajaus, and certain Atvlofiia and species.

2. Devel-t of methods to inprove species crossability.

3, Assessment of genome relationships between c. and

its wild relatives.

4. Screening for haploids produced by selective c h t m m

eliminathn in Ujmus - Atvlofiia crosses.

5. Study of the inheritance and variation for qualitative

and quantitative t rai ts in the intergeneric hybrids and

6. Develop& of in vitra regeneration techniques in

pig-npea*

S- m: The Genetic Resources Unit, I(IRISAT was the swrce

of seed for all the &Q&a and &yncb&a species while the

seed of the cultivars of C. w a s obtained from the pigeonpea

breeding subprogram a t IW-. The salient morphological

features and eammic inprtance of the species used in this

study are presented in Tables VIII, M and X I and Table I1

respectively.

PLANP CULTURE: The f ield investigations in the present study

were carried out a t the IClUW research farm which is situated

180N, 78 E a t an altitude of 536 m. Prior to planting the land

was ploughed, harrowed and ridged. The f ie ld was divided into 4m

plots and the seed was sown rnanually a t a spacing of 3 0 6 0 an

between plants depending upon the plant habit and 75 rn between

rows. Support w a s provided wherever necessary for the clinbing

types. In ckses where plants were gram i n pots the potting

mixture consisted of three parts of m i l and one part of farm

yard manure.

Seed of the wild species and also the F18s possess a hard

seed coat, hence their seed w a s sacrified with a scalpel before

eowing to fac i l i ta te easy germination. Irrigation was provided

whenever necessary. In f ield plantings hand weeding was done

tvice to keep the crop clean. msulpban (0.2%) was sprayed a t

regular intervals to check insect damge.

E Y B F U D I ~ O N : In C. G&ZJ flower opening begins i n the morning

a t about 7 A.R. with Li.6 anthesis continuing unti l l a t e in the

Table 11. Or ig in and s a l i e n t economic features o f t h e AtvloE(a species used i n t h e study.

Species Acc. No. Or ig in Important Charac te r l s t i cs --------------------------------------*--*---*-----------------------------

A. alblcans NKR-177 Asia S t e r i l i t y mosaic r e s i s t a n t and high seed p ro te in content

A. calanlfolla PR-4876 Asia High seed p ro te in content

A. g C a U f d a PR-4221 Austra l ia High seed p ro te ln content

A. lanceolata CQ-1619 Aus t ra l ia Frost and drought t o l e r a n t

A. J d L b g a h CQ-1618 Aus t ra l ia F ros t and drought t o l e r a n t

A. JiL-mh JM-3366 Asia S t e r l l l t y mosaic r e s i s t a n t and h igh seed p ro te ln content

A. rnnuls JM-4331 Asia

A. @&SUQA PR-4557 Asia B l l g h t res is tant , annual. high seed p ro te in content

8- LuQQ= KM-4180 Asia An t lb ios is t o W&&t& armlaera and h igh seed p ro te in content

A. scarabaeoCdes JM-2367 Asia An t fb ios ls t o srmlaera and h igh seed p ro te ln content

A. sericea JM-1961 Asia B l i g h t and s t e r i l i t y mosaic res is tan t and high seed p ro te ln content

A. Yolublllr JM-4208 Asia S t e r l l i t y mosaic res is tant ' and high seed p ro te in content

afternoon. The flowers raMin open for &xut 20-24 h and the

anthers dehisce before flower opening. The species of

have a similar floral biology except that they have delayed

flower opening in some cases. Anther dehiscence in all the

Atyhs ia species occurs before flower opening.

FOE hybridization, buds of the awropriate size were opened

with the help of a forceps and anthers -re rerroved without

injuring the stigma. The forcep was dipped in spirit after each

emasculation. Initially two methods of pollination were

attanpted.

1. ESMsculation and imnediate pollination and

2. Ebsculation and pollination on the next mrdng.

The first method proved to be muginally better and it was

followed in all the later studies.

EXMXE -: Cultivar Pant A2 was us& as the Gajaws

parent for ho-ti: treatment studie6. Gibberellic acid (GA3) and

kinetin were used independantly at concentrations ranging from 19

to 80 p or as a 1:l mixture keeping the net honmhe

concentration in the szaw range (10 to 80 Am) . A ~o:22

hypodermic needle was used to place the hormone solution so as to

fill the bud cavity surrounding the pistil. The tredtments were

given twice, 24 and 48 h after pollination. Care was taken to

avoid physical injury to the buds &ring hormone application.

Cne hundred pollinations were made for each treatment. Bud drop,

p d set, pod length, and seeds per pod were recorded for each

cross and treatnent .

CX'KWSZCAL lEVE9lXGRTI(TZS: Por cytological studies buds of the

eropriate size were fixed between 9 to 12 h in Carnoy's fluid

(6 parts ethyl alcohol: 3 parts chloroform: 1 part glacial

acetic acid). After 48 hours in the fixative the buds were

transferred to 70% alcohol and stored in a cool place. For

chromsoml studies the anthers were squashed in 1% acetocarmine.

For nucleolar -ring the anthers were first squashed in 1%

acetocannine followed by destaining with 459 acetic acid and

restaining with 2% acetocarmine. Detailed observations were made

on metaphase configurations, anaphase disjunctions and nucleolar

size and distribution in the parents and hybrids. Chi-

frequencies were recorded in both parents and hybrids.

Observations were also made on pachytene chromosome pairing.

KELEN FERJ!ZITY: Pollen stainability in 1% acetocannine was

taken as an index of pollen fertility. All shrivelled, unstained

and poorly stained grains were counted as steriles, Pollen

fertility was recbrded for all the parents and hybrids.

-: Desirable -11s were photcgraphed using .a

piwtomicro8~0pe with oil intersion lens end a green filter.

Kodak high contrast copy film (ASA 64) was used for micro

EuK?lXm WICKSaXY: Laaf bits were made to adbere over a thin

film of dolite silver paint on specimen stubs, dried and were

sputter coated with a thin film of gold. Photograph were taken

on a JOEG35 scanning electron microscope.

FEWlXIN ESFDMTON: Seed protein percentage was determined

with the aid of Technicon Auto Analyzer. Fifty grsms of seed

q l e was needed for the estirmtion of protein content.

GENETIC STUDIES: The F2 poplation was raised from the croases

C. G&II X A. db,h~IS, C. & x 8. caianifoliat C. X

A. sericea, C. rajan x 8 . - and C.- x

A. UneaLa. The germinability of F2 seed is presented In

Table 111. In all the above five hybrids the Ca.ianus parent used

was Pant A2. The F2 populations were scored for seed strophiole

(presence, absence) , seed mttles (presence* absence), pod

hairiness (short hairs, long hairs), leaflet shape (lanceolate,

intermediate, type) and twining nature (ere,

intermediate, twining). The plants were classifie into distinct

groups for the above characters and the goodness of fit of the

&served segregation was calculated by the standa~d chi-square

test using the following formula

Where T = Sumnation

Observed frequency and

& Expected frequency

The variation in the F2 popllation was oorrptted for the

characters, mid-leaf length, mibleaf width8 @-length8 seeds

per pod and ICr-seed weight. Pie leaf measurmcnts were I X ~ C :r.

Table 111. Seed germinabi l i ty I n F2 populat ion of hybr ids between C. and Atvfasla species.

Populat ion No. sown No. germl- Germlnatlon source nated percentage

the terminal leaflet and the megsurements were on the average of

measurements based on five -lea. Observations on pod and seed

characters were from the meture pods.

IN VITFQ -ON SIODIES: Ms medium (Murashige and Skoog,

1962), B5 medium (Ganbrg & & 1968) and White's d i m (White,

1939) &re used as the basal media. The capsition of these

media is' presented in Table IV. Apart fran these media, potato

starch extract d i m (Anonymus, 1976) was used in anther

culture experbts. Stock solutions were prepared for the

different hormones used in the study and used to swlenent the

basal media depending upon the requirement. Sucrose at 39

concentration was used as the arbon source. Solidification of

the medium was achieved by Difco grade agar (8%) .. The gS3 of the media was adjusted to 5.6-5.8 by means of 0 . N IKZl or O.lN m. The media were sterilized by autodaving.

In uitrp .&eneration response was evaluated from the

cotyledonary explants of the fdjaua cultivars I B 4726,

I B 7035, Pant A2 and GS 4 and from A. caianifalia, A. aUk=am

and A. m. The regeneration potential of distal Md nodal

halves of cotyledons was evaluated. The Gajama cultivars,

Pant A2, ICP 7035, Prabhat and C 11 were used to stfvldardize the

mlture conditions for regeneration from h t u r e errbryos. The

in yifrP response of the anthers of C. &an (Pant A2),

A. rlbicans. A. atandFfdlia and A. Mlubilis yea tested.

GZXXE C(;W)ITIQB: S& (for c,.,;&ri and enbryo u r i i u r u )

Tablm I V . Constltumnts of dlffmront basal w d l a used In W ragenration rtudlms.

Constituents Murrshlgm Whltm Gamborg (mg/ll and Skoog (19191 SZ dl

(1962) MS (1968)85

and flower buds (for anther culture) were surface sterilized for

15 minutes in 10% chlorox ((BL(3WaX, USA), washed thrice i n

s ter i le water and retained for about half an bwr in sterile

water and dried before inoculation. For ootyledon cultures, the

seed coat was rem~ved and the seed was spl i t vertically to get

t m distal and two nodal halves. A l l the operations were

conductd under asceptic ~ 0 n d i t i 0 ~ in a laminar flow hood. The

a l t u r e s were maintained a t a tenperature of 25 G ' C under cool

fluorescent llght (6.8 watts m-*) .

Eight species of (8. -, A. caianifolia,

A. lineatat A. sericea, A. scarabaeoides, A. orandifolia,

A. lanceolata and A. latisepala) hybridized successfully with

C.- while the other four AQAda species (A. mollis, A.

M1UbiliR. A. w- and A. ruaosa) and the two &ynch6h

species (B. rothii and R. minirrra) used in this study failed to

cross with the cultivated pigeonpea. The - crossability was influenced by the gemtype of the Cajanu parent

( 9 ) and the cross anrbination and the hybridization attempts in

all resulted in 24 successful corrbhations (Table W .

Fumng the 8 successful Cajmm - A t y h a h crosses, the

crossability was highest when A. lineata was the male parent with

19.5%, 12.30%, 2.37% and 15.20% of the pollination resulting in

hybrids in crosses with Pant A2, Bdigani, ICP 7035 and C 11

respectively. This was followed by crosses involving A. rlbicans

ae the male parent with the successful pollinations averaging

9.16% with Pent A2, 7.23% with Baigani, 1.09% with I B 7035 and

5.12% in crosses with C 11.

The degree of success was low in crosses involving

a. A. scarabaeoides and A. caianifolia as d e parents.

A. scricea as a pollen parent yielded hybrids in 2.36%, 3.03%,

0.75% and 1.7% of pollinations with Pant A2, Baigani, ICP 7035

and C 11 respectively ar,~ when urr polirri sour-cr w a s

A. scaraboeides the crossabi l i ty with the respective C&ws

parents w a s 4.61%, 2.35%, 0.47% and 1.46%, while in crosses

hVolv.ving A. caianFfolia 2.70, 3.05%, 0.58% and 2.09% of t he

pollination with Pant A2, Baigani, ICP 7035 and C 11 respectively

resulted in pod set. Crosses involving the genotype I B 102 with

t he above mentioned species of fai led. In crosses

involving the three Australian the crossabi l i ty was low

with a pod s e t of 1.72% and 3.07% in Pant A2 x A. and

C 11 x A. arandifolia crosses respectively, 0.9% and 1.96% in the

crosses Pant A2 x A. and C 11 x A. and

1.69% and 0.93% in PantA2 x 8.- and C 1 1 x

A. lanceolata conbinatiow.

Reciprocal success of a very low frequency was obtained in

two cross oonbinations viz. A. albicans x S. &.an ( .22%) and

A. aericea x G. & (.IS%) (Table V) . In the crosses A. mollis

x C. rajr7n and A. volubilis x S. pod dwelopnent in the

crossed buds was mrmal but the seeds from such pods were

extremely shrivel& and inviable.

Seed set per pod also varied with the f e e genotype, with

ICP 7035 m i s t e n t l y averaging a higher n m b r of seeds (about 3

per pod) (Table V) . In the study in which Ii-aUkana and A-serfcea were used a s

the male parents in crosses with 1- 32, ICPL 47, ICPL 59 and

1- 95 all of which have a commn female background in t h e i r

parentage variation in the crossabi l i ty was narrow ranging from

2.5% to 5.5C m crosses k l t n A. slblcans and 1.5 t o 4% i n those

Table V I . Crossabi l i ty o f specles with G&nus having a comnon female background*.

Cu7 t i v a r Parentage A. albLanS A. adGm -------------- -------------- a b c a b c .................................................................

ICPL 32 T 21 x B r a z i l 1465 200 5.5 31.6 200 3.5 32.9

ICPL 47 T 21 x Ja 2772 200 5.0 36.2 200 1.5 34.7

ICPL 59 T 21 x EC 100467 200 4.5 34.9 200 4.0 29.8

ICPL 95 T 21 x NP(WR)lS 200 2.5 33.2 200 2.5 31.4

--

*a: ~urn* o f p o l l lnat lons attempted b: Percentage o f successful p o l l l n a t l o n s c: P o l l e n s t e r i l i t y I n t h e F1 hybrids

3 1

involving A. (Table VI) .

C. cv Pant A2 was the fanale parent in the hormone

studies. The successful conbinations exhibited a unif o m

response to hormone treatments. GA3 was found to be suprior

irrespective of the cross &ination. For e x q l e when

A. arandifolia was the pllen parent, *set increased from 2%

in the control to 14% in treatments with M p of GA3. The

optimum concentration of GA3 was found to be 40-50 m. Concentrations above 50 @ were detrimental with ocmplete

failure of pcd-set at 80 m or above. Kinetin trf?atments showed

no oonsistent response at the concentrations tested. Treatments

with GA3 + Kinetin mixture also did not inprove pod-set but were

detrimental at higher concentrations. Similar trends were .d

aparent in crosses of C. s&an with A. albicaw,

A. s+d&lh, or A. sericea as m l e parents (Table VII) . In the four successful crosses studied for hormone response,

GA3 alone or in dination with kinetin increased pod length and

seeds per pod (Fig. 1). In these crosses the average pod length

at physiological maturity increased from 4.5 to 5.5 an in the

control to 7 cm. Furthemre, the seeds per pod increasd from

the range of 1.6-2.2 in controls to 3.5-4 when Gh3 or GA3 +

kinetin treatments were given. W w e r , honmne treatments did

n: t i~fluarce reed size.

F i g . 1 . Effect o f gibberel l ic acid and/or kinetin on percent pod and seed se t in four C q j a m s - A t y l o s i a crosses .

norane concentration

F i p u r c 1.

AmDng the wucceesful crosses bud drop comnenced within two

days after pollination (Fig. 2) . GA3 treatments prolonged wary

Wel~pnent and delayed bud drop by varying periods depending

upon the cross carbination and the homne concentration (Fig. 2).

For wmqle, when A. and A. Mlubilis were the pollen

parents, bud drop cannenced two days after pollination in the

control. GA3 prolonged this period up to five days at 60

concentration in A. volubllls . . and at 50 ppn concentration in

A. -. When A. nallis was the male parent bud drop was

delayed by 3-4 days. Increase in ovule size was evident in cross

cabinations where bud drop was delayed following hormone

applications.

In all the reciprocal crosses there was variation in

response to hormones (Fig. Zb) . A. sericea and A. arandlfolla . . failed to respond to any of the three treatments. In

A. GA3 delayed bud drop while GA3 or GA3 + kinetin treatments were ineffective, a trend which was similar to the one

observed in unsuccessful f&ws crosses. Kinetin at a low

concentration (10 Hm) prolonged ovaq developmnt by 3 days. in

A. platvcarDa and 4 days in A. -.

Fig. 2. Effect of gibberellic acid and/or kinetin on delaying bud abscission.

a . Cajanus cajan as female parent b. Cajanus cajan as male parent

Days GA?+Kl n Kin

In gross morphology the F1 hybrids in general tended to be

intermediate. Atvlosia characters like seed strophiole, seed

mottling, pod hairiness and persistence of petals were expressed

in the F1. A oarparison of parents and their hybrids for

qualitative and quantitative traits are presented in Tables VIII,

IX and X, and a hybrid wise description is given below.

The hybrid was a twiner and had a leaflet shape intermediate

between the parents (Fig.3B) in ita initial stages of growth.

'Ihe intermediate leaf shape in the hybrid continued for about 100

days. LXlring this period the leaf tip was obtuse.. The leaves in

some of the branches (Fig.Sa-leaf 2) which pea red after 100

days resabled the Cajanw parent, having an acute tip

(Fig.5C:leavess 4-6). However, as the plant grew further the f

remaining branches aleo developed leaves similar to that in the

Qijanus parent. Although the change in leaf shape was observed

in all the three leaflets it was most p r o m & in the terminal

ones. The texture of the leaves produced after 100 days atso

reserrbled the C ~ ~ B U I E parent. Scanning electron microscopic

studies of the upper surface of the terminal leaflets revealed

slniluities in the leaf surfaces of the Sajanu parent (Fig. 5d)

a d the --like leave6 produced in the hybrid (Fig 59) . m t h ihe leaf surfaces exhibited long trichomEe with uni£orm

spread. I*?zv,es of the A. albicans -rent had a very dense

population of tr ichoims (Fig. Se) , whereas the initial

Table IX. Expression o f quant i ta t iave t r a i t s I n parental . p r i e s and F1 hybrlds batweon C. a c v . Pant A2 and A w Q A A sp.ci.s.

Character Specles/Hybrld ------------------,-------------------

Midleaf Midleaf Pe t i o l e Days t o Durat ion length width length f lower of f l o - (cm) (cm) (cm) r e r f ng .................................................................

C. a 7.13 2.85 4.1 65 Sep-Jan cv Pant A-2 - 8. alblcans 4.41 3.82 4.3 155 Dee-Mar

C. ralna x 5.3 3.3 6 .O 140 Nov-Mar A. - 8. SBJxba 2.51 0.63 1.52 126 Nov-Mar

115 Nov-Mar

76 Sep-Mar

85 Oct-Feb

118 Oct-Ma r

123 Oct-Mar

160 Dec-Mar

l30 Nov-Ap r

Maintained as perennl a1

C. U x 7.7 4.15 5.8 155 Dec-Apr A. gLum-uA

S. Gahn x 6.34 3.96 4.6 172 Dec-Ma r A*

A. h x ~ ~ k t ~ 9.4 0.82 1.5 222 Fob-Apr

Table X. Expression o f quantltat iave t r a l t s In parental . p a l e s and F1 hybrlds between C. & cv. Pant A2 and Atvlosla species.

Character Species/Hybrld ...............................................

Pod Chambers Seeds/ 100-seed Seed pod weight p ro te ln

(cm) ( X )

c. a d n cv Pant A-2 5.93 3.8 3 .7 6.65 23.95

I;. r;aiaa x A. P 3.74 3.5 2.6 4.25 28.33.

C__

intenoediate type leaves had a eparse poprlation of short

trichomes (Fig-Sf) when caqxued to the leaves of the r;iianus

parent and the caianus like leaves in the hybrid. Floral

initiation took place only on tk branches which had developed

leaves similar in shape and texture to the parent. The

pods in the hybrid were flattened and glabrous with an average

length of 3.91 an. The chaxbers per pod averaged 4.2 and the

seeds were strophiolated and mottled. The seed weight (4.92 g)

was in between the parents with protein content (28.95%) close to

the &yb&ia parent (30.09%). Petals in the hybrid were

persistent and the hybrid flowered from N o v ~ r ~ c h with the

first flush a~pearing 140 days after sowing.

The F1 hybrid was erect with a leaflet s h w towards the 4

Akybsh parent (Fig-4D). The leaf length (7.97 cm) and the leaf

width (3.34 cm) in the hybrid were higher than the better parent

(Table MI. The first flush b. the hybrid a m e d about 130

days after sowing with the peek flowering period falling between

Mvenber and April. Petals in the hybrid were persist&. Rd

mrphology was cloee to the Uyhaia parent. The pods were

reddish green in colour with dense hairs and had 2-3 chsmbcrs

with an average of 1.6 seeds per pod. The pod length averaged

3.05 an. Tho seeds were mttled and strophiolated. A 100-scsB

weight of 5.15 g, and a protein content of 27.39% was recorded

(Table X)

F i g . 3 . Twigs and l e a f l e t s o f parents and h y b r i d s .

A) a . Cajanus cajan

b . Hybrid between Cajanus c d a n and AtyZosia scprabaeoidt

c . Aty Zosia scarabasoides

8 ) a . Cdanus cqjan

b . Hybrid between Cajanus cajan and AtyZosia aZbicans

c. AtyZosia aZb icas

C) a . Ca,janus cajan

b . Hybrid between Cajanus cajan and AtyZosia sericea

c . Aty Zosia sericea

D) a . Cajanus cajan

b . Hybrid between Cajanus cajan and AtyZosia cajanifo:

c . Aty Zosia cajanifoZia

F i g . 4. Twigs and l e a f l e t s of parents and F1 hybrids.

A ) a . Cajanus cajan

b. Hybrid between Cqianus cajan and AtyZosia grandifotia

c . AtyZosia grandifozia

B ) a . Caj;~us cqjan

b . Hybrid between Cqianus cajan and AtyZosia Zatisepata

c . Aty Zooia Zatisepata

C) a . Cajanus cujan

b . Hybrid between Cajunus cajan and AtyZosia ZanceoZata

c . A t y Zosia tanceotata

D ) a . Cajanus cqjan

p. Hybrid between Cajanus ca;'an and Atytosia Zineata

c . AtyZosia Zineata

Fig. 5 . Varation f o r leaf shape and u l t r a s t r u c t u r e i n hybrids between CaJanus c&an and AtyZosia atbicans,

a . Leaves from d i f f e r en t branches (1 t o 6 from t h e bottom) of a t yp ica l Cajanus cqjan-Atytosia aZbicans hybrid. (Note d i s t i n c t leaf shape from t h e branch two).

b . Leaves from bottom t o top o f a branch, which d i d not show d i sce rn ib l e va r i a t i on upto 5 months.

c . Leaves from bottom t o top of a branch which showed d i f f e r e n t l y shaped leaves a f t e r 100 days.

d. Leaf surface scan of Cqjanus cqjan.

e . Leaf surface scan of AtyZosia azbicans.

f . Intermediate type leaf surface scan.

g. Cajcmus type l ea f surface scan.

lh hybrid was e rec t in Wit and close t o the Cajauus

parent in leaf shape (Fig.3C) . The mid-leaf length (5.34 cm) , mid-leaf width (1.85 on) and the petiole length (3.55 cad were

all i n t e d i a t e t o the pazents (Table IX). Floral i n i t i a t i on

t o o k place 126 days a f te r sowing and the peak flowering was

recorded between Mv&xr and March. The pztals were persis tent

and the pods were hairy. Pod length in t h i s hybrid averaged 3.83

an with an average of 1.8 chatrbers and 1.2 seeds per pod. The

strophiolated and mottled seeds of the hybrid had a 100 seed

weight of 4.75 g and a protein content of 28.42% (Table X I .

The hybrid was intermediate i n its habit with t he base being

bushy and the u w r portions showing atwining tendency. The

l e a f l e t shape was intermediate (Fig. 3A) as were also the mid-leaf

l ~ g t h (4.9 an), mid-leaf width (2.0 d and the pet iole length

(3.8 on) (Table I%) . While the average pod length (3.74 ad in

t h e hybrid was in between the two parents the n a b e r of &tubers

p r pcd (3.5) and seeds per pod (2.6) were less than those of

e i t he r parents (Table X) . The seeds were mottled and

strophiolated and the seed weight and protein content were 4.25 g

and 28.33% respectively. The hybrid flowxed 85 days after

wming (October to February) as against 75 days i n E&h&a and

65 days in the !2&nu parent.

This hybrid unlike others was irrjistinguishable f r m the

parents. It has an erect habit and a lanceolate leaf shape

(Fig. 3D) similar t o the parents. The mid-leaf length (6.5 an) , mid-leaf width (2.27 an) and the petiole length (4.4 cm) were

also close t o the parental values (Table IX) . The pod length in

the hybrid averaged 4.5 cm with an average of 3.7 chaJrbers and

3.4 seeds per pod. The seed was strophiolated and mottled with a

100-seed weight of 5.86 g and a protein content of 32.95%. The

hybrid bloomed i n 125 days after sawing and the flowering which

intiated in October c~ntinued unti l March.

The hybrid resenbled the a parent in gross morphology.

The leaves were ovate to rounded i n shape (Fig.&) and the

mid-leaf length (7.7 cm) was greater than that in either of the

parents while the leaf width (4.15 cm) was intermediate between

the parents (Tablw IX) . Pods being hairy and yellwish r e s d l e d

the AtykxLa parent and the petals were persistent. The pods had

an average of 3.5 chanters and 1.8 seeds with an average l q t h

of 3.54 cm. The seeds were mottled and strophiolated and had a

100 seed weight of 4.65 g as against 1.58 g i n A. clrandifolia and

26.8% protein (Table X I . The hybrid which flowered between

Decerrber and April had its f i r s t flush 155 days after sowing.

In gross tmrpblogy the hybrid was intenrediate to the

parents. The leaves were intermediate in shape but had a m r e

pointed t i p (Fig. 4B) than those in A. and were much

l e s s densely haired than the leaves of A. m. The hybrid

leaves were 6.34 an long, 3.96 an wide with a 4.6 an long

petiole. The pods in the hybrid, which were less densely haired

than those in A. averaged 3.11 an in length w i t h an

average of 3 chanbers and 1.6 seeds per p d . The 100 seed weight

(5.27 g) was intermediate t o the parental values and the seed

protein content was 23.86% (Table X) . The hybrid flowered

between Decerrber and March with the f i s t flush a p r i n g 175 days

af ter sowing.

The hybrid, which in general was intermediate i n its gross

mrphology, had leaves shorter and broader than those of .f

A. lanceolata (Fig. 4C). The hybrid did not flower wen af ter 200

days of growth.

Eight species of Atvloaia, A- albicans~ A. aericea, A.

scarabaeoideet A. caianifalia, A- A. arandifolia, 8.

and A. latisebala hybridized w i t h G. &an with

varying degrees of success. The Australian species of Atvloaia.

8. -, A. lancealz+r a d A. have been crossed

w i t h cultivated pigeonea for tbe f i r s t time. Studies on a few

other epecica of Australian were conducted in a

parallel study at the University of Weensland (Ian mndas,

pers. cam.). Earlier atteapts at Lntergeneric hybridization of

pigeonpea were mstly concerned with crosses involving 8. lineate

(Deodikar and Thakur, 1956; K w & dl, 1958; K m and

llrrrbre, 1958; Reddy and De, 1983). There have been a couple of

successEu1 attanpts at crossing A. aericea and A. scarabaeoides

(Sikdar and De, 1967: Reddy, 1981; Pundir, 1981) and Alndir

(1981) used 8. 8. trinervea and A. caianifolia in

intergeneric studies.

Mnng eight successful fajanm - &ybsh crosses in the

present study the degree of species crossability was highest in

crosses involving A. lineata as the pollen parent followed by

those imrolving A. albicans. The crossability was.relatively low

with the other species. A. caianifolia. which is ~rorphologically

Mistinguishable from Cajxiua except for the strophiole on the

seed, did not hybridize with the orltivated species as freely as

8. lineata or A. elblcans j . . The rate of success in intervarietal crosses of pigeonpea varies from 6% to 70% depending upon the

genotypes involved. Thus the degree of species crossability in

itself is not an index of species relationship.

A wide variation in the crossability of a given &yk?sia

species with different cultivars of pigeonpa was wident.

Similar gamtypic variations for crossability are already knawn

in intervarietal crosses of pigeonpee (Singh & nl, 1980; Saxena

& b;L, 1984). The wnplete failure of the cultivar ICP 102 to

cross with any of the Atylosia species is not ~urprisir~g in the

light of its poor performance in intervarietal crosses of

pig-npea (K.B. Saxena, pers. cam.) . The w r o w range of

variation in the crossability of species with four

different gemtypes (IBL 32, ICPL 47, ICE% 59 and ICPL 95) of effect

f. is a reflection of a possible cytoplasmiclsince each of

these genotype originate from the azure f a d e parent.

Differences in the crossability of different varieties of

heat with rye (Rao, 1968; Zeven and Van Heeuert, 1970; Lange --

and Wojciechowska, 19761 W s s and Jalani, 1980) and with Hordeurn

bulbasurn (Snape SL pl, 1979) are known to be under the control of

crossability genes (Moss and J a s l , 1980; Falk and Kasha,

1980). With the developrent of genetic stocks of pig=-, it

should be possible in Cajmus - &yk&a crosses to elucidate the

inheritance pattern and the nature of crossability 'barriers.

The failure of as a male parent to cross with most

of the species might reflect a pre-fertilization barrier

as known in some bf the interapecific crosses of AraAh (Sastri

and Mae, 1982). The differences in the reciprocal crosses might

originate from the differences in the ccmaenceaent and progress

of division of endosperm and dryonic cells which has been shown

in (Robakoarihanta et & 19791 Shii & aL 1982) or

as a result of the failure of the pollen tube to grow through the

style (Muller, 1960) . In incmpatible S & ~ U E - A Q ~ M ~ CrOBsCB of the present

study our preliminary observations on pollen germination and

pollen t.:).: c,:o-,,th with tk,i: aid of a light iiicroscop usincj

acet-e s t a i n Indicated t h a t the pollen germinates and

en te rs the s tyle . Unfortunately this technique did mt help in

probing further t h e develo-t of pollen tubes. Limited

investigations on flourescence microscopy using Aniline blue did

not provide any further information because of t h e limited

clearing of t h e s ty la r t issue. I n the crosses A. voltlbilir x

C. rajan and A. rrollis x C. sajau pod dwelopnent was normal but

t h e seed w a s extremely shrivelled and w a s inviable. In these

crosses the bud drop occurs 6 days a f te r pollination as against

t h e usual 2nd or 3rd day a f t e r pollination observed in t h e other

crosses. These observations indicate t h a t t h e bar r ie r s t o hybrid

production in these two cross corcbinations might be operating a t

o r a f t e r fe r t i l i za t ion . But the fa i lu re t o recover any viable

seed w i t h honmne treatments o r errbryo cul ture in these crosses

could be taken as an indication for p r e f e r t i l i z a t i o n barr iers .

Developrent of mature pods with no seed d e v e l o m t has also been

reported in crosses involving Ebm arvense as female and

. . E. -and E. as pollen parents (Foujdar and

Tandon, 1976) . The crossed pods were shorter in length with fewer seed per

pod than selfed pods. The r a t e of pod developnent was also

slower in the crossed pods. I 8 7035 consistently averaged a

higher n d r of seeds per pod i n crosses with a l l t h e Atyks ia

species. Seeds per pod in ICP 7035 is greater than that in any

other cul t ivar used in this study inplying the predisposition of

a greater n w t r r of ovules per wary for t h e a l i e n pollen t o

f e r t i l i z e != ; i ?.; tc a k,igher seed se t i n t h e mature pods.

The results of h o m e treatmpnts indicate that in the

intergeneric crosses of and the rate of success

can be increased with hornvne treatments which also increase the

percentage of pod-set, pod length and nlmber of seeds per pod in

the cross cabinations. !R-e success rate in the untreated

wntrols was law. Moreover, the onset of flower drop amng the

unsuccessful crosses was delayed following hormone treatments.

The increased pod set associated with most of the W or

W+kinetin treatments is likely due to enhanced post-

fertilization developklt of both the ovary and ovule(s) since

treatments ccsrmenced one day after pollination when fertilization

would have been completed. The increase in pod length and ncmber

of seeds per pod following hormone treatments further

substantiate such an interpretation. Higher concentrations of

GA3 and GA3 + kinetin rdced the rate of success, but promted pod length and the nuher of seeds per pod which suggests the

role of hormones Fn post-fertilization developnent . Kruse (1967) &d Kasha et BL (1978) attributed increased

success in obtaining progeny from intergeneric or interspecific

crosses to the check by emgemus GA3 supplied to the florets on

post-fertilization break-. The increase in pd length in our

C a j a n m - m crosses may reflect increased cell n m e c and/or

cell elongation in the ovary wall after hormone treatments.

Kasha & al. (1978) observed increased mitotic activity following

GA. treatments in interspecific crosses of &)dam. Sastri and

EIoss (1982) found increases in peg nm&r as we7 1 as peg length - following GA3 treatments in Arachjn intersyeclfic crosses.

Al-Yasiri and Coyne (1964) alm rcmrded a positive influence of

honrones on pod length and diameter in a Phaseolus species cross.

(Xlr findings in pigeonpea intergeneric crosses are consistent

with these results.

The fai lure to increase further the nunher of successful

cross &inations erphasises the homna l role in the

post-fertilization processes only. Since the honmnes did not

improve the size of the mature seeds, the increase in ovule s ize

in the unsuccessful crosses is likely due to delay in bud drop

thereby permitting the ovule to develop for a longer period. In

the unsucce%sful carbinations GR3 treabnents helped to delay the

comnencement of flower bud abscission. In crosses between

P~SEQUE and E acutifolius. a ambination of G.3 and

NAA was wed to o v e r m pod abscission (Al-Yasiri and Coyne,

1964). Rnsweller and Stuart (1948) reported the Mluence of

grawth regulators on retarding the senescence of the arbryo sac,

and on the length and diameter of capsules in Lililrm LmgUhum. d

Although kinetin treatments (10 t o 80 FFn) did not show any

inprovement in *set or the nmber of seeds per pod in our

study, treatment a t the lowest concertration (10 Rn) w a s

effective in delaying bud drop in the A. albicans x C. ~ & n

and A. x C. crosses.

The data show that , amng the various crosses studied,

reeponse t o horrrone treatments is determined by the f d e parent

irrespective of the pllen parent tllsed (Table VII, Figs. 1 and

2) . The response of a, caianifalfa as a female parent was

6 - 1 ~ to that G -. These two species have more

morphological similari t ies than do the other species uaed in this

stucly and are considered one genus by mnre twonomtste (van &r

Maesen, 1980). In the unsuccessful cutbinations in spi te of

increased ovule size followhg honmne treatments, our at terpts

to culture treated ovules asceptically have failed. Furthermore,

cultures of pr-ture (< 11 day old) @fed embryos on defined

media have not developed into plants. (Details of d r y 0 culture

are presented in Chapter 7).

The present study shows that hormones can increase the

percentage of success in the crosses where the degree of success

is otherwise low. Cm the basis of the present results, it is

suggested that it would be worthwhile to t ry other hormones and

the i r cabinations in an attempt to increase the n h r of

successful crosses through further refinemnts' i n the a r y o

culture technique.

The hybrid nature of the F1 plants w a s very rmch wident

from &serving d e i r plant mrphologr. &yJ,c&a characters l i ke

seed strophiole, seed mottles, pod hairiness and twining nature

are dominant and their expression in the hybrids permitted an

ummbigous identification of hybrids and selfs. The leaf shape

in the hybrids, which' in mat of the cases was either

intenaediate or tending t o r e s d l e the male parent, helped i n

easy identification of the hybrids. MaXmb (1975) raised doubts

about the genuinity of intergeneric hybrids in l a s a e wing

to epcmictic developnent of seeds in same 1egrmLinous species but

in the present material wit? the expression of the ndle parent

t r a i t s the problem of identifying true hybrids is ciramnrented.

In the C. cajm x 8. abkma hybrid an interesting feature

was the variation for leaflet shape and texture (Fig.5) . The

deklopwnt of leaves similar to the parent after about

100 days led us to lodc for the possibility of selective

chromsome elimination similar to that of Nicotiana (Gupta and

Gupta, 1973) or Hordeum (Subralnnanym and Kasha, 1973). But our

cytological studies presented in Chapter 4 revealed eleven

bivalents and regular didunction. This could be taken as

evidence t o rule out the possibility of chromsome elimination or

in support of chrmsome doubling following chromsane

e lh imt ion . A detailed meiotic analysis (Sateesh K ~ r m a r et al,

1984) revealed the hybrid nature of the material. Thus it was

concluded that the variation in leaf mrphology accompanied by

flowering is a consequence of differential gene expression in

different branches. Since floral ini t iat ion occured only in the

branches on which !2jauu l ike leaves started developing, it is

likely that these two processes are temporal events in gene

expression . -tic variation in C. c&n was reported to be chimeral

(Rao and Reddy, 1975) which qpeared from the seedling stage.

Thus it is different from, the type encountered in the present

--&yl~& hybrid.

Qltoa~some nunber in and all the species of

atvloeia and Rhvnchoeia used in this study was found to be n=ll

or 2n=22,. Meiosis in pigecxlp (Fig.6AzG) was highly organized

with r-ar bivalent formation and normal disjunctions at both

divisions irrespective of the cultivars used. Species of

Akyb&ia and also formed regular bivalents (Figs. 7 and

8 ) with normil disjunction except for occasional asynchrony at

division-I1 in A. lancealata and A. serice9. Different cultivars

showed minor variations in their chi- frequmcy (Table XI).

In all the nine cultivare of fajmus the chiasma frequency was

around 20 per cell ranging from 19.75 i n C 11' to 20.75 in

Baigani. The chaismata per cell in A. sericea (20.85) , A. (20.50), A. (20.501, A. lanceolata

(20.301, A. acarabaeoides (20.52) and A. lirrPata (20.42) were

close to those in but different from A. (17.75)

and A. arandifolia (17.72).

In and &ykasia ~pecies studied, pollen mther

cells (PMCs) containing nucleolus aslsociated with two bivalents

were m m n (Fig&6B, 7G, 75 , 8D, 8.7) at diakinesis while two

nucleoli per Pm: (Figs.7A and 7J) were less frequent at

diakinesis and rare at pachytene (Pig. 2OA) . In C. &an the

n m r of nucleoli per PMC varied from 2 to 4 at telopha6e-I

(T-I) (Fig. 9A) and 4 to 8 at T-I1 (Fig. 9B-P) irrespective of the

cultivar. Similar variations a r e evident in A. albicans,

Tabl r XI. Ilrt.phas6-I conflguratlons* and po l l on f o r t l l l t y I n G. W c u l t l v r r r and Atvla.(l s p r l r s .

B l va l rn t s Av. Cult lvar/sprclos (Range) Pol 1 en ---------- r t d c e l l f o r t l l l t y

Ring Rod (I) ........................................... fi. tplM cv. ICP 7035 9.4 1.6 20.40S.09 96

(9-10) 10-2)

C. rPiPD cv. Bafganl

G. m cv. C 11

C. U cv. ICPl 32

G. raLnn cv. ICPL 47

C. rPL.n cv. XCPL 59

fi. rplna cv . ICPL 95

C. rntnn cv. ICP 102

A. Alhkma

A. lrneata

A. - A. - A- c&mlid-

*30 c e l l s were scorad for rach s p s l e d c u l t l v a r .

Fig. 6. , Meiosis in C. c(2vian cv. Pant A2 and metaphase I in cultivars of C. cajan. (Bar indicates 10 u).

A-G: Meiosis in C. cajan cv. Pant A2.

A) Pachytene showing regular pairing.

6) Diakinesis showing two bivalents attached to the nucleolus.

C) Metaphase I with 11 bivalents.

D) Equatorial view showing chromatic associations at late metaphase I.

EEF) Anaphase disjuncrions.

G) Late anaphase I1 showing regular disjunction.

H-0: Metaphase I in cultivars of pigeonpea showing 11 bivalents.

H) ~aigsni. I) C 11. J) ICP 1 0 2 . K) ICP 7035 .

L) ICPL 32. M) ICPL 47. N) ICPL 59. 0) ICPL 95.

- &." , . , Y*

! .fr --.... '.; 4 .,#

4, .. * 6 , ,.$; : ' ... .$, " I '9 'a ':.:, . , < , - y

, , .> ". * .'* tV . "5 . . . ' 0 " ' U."?," P -. A . B -

L b . - C - - a m , <> -JW.

- # ' .=r -e 5

-'-e ,a a- CZ

-2% m. 1'6 "A, .- D ,, !*m$g F

\ k C

hb , & dV

)r e t - H

Z . 1-

Fig. 7. Meiosis (Diakinesis, metaphase I and anaphase I) in AtyZosia species. (Bar indicates 10 u)

A-C: AtyZosia zanceotata

D-F AtyZosia grandifot ia

G - I AtyZosia Zatisepata J-L Aty los ia aZbicans

Note two nucleoli in A and J.

Fig. 8. Meiosis (giakinesis, metaphasc I and anaphase I) in AtyZosia species. (Bar indicates 10 u)

A-C: AtyZosia sericea

D - F : AtyZosia ca jani fo t ia

G - I : A t y Zosia Zineata

J - L : At? Zosia scarabaeoides

A. lineeta and A. Ptanairalla w h i l e in A. caianifolia,

A. lanceolata and A. scarsbaeoides, the nvutirnna nlmber of

nucleoli per FfC vks seven (Fig. S O L ) . The maxinun n m r of

nucleoli in each daughter nucleus at T-I or T-I1 never exceeded

two (Fig. 9) one of which was always bigger than the other. The

variations in frequencies of PMCs with different nlmbera of

nucleoli at -11 in different species k e presented in ~ig. 10.

The nost, freqc,--.t types were FWS with four nucleoli while the

least frequent czes were those with eight nucleoli.

The present study on the chromsome nwr&r. in C.

A. linaata. A. caianifoliat A. serice9, A. scaraboeides,

8. MlubilFs and A. confirms the earlier reports ( K m

ale 1958; Sikder and De, 1967; Shrivastava eL aL, 1973; Reddy,

1973; Bir and K L ? ~ 1973; 19773 Pundir and Singh, 1978; Rao,

1978; Sanjappa and Satyananda, 1979; Pundir, 1981) as 2n=22.

The chranosome nwrbers of A. atanalfolla . . , A. latisepalat

A. lanceolata, A. mllia, B. rathii and B . minima which have not

been r-rted eazlier has been found to be 2n=22.

The mwimum n h r of nucleoli in each daughter nucleus at

T-I or -11, never exceeded two (Fig.9) irrespective of the

cultivar or species studied suggesting thereby the presence of

two nucleolar or~anizers (N.0.) per genome in pigeonpea.

i r . L l t e ~ n ~ ~ i , .;.= b r L - ~ ~ n ~ ~ e of a naximum of two nuciwri at

Fig. 9. Nucleolar distribution in pollen mother cells of C. ca.jan and species of AtyZosia. (Bar indicates 10 m).

A-F: Nucleolar distribution in 'lollen mother cells of Cajanus ccqian.

A) Telophase-I nucleolar distribution, 2-2. Ti lophase-II nucleolar distribution. 8) 2-2-2-2.

C) 2-2-2-1. D) 2-2-1-1. E) 2-1-1-1. F) 1-1-1-1.

G-L: Nucleolar distribution in pollen mother cells of A t y Zosia species.

G) Telophase-I nucleolar distribution in A. grandifozia, 2-2. H) Telophase-I1 nucleolar distribution in A. grandifotia,

2-2-1-1 (Arrow indicates two fusing nucleoli). I) Telophase-I1 nucleolar distribution in A. ca jan i fo t i a ,

2-2-1-1 (Note the difference in the size of fused and primary nucleoli).

J) Telophase-I1 nucleolar distribution in A. t i nea ta , 2-2-2-1. K1 Telouhase-I1 nucleolar distribution in A. ZatiseoaZa. 2-2-1- ~j ~elophase-11 nucleolar distribution in A. a ~ b i c & s , i-2-2-1.

Fig. 10. Frequency distribution of nucleoli at telophase-I1 in pollen mother cells of C. cw'an and A t y Z o s i a species.

pachytene and the presence of two nucleolar bivalent8 at

diakinesis is also indicative of two pairs of nucleolar

organizers in the Qj&us and species studied.

The variation in the frequencies of PMCs at T-I1 with

different nunbers of nucleoli in different species are presented

in Figure 10. Invariably the most fr@ent type were PEW3s with

four nucleoli while the least frequent ones were those with eight

necleoli. However, the frequency of FtKb with at least one

daughter nucleus possessing the rcaxSmrnn nunber of nucleoli was

>Ma in all the species which showed up to 8 nucleoli per T-I1

PMC (Fig. 10) while in the other species that had a r m x h of 7

nucleoli, the frequencies of PMCs containhg at least one

daughter nucleus with two nucleoli was -30%. Such variation in

the frequencies of PMCs with different nunbers of nucleoli (4 to

8) is a reflection of the nucleolar fusion which is very ccmmn

(Sybenga, 1972; Bennett a&, 1973; Darvey and Driscoll, 1972;

Flavell and O'Dell, 1979). The prepondrance of PMCs with 4 /

nucleoli (1 nucleolus per daughter nucleus) at T-I1 is indicative

of the frequent occurance of the fusion of two nucleoli in each

daughfxr nucleus and the accunulation of cells with 4 nucleoli.

Miotic studies, in a spontaneous tetraploid of C. s&in

(isolated by Dr. K.B. Saxend revedld eight prinvlty nucleoli

at P I (Fiq 21 in chapter 4.2.2) further substantiating the

presence of two nucleolar organizers per genome.

!fhe s i z e difference in the two nucleoli in each of the 'ICII

nuclei (Fig-9B) indicates that t h e two nucleolar organizers (in

each of t h e gemmed d i f f e r i n t h e i r ac t iv i t i es . Cultivated

barley is a well known example f o r such differences

(Anastassova-Kristwa &AL, 1977; S u b r ~ y a m and h a d , 1978a;

Linde-Laursen, 1984; Jessop and Subrahmanyam, 19E4). Acoording

t o Beitz (1931). t h e nmber of telophase nucleoli is constant, as

well as t h e nwrber of nucleolar organizers and Sw. chromsomes

f o r a given karyotype. These observations aoquire a new meaning

i n the l i g h t of the evidence t h a t genes d i n g for riboaoual RNA

a r e located in t h e nucleolar organizer region (NOR) and t h e

m a x m nimber of nucleoli per nucleus correspond t o the n-r

of rPNA synthesizing l o c i (Anostasswa-Kristwa, 1977; Nicoloff

ge & 1977; Subratnnanyam and Azad 1978ar b; Flavel l and

OIDellr 19791 Linde-Laursen, 1984; Jessop and Subrahrnanym.

1984). For t h e karyotype of pigeonpea, there have been

mntrovers ia l reports on the n-r of SAT c h r o m m . For

instancer Sinha and K M l a r (1979) reported lack of SAT chronosomes

in certain cultivars of C. and Shrivastava e.t al (1973)

remrded one SW. drromsome per genome in Qiarus while Pundir

(1981) found two chrorrpsomes per genome in Cajaws. ard

U o - species. The present data (Figs. 9 and 10) on the

nucleolar behaviour in PMCs conclusively establ ish two nucleolar

organizers per gemme i n Caianufi and the A W l ~ s b species

studied. Ths differences i n the s i z e of t h e two nucleoli in

daughter nucleimay r e f l e c t differences i r the degree of a c t i v i t y

andlor differences in t h e mult ipl ic i ty c f - R N A cis trons in t h e

two nucleolar organizers i n each of the species.

Controversies in the nucleolar organizer nuu&r bave oft-

been resolved using the mitotic wcleolar nMber as an index of

N.O. activity (Anastasmva-Kristeva rt &.I 1978; Jessop and

Subrahmmyam, 1984: LMeLaursen, 1984). In the present study

nucleolar behaviour during meiosis has been used in dar~nstrathg

the nlmber of nucleolar organizers in a wide range of species.

All the ! 3 j a u s - ~ hybrids that flowered were analyzed

cytologically. Meiosis was studied in detail with enghasis on

metaphas-I pairing, and amphase disjunctions. The results show

the following:

Erms of this hybrid exhibited regular fonration of eleven

bivalents which were predominantly rkrgs (Fig. llE) . There were m significant differences in the chaisma frequency between

cultivar combinations (Table XII), the rwrimnn being 20.75 per

cell in Pant A? x 8. albicans and the minimrm 20.00 per cell in

hybrids with ICP 7035. A low frequency of preoocious separation

was evident in all thg cultivar colrbinations. Diplotene cells

displayed nucleoli with three or four bivalents attached

(Figs. llA-B) , while at diakinesis 20 of the 64 cells contained

nucleoli with three or four bivalmts (Fig. 11C-Dl. Ana&aae-I

and I1 separations in all b e cultivar cabinations were regular

(Fig. lU4) . Pollen sterility in these hyb~ids rang& from 27.3%

in Baigani x A. rlbicans to 46.1% in ICP 7035 x A. rlbicans while

ovule fertility ranged from 21.6% in Pant A2 x A. aU&Scl5 to

32.2% in Baigani x A. albicahs hybrids.

Fig. 11. Meiosis in the hybrid between Cajanus cujan and A t y Z o s i a aZbicans (Bar indicates 10 u): (A) Diplotene nucleolus with four bivalents attached. (8) Diplotene nucleolus with three bivalents attached. (C) Diakinesis nucleolus with four bivalents attached. (D) Diakinesis nucleolus with three bivalents attached. (E) Metaphase-I with eleven bivalents. (F).Regular disjunction at division-I. (G) Regular disjunction at division-11. (H) Quartet showing nucleolar variation, 3-2-1-1.

0 - -- WE; RE; W E ; '" 28 && d & d & 0 0 0 0 0 O O O Q

~iv&&t fonmtion was a general feature in these hybrids

with the e x m i o n of the hybrids between ICP 7035 and A. sericea

which displayed a low frequency (0.4 per W of univalents.

Chiasma frequency varied from 19.38 to 20.37 (Table XI11 . Cme or two bivalents were attached to the nucleolus (Fig. lZA) . Anaphase disjunctions were regular at both the divisions (Fig.12CID))

except h hybrids with ICP 7035. Laggards were observed in 8.6%

of the PMCa. Pollen sterility of 29.7%, 33.6%, 48.4% and 36.1%

and a owle fertility of 54.3%, 61.5%, 46.6% and 52.5% were

recorded in hybrids of A. sericea with Pant M I Ehigani, ICP

7035, and C 11 respectively (Table XII).

Hybrids of Cajaax with A. scarabaeoidea 'showed a low

frequency of univalents (Fig. 13C) (0.264.34lcell) with a nraximum

of two per cell. The chi- frequmcy varied between cultivar

carbinations rangw from 19.48 in Pant A2 x A. scarabaeoides to

20.35 in hybrids with C 11 (Table XI11 . Diplotene/Diakinesis

nucleoli were found associated with up to t m bivalents. A low

frequency (3.8 to 8.6%) of **I cells displayed laggards

(Fig.13D) and irregular disjunction irrespective of the cultivar

used. Pollen sterility ranged from 22.7% in hybrids of

A. scarabaeaides with Baigani to 41.5% in hybrids with 1 8 7035.

Ovule fertility varied from 29.3% in hybrids with ICP 7035 to

52.8% in those with C 11 (Table XII) .

Fig. 12. A-D: Meiosis in hybrids between Cajanus cajan and AtyZosia ser icea .

A) Diakinesis showing 11 bivalents. B) Metaphase-I showing 11 bivalents. C) haphase-I showing regular disjunction. D) Anaphase-I1 showing regular disjunction.

E-H: Meiosis in hybrids between Cajanus cajan and Aty2osi.a cajanifoZia.

E) Diakinesis showing 2 bivalents attached to the nucleolus.

F) Metaphase-I showing 11 bivalents. '

G) Regular disjunction at division-I. H) Regular disjunction at division-11.

Fig. 13: A-F: Meiosis in hybrids between Cajanus cqjm and Aty Zosia ~carabaeoides (bar indicates 10 p) .

A) Diakinesis. B) Metaphase-I showing 11 bivalents. CJ Metaphase-I showing 2 univalents (Arrow) D) Division-I showing laggards. E) Unequal chromosome distribution at division-I. F) Regular disjunction at division-11.

G-J: Meiosis in hybrids between Cajanus cajan and Aty Zosia Zineata.

G) Diakinesis showing 4 bivalents attakhed to the nucleolus.

H) Metaphase-I showing 11 bivalents. I) Regular disjunction at division-I. J) Regular disjunction at division-11.

Hybrids with 8. exhibited eleven bivalents in

the i r FMCs (Fig. la) w i t h a relatively high chiasma frequency

ranging from 20.49 tc, 21.20 depending on the cultivar involved in

the cross (Table XII). Mostly two bivalents were seen associated

with diplotene nucleolus. Anaphase separations a t both the

divisions were regular (Fig. l X , H ) . Pollen s t e r i l i t y in these

hybrids was relatively low (14.6% to 29.1%) with high ovule

f e r t i l i t y (63.7% t o 85.2%) (Table XII) .

These hybrids shaved regular bivalent formation (Fig 13H)

except in combination with ICP 7035 where a l o w frequency

(0.09/cell) of univalents were found. Chiamata per cell varied

from 19.70 t o 20.34 depending on the Caimu genotype

(Table XI11 . A nmximum of four bivalents attached t o the

nucleolus (Fig. l3G) were found a t diplotenddiakinesis. Amphase

segregation was r w a r a t both the divisions (Fig.131,J).

Pollen s t e r i l i t y varied from 27.5% in hybrids with C 11 t o 46.2%

in those with I B 7035 while ovule f e r t i l i t y ranged from 31.3% in

hybrids with ICP 7035 against the rwtimum of 72.7% recorded in

hybrids with C 11 (Table X I I ) .

A range of w i o t i c abmnmli t ies were encountered in

C. &an x B. hybrids (Fig. 14) . These hybr idk

disp1i:wJ a relatively hiqh frequency of univalents (up t o

(Fig.14D) and rod bivalents (3/cell). The chaiana frequency was

Fig. 14. Meiosis in hybrids between Cqianus c a j m and A t y Z o s i a grandifozia.

A) Diakinesis showing 4 bivalents attached to the nucleolus.

B) Diakinesis showing univalents (Arrow). C) Metaphase-I showing 11 bivalents. D) Metaphase-I showing 6 univalents. E-H) Laggards at division-I. I] Bridge at division-I. J) Division-I1 showing chromatic separation but

no disjunction in one of the telophase-I1 daughter nucleolus.

K] Telophase-11. L) Five daughter nuclei at division-11.

low (1Wcell) (Table XI11 . The diplotene nucleolus wae often

found with t ~ ~ r three or four bivalents attached (Fig 14A).

Bridges (Fig. 141) were found in 8.2% of the cells a t

anaphase1. Bridges were rarely accorrpenied by fragments. Other

Anaphase irregulari t ies included laggards (Fig.14EIF) mz l t ip l a r

disjunctions (Fig.14L) and occassional restitution a t division-I1

(Fig. l 4 J ) . Lagging chromosomes were commn in t h i s &ination

(Fig. 14E-H) . Division-I laggards ranged from 2 t o 6. A t

division-I1 chromosom segregation t o f ive poles was frequent.

Restitution i n one of the T-I daughter nuclei was occassionally

found (Fig. 14.3.

These hybrids whibited a high degree of pollen s t e r i l i t y

and poor ovule f e r t i l i t y . While the pollen s t e r i l i t y was 54.8%

and 48.3% in hybrids with Pant A2 and C 11 the corresponding

figures for ovule f e r t i l i t y were 23.4% and 19.9% (Table X I I ) .

Univalents were m r e frequent (Fig.15B) than in hybrids with

Asian but the maxirrPlrr1 nrmber of univalents recorded was

4 with an average of 1.12 in hybrids with Pant A2 and 0.75 in

those with C 11. R d bivalents averaged 2.89 and 3.89 per c e l l

in cabination with Pant A2 and C 11 respectively. The chiasma

frequencies were 17.97 and 17.33 involving cross6 with Pant A2

and C 11 as farrale parents.

Fig. 15. Meiosis in hybrids between Cly'anus cqjan and A t y Z o s i a Z a t i s w p a k (bar indicates 10 y) . A) Diakinesis . 0 ) Metaphase-I showing 11 bivalents. C ) Metaphase-I showing 4 univalents. D) Laggards at division-I. E) Three chromosome groups after division-I. F) Five daughter nuclei at division-11.

AMphaseI laggards were frequent (Fig.15D). A comrrcn

feature observed in these EMCs was congregation of the separated

chrwosomes at the center of the cell and subsequent segregation

Occasionally into m r e than two groups (Fig. 153. Five groups of

ChrOImsomes were often seen at division-I1 (Fig.15F).

In general precocious separation of bivalents was a general

feature in all hybrids with all A t y U s b species where ICP 7035

was the female parent and heteromrphic bivalents were c o m n in

all the hybrid conbinations. Secondary chromsome associations

were found in hybrids with the Australian At-. Differences

in staining intensities in partners of bivalents was detected in

several hybrid cahinations.

A wide variation in nucleolar nmber, size and distribution

in EmCs at division-I and division-I1 (Fiy.16.17) was recorded.

Such a variation w& detected in the hybrids between C. and

A. albicans (Fig. 161, C. and A. (Fig. l7A-D) and

C. s&dn and A. aranclifolia (Fig. 17E-H) . In these hybrids the nucleolar n&r varied from 4-8 at

-1. At T-I1 nucleoli in daughter nuclei varied from 0-4

(Fig. 18) wfiile the total nwber of nucleoli per PMC varied from

4-8. Consequently in 14% of the Pm38 of C. .&an x A. lllbicans,

6% in the FWa of C. caj,an x A. lineata and 4% in the FtCs of

C. a x A. w r d u hybrids the nucleolar distribution at

Fig. 16. Nucleolar variation in pollen mother cells of the hybrid between Cajanus cajan and A t y Z o s i a aZbicans (Bar indicates 10 PI.

A) Telophase-I nucleolar distributions, 4-4. B-I: Telophase-I1 nucleolar distribution B) 1-1-1-1. C) 2-1-1-1. D) 2-2-1-1. E) 4-2-1-0.

(Arrow indicates nucleus devoid of nucleoli). F) 2-2-2-1. G) 3-2-2-1. H) 4-2-1-1. I) 2-2-2-2.

Fig. 17. Nucleolar variation in pollen mother cells of hybrid; between Cajanus cajan and AtyZosia Zineata and Cajanus cqian and AtyZosia grandifoZia.

A-D: Cajanus cajan x AtyZosia Zineata.

A) Telophase-I nucleolar distribution: 4-4. Telophase-I1 nucleolar distribution.

B) 3-1-1-1.

C) 3-3-1-1.

D) 2-2-2-2.

E-H: Cajanus cw'm x AtyZosia grcmdifotia.

E) Telophase-I nucleolar distribution: 3-1. Telophase-I1 nucleolar distribution.

Fb3-2-1-1.

G) 2-2-2-1.

H) 4-2-1-0 (Arrow indicates nucleolus devoid of nucleolus'

Fig. 18. Frequency distribution of nucleoli in telophase-I1 daughter nuclei of hybrids between C a j a u s cajan and Aty Zosia species.

0 1 2 3 4 0 1 2 3 4 0 1 2 3 4

NO. OF NUCLEOLI

T-If w a s amfined t o three wt of the f w r daughter nuclei. The

frequencies of daughter nuclei devoid of nucleoli were 3.5% in

C. m x A . albicans, 1.5% inC. a x A. UneaUi and 1% in

C. x A. aranalfolla . . . A majority (65% in C. &an x

A* nlbicans, 59% in C. a x A. lineata a d 54% in C. &an x

A. arandifolia) of daughter nuclei contained one nucleolus while

the remainder had 2, 3 or 4 nucleoli. The frequencies of

daughter nuclei with two nucleoli were 22% in the hybrid C. a x A. albicans, 29% in tha t of C. &gm x A. linsata and 36% in

C. x A. arandifolia, while 6% in C. x A. Rlbicans, 8%

in C. c&an x A. lineata and 9% in C. Egjan x A. arandlfolra . .

included 3 nucleoli. Least frequent (2% i n C. a x

A. albicans, 3% in C. a x A. lineata and 3% in C. x

8. were the daughter nuclei w i t h 4 nucleoli.

The cells with variation i n nucleolar nrmber also showed

variation i n nucleolar size. The nucleoli could be classif ied as

large or -1. In the R3Cs with 4 nucleoli a t FII, (one in J'

each iiaughter nucleus), the nucleoli were uniformly larger

(Fig.16B) than those in the daughter nuclei w i t h 2 or more

nucleoli. There was no discernible variation in the s i ze of the

larger nucleoli, while variation was evident i n the s ize of the

wlaller nucleoli. A wide variation was recorded for nucleolar

distr ibution a t F I X (Fig. 16 and 17) as against the regular

2-2-2-2 distr ibution recorded in the parents.

8 1

4.2.1.2. BIgAVTm IR P2

F e r t i l i t y of the P2 segregants in the f i v e pplations,

X As-, C.- X A*-, C . U X

A. scarabaeoides, C. &~II x A. lineata and C. &dt~ x

8. is presented in Fig.19. The f e r t i l i t y in t h e

segregants ranged from 18 to 81% except i n those of C. &dn x

A. caianifol.ia where the segregants were relat ively m r e f e r t i l e

with 106 plants showing >71% f e r t i l i t y , 95 plants with >56%. In

t h e other four p p l l a t i o n s (Fig.191, the majority of plants

showed 41-70% f e r t i l i t y w i t h a few plants i n t h e lower (10-25%)

and t h e u p r (>71%) ranges. Meiotic analysis, such as metaphase

pairing studies, were conducted on randomly selected plants from

t h e various groups of segregants.

I n randomly analyzed plants in t h e higher f e r t i l i t y group

(>56% f e r t i l i t y ) , chromsome pairing was m s t l y mrrrurl with the

formation of eleven bivalent6 which predominantly were of t h e

ring type. Unpaired chromosomes were noticed occassionally i n

very few PMCs. Unpaired c h r o m s o m were m r e frequent in the d'

lower f e r t i l i t y g roup . Sane rrmltivalent configurations were

found in some of the segregants of t h e C. x A. sericea

p o p l a t i o n while secondacy chrom~sow associations were c o m n in

the segregants of C. sajan r A. scarabaeoiaes and a l so to a less

extent in other ppulat ions. Chrommm pairing was regular in

al l t h e analyzed segregants of the f. &au x A.

popla t ion . In general pairing was nosml in the segregants of

the popllations of C. && x A. and C. x

A. lineata except i n the i r lower f e r t i l i t y group. H ~ t e r ~ r O r p h i ~

bivhlents were camon in several cases.

Fig. 19. Frequency distribution of F2 segregants in different fertility groups.

.ty Zosia aZbicans

sericea

30

20 A t ; i Zosia cqianifoZia

10

0 -25 tC-(DILSl -lo w m

FERTILITY ( V r )

Meiotic studies in the intergeneric hybrids revealed regular

chromsome pairing in hybrids of Cajams with Asian m. In these hybrids eleven bivalents were c o m n a t uetaphase1. A s

in tbe parents ring bivalents predominated in these hybrids.

Chi=& frequencies in hybrids with Asian &yba&as was high and

m w - a b l e t o the values in the parental species. Fedak (1982)

has shown significant variation for chiasrrra frequency between

cult ivar cubinations in Barley-wheat hybrids but such

differences were not evident in the present f&aws--

hybrids.

knother interesting feature in these studies has been the

behaviour of the Cajxus cultivar I 8 7035. .Invariably in

hybriiis with all the ALykaia species where it w a s used as the

£ m e parent the pollen s t e r i l i t y w a s consistantly higher.

Chiasra frequency w a s also found to be lower in hybrids involving

this cult ivar and the occassional univalents and anaphase

i r r e g d a r i t i e s en'countered were also in hybrids w i t h this

genotlpe. In fajaws intervarietal crosses also the behavaiour

of I B 7035 is reported to be distinct (K.B. Saxena, Pers.

corn.) . Shrivastava aLr (1973) and Sinha and K- (1979)

found significant differences in k a r y o t p of G&nu6 cultivars

and -3uivastara & a, (1973) who conpaced the karyotype of

A. lir.eata with 15 cultivars of Sajmu6 found that the karyotype

of &. lhe&a was closer t o karyotypes of some !Xjanm cultivars

than t o othels. It is likely that some differences ir the

chramoscme conplement of I 8 7035 are responsible for the

distinct behaviour of this cultivar. The differences between

cultivar ambinations for pollen sterility and wule fertility

are V t e d and attributed to the effect of the genotype. A

parentage dependent pollen sterility was also reported in

interspecific crosses of (Xiazuma and Onor 1980).

In general chromsome pairing and disjunction is regular in

hybrids between and species of Asian m. Meoitic studies suggest that the Asian share a cnmron g m e

with C. &. Absence of univalents in hybrids is an indication

of chrornsome to chromosome -logy between the parental

genomes. Pairing at diakinesidmtaphkse gives only an

indication of the gross-chrorrosorce -logy but does not provide

information on the extent of homology. Pachytene FUCa of the

hybrids (Fig. 20B-F) also did not Bbow univalents indicating a

hornlogy of a high degree between C. &an and species of

m. H?wwer, intercalry unpaired regions (Fig.20B,C8E)

were evident in the pachytene chromsornes. Similar observations

were made by (1981, a,b,c).

It has been shown in som cases that chromsome horrology is

not a prerequisite for pachytene pairing. For -1e in

mna-haploids of -ley the seven chrornsoms participate in

pairing at pachytene thereby iPplying the occurrence of

Won-Hcuvlogous pairing" that does not persist until wtaphase-I

( W i v a i a h and Rasha, 1971). Such non-homlogons associations

at pachytene have also been reported inmonoploids of rice (Chu,

19671, tomto (Ecochord e% al, 1969) and maize (Ford, 1970;

weber and Uexandrr l5W.l Lr mlvhar,!c~ids, e substantial

Fig. 2 0 . A) Two nuc leo l i a t pachytene i n Cqjaus cajan.

BBC) Pachytene i n hybrids between Cajaus cajan and Atylosia grandifotia showing inversion loops ( s o l i d arrow) and unpaired regions (dotted arrow).

D) Pachytene i n hybrids between Cajanus caJm and AtyZosia ZatisepaZa showing cross shaped con- f iguration (Arrow).

E) Pachytene in hybrids between Cajanus o a j m and Atylosia l ineata.

F ) Pachytene i n hybrids between CaJ'anus cojan and AtyZosia c a j m i f o l i a .

reduction in chramb.aaal pairing from prophase-I to metaphase-1

is seen and such a raction in pairing has been attributed to

non-homologous pairing (Subrmyam, 1978).

In species hybrids univalents and other higher chromosome

associations are usually frequent and these japairments in

pairing are taken as an index of genome affilities or the lack of

it. Meiotic studies in distant hybrids have helped in

establishing evolutionary and species relationships in several

crop species which include m y leguminous species (Foujdar and

Tandon, 1976: Machado &dl, 1982; Newell and Hymwitz, 1983).

In the hybrids of Cajaui and the Asian Atvlosias.

chronvsome pairing at metaphase has been perfect implying a very

high degree of genome homlogy. A. which is

nvrpbologically very close to Qjau,s shares ' a close genome

relationship as well with the cultivated species. A. -, A. lineata and A. sericea though morphologically distinct from

pigeonpea also haves close genome relationship with fajau&. It

was only in the hybrids of C. G&U x A. scarabaeoides that

consistently univalents were found irrespective of cultivaz

ombination. This distinctly suggests that one drromsome of

A. has diverged to a relatively greater extent from

its mterpart in the Cajams wrrplerrent. The differences in

staining intensities between partners in a bivalent and also the

qpearance of heteromrphic bivalents in the hybrid PElCs is an

indication of intergenomic hornlogy .

-phase separations were highly organized in all the

CajaDU cultivar. curbinations involving A. alblcans (Fig. 11F),

A. aericea (Fig. 12C), A. lineata (FIg. 131) and A. caianifolia

(Fig. 1x1 with the hybrids with I 6 7035 background being the

exception where a low frequency of anaphase abmnmlities rmstly

laggards were observed (Table XII). Anaphase irregularities at a

low frequenoy were encountered in hybrids with A. acKabaeoiden

in all the cultivar oorbinations. w e n t l y the sparse horology

between one of the chsonvsomes of C. and A. scarabaeoides

as evidenced by univalents at metaphase contributes to these

irregularities at anaphase-I. AMphase-I1 separations were

aU?ost regular (Fig. 11G, 12D, lW, 13F, with occassional

asynchromy in disjunction in a few cases. These findings, i.e.

regular chromosome pairing and disjunction in the hybrids between

f. and Asian Btvlosias. are in line with those of earlier

studies on hybrids of Saiaws-Atyhxia hybrids (K- et aL,

1958; Pundir, 1981) . As against the regular meiosis encountered in the hybrids

with Asian the hybrids of fajaws with the Australian

species of for which no previous reference was found

exhibited a relatively high degree of pairing and disjunctional . .

abnonmlities. Univalents hybrids with both A. arandlfolla

end 8. were far nvre frequent. As many as 6

univalent6 were recorded in hybrids with A. arandifalia and up to

4 in those with A. -. These findings suggest that at

least 3 chromsomes of A. clrandifalia and 2 of A. have

d;-;cr.ged frop their wunterrsrts ir? thc L&i,iii& guiOl!,l!e.

m e n t l y less effective crossing w e r due to lack of adequate

hmlogy has contributed to the higher frequency of univalents.

However, from the present findings it is difficult to ascertain

whether the chsorrommes of A. and A. orandifolia that

have diverged from C. &an are similar or different from each

other. Hybrids between these species of can help in

resolving this question.

A significant obsenration has been that the univalents in

the hybrids of with A. ~ & a d & s , A. orandifolia or

A. 1- were always even nunbered (2, 4 or 6) which is

indicative of a low n m b r of pairing initiation sites

[ (Zygomeres) Sybenga, 19661 . Studies on pairing in tetraploid

C. & revealed a high degree of bivalent formation (16.54 per

cell) and a fertility of around 60%. Quadrivalents averaged only

2.73 per cell with the maximrm being 4 per cell. The selfed

progeny of the tetraploid exhibited regular disjunction (Fig.21E)

and high fertility (70%). The high freequency of bivalent

forination in the tetraploid also inplies a few pairing initiation

sites in pigeonpea similar to that found in &edkag~ sativa

(Bingham and Gillies, 1971).

An interesting feature in the PMCs of C. cajza x

A. arandifalia hybrids is the fonmtion of chromtin bridges at

division-I with or without fragments. Meiotic bridges are mstly

a consequence of reconbination within a paracentric inversion

(KKlintock, 1933; 1938) or alternatively in very rare instances

d : t to pruphase breakage of chrorasoms often referred to as 'U'

t i ~ c exchanges (Lewis and John, 1966). The distinguishing

Fig. 21. Meiosis in tetraploid Cajanus cajm.(bar indicates 10 p)

A) Diakinesis showing quadrivalents (Arrow). B) Metaphase-I showing quadrivalents. C) Irregular disjunction at division-I. D) Progeny of tetraploid showing regular bivalent

formation. E) Regular disjunction in the progeny of tetraploid. F) Eight nucleoli at telophase-I. G) Pollen grains of diploid Cajanus cujan. H) Pollen grains of tetraploid Cajanus cajan.

feature of the *U1 type excharqes fran those arising out of

crossing wer within a paracentric inversion is the presence of

fragments of variable size following 'U' type exchanges.

Observations on pachytene rn of the C. caianus x A.

arandifolia hybrid (Fig. 2OB,C) also revealed loops which indicate

that bridges at anaphase are a consequence of inversions.

Further ' both the parents involved in this cross have exhibited

regular bivalent formation and disjunction. The limitations in

chrormmme size and the inconsistent a e r a n c e of the fraqments

in the present material did not permit precise fragment size

measurements. Chromatin bridges are comaon in species hybrids

(Ahmed & dl, 1977; 1979; Mdlik and Mary, 1975; Chaudhuri gt

a, 1976; Reddy and Subrahmanyam, 1985; Sprirger and Buckner,

1982) but this is the first report in the Cajauw - Atybaia

hybrids.

These results indicate that structural variation in the form

of inversions have played a role in species differentiation

between $2. &an and A. crrandifolia. Meiotic bridges are only a

qualitative index of inversions but not quantitative. Not all

paracentric inversions mnifest themelves in the form of

bridges. A minimum of one crossover in the inverted 6-t is a

prerequisite for bridge formation and the possibility of a

crossover is directly proportional to the size of the inverted

sepent .

The structural differences between the chraw- of

C. cdjdn and A. arandifolia as evidenced by the a n a m bridges

and the relative lack of hamlogy bet- C. and

A. arandifolia and A. which is -rent from the

higher frequency of univalent6 and anaphase abnormalities in

hybrids between C. and these species explain the relatively

high deg'ree of sterility encountered in these h*rids. This

suggest that the genomes of the Australian species of Atyksja

are m r e diverged from Sajmw than tbose of Asian Atyhshs.

The regular foemation of elwen bivalents in the hybrids of

Gajanu with Asian without any detectable conventional

meiotic irregularities might be interpretted to indicate caplete

gemme b l o g y between these species which can be misleading as

will be wident from the following discussion of,our results on

nucleolar variation.

The most intriguing feature in the hybrids of S&rui with

A. dbkana, A. lineata, and A. was the association

of up to four bivalents with the nucleolus at

diplotenddiakinesis and the wide variation in nucleolar nlmber,

distribution and size. Karyotypes of L saj.au and tbse of

A. -" and A. have two pairs of satellite

chromsomes in each of their gem= (Pundir, 1981). Cur studies

on nucleolar nmbers at telophase have confirmed this finding and

have revealed a similar situation for A. crrandifolia. Thus an

association of nucleolus with a rnaxinum of two bivalents at

prophase-I and a mxinnnn of four nucleoli per PMC at telophs-I

are a-.- = i ? t h c : ~ bit*-???. The asscci~ticc sf ZG;; i. -. t,b

bivalents with the nucleolus in the hybrid -11s is suggestive of

pairing between the N.0.s of Cajmw s&an and non-N.0.s of

Atvlosfa species or a ~ e ~ s a and the variation in nucleolar

nuher and distribution at telophas-I and 11 is interpreted to

have originated from allosyndetic recombination.

The possible nucleolar distributions with 4 N.O.s, with and

without reoombination, are presented in Fig.22. If one assumes

no ~~nbhtion between N.O. and a non-N.O. (Fig. 22:I), a

meximum of 4 nucleoli can be expected at telophas-I. The

presence of 4 to 8 nucleoli at telophase-I ccrrpels us to suggest

allosyrdetic reconbination. Furthemre, if the N.O.

chromsomes are not involved in reconbination there are only

three possible telophaseII distribution patterns. However, the

observed deviation in nucleolar distribution at telophase-I1 fits

well with the theoretical expectations assuming crossing over

involving 1, 2, 3 or 4 N.0.s with non-N.0.s (Fig. 22:II-V). Our

interpretation that variation in nucleolar nuuber at telophase-I

and telo@ase-11, &d the variation in nucleolar distribution at

telophase-I1 result from reconbination between a N.O. of

c. and non-N.O. of A t y h ~ b species or vicwersa is

su~ported by the fact that the nucleolar nunher never exceeded

eight, which is the maximrm that can be expected with 4 N.0.s

(i.e. 8 satellite chromatids). Nucleolar distribution to only

three of the four daughter nuclei in a given Pm: (Fig. 16Ej 17Cr

H and Table XIII) can occur only when the N.O. chrorroEune6 are

involved in reconbination with mn-N.0.s. Additional proof for

recok4inatlnn core. ' , . . v the telophase-11 ! ,- 'ri which have L'

Fig. 22. Possible nucleolar variation in pollen mother cells of the hybrids between Cujanus cnj'an and A t y Z o s i a species without recombination and following recombination between nucleolar organizing and non-nucleolar organizing chromosomes.

nucleoli (Figs. 16Et 11 and 178) . Frcm Pig. 22 it is evident that

without reanbination there nwer exists a possibility for more

than three nucleoli per nucleus at telophase-I1 when there is

nucleolar distribution to either three or four nuclei. The

occurence of three or m r e nucleoli per telophase-11 nucleus

might suggest a possible expression of otherwise latent N.0.s of

the parehts. But the presence of a total of eight nucleoli per

EmC with a regular 2-2-2-2 distribution in the parents and the

absence of PMCs with m r e than eight nucleoli in the present

hybrid rules out such a pssibility.

A nmjority of the PMCs in the hybrids exhibiting nucleolar

variation had only 4 nucleolit one in each daughter nucleus at

telophase-11. Hence it can be presumed that the no& N.O.

chromosome disjunction without reconbination is 2-2 (Fig. 22: I-C)

resulting in 2 nucleoli in each daughter nucleus thaton fusion

give rise to one nuclwlus. Nucleolar fusion is m n in

several plant species especially in meiosis (Darvey and Driscollt

1972; Sybenga, 1972; Bennett SL al, 1973; Darvey gL al., 19731

Flavell and O'Dell, 1979; J e s q and Subrahmnyamt 1984). The

remaining P.M.C.8 undergo allosyndetic reconbination with respect

to the N.O. involving a minimrm of one N.O.-chromosome and thus

deviate from the expected nucleolar n W r at telo@mseI and

distribution pattern at telophaseI1 assuming no reconbination.

The total n-r of nucleoli at telophase1 in a Fm3 is a direct

indication of the n m b r of N.O. chromss011~~ involved in

recohination. Although we obtained r variation ranging from 4-8

?q7c!ml i at telo@s=cT (Fi? 16A, I - ' it b'r= r~r't possihl~ tc~

score emugh c e l l s a t this stsge to mable u s to arrive a t the

frequency of reconbination p r a c t s . This w a s because it is

d i f f i cu l t t o get a PMC a t telaphese-I w i t h p r h s r y (unfused)

nucleoli owing to the short meiotic cycle in pigeonpea. In the

cells containing 8 nucleoli a t telophase11 the distr ibution

pattern of 4-2-24 (Table X I I I ) is only possible when a l l the

four N.O'.s a re involved in reombination with non-N.0.s.

The presence of 5, 6 and 7 nucleoli a t telophas-I1 many a

time w i t h variable s ize is due t o nucleolar fusion. In the l ight

of the different possibi l i t ies (Fig.22) the pattern of nucleolar

fusion could not be assessed. Although in the daughter nuclei

nucleolar variation of 0-4 existed a t telophase-I1 (Figs 16 and

17), rrost of the daughter nuclei a t the quartet stage showed a

single nucleolus with a few quartets (Fig.11H) ahowing 2 o r 3

d i s t i nc t nucleoli. Since the overall variation of nucleolar

nurrber can be attributed to both fusion and/or allosyndetic

reconbination, an estimate of such recorrbination a u l d not be

obtained. r evert he less, for any given nurrber of nucleoli a t

telophase-I and nrnrber and distribution of nucleoli a t

telophaseI1, the minimin n-r of N.0.s involved i n

allosyndetic recurbination could be given.

Another interesting feature in these hybrids was the

varist ion in nucleolar size. Variation in the nucleolac size,

e s ~ ~ i a l l y the presence of BMYer nucleoli than the nonnal

f o r r , 4 by a single N.O. chromatid (Fig.161), can be attributed

t c cither a crossover involving a break within the secondary

cori;triction or a crossver between two N.0.s resulting in the

Table YIIINucleolar variation at telophase-I1 in a hybrid between C. %?a and A. albicms.

I No. of nucleoli I , -

I I

4 / 5 \ 6 ; 7 1 8 I I

I I I I t

I 11 1 26 62 1 14 I 46 I

I I I

I I I I 39 8.8 [ 28.9 6.9 16.4

I I I 1

P.M.C1s NO.

0)

formution of a chromatid with tvo N.O. termini leading to

mcleolar domFnance (differential anphiplasty). If the first

alternative is assumed one wuld expect more than 8 nucleoli.

Absence of PMCs with more than 8 nucleoli rules out such a

possibility. Thus we are inclined to suggest that nucleolar

cbminance is operative in the present h*rid. There have been

several 'reprts on differential anphiplasty in interspecific

hybrids, wherein nucleolar expression of one of the ~pecies

involved in the cross is supressed (Navashin, 1934; Keep, 1962;

Subralnmnyam and Azad, 1978a.b). Partial supression of a N.O.

leading to a reduction in nucleolar size has been reported by

Nicalof f & & (1979) in their barley translocation lines.

As indicated earlier in spite of the profuse flowering and

regular meiosis the high degree of pollen sterility and low seed

set in the hybrids of Sajaus with Asian AtyUshs is surprising.

Structural heterozygosity in the cluommmes of the parents as

evidenced by the allosyndetic recorrbination in the present

hybrids could b'e the major reason for the sterility, since the

pllen fertility was mch lower than expected on the basis of

m& bivalent formation and disjunction suggesting thereby,

complete h l o g y is not a prerequisite for regular bivalent

formation. The absence of nucleoli in som of ,the daughter

nuclei clearly indicate difficiency for the nucleolar organizer

while the presence of four nucleoli in a daughter nucleus is

suggestive of duplications for the nucleolar organizer. AlthDugh

these two are the exclusive conditions which indicate deficiency

L-t.;i..ptidi~, d . ~ .d,(je ~i k d i a t ; - . . L ~ U O L B ~ EJ;LSU inclucirs

such possibilities. It has already been demonstrated (Burnhw,

1950) that translocations involving satellite c h r o m s m ~ ~ result

in duplication-deficiency aberrations leading to pollen

sterility. Stebbins (1971) disassed the causes of sterility in

distant hybrids and proposed that "cryptic structural hybridity"

could be the major contributing factor for sterility in the

hybrids 'kssuming that the parental species differ with resped to

many .mall chrorrpx,mal rearrang-ts such as interstitial

translocations. Since then several workers on hybridization have

attributed sterility in spite of normal bivalent formation and

disjunction in their hybrids to a series of gmall structural

differences in the parental chromsoues. Our results provide a

conclusive evidence for that possibility.

The cryptic structural alterations involving .the satellite

chromosomes and chronosomes that pair with the satellite

ch~omsmes were discernible in view of the advantage of the

nucleolus act- as a marker for the products of reconbination,

while reexbination'arising out of structural alterations in

other chrcmvsomes is not traceable at later stages in the absence

of any such cytological marker and hence the structural

alterations traced with the aid of the nucleolus form only a part

of the total.

Regular meiosis in the ~-~ hybrids Micate a

dose relationship between the cultivated and the wild species.

The high frequency of recabination in the hybrids further

confirms the proximity c>f the parental species and suggests that

the species have differentiated through structural alterations of

chrowsomes. Cur findings s w r t the carclusions of - other

workers that the taxonomic separation of and

should not be wintained ~~, 1975; van der Maesen, 1981) . Cur findings on the chromosome pairing and reambination in

the S a j m u s - m hybrids clearly indicate that A.

is the closest relative of G. e. The regular ch ro l l ~sa~e

pairing in the hybrids of Sajaus with A. caianifolia and a

relatively high degree of f e r t i l i t y inplies a close genome

relationship between these two species. "Cryptic structural

differences" i f any between these genome6 are less than i n the

other species. On the other hand the relatively high degree of

pollen s t e r i l i t y in the hybrids of C a j a u s with the other species

of Asian m, which is unaccountable on the basis of

n-eiotic pairing and disjunction in their hybrids, is a

consequence of gametic duplications and deficiencies resulting

from structural differences in the chromo&me~ of the species

involved and consequent reconbination which has found clear

cytological evidence in the pairing of N.O. chromosones with non

N.O. chromsomes and the data on nucleolar variation. Hence the

four A t y k s b species, A. nlbicans, 8. Ilneata, A- Bcarabaeoidefi

and A. sericea which have differentiated through structural

heteroygosity are closely related to but m r e distantly

than 8. caianifolia. &mng these four species, a l l of which

differ from on the structural basis, it is difficult to

establish the hierarchy of relationship with respect t o CaianuR

since in th i s material the limitations in chromsome size dces

2 . . -. 1.-2::: :i t: ,-L-.tify ttre s t r b c t u r a l difftit, .~,. L s o the

differences in sterility between hybrid ombinations are not

different enough to delineate their closeness of relationship.

Since univalents though at a low frequency, acconpanied by a low

frequency of anapbase abnormalities were found in all the

cultivar cabinations of - A. Bcaraboeides hybrid it is

possible that the genome of A. scaraboeides is relatively more

distantly related to than those of the other three

species. A. albicans and 8. lineata share a om-on feature i.e.

structural heterozygosity with respect to the nucleolar

organizing chrmmnes. Both the Australian & y l ~ & ~ used in

our study are cytologically distinct £ran with respect to

the Asian &y l~& species. The structural heterozygosity

between the genomes of Qjanw and the Australian

discussed earlier in this chapter contribute to the high degree

of sterility in these hybrids.

Our results clearly krdicate that pollen sterility and ovule

fertility in the hybrids is a reliable index of genm

affinities. FO; example the hybrids with A. caIanifolia which

were cytologically mst stable had the highest pollen and ovule

fertility whereas hybrids with A. clrandifolia and A. J&bp&i

exhibited the highest degree of meiotic abnornvllities and the

highest pollen sterility 'thereby inplying that the levels of

garretic sterility reflect the degree of divergence of the

species fran C. &an.

Pgatt from the W e major general trerds the hybrids also

exhibited a few cgtological features which merit discussion in

stmne detail. OccassionaZ meiotic restitution was observed at

division-= in the figbrid C. x A. w. Restitution has been rwrtcd in several species hybrids (Li 9t Blr 1964;

Witgenarr, 1968a, b; Hann and Sasakuim, 1977; Riley and Chapan,

1958; Islam and Shgherd, 1980). Meiotic restitution could be a

result of delay in spindle fonmtion or due to spindle fusion.

Restitution is also possible when the t h + required to reach

univalent accrrrrulation exceeds the period of activity of

kinetochores resulting in failure of anaphase movanents as has

been suggested by Islam and Shepherd (1980). In the present

instance the occassional restitution might be h e to either

spindle fusion or delayed spindle fonmtion. If $he restitution

nucleus h a w to form a fertile gamete as has been reported in

species hybrids of Arachis (Singh and b@ss, 1984) a faw triploids

can be expectd in the F2 progeny of C. &an x A. m. . .

(hrormsome ~mvemmt to five poles in hybrids of !2ajaum with the

Australian is also a consequence of spindle

abnonmlities. Fosmation of chromosome sub groups resulting in

m r e than four spocade has been reprted in allopolyploids, for

exanp?le Buhus. (Bwi, 1965) and GUl&a (Lh, 1973). Such a

situation has also been reprted in a trihaploid of Hordeurn

(w m d Subrahmanym, 1985). In this case the

plyads w r e a -ace of genome specific organization at

division-I. In the present hybrids the occurrcnoe of ~ m r e than

four sporads result from alte s?indle organizatior.

In all the five --- hybrids from which a F2

population was raised, a majority of the segregants showed an

inproved level of fertility over the Fl hybrids while a few

segregants did show fertility less than that in the F1.

Redovery of fertile segregants fran the progeny of partially

sterile interspecific hybrids has been acamplished in genera

like Nicotiana, Tm&xam . . , (Stekbins, 1950) and also in

species crosses of wlilotus (Webster, 1950) and

(Steins, 1958). Such results have also barn reported in the

segregants of hybrids between x E. coccinw

(Ilaq & dl, 1980) and in crosses involving lonaialllmic; and

puaa (Yamaguchi gt Bl, 1976).

Stebbins (1958) suggested that the F2 segregants which show

inproved fertility over the Fl have a~pacently recovered a

relatively higher degree of genic balance than tbe one existing

in the F1. A failowup of this interpretation would be whether

the opt- genic balance corresponds to the balance that existed

in the parents or whether it denotes a new balance. In the

present case several of the F2 segregants which shcrwed -roved

fertility were wrpholqically distinct fran the wents, with

the F2 poprlations showing a high degree of vaciability for

different characters, a point which will be discussed in mre

detail in the cbapter 6. The remvery of fertility in the

segregants without recovery of parental morphology suggests that

rj-n recover& pciiic t~ , .~ . - .e A&. a new orrs. lnlr can be oonfrrmfu

by crossing the f e t i l e derivatives to the or ig ina l parents i.e.

back crossing and i n case t h e derivatives have acquired a new

balance, the progeny of t h e back CIOSS w i l l again be expected t o

be p a r t i a l l y s t e r i l e .

Secondary association of bivalents w a s evident in some F1

hybrids and w a s prevalent in e3me F2 segregants. Secondary

associations appear due t o the presence of genetically and

s t ruc tura l ly similar chrorro50rpes (Riley, 1960) and t h e bivalents

involved i n such associations have "residual homology"

(Darlington and Moffett, 1930; Thonvrs and Rwel l , 1946).

Kenpanna and Riley (1964) with the a id of te locentr ic chromsoms

danns t ra ted t h a t secondary associations occur between

genet ical ly related bivalents and that they a r e independent of

s imi la r i t i es in t h e s i z e of bivalents. I n t h e present material

t h e secondary associations further re f lec t t h e presence of

s t ruc tura l a l t e ra t ions in the related genomes.

The aperance of rmltivalents in some of t h e segregants

could be an a r te fac t of chromsorne s t ickiness s ince never could

we observe any clear corifigurations of higher associations.

As is evident from t h e resul ts and t h e above discussion

there is no chromosome elemination in any Saimu-Atybsia

crosses. Kasha and Kao (1970) reported recovery of haploids from

the cross Hs&em Y U ~ I ~ E x H. buEQsm and Sbr-yam ancl

Rasha (1973) reporte t h a t the haploids from this cross a r e a

consequence cf gra3,rl ant! selective elimination of the b l b ~ s u m

chrcrc.sc~r,~r frorr, t v t,ric' ert -yo-. In the last ! YePr? si.?r<

the se lec t ive chromosom elimination wthod oi haploid production

-nly kKmn as the "Bulbosun methodm has caw to l i g h t there

has been no r e p r t of haploid prcduction by this method without

t h e involvenent of a I u d a m species. I n both wheat (Barclay.

1975) and (Bother & & 1984) two other species

where haploid have been rewvered & the Bulbosum method,

species ' of tIordeLln a r e involved. In t h e genus Nicotiana

s e c t o r i a l elimination was recorded in interspecif ic m r i d s

(Gupta and Gupta, 1973) . A systanatic screening for select ive

chromosome elimination both in the present study in pigeonpea and

also i n the genus &&&is (Subralnnanym, unpblished and Singh

and Moss, perrs. comn.) there has been no evidence of

elimination. Thus select ive chrormsome elimination appears t o be

of limited w l i c a t i o n in haploid production.

The segregation and inheritance of the following qualitative

t r a i t s was studied in the hybrids of C. rainn x A. - I

C. S&II X A* lineatat C. x A. -, C. X

A. sericea and C. &an x A. caianifolia. The fanale Sdjmu

genotype i p a l l the cases was Pant A2.

SEED sl'mPHIOLE:

Strophiole on the seed is rudimMtary in C. cv Pant A2

while a prominent strophiole is present on the seed of all

BtylPsia species. In all the five hybrids the seed on the F1

plants invariably had a prominent strophiole. In the F2

generation the segregation for seed strophiole found ,a good f i t

for the ra t io 13:3 (Table X I W except in C. a x

A. caianifolia where the segregation found a good f i t for 15:l

ratio.

While the seeds of &yl.~&i species are mt t led , in cv Pan t

A2 the seeds are not rrottled. Seeds produced on the f ive F1

hybrids were mottled. Fp segregation i n C. sajm x A. &Usas,

C. &an x A. C. &an x A. scarabaeoiaes and C. &an x

A. lineata found a good f i t for 9:7 rat io (Table XV) . Whereas

the segregation pattern in the FZqs of C. &an x A . m did

not f i t into any of the standard genetic ratio's.

Table X I V . Inheritance pat tern o f seed strophiole I n C. mJm x specles hybrids.

F2 segregation Proba- Cross o d F1 ----------------- X2 b l l i t y

Pre- Ab- Rat to sent sent

C. LalM x A. &&au Absent Present Present 154 28 13:3 1.35 0.5-0.25

C. c&.l x A. Absent Present Present 189 18 15:l 2.11 0.25-0.1 - Absent Present Present 140 23 13:3 2.30 0.25-0.1 A. SeLUa

I;. r;pbn x A. Absent Present Present 165 46 13:3 1.28 0.5-0.25

C. ralan x A. l h & a Absent Present Present 245 46 13:3 1.65 0.25-0.1

Table XV. Inheritance pat tern o f seed mottles i n E. x At~ lo& species hybrids.

Cross FZ segregation Proba-

P d F1 ----------------- $ b i l i t y Pre- Ab- R a t i o sent sent

E. c&n x A. Absent Present Present 101 81 9:7 0.04 0.9-0.75

C. rnipp x A. ~ I f o l l a Absent Present Present 127 80 9:7 2.19 0.25-0.1

G. l a a n x A. zmkm Absent Present Present 110 53 .9:7 8.36 < 0.005

C. S A j m x A. Absent Present Present 118 93 9:7 0.009 > 0.9

C. !abn x A. llneata Absent Present Present 170 121 9:7 0.55 0.5-0.25

Although the pads of C&nw have hairs, the hair being very

short the pods a ~ p e a r almost glabrous, while the pods of

8. scarabaeoides, A.lineata arid A. sericea have very prominent

and dense hairs. In the Flts involving all these three BtylPsin

species, pods had dense and long hairs similar to those in the

A t y b h parents and in all the three cases the F2 segregation

found a good f i t for the s-le 3:l ratio (Table XVI ) .

!bo (A. n?bicans and 8. -) of the five &&&,a

species under study are clinbers while the rest are erect types

as in C&nw, hence the inheritance of twining nature w a s

studied i n the hybrids of C. cajm x A. albicans and C. & x

A. --ides. Hybrids with A. all&ma were twiners in their

gross morphology. In the F2 generation although minor variations

were recorded for the erect or twining nature, basically the

segregants were either twiners or erect types and the segregation

found a good f i t for 13:3 ratio (Table XVII) . The hybrids of

Cajau~ with 8. sericea were intermediate with a bushy base and

twiny nature in the upper pr t ions and the F2 segregation in this

case did not f i t into any s-le ratio.

Table XVII. Inher i tance p a t t e r n o f tw ln lng nature I n C. x dtrlrr.la rpec i hybrids.

F2 generation CMSS O d F1 "-"'--"--'-"--"--"'- Proba-

Twin- Erec t I n t e r - R a t i o x Z b i l i t y f ng mediate

------------------------*----------------.------------------------------------------

C. rPlan x A. alhlcans E r e c t Twining Twining 150 32 - 13:3 0.16 0.75-0

C. SLbn x A. scarbbasoldel Erec t Twining Twining 35 74 102 - - -

( I n t e r - mediate)

Table X V I I I , Inher i tance p a t t e r n o f l e a f l e t shape i n C, faja x &h& speci hybrids.

F2 generation Cross P u F1 --"-""""""--'-"'- X1 Proba-

I n t e r - Rat io b i l i t y type mediate t y p e

C. rninn x A. rlblcans Lancec- Obovate I n t e r - 39 90 53 1:2:1 2;16 0.5-0.;

1 a te mediate

C. rPlnn x A. shu&kd% Lanceo- Obovate I n t e r - 48 104 59 1:2:1 1.18 0.75-0.

l a t e nediate

The leaflet Bhape in C. Eajan is lanceolate while that in

A. alblcma and A. scarabaeoides is &ovate. The leaflet shape

i n the hybrids involving A. albicans or A. scarabaeoidee was

intermediate to the parents. The F2 generation segregated for a

1:2:1 ratio for leaflet shape in both the goplations

(Table XVIII) .

SEED mPH1OLE:

Our results clearly indicate that the seed ~triopbiole in

the A t y l ~ & species is a dominant trait and is controlled by two

genes. Even in the hybrids with the two Australian species where

genetic studies were not conducted the hybrid seed had a

strophiole, unequivocally establishing the dominant nature of

this trait. Re* gk al (1980) reports three ratio's, 3:1, 9:7

and 13:3 for seed strophiole expression in crosses involving

~pecies of A. sericea and A. scarabaeaides while Pundir (1981)

indicates that two genes with a duplicate gene action (15:l)

govern this character. In the present study four out of the five

hybrids segregated for a 1333 ratio indicating a inhibitory gene

action while the hybrids of C. x A. caianifolia segregated

in to 15 strophiolated to 1 nonstrophiolated types. A

significant cbservation is that the seed geminability in the F2

poplation$ of C. & x A. caianifolia which has deviated from

the 13:3 ratio has been poor (Table 111) when compared to others.

This inplies that the genetic factors reqonsible for the absence

of strophiole are linked with certain lethal factors affecting

sed germinability.

8EPDlwmtINs:

Seed mtt l ing being invariably expressed in all the hybrids

is a dauinant t r a i t . Re* & a (1980) i n hybrids of

with A. scarabaeaiaes reported a 927 ratio when I B 6915 and

ICP 7035 were used as ferrale parents and a 15:l rat io when

I B 6997 was the female parent. In our studies 4 out of the 5

ppl la t ions found a good f i t for 9:7 ratio indicating a

co11@1ement&~ gene action. The segregation in the hybrids with

8'. sericea did not f i t into any of the standard genetic ratio 's

although it w a s nearest to the 9:7 ratio but it finds a good f i t

for 2:l ra t io suggesting a close linkage with a d d o m i n a n t

lethal of the carplimentry gene(s) responsible for the

experession of this t r a i t wherein the lunmzygous daninant

genotypes do not survive.

m HAIRINESS:

Pod hairiness of the Atyk& species is dorninant over the

relative plbescence of C. s&a. Reddy (1973) and Pundir (1981)

found that pod hairiness of E & y h h species is a mnogenic

dominant t r a i t . Cur results for this character agree with the

two earlier reports since in all the three hybrids involving

p i e s of &&& with hairy pods the pis in the Flls were

hairy and in the F2 generation they segregated in a simple 3:l

ratio.

The segregation pattern of twining nature in hybrids

involving A. rlbicans indicated tbat the character is gwerned by

two genes with a inhibitory gene action. In r. a x A. the F1 was intermediate to the parents in its

habit and in the F2 generation all the three types i.e. twining,

intermediate and erect types were recovered and the pattern of

segregation did not fit into any of the genetic ratio's.

Accurate classification of segregants in to distinct groups was

not possible for this character due to overlapping of characters

between the groups.

LERFLFT SHAPE:

Leaflet shape in both the hybrids studied was found to be

controlled by a single gene with a pactially &&ant gene

action. Reports on genetic studies for leaflet shape in S&au~

are variable. Deshpnde and Jeswani (1956) reported both 15:l

and 3:l ratio for lanceolate and obcordate types and the studies

of Jeswani and Deshpande (1962) and D'cruz and Deokar (1970)

indicate a 3:l ratio, while the studies of Patil and M a r

(1980) and Chaudhary and Thorrt,re (1977) indicated that m r e than

m e gene controls leaflet shape.

In intergeneric crosses involvhg C. and

A. srrarahneeides, Reddy (1973) and Pundit (1981) reported a 1:2:1

ratio which is in agreement with cur results.

Variation f o r length (Table XDU , 100-seed weight

(Table W) , seeds per pod (Tabla WI) , mibleaf lergth

(Table XXIII) and *leaf width (Table XXII) were studied i n t h e

F2 generation o f the hybrids of G -(Pant A21 w i t h 4,

a U k ~ & , A . s e r k t s a , A . -,A.lineata,andA.

The mean pod length of the F2 segregants in all the f i v e

popllations was clcse t o t h e mid parental value (Table X I X I . The

w a n f o r the p o p l a t i o n of C. a x A. rlbicans w a s 4.71 an

with a range of 2.8-6.3 an and a coefficient of var iat ion (c.v.)

of 19.5% as again& the parental values of 5.93 cm (C. -1

and 3.52 m (A. albicans). The difference between t h e parental

values was minimmar between C. c&n (5.93 cm) and A.

&mi&Ua (4.31 da) . The P2 mean in t h e p o p l a t i o n derived

from the cross between these species was 4.82 cm, t h e F2 range

being 3-86.1 an w i t h a C.V. of 13.99. The difference b e t w e e n

pwental valuesuasraximum i n C . rajnn (5.93 cm) x A. aericea

(1.35 an). The variation for p d length in fie F2 a p l l a t i o n of

this hybrid was 29.2% with the length ranging from 1.2 t o 5.4 an

w i t h an average of 3.59 m. Aln-ost ident ical resu l t s were

obtained in the ppula t ion of C. s&au x A. Jheata where the

average p d lengtbwas 3.35 an with a range of 1.3-5.1 an and a

C.V. of 29.9%. A variation of 22.47 i. .:: recorded in the F Z 1 s of

. s;aian x A. -e with t;,e pod length ra-:jing from

Table XIX. Variation fo r pod lmngth (a) i n the Fz population of hybrlds between E. mjm and spu i rs .

Mid- F2 population CFDSS 0 parental .........................

value Mean Range S.D. C.V. -----------------------------------------------*------------------------

Table XX. Variation f o r 100-seed weight (g) i n the F2 population of hybrids between C. a and &t&sh species.

Mid- FZ population Cross P parental -----------------------------

value Mean Range S.D. C.V.

Table XXI. Variatlon fo r seeds per pod i n the F2 population of hybrids between C. ajan and Atvlosrr species.

~ lb - F2 population Cross a .........................

value Mean Range S.D. C.V. ............................................................................

Table XXII. Varlatlon fo r mld-leaf wldth (cm) I n the F2 population o f hybrids between C. & and specles.

Mid- F2 population Cross. 0 parental ..........................

value Hean Range S.D. C.V.

Table XXI I I . Var lat ton f o r rnfd-leaf length (a) i n the F2 population of hybrtds between C. and spectes.

Ut d- F2 popul at ton Cross parental ..........................

value Mean Range S.D. C.V.

2.5-5.5 an with an average of 3.62 cap.

SEED WEIGHT:

A rmch greater variat ion ranging from 21.1-33.3% was

recorded for seed weight (Table XX) in the f ive p o p l a t i o m but

t he F2 mean w a s again mostly close t o t he mid parental value.

Seed weight in Pant A2 is 6.65 g. The 100 seed weight in the

~ O U K Atylosia species, A. irlbicans (2.52 g) , A. sericea

(2.28 g ) , A. scarabaeoides (2.10 g) and A. lineata (2.35 g) were

close t o each other while that in t he other species, A.

-was 4.46 4. I n the F2 generation the 100-seed weight

ranged from 3.00-7.18 g in the popllations of G. u x A.

t 4.71-6.77 in the pop l a t i on of

G. sajm x A. s & u U d h , 2.13-6.45 g in that of C . ' u x A.

sericea, 2-15-6.13 g in C. Eainn x a. scarabaeoiaes and

2.23-6.19 g in G. m j a ~ x A. lineata with 4.16 g, 5.4 g , 4.3 g,

3.7 g and 4.09 g being the respective means.

SEEDS PER POD:

None of t h e segregants in the f i v e F2 p o p l a t i o m exceeded

t h e be t te r parent value for seeds per pod (Table X X I ) .

Segregates w i t h fewer Fseeds per pod than t h e poor parent were

c o m n as is evident from the range (Table IW). The mean in the

F2 p p i l a t i o n of C. c&n x A. mjaUsUa (3.311, C. G&AR x

A. eericea (2.51) and C. G&AD x A. lineata (2.71) were close

to the mid parental value while tk~sse of C. rajan x B . a l r i w

(2.61) and C. x A. scarabaecides (3.05) were l e s s than the

mid parental value. The coefficient of variation for this

character was maxirnrm (32.6%) in the popllations of C. &an x A.

and minm (6.3%) in that of S. s&n x A. . . m, while in tbe other pgulations the variation was

21.4% in C. a x A.lineata, 22.3% in C. x

A. and 30.7% i n C. @an x A. aericea.

F2 variation for mid-leaf width was maxirnrm (31 .I%) i n the

population of C. &an x A. sericea, while the other four

poplationsr C. 4 . a ~ x A. c&dbU (5.181, C. Edjan x

A .albicans (11.4%), C. x A. scarabaeoids (17.1%) and

C. a x A. lineata (16.3%) shmed much less variation

(Table XXII) . The F2 means for mid-leaf width althdugh close t o

the mid-parental valuer fractionally exceeded that value in G

~ x A . albicanstC. b X A . scacabaeoiaesandc.

x A. sericea. The width ranged from 2.4-5.3 an in the poplation

of S. x A. w, 0.7-2.9 an inC. u X 8 .

sericear'1.4-3.3 an in C. x A. scarabaeoiaes and 1.4-3.2

an in the population of C. G&AII x A. lineata while the range

(2.4-2.8 ad was very narrow in the p p l a t i o n of C cajan x

A* caianifolia*

Studiea on F2 variation for the charactera pod lqth, seeds

per pod, 100-seed weight, mid-leaf width and mid-leaf length have

shown interesting trends. In general the Fq means with some

exceptions have been close to the mid-parental value but

transgressive segregants, i.e. those exceeding the better parent

values have been recorded for all the characters except seeds per

pod: Transgressive segregation has been most pronounced for the

leaf characters especially in the population of C. a x A.

albicans. Transgression of a marginal msgnitude has also been

recorded for pod length and 100-seed weight in the F2 '6 of s. raian X A- albicansdnd C. &an x B . c a i a n i f o l i a . A f a

-regants with leaves wider than the better parent have been

obtained from the poplation of C. x A. seric9a. As is

evident from Tables XXII and XXIII leaf length and width in some

of the F2 segregants of 6. &an x 8. is considerably

higher than that of the better parent.

Another striking feature in these studies is that the

variation m n g the five F2 populations is invariably mininun for

all the characters studied in the cross C. cajan x A. . . -, the variation being as low as 5.1% for leaf width,

7.1% for leaf lergth, 6.3% for seeds per pod, 13.9% for pod

length and 21.1% for 100-seed veight. FOE 100-seed weight and

the leaf characters the variation has been mxinunn in the

poplation of G. c&an x A. sericee while for seeds per pod it

is highe?' m the population of C. x A. abiwns. In the

h ;, ~i LA ciuan_x Pu -egreqmts wltn values

better than the better parent have been recorded for all trait6

except seeds per pod. F2 segregants poorer than the poorer

parent have been recorded in several cases.

Transgressive segregation for most of the characters

revealed the potential of the A&b&a species for pigeonpea

inprov-t. . BarBacki & al (1976) has enphasized the role of

transgressive segregation in crop improvement. Stebbins (1977)

and BarBacki & a;L (1976) have sham that transgressive

segregation front distant hybrids can give rise to extreme

characters and a range of new forms. The exploitation of the

transgressive segregantv would be a viable method for the use of

wild v i e s in the inproverrent of the cultivated species

especially when the cultivated and the wild f o m share a comron

chrcmsome n-r as in pigeonpa. Such studies i.e. those

aimed at the exploitation of transgressive segregants have been

~ ~ ~ e S 8 f u l l y attempted in other crop species (Reeves, 1950;

Raeves and BocWlolt, 1964; Efron and Everett, 1969; Harlan and

Dewet. 1976; Lawrence and Frey, 1976; Frey, 1976; Frey & al,

1983).

In the present study as indicated earlier trangressive

segrega~ts of considerable significance especially for leaf

characters have b- recorded in the C a j m u s - u F2

popllations. Such segregants for leaf characters assume greater

significance in the light of the findings of Shacm and Saxena

(1982) who have shown from intervarietal cross of pigeonpea that

lesf characters ere, k '.;hly heritable with a positive irfluence on

grain yield. The ~arginal hiproverent over the better parent in

the eeed weight in aome of the F2 populations is also a point of

significance. In a nutshell these findings indicate that the

species of -can be exploited for the inproverent of

certain quantitative t r a i t s of Chianus apart from their u t i l i t y

in incorporation of specific t r a i t s l ike disease and insect

resistance.

Further the wide range of variation recorded for the

chdracters studied with segregants better than the better parent

and poorer than the poor parent is indicative of the substantial

degree of reconhination between the gerromes of and

m. In these studies the variation recorded has been least

in the population of (laianus x A. . . which further

swstantiates the uor#mlogical and cytological evidence for the

relative genetic proximity of these two genomdspecies.

The limited investigations made on seed protein c o n t a t

reveal a dist inct trend. In all the F1 hybrids the seed protein

content is close to the values recorded in the better parent

!&y&&al w i t h the lone exception of 6. a x A. aranaifolia

where the protein content was intermediate to the parental

values. These results i.e. the proximity of the protein

percentage values in the hybrids to the values in the Aty&sia

parents indicate that the high protein content in the E Q h s h

parents is a dominant t r a i t . W h r (19%) re~or t ed part ial

domFnance for high protein content in crosses involving wild and

cultivated saybcans. It has not been possible t o analyse the

protein content in all the F2 segregants due t o limitations in

s& sar.p~r; wt analysis m sane segrqates silowed values

mrginally better than the better parat values. Haever,

without sufficient data it is not appropriate to conclude that

these are transgressive segregants since environment exerts a

strong effect on protein percentage.

Certain characteristic and interesting segregants were

&served in some of the F2 populations. In the poplation of

C. SWXI x A. pods in one of the segregants were with

and with out streaks on the sane plant. A few segregants in the

ppllations of C. sajm x A. and !2. x

&. scarabaeoides were found to have seeds of different colours.

A segregant each in the poplation of C. i&m x A. lineata and

C. C ~ J . ~ D x A. nlbicans was found to be non branching.

The occurrence of pods and seeds of different types on the

same plant is a possible case of chimera. Such chimera1

ir.dividuals especially for pod colour were also observed in the

progenies of Ca.ianus intervarietal crosses (K.B. Swena, 1984).

A non branching single stem mutant of the type observed in so=

of the F2 pplations was earlier reprted (Dahiya and Sidhu,

1979) as a spontaneous mtation in the F2 generation of a Sajaru

intervarietal hybrid. These aberrant types in the F2 popllations

l r ~ g h t also be a consequence of some freak genic constitutions as

a result of recarbination and segregation in the F, hybrids.

A t t m t s to standardize culture conditions for regeneration

of Q&aus and EQl.&a plants f r m different explants such as

a t y l e d o n s from mature seeds and leaf and epicotyl s-ts from

one week old seedlings resulted in different degrees of success.

~ o t ~ l e d o r . t i s s u e wss found t o be the best responding explant in

our i n i t i a l exploratory a t t enp ts and further detailed s tudies

were conducted on t h a t explant. The i n i t i a l a t t q t s a l s o

reveal& the superiority of H.5 M i u r n over the others hence, t h e

r e m n s e of cotyledons was studied on MS medium.

B&s.zl ( V S ) W i m su~le inen ted w i t h 2, 4 -0 ( 2 q/l) induced

copious mounts of heaithy ca l lus from all e x p l a t s i r respect ive

of the r q i o n of the cytoledon used. kaole cotyledons and nodal

halves of the qotylfdons on MS medim. su&plenented with 2 , 4-D

(0.5 q/l) and BA ( 2 q/1) developed rmlt iple W t s (3-7) in

addition t o mall m u n t s of callus (Fig.23C) in 2-46% of t h e

cul tures depending on t h e cu l t ivar (Table XXIV) . &mng the wild

re la t ives , m l t i p l e shoots developed from 21% of A. caianifolia

cul tures , while in t h e ccl tures ..of 8. and A. sericea

n.=inly s ing le shoots developed (Fig. 23B) and multiple shoots were

rare . In tkie cul tures of the d i s t a l s-ts of the cotyledons,

only shoot bud I:.itiation was observed i n l o w frequencies a f te r

profuse ca l lus i r~g . From t h e explants with nul t iple shaots,

r o o t k g w a s &t:-:: +.: cn b5 d i m supplerented with NAA ( 2 rq'l)

acd EA (0.5 &1:.

Fig. 23. In vitro regeneration of Caj'aras and A'y ios ia plants from cotyledons and immature embryos.

A) Sequence of plantlet regeneration from whole cotyledons of C d n u s ccjan.

B) Shoot regeneration from whole cotyledons of AtaZoslc sericec.

C ) Mu!tiple shoot formation in whole cotyledons of Cajcms @Gun.

D) Sequence of plantlet regeneration from immature embryos (15 day old) of Cc,icn.us ccj'cn.

T a b l e ~ x ~ \ ~ . ~ e r c e n t a g e s h o o t r e g e n e r a t i o n from c o t y l e d c n c u l t u r e s o f d i f f e r e n t c u l t i v a r s of C-s and s ? e c i e s of u c s ; e on HS rr.edium supplemented w i t h 2 ,4 -3 ( 3 . 5 mg/l) and & A t 2 n q / l J .

-- ---------------- Whole Cory1ec:n seqrnents

Spec les /cu i : i ' ; a r5 COty- ------------------ l e d on Nodal E l s t a l -----------------------------------------------------------------

G G ~ ) E D cv ICP 7335 4 6 3 1 1 3

Basal W i m n sqpl-ted w i t h 2, 4-D (0.5 u ~ / l ) , 54 (2

mg/l) and NAA (1 nq/l) also meed rml t ip le shts from whole

cotyledons and r d a l cotyledonary seguents. In this honmne

d i n a t i o n both shoots end rwts developed from 2 t o 14% of

wble-atyledon explants of C. &an and from a b u t 2% of

A. caianifolia (Table W . A l o w frequency of plant

rweneration was obtained from cul tures of IKXW cotyledctlary

s-ts i n C. &. In t h e cul tures of &. and

A. sericea only shoot regeneration was obtained.

Of t h e four C. cu l t ivars tested, three cu l t ivars

showd varying leve l s of regeneration, while the fourth cul t ivar

GS 4 , which had the smallest seed s ize , dLrrPst mmpletely fa i l ed

to respnd . The r e s p n s e of A. ca'lanifolia, which is

m r p b l o g i c a l l y q u i t e similar t o L;. e, c o n p r & well w i t h the

response of C. & cul t ivars .

A g e n e r a observation i n cotyledon cul tures has been tha t ,

wherever there was m l t i p l e shoot f o m t i o n , only one or two

shoots developed into f u l l shoots while t h e others r a i n e d

su~pressed.

Respnse of errbryos from four genotl-5 of pigeonpea

(Pant A2, ICP 7035, P r a t b t and C 11) w z s evduated on B5 nediw

t;ug.@mted with 2, 4-D (1 rrq/l). An age d-ent d r y 0

resporse was evident (Table XXVI) . Eleven t o fourteen day old

e:trvos i=-,.c:c:.<-c- r : :?;? f r c : wt.:.:? r- ICK f r x , . e=r_ , ?f p l m t l e t .

T a b l € \ u i . P e : c e i i a q c p l a n t l e t and s h o o t r e c e r . e r + t ~ o n f rom cct)!cdar , c u l t u r e s of different W ~ S c ~ l t l v a r s a r d s p e c l e s of &-8 on fi5 m e d l ~ a s ~ p p ? e - e n t e d w l t n i , 4 - C ( 0 . 5 m g / l ) , BA ( 2 mg/l) and NAJ (1 nq / l ) .

Whole C o t y l e d o n oegn ,en t s c o t y l e d o n -----------------------------

-------------- Kodal D i s t a l Cul : lva r /apec les s h o o t s p l a n t - -------------- --------------

l e t s S h o o t s P l a n t - S h o o t s P l a n t - l e t s l e t s ---------__________----------------------------------------------

regeneration wa.~ obtained. I n 15-19 day old ent~ryos p lan t le t s

were recovered d i rec t ly a c a q m i e d by d l a m t s of ca l lus a t

the base ( F i g 23D) . Direct plant let recovery and occasional

ca l lus forrration w a s observed in a b r y o s older than 19 days.

m r y o s .younger thar, 11 days fa i l ed t o r e s p n d on both the d i a

and b-1 media sup~lemented w i t h several h o m n e a r b i n a t i o n s

fa i l ed t o extract any r e p n s e from such d r y o s .

Though there was no s t r ik ing influence of genotype on

p lan t le t recovery, ICP 7035 performed marginally bet ter in all

t r eamts (Table XXVI) . ICP 7035 has the l a rges t seed s i z e of

t h e cu l t ivars used, in this study.

Cdllus w a s obtained from the mthers of C. c&n and

A. alhicuLs on M.S rredim supplimnted with 2 4 1 of 2, 4-D.

Potato s tarch extract sd iun : ~ r o m t & a l l u s developnent from 'he

anthers of A. ora.ICifolia md A. volubllls . . . Callusing was m r e

profuse from the anthers of A. aJ&ms and A. ~ Q W U than from

the oL&r slpcies . A t t q t s t o induce different ion by

s k m l t u r i n g t h e callus on k a l media supplimentd with various

horrrones and t h e i r coribiiiations did not meet with success. C k

s , a l t u r i n g the c i l l c s turn& trow. and dqu .e ra te2 .

Table SXVI. Percent plantlet r e c o v e r y i n embryo c u l t u r e s of p i g e o n p e a o n - H S and BS .media supplemented with 1/1mg o f 2,4-D.

Embryo age Ca,enus c u l t i v a r s ............................ P a n t A2 Prabhat ICP- C-11

7035

ns - <11 days

B 5 -

HS 11 11-14 days

B 5 17

HS 7 3 6 9 8 1 6 7 15-19 days

B5 7 4 8 8 93 8 4

US 5 7 6 6 7 1 5 5 >19 days

B5 8 7 9 0 8 9 8 4

I n cotyledon cul tures bksal medium supplerented with 2, 4-D

induced only ca l lus while the rredium su~plemented w i t h both 2,

4-D and FA induced shoots. Robting of such explants was'achieved

on the medium when it was su~planented with W.&. When the basal

medim was scgpl-ted with bath BA and NAA in addition t o

2, 4-D a low frequency of whole plants were recovered. These

findings suggest thet while BA plays a role in s b t regeneration

while tGA helps i n rcot regeneration. CUtivar differences with

respect t o t h e percentage of regeneration (Table X X M is a

possible consequence of the genotypic e f fec t and such resu l t s

have been r e p r t e d in other species. This conclusion is

strength& by t h e f a c t t h a t the response of 8. caianifolia which

is genet ical ly close t o C. w a s similar t o the' response

obtained i n the a l t u r e s of C. -while t h e prforrrmce of

A. and A. m, w i e s Mich a r e rrore dis tant ly

related t o Caianc-+ was distinct.

Variation in respnse kas recorded w i t h respect t o the

cotyledonary s e g e n t cultured. The r e s p p s e of &dl s q e n t s

w a s c lose to t h e resLwrse ob'iained from t h e m l e cotyl&ns

while t h e r e s p r s o £LC,;: t:w d i s t z l segrmts was poor. The

irproved r.=sF<rrse cf the r.:dal s e p s n t s night be due t o the

presence of m r y c : i c cells in the nod& half.

Culture ccr.dl:ions for the regeneration of imature

pigeonpa erbrycr : rve tw scmdardized for the f i e s t tire. PLI

.:;: 5'; c-r'cnt cr.: - : c : ; . : ~ . : . \.;r cta i r .62 in t h e c:,tryc c l t - r c . -

~t vas mt possible t o regenerate plants from erbryos younger

than 11 day old. This has been a m n fea ture in other

leguminous species ( G o d and Bajaj, 1983: Newel1 and Fiymi tz ,

19821 Qlbero, 1981). Genotypic effect on percentage of

regeneration in d r y 0 cul tures was less pronouncd than Fn

cotyledon cultures.

Anther Eulture s tudies were not successful l beyond the s tage

of callus induction insp i te of a t t enp ts t o induce regeneration by

s u ~ l i m e n t i n g t h e basdl media with several b r a o n e carbinations.

Anther cul tures have been successful t o a la rge extent in

cereals , solanaceous crops e tc . but have not made an impact on

any of t h e leguminous crops. An e a r l i e r attempt on pigeonpea

anther a l t u r e s also did not progress beyond the c a l l u s induction

stage (Bajaj & a, 1980) .

8- GENERAT, DISCUSSION AND 00NCLL1SI<3N

present hestigation m Mtiated in to

understand the q=c-me relationahi~e bet= C. arrd

AQhsia species, screen -- crosses for selective

chrmaome elimination, develop methods to overcane barriers to

species crossability, study the inheritance and variation for

important traits and develop h YitEn rgeneration techniques for

pigeonpea. All these objectives have been met.

Out of the twelve species of Atyksia and two species of

used in the study eight species of hybridized

with the cultivated species. Thepreeent study has aho*m that the

species crossability is significantly Mluenced by the genotype

of the Cajmu pacent. Studies on h o m e treatments have

revealed that post-pollination hormone applications -rove

species crossability by checking the post-fertilization barrires.

Reciprocal success in these crosses is very difficult. Four of

the twelve Atyhs ia species failed to crose with Caiauu in any

direction. In the unsuccessful crosses post-pollination hormone

treatments delayed bud/@ drop but it was not sufficient to get

ovuledenbryos responding to in yitLp rgeneration. In YttEP

rescue of dry06 could be achieved only in embryos older than 11

days. Both hormone a~plications and d r y o culture techniques in

pigeonpa have been developed for the first time. The results

indicate that h o m n e amlications in conjuncture with &ryo

culture technique offer potential for obtaining new

~~ hybrids with further refinements of these

techniques.

The present study harr resolved the longstanding controvep

regarding n-r of nuc$lar organizer chromeomes in pig-.

In spi te of the limitations of short meiotic cycle and

di f f icul t ies in cbtaining good cytological preparations in

pigeonpe a detailed information on the nuker, size and

distribution of nucleoli a t T-I and T-I1 was obtained. Evidence

in the form of nucleolar n m r a t T-I and 'lr-I1 in diploid and a

spontaneous tetraploid of pigeonpea establishshed beyond doubt the

presence of two nucleolar organizer chromosomes in pigeonpea. A

similar situation exists in the species of -. This is the

f i r s t study t o use meiotic activity of the nucleolaf chrom- e

as an index of the nucleolar organizer nuker. The preqnt study

has also shown the attachment of two bivalent6 t o the

diplotenddiakinesis nucleoli and also the p r v c e of two

p r i m nucleoli a t pachytend diplotenddiakinesis t o

substantiate the presence of two nucleola I organizing

c h r - m . The chromsome n-r i n the Australian $ies of

&ylg&a is being reported for the f i r s t time.

The major accorfplishmt of t h i s investigation is the

detailed insight provided into the g e ~ m e &ationship between C.

sajan and the species,, A t y h d a . These investigations suggest

that C. &an and the spxies in the genus A t y l d a basically

have a similar genome or i n other w r d s they have evolved from a

c a m n gene pool and the differentiation has occur& as a result

of structural alterations. The closest relat ive of pigeonpea . .

among the specie6 studied is A. ca,nnlfolla. The major

oist inguishirq feat1 9 I r t w e w these t r q-~ccier is the prccc:: e

of seed strophiole. sane of the recent collections of

&an by the Genetic Resources Unit a t ICRISAT ,ahow a promihent

strophile (van der Maesen, pers. am.). In view of tbese

findings it i s likely that C. mj.an might differ fran A.

. . by only a few gene uutations. This was further

substantiated from the data on variation for quantitative t ra i t s

from the F2 populations. For a l l the characters studied the

variation .has been the lowest in the populations of C. x

A. caianifolia. The other species of that have

hybridized with C. &an are mrphologically mre distinct.

Meiotic studies of their hybrids reveal that they F e genomically

close to pigeonpee and have differentiated through structural

alteratiohs and the results have shown that the levels of gametic

s ter i l i ty reflect the degree of species divergence. Australian

species of A ~ Y ~ Q & are more diverged from than the Asian

species.

The predominantly one way success of the --&yl~&&

crosses suggest that the structural alterations and/or gene

nutations that have led to the species differentiation are

acconpanied by the differentiation of p l m n . There is no

evidence for chromosome elimination in the intergeneric hybrids

of pigeon*. The infomktion gathered in the present study is

rot sufficient to comnent upon the relatianship of C. Eajan

with the species of Fhyshsh except that they share the same

chromomme rumber.

The study on inheritance of qualitative t r a i t s has shown

that these t r a i t s are controlled by one or t m genes. Sweral

w e 1 segregants were recorded in the F2 generation and

transgressive segregants of considerable value have been

reomered in the F2 generation for characters l ike leaf length,

leaf width, seed protein content e tc underlining the importance

of species for the hprovement of the cultivated

' species.

This study for the f i r s t t h e has provided direct

cytological evidence for reambination between g a m e s by way of

studying the pairing of nucleolar organizer chromsomes and

tracing the products of recabination using the nkleolus as a

mrker. This technique can be a potential tool Fn reambination

studies in distant hybrids. These findings have also provided

the f i r s t cytological evidence for the structural hybridity

between Cajaus and B f s ! b s i a species.

'Ihe present investigation has provided anple cytological and

geneticdl evidence for reconbination between the genome6 of

Ca.ianus and A t y l ~ & species and has proved that the species of

A t y k s i a possess all the requisites (close genome relationship

with the cultivated species, a high degree of recarbination

between the genosrres and recovery of transgressive segregants from

the progeny of hybrids with the cultivated species) for effective

introgression.

In a overview the present imrestigaticn has shovn that the

species of Uyhda can be of potential in pigeonpea hprwment

programnes and these species deserve m r e attention than they

have received so far.

In view of these findings future attempts rmst be directed

a t obtaining hybrids between and species of

which have not been used so far in intergeneric studies. Further

refinements in unconventional techniques l ike d r y 0 culture and

hormone treatments and developrent of new techniques l ike

r-ition and mentor pollen techniques should be given

importance t o achieve success in otherwise incompatible crosses

in an attenpt to widen the genetic base. There is also a need t o

obtain hybrids between I&yh& species to be able t o gain more

knowledge about the divergence of these species from -. There is a need t o screen the progeny from the i n t e r g e r i c

hybrids and select t h e desirable segregants, evaluate them and

incorporate thm into pigeonpea inprovenrent p r o g r m s t o

achieve introgression of the deisred character by adapting the

relevant breeding mthcdologies.

The present study has shawn tha t tetraploid pigeonpea is

cytologically stable and hence long term attenpts must also be

directed a t crosses between tetraploid C&m& and species of

tO Obtain addition andtor substitution lines of

pig=%=.

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PUBLICATION FROM THE THESIS

1. P. Sateesh Kumar, N.C. Subrahmanyam and D.G. faris, 1982. Studies on developing pigeon-pea haploids. International Pigeonpea llewsletter 2 : 14-15.

2. P. Sateeah Kmar, N.C. Subrahmanyam and D.G. Paris, 1984. Nucleolar variation in a pigeonpea intergeneric hybrid: Evidence for allosyndetic recombination. Can. J. Genet. Cytol. Vo1.26(5): 499-505.

3. P. Sateesh Kmar, N.C. Subrahmanyam and D.G. Faris, 1984. Inter- 'generic hybridization in pigeongea: I. Effect of hormone treatments. Field Crops Research (In Press).

4. P. Sateesh Kumar, N.C. Subrahmanyam and D.G. Faris, 1984. In vitro regeneration of pigeonpea from cotyledons. International Pigeonpea Newsletter 3:15-16.

5. P. Sateesh Kmar, N.C. Subrahmanyam and D.G. Faris, 1983. Allosyn- detic recombination in CAJANUS-ATYLOSIA hybrid. Abstracts XV International Congress of Genetics, New Delhi. pp 671.

6. P. Sateesh Kmnar, N.C. Subrahnyam and D.G. Faris, 1984. Plantlet regeneration from inmature embryo s of pigeonpea, International Pigeonpea Newsletter 4 (In Press).

7. P. Sateesh Kumar, N.C. Subrahmnayam and D.G. Faris, 1985. Morpholo- gical variation and inheritance in a pigeonpea intergeneric hybrid. Current Science (In Press).

8. N.C. Subrahmanyam. P. Sateesh Kumar and D.G. Faris. 1984. Genome relationships in pigeonpea and its implications in crop improvement. Symposium on Advances in Genetics and Crop Improvement, Meerut. Abstract. p 11.