Indian Journal of Experimental Biology Vol. 39, November 2001, pp.1080-1095

Review Article

Citrus tissue culture employing vegetative explants

H C Chaturvedi*, S K Singh, A K Sharma & Savita Agnihotri Ti ssue Culture Laboratory, Nati onal Botanical Research Institute, Lucknow 226 001, India

Citrus being a number one fruit of the world due to its high nutritional value, huge production of frui ts and fru it products, the citrus industry may be considered a major fruit industry. Though citrus orchard area in India is comparable to USA, the produce is far less, while its export is nil. Biotechnology has played an outstanding role in boosting the citrus industry , e.g. , in Spain , which is no w the biggest exporter of citrus fruit with the application of micrografting. Amongst the fruit trees, perhaps the maximum ti ssue culture research has been done in citrus during the pas t four decades, however, the results of practical va lue are meagre. T he short fa lls in citrus tissue culture research and some ad vancements made in this direction along with bright prospects are highli ghted, restricting the review to vegetat ive ex pi ants only .

Whilst ut ili za ti on of nucell ar embryogenesis is limited to rootstocks, the other aspects, like, regeneration and pro li fe ration o f shoot meri stems measuring 200 !lm in length - a global breakth rough - of two commercially important scion species, Citrus auranrifolia and C. sinensis and an important rootstock, C. lilllonia, improvement of micrograftin g technique, cloning o f the same two scion species as well as some Indi an/rootstock species, employing nodal stem segments of mature trees, of immense prac tical va lue have been elaborated . A rare phenomenon o f shift in the morphogenetic pattern of differentiation from shoot bud di ffe rent iation to embryoid formation occurred during the long-term culture of stem callus of C. grandis. Ste m callus-regenerated plants of C. auranrifolia, C. sinensis and C. grandis showed variation in their ploidy levels and a somaclonal va riant o f C. sinensis, which produced seedless fruits was isolated. Tailoring of rooting in micros hoots to a tap root-like sys tem by changing the inorganic salt composition o f the rooting medium, resulting in 100% transplant success, and germplasm preservation through normal growth culture of shoots o f C. granciis without loss of regenerati on capac ity duri ng 31 years, observed so far, are some other significant results. Plants of C. aurantifolia and C. sinensis raised from shoot meristem and micrograft ing were grown in a net house and those from nodal stem segments in the fie ld along with the in vitro-raised pl ants of rootstocks, namely, C. jambhiri, C. kama and C. limonia. All the plants showed normal healthy growth . Significantly enough, the meriste m regenerated plants o f C. auralllifolia attained the reproducti ve phase just in I year of transplantation to soil simil ar to those raised from nodal ste m segments o f mature trees, which also produced normal fruit s in the subsequent year while growing under fi eld conditions. Thus, a significant fundamental concept of a maturity fac tor, carried over th rough as small a shoot meristem as 200 !lm in length to cloned plants has been demonstrated . T he concept is o f far-reaching signi fica nce in ci trus industry besides production of pathogen-free orchards.

Considering the nutriti onal value, the magnitude of fruit production and an array of commercial products made, the citrus may be considered as a number one fruit of the world. Besides being a rich source of vitamin C and of minerals, it is a rare source of vitamin P, the anti-haemorrhage vitamin ' .

The citrus orchard area in India may be comparable to that of U.S.A. and of Brazil. Also, the North-East region of India is considered to be the place of Citrus origin and it has a very rich heritage of Citrus species, its producti on is not at all comparable to these countries and is even less than that of Spain or Italy, while the export of citrus fruits is nil (Table 1)2.

Regrettably, there has been a gradual persistent decline in citrus fruit production in India mainly because of two reasons. First, virus diseases affecting the orchards and second , practice of conventional approach bereft of Tissue Culture Technology

*Correspondent author

(Biotechnology) for propagation with the result that per plant production in India is quite low amongst the citrus growing countries. On the contrary, with the application of Tissue Culture Technology, a small country, like, Spain has become the biggest exporter of citrus fruit in the world, whereas Israel is stri ving to follow the suit. It is only appropriate to mention that the consistent scientific studies pursued in US A for about four decades have given substantial dividends so that it has come to be a major producer of quality citrus fruits in the world. Such scientific pursuits have also given advantage to Brazil to become the biggest producer of citrus.

Paradoxically enough, though Citrus Ti ssue Culture of practical application , i.e., in vitro nucellar polyembryony has been initiated in India) and the first trees of commercially important Citrus species, i.e., C. sinensis (L.) Osb. (sweet orange) and C. grandis (L.) Osb. (shaddock) were produced in India4 (MS submitted in 1972), the fruits of moclern methods of

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CHATURVEDI et at.: CITRUS TISSUE CULTURE 1081

Biotechnology remain un-harnessed. On the contrary, the achievement of Spain to excel in Citrus Industry is totally based on application of in vitro shoot meristem grafting, better termed as micrografting, the second best method to produce clean plants (pathogen­free)5.6. However, the persistent efforts made in Citrus Tissue Culture at the Tissue Culture Laboratory, National Botanical Research Institute, Lucknow, have led to realization of the global best in production of pathogen-free plants of Citrus by a recent breakthrough of successful shoot meristem culture, employing meristems as small as 200 /lm7.

Perhaps the maximum Tissue Culture research pursued amongst the fruits has been on citrus, but the work of real practical value to Citrus Industry is quite limited, which has been highlighted in this review. The various facets of Citrus Tissue Culture include: Callus culture; nucellar embryogenesis; culture of pistil and ovules8

; culture of anthers for production of androgenic plants9 and of endosperm for triploid production 10; culture of segments of leaf, stem, root,

hypocotyl, epicotyl and cotyledons; shoot meristem culture (ca. 1 mm in length) and micrografting, albeit with no success in the former case6; somatic hybridization and genetic transformation II and

I . 12 germp asm preservatIon . A very significant phenomenon of far-reaching

practical importance was observed in the morphogenetic pattern of differentiation in freshly isolated stem callus of C. grandis and the same after prolonged subculture. Whilst in the former the usual shoot buds were formed, in the latter embryoids were differentiated 13 . The crucial point is that vegetative calli of Citrus differentiate through caulogenesis and those from reproductive organs through embryoidogenesis, but in the present case, a shift in the morphogenetic pattern of differentiation has taken place along with certain other associated phenomena, from shoot bud differentiation to embryoid differentiation. Regeneration via somatic embryoge­nesis is to be preferred, not only because of high rate of regeneration, but due to availability of a ready­made root system for plantlets to be formed .

Table I-Major producers and exporters of fresh citrus in the world2 (Metric tonnes)

Country Total Citrus Sweet orange Tangerine/Mandarin Lime/Lemon Grapefruit Prod Prod Exp Prod Exp Prod Exp Prod Exp

Brazil 17872 16973 82 670 9 780 62

USA 14689 10761 601 539 23 768 123 2619 440

China 7991 1850 17 5730 135 203 207

Mexico 4950 3500 260 960 150 230

Spain 4418 2248 1162 1509 1109 641 386 20 17

Italy 3401 2180 89 504 27 713 31 2

India 3152 2080

Egypt 2817 1910 258 360 544

Argentina 2126 703 89 336 871 178 216 34

Turkey 1795 840 60 490 131 385 123 80 54

S. Africa 1221 970 68 74 44 177 105

Morocco 1192 766 301 403 212 20 3 Prod = Production; Exp = Exports

Table 2- Regcneration of Citrus plants through in vitro nucellar embryogenesis. grown ex vitro

Citrus species C. reticulata x C. sinensis C. grandis C. sinensis

C. al/rantijolia C. sinensis C. sinensis C. paradisi C. retiCtilata C. al/rantijolia C. lilllOIl

C. paradisi x C. reticulata

Explant Reference Nucellus Rangan et al. (196 8)16

Unpollinated and unfertilized ovules Button and Bornmann (1971 )17

Unpollinated ovaries and from ovules (pi stil) Mitra & Chaturvedi (1972)8

Undeveloped ovules and embryogenic calli Gmitter and Moore ( 1986)18

1082 INDIAN J EXP BIOL, NOVEMBER 2001

The present review is confined to the aspects involving vegetative explants, including nucellar ti ssue bei ng somatic.

Nucellar Embryogenesis Nucellus is a somatic ti ssue having a genetic make­

up simjlarly to the vegetative plant body. The cu lture of nucellar ti ss ue and augmentation and induction of nucellar polyembryony have initially been the main focus of research I4

.15

. However, the associated ex tended juvenile characteri stics of I1Llcellar plantlets, if not slightly poor quality of fruit , restri ct the appl ication of in vitro nucellar embryogenesis to rootstocks on ly, si nce both such characteristics wi 11 not be of any consequence while virus-free rootstocks will be produced at a rapid rate throughout the year, which in turn is imperative fo r a ll the year round production of micrografts. When critically examined, the I1Llcellar embryonal proliferation, i.e., embryo-to­embryo multiplication is an uncommon phenomenon. Instead, there is cotyledonary proliferation forming rosette-l ike structures having a single radicle with pluricotyly, which is a big snag in production of nucellar pl:lI1tlets. Nucellar embryogenic tissue provides the best system for genetic transforma ti on studies at a single cell level avoiding chi meri c prob lems.

Some of the important examples of ill Fitro plantlct production th rough nucellar embryogenesis are given in Table 2.

Clonal Multiplication The lacunae men tioned for the ill I'itro nucellar

embryogenes is al so exist in respect of micropropagation with vegetati ve explants. Whilst in a horticultural fruit crop, like, citrus where maintenance of characte ristic traits of different varieties has to be strictly fo llowed by vegetative propagation , in tissue culture of citrus mostly the seedli ng explants have been used fo r multiplication (Table 3). Citrus being a heterozygous and open pollinated tree having severa l important spec ies and variet ies with low or less viab le seeds, such studies ar~ of no relevance so far as cloning fo r raising qual ity orchards is concerned.

i. Culture of ShOOT Tips and nodal sTem segments Production of clonal plants of Citrus through

nucellar tissue has been so convincing that perhaps there has been lack of efforts made for cloning it th rough vegetative explants of field-grown mature

trees. However, intractab le-to-regenerate nature of vegetative explants of field-grown trees as compared to seed ling explants also greatly contributed for limited progress made in thi s important aspect33

.34 .

The initial success achieved in transplantati on of ill vitro-raised plants of C. grandis, C. al/ralllifolia (Christm.) Swing. and C. sinensis, albei t regenerated from somatic callus, to soi l and ultimately to fi eld paved the way for cloning Citrus through vegetative explants of field-grown trees. And for the first time by 1983, the results were presented in a National Ti ssue Cu ltu re Conference on producing complete plants of C. aurantifolia and C. sinensis throu gh regenerati ve cul ture of ax ill ary buds of nodal stem segments obtained from field-grown trees24

. However, proliferation of shoots was poor, more so in C. sinensis, which in subsequent experi mentati on could be increased (Fig I). Amongst the three important rootstocks, namely , C. jall/blli ri Lush., C. kwna Raft. and C. lill10nia Osb. rai sed from ill vitro culture of nodal explants and grow n under field cond itions, the plants of C. jalllbhiri showed exce tiona lly good growth attaining a height of 3.5 m in 20 months fo llowed by C. kama 2. 1 m and C. iiI/IOnia 1.7 rn .

The in vitro-raised plants of C. auralllifolia grew vigorous ly on their own roots, bu t not of C. sinensis , albeit in the former case also the plant growth was strikingly less than that of rootstocks (Fig 2). There are so me other reports also on regeneration of shoots from vegetative ex plants of mature trees (Table 4) . However, these are not withou t ambigui ty in respect of the fate of in vitro-regenerated shoots leave alone their successful transplantation to soil Jl.36. Regeneration from mature tissue (i nternodal segments) of C. sinew;is reported by Cervera ef al.52 is in fact a report on micrografting, where the transformed shoots were not rooted direct ly. The same ambiguity ill regeneration of complete plants exists in a report published on in Fitm propagation of C. reticulata Blanco and C. limon Bunn.f.50

. The fa te of poorly rooted plants produced in the presence of a high concentration of BAP is not known as to how long they survived after transp lantation and whether they ever came to flowering. On the contrary, the plants raised from cu ltures of proliferating shoots, employing noda l stem segments from mature trees of C. Qurantifolia came to flowering and produced fruits in just one year of ex vitro growth, albe it the fruits of the first year abscised48

. However, during the second year, there was normal fruit set (Fig 3a, b) . Also , Cervera et al. 52 reported flowerin g and fruiting of

r .

CiTrus species Common name

C .aurallli{olia Lime C. aurantifolia Lime

C. aurantifolia Lime C. aurantifolia Lime C. aurantifulia Lime

C. aurantifolia Lime

C. aurantifo lia Lime

C. aurantium Sour orange C. aurantium Sour orange C. auramium Sour orange C. grandis Shaddock C. grandis Shaddock

C. grandis Shaddock

C. grandis Shaddock

C.jambhiri Rough lemon

C. kama Kama khatta

C. limeuuides Sweet lime C. limoll Lemon C. Iimonia Rangpuf lime C. macropiJylla Alemow C. macrophylla Alemow C. madurensis Calamondin C. medica Citron

C. microcarpa Calamo ndin

C. mitis Calamondin (e. microcarpa) C. paradisi Grapefruit

Table 3-Regeneration and micropropagation o f Citnls employi ng seedl ing explants

Explant R.:spon se

Stem & leaf segments S te m & leaf segments

Root segments Stem & root segments Leaf & stem segments

Internodal. nodal & root segments

Internodal seg ments

Internodal segments Internodal segments Epicotyl segments Stem & leaf segments Stem & leaf segmenl~

Stem call us: a. Freshly isolated. compact b. 3-year-o ld. friabl e (autotroph ic for aux in) Epico tyl, leaf, root segments, co tyledo n. shoot tip & nodal segments Leaf, stem & root segments

Leaf, ste m & root segment s

Stem segments Root meristems Internodal & nodal segments Internodal segme nt s Epicotyl segments Stem sectio ns Internodal , nod al & root segments

Shoot tip & internodal segments

Epicotyl. leaf, root. shoot ti ps & nodal segments Ste m &Iea f segmen ts

Shoots from somati c calli. Plants ex vitro Shoots from somatic l:a ll i. Pl ants ex vitro attai ned reproductive phase Shoots from root callus, defi cient in rooting Ad ventitious rooted shoots Shoot differentiation in somatic cal li . Axillary sboot proliferation. Plants ex vitro S hoot & root regeneration from somatic calli. Axi llary shoots. Plants ex vilro Adventitious shoots. Plants ex vitro

Adventitious shoots Adventitious shoo ts. Plants ex vitro Adventitious shoot regeneration Shoots fro m somatic calli. Plants ex vitro Shoot differentiation in somatic calli . Adventitious & axi llary shoots. Plants ex vitro Caulogenesis (shoot bud fo rmatio n) Embryoidogenesis (embryoid differentiation)

Shoot differe ntiati on in somatic & cotyledonary call i. Shoot tip & axi llary shoot regeneration . Plants ex vitro Shoot d ifferentiation in somatic calli. Ax illary shoot proliferation. Plants ex vilro Shoot diffe rentiation in somati c call i. Axillary shoot proliferation. Plants ex vilro Adventitious rooted shoots Shoot primordia. Plan ts ex vitro Adve nt it ious & axi llary roo ted shoots Adventitious shoots . Plants ex vitro Adve ntitious shoot regeneration Adve ntitious shoots Shoot &root regeneration fro m somatic calli. Axillary shoots. Pla nts ex vitro Shoot tip growth . Advent itious shoot & root formation

Shoot regeneration. Plants ex vitro

Adventiti ous shoots

Refere nce

Chaturvedi & Mitra (1972)19 C haturvedi ( 1978, 79) 20. 21

Chaturvedi ( 1978, 79) 20. 21

Raj Bhansa li & Arya (1978)22 Chaturvedi & Sharma (1987, 88)23.24

Duran-Vila et al. ( 1989i5

Perez-Molphe-B alch & Ochora-Alejo ( 1997)26 Moore ( 1986)27 Ghorbel et al. (1998)28 Bordon et al. (2000)29

Cbaturvedi & Mitra (1972)1 9 Chaturvedi & Mitra ( 1974)4, Chatufvedi ( 1978. 79) 20. 21 Chaturvedi & Mitra (1975)13

Goh et al. (1995)30

Chatufvedi & Sharma (1987. 88i3.

24

Chaturvedi & Sharma (1987, 88)23.24

Raj Bhansali & Arya (1979)31 Sauton et al. ( 1982)32 Barlass & Skene ( 1982)33 Ghorbel et al. (1998)28 I3ordo n et al. (2000)29

Gnnblat ( 1 972)~ Duran -Vila el al. ( 1989i 5

Rangaswamy ( 1975i 5

Sim et al. ( 1989)36

Raj Bhansa li & Arya ( 1978)]7

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Table 3-Regeneration and micropropagation of Citrus employing seedling explants -Contd

Citrus species Common name Explant Response Reference

C. paradisi Grapefruit Internodal segments , Adventitious shoots. Plants ex vitro Ghorbel et aI. (1998/8

C. reshni Cleopatra Shoot apex. internodal & nodal Adventitious and axillary shoots Barlass & Skene (1982)33 mandarin segments

C. reshni Cleopatra Internodal segments Adventitious shoots Moore (1986)27 mandarin

C. reshni Cleopatra Epicotyl segments Adventitious shoot regeneration Bord6n et ai. (2000)29 mandarin

C. reticuiata Mandarin Internodal segments Adventitious shoots. Plants ex vitro Perez-Molphe-Balch & Ochoa-Alejo (1997)26

C. sinensis Sweet orange Stem &leaf segments Shoots from somatic calli. Plants ex vitro Chaturvedi & Mitra (1972)19 C. sinensis Sweet orange Stem &Ieaf segments Shoot differentiation in somatic calli. Adventitious and Chaturvedi & Mitra (1974t.

axillary shoots. Plants ex vitro Chaturvedi (1978. 79) 20, 21 Z C. sinensis Sweet orange Stem segments Adventitious rooted shoots Raj Bhansali & Arya (1978)38 0 C. sinensis Sweet orange Internodal. nodal segments & shoot Adventitious & axillary shoots rooted shoots Barlass & Skene (1982)33 ;;

apex Z -C. sinensis Sweet orange Root meristems Shoot primordia, Plants ex vitro Sauton et ai. (1982)32 tTl C. sinensis Sweet orange Epicotyl & root sections Adventitious shoots Burger & Hackett (1986i9 :><:

'"0 C. sinensis Sweet orange Leaf. stem & root segments Shoot differentiation in somatic calli. Axillary shoot Chaturvedi & Sharma (1987.88)23,24 0::1

proliferation, Plants ex vitro 0 C. sinensis Sweet orange Internodal. nodal & root segments Shoot & root regeneration from somatic calli. Plants ex Duran-vila et aJ. (1989/5 ..r

vitro Z 0

C. sinensis Sweet orange Nodal &Intemodal segments Axillary & adventitious rooted shoots Duran-Vila et al. (1992)40 < C. sinensis Sweet orange Hypocotyl & epicotyl explants Adventitious shoot regeneration Maggon & Singh (1995)41

tTl 3::

C. sinensis Sweet orange Shoot meristems & nodal segments Shoot proliferation. Rooted shoots Mohanty et aI. (1998)42 0::1 tTl

C. sinensis Sweet orange Epicotyl segments Adventitious shoot regeneration Bordon et ai. (2000)29 :;0 tv

C. paradisi Citrumelo I em-long seedlings stem with node Axillary & adventitious shoots. Plants ex vitro Grosser & Chandler (1986)43 8 x Poncirus trifoiiata C. sinensis Carrizo citrange Shoot tips Shoot proliferation, Plants ex vitro Kitto & Young (1981)44 x p, trifoiiata C. sinensis Carrizo citrange Internodal. nodal stem segments & Adventitious & axillary shoot. Plants ex vitro Barlass & Skene (1982)33 x shoot apex p, trifoliata

C. sinensis Carrizo citrange Internodal segments Adventitious shoots, Plants ex vitro Moore (1986)27 x p, I rifoliala

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plants raised from micrografting of mature shoot apices of C. sinensis in 14 months of transplantation in glasshouse conditions. It is a well-known fact, not only in Citrus, but in case of trees as a whole that maturity factor/ s are carried over through an explant and expressed accordingly as in vegetative grafting in the conventional method of plant propagation . The aspect of maturity versus juvenility IS most pronounced in Citrus, where grafted plants come to flowering in just 3 to 4 years and those from seedlings or raised from cuttings of juvenile shoots attain reproductive phase after 6 to 20 years. This aspect has been discussed later in some detail.

Similar to C. aurantifolia, the plants raised from nodal stem segments of adult trees of C. limonia and C. kama came to flowering and fruiting during the second year of transplantation to soil. However, in case of C. sinensis and C. jambhiri, attainment of reproductive phase was a little delayed compared to other three Citrus species, which fact makes the maturity issue species specific .

ii. Micrografting Micrografting provides cloned plants, which are

also largely free from pathogens, including viruses and virus-like pathogens. Though micrografting is a tedious and time consuming technique requiring also exceptive dexterity, it provides a practicable method, whereby very minute shoot meristems (less than 1 mm in length) can be regenerated even in those plant species where such small excised shoot meristems cannot be regenerated as has been the case in Citrus. Micrografting has been most extensively and usefully applied to Citrus amongst all the fruit trees. The most glaring example of improvement of citrus orchards leading to many-fold increase in fruit production has been demonstrated in Spain by producing about 31 million healthy plants originally recovered by micrografting6.

Since the size of shoot meristems used has been very small (200-500 /lm In length) all the major viruses and virus like organisms could be eliminated, which include the most virulent pathogens causing citrus decline In India, namely, citrus tri steza closterovirus, citrus exocortis viroid and the citrus greening caused by Liberobactor asiaticum. In Citrus, mostly the seedlings of Troyer citrange have been used as a rootstock besides some other Citrus species and hybrids, which include, Carrizo citrange, trifoliate orange, rough lemon , Rangpur lime and Kama khatta. The micrografting procedures In Citrus, first demonstrated by Murashige et al.53

In 1972 have

1086 INDIAN J EXP BIOl, NOVEMBER 2001

furth er been modified in a number of ways, but in a preferred method , a particular length of the decapitated ep icoty l was planted with a shoot meristem at its cut face in such a way that the tiny shoot meristem has been in contact with its cambial tissue. J n the second best method . the shoot meristem has been placed in an inverted 'T' incision made in the decapi tated epicotyl. The subject has been rev iewed by Navarro54.56.6 and Jonard57. Some selected successful reports on micrografting are li sted in Table 5.

otwithstanding the revoluti oni zation of Citrus production by micrografting in Spain. the tech nique required some improvements, wh ich have been accomplished to a great extent by Singh and Chaturvedi (unpublished data). A major drawback cxisted in the use of seed lings. wh ich is limited by the availability of seeds of roobtocks on ly once during a year, compounded with their short- li ved viability. The lacuna could be filled by employing the isolated rooted shoots obtained throughout the year from cu ltures of prol i I' erating shoots. Remarkably, shoot mcri stems measuri ng as <; ma II as 200 ~lIn in length regenerated, which could then be developed to active ly proliferating and growing shoot cultures. The details of meristem culture have been described in the section on meri stem culture. Merit of the micrografting process can be greatly improved if the proliferating shoot cu ltures are raised from meristem culture of rootstocks, in which case the rootstocks will also be pathogen-free besides being clonal.

Micrograft ing was accompli shed em ploying excised shoot meri stems, measuri ng from 200 )..tm to I mm in length, of C. allrallt(jolia and C. sillellsis and us in g ill vitro-grown seedl ings of some important Indian rootstocks, namely, C. jall1bhiri, C. kama and C. filllollia. Of the differem manners, in wh ich a shoot Inerisrem was planted on the cut face of epicotyl , its direct planting in contact with the cambial tissue of ep icoty l gave the best results (Fig 4) . However, the percentage success of micrografting varied not on ly with two scion species, but also with their respecti ve rootstock combinations. The best results were obtained with C. limonia for both the scion species (shoot meri stems of ca. 500 )..tm length were used) leading to 57.14 % success in C. aurantifolia and 42.85 % success in C. sinensiio.61

• The percentage success was further improved by the topical application of GA solution on moribund meri stem grafts, as the growth was evoked.

The use of isolated rooted microshoots of the best Citrus rootstock species, C. limonia in pl ace of its aseptically-grown seedlings was found to be feasible. The per cent success of micrografts was a little lower than obtained in both C. aurantiJolia (65 %) as well as C. sinensis (50 %) using seedlings (70% and 60%, respectively), while the improvement in percentage of success was due to the use of bigger explJnts of shoot meri stem (ca. I mm in length ) excised from ill vitro­grown prol i fe rati ng shoots raised fro m shoot meristem culture. However. the deficit was more than compensated with the unlimited a\ailability of rootstock shoots all the year rouIld49 (S ingh and Chaturvedi, ullpublished data) . Furthermore, the advantage of using excl usively nucellar seedlings of rootstocks th an thc zygotic ones also has not been eva lu ated earli er. Al though growth of grafted shoot meristems was sl ightly less in case of nucellar seedlings than in zygotic seed lings as observed in C. kama, the genetic uniformity and freedom from pathogens of the fo rmer scores over the use of zygotic seed lings of rootstockS60

·61

. It took about 10 days for grafted shoot meristem to pick up growth and during subseq uent 20 days, the meristem attained a respectable length of about 1.5 cm (Fig 5).

A number of significant observations were made while evaluating fie ld performance of micrografts of C. auranti/o/ia and C. sinell sis on C. Iil1lollia anel their co unterparts grown on their own roots. Whil st C. {/UrLlllt(jo/ia grew far better on its own roots than whe n Illicrografted on C. fill/ol1ia under nethouse as wel l as field conditions, C. sillensis grew far better as micrografts on rootstock than on it. ow n roots. However, in both the spec ies, the plants grown in field registered better growth than their cou nterparts grown in a nethouse (Fig 8) .

iii. Shoot MeristclII Cll/ture Efforts to grow excised shoot meristems of mature

trees, measuring 500 ~m to I mm in length and compris ing the meri stem dome and 2 to 3 youngest leaf primord ia date more than past 3 decades65 . The importance of culture of shoot 01eristem of Citrus lies in the fact that Citrus is infected by a number of viruses besides other pathogens. Shoot meri stem culture using explants of critical minimum size of 200 to 500 )..tm is the best method to eliminate Citrus viruses, most of which are mechanically transmitted. A breakthrough was made recently in regenerating excised shoot meri stems of mature trees of C. aurantifolia , C sinensis and C. limonia7 (Fig 6).

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CHATURYEOI et al. : CITRUS TISSUE CULTURE 1087

Figs 1-5-Cultures and in vitro-raised fi eld-grown pl ants of Citrus species. (I) A culture of proli ferating shoots establ ished from a nodal stem segment taken from a mature tree of C. sinensis x 0.9; (2) A view of plants of Citrus scion species and rootstocks raised from culture of nodal stem segments and grown under field conditions as seen after 20 months of transplantation x 0.0 16; (3a) An in vitro-raised 20-month-old C. auranti/olia pl ant in fruiting stage under fIeld conditions x 0.036; (3b) A magnified view of a bunch of young fruits as seen in Fig. 3a x 0. 13; (4) A shoot meristem of C. aLiranli/olia, measuring 500 11m in length, top grafted 4 days before on the decapitated cpicotyl of C. kan:a x 27.7 ; (5) A 30-day-old micrografted plantlet of C. aurantifolia x 1.2.

1088 INDIAN J EXP BIOL, NOVEMB ER 200 1

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Figs 6- II -Cultures and nethouse-grown in vitro-raised plants of Citrus species. (6) An exci sed shoot meri stem ex plant o f C. aurantifolia as seen after 10 days of inocul ation x 56; (7) Pro lific multiplicati on o f meristem­regenerated shoots x 0.9; (8) A view of shoot meristem-regenerated plants as well as o f micrografted pl ants o f C. aurantifolia and C. sinensis grown under nethouse conditions x 0 .0 15 ; (9) A microshoot of C. grandis with several weak roots and intervening callusing x 1.8; (10) A microshoot o f C. grandis with a tai lored tap root-like syste m without intervening callusing x 2; ( II ) A 3 1-year- old normal growth culture o f pro li fera ting shoots o f C. grandis x I .

,

Citrus species Common name

C. aurami/olia Lime C. al/ranti/olia Lime C. auranti/olia Lime

C.jambhiri Rough lemon C. kaT7Ul Kama khatta

C. limonia Rangpur lime

C. '1imonia Rangpur lime

C. limon Lemon C. milis Calarnondin

C. reshni Cleopatra mandarin C. retictllata Mandarin C. sinensis Sweet orange C. sinensis Sweet orange C. sinensis Sweet orange C. sinensis Sweet orange C. sinensis x Carrizo citrange P.lri/oliara

Table 4--Cloning through shoot apices and nodal segments of mature Citrus trees

Explant Response

Nodal segments Axillary shoot proliferation. Plants ex vitro Tiny shoot apices Shoot growth. Plant ex vitro Nodal segments Axillary sboot proliferation. Plants ex vitro.

Attained reproductive phase after 1 year of transplantation

Nodal segments Axillary shoot proliferation. Plants ex vitro Nodal segments Axillary shoot proliferation. Plants ex vitro.

Attained reproductive phase after 2 years of transplantation

Nodal segments of glasshouse-grown plant Multiple shoots. Rooted shoots

Nodal segments Axillary shoot proliferation. Plants ex vitro. Attained reproductive phase after 2 years of transplantation

Shoot tips Multiple sboots. Plants ex vitro Shoot tips & nodal segments of glasshouse Shoot regeneration. Plants ex vitro grown plants Nodal segments of glasshouse-grown plants Multiple shoots Shoot tips Multiple shoots. Plants ex vitro Dormant buds Axillary shoots Nodal segments of glasshouse-grawn-plants Multiple shoots. Rooted shoots Nodal segments Axillary shoots. Plants ex vitro Nodal segments Axillary shoot proliferation. Plants ex vitro Shoot apices & Nodal segments of glass Multiple shoots. Plants ex vitro house- grown-plants

Reference

Chaturvedi & Sharma (1987.88)23. 24 Chaturvedi & Sharma (1987. 88)23. 24 Singh & Cbaturvedi (1999)48

Singh & Chaturvedi (1999)49 Singh & Chaturvedi (1999)49

Barlass & Skene (1982)33

Singh & Chaturvedi (unpublished data)

Singh et 01. (1994)50 Sim et 01. (1989)36

Barlass & Skene (1982)33 Singh et 01. (1994)50 Altman & Goren (1974)51 Barlass & Skene (1982)33 Chaturvedi & Sharma (1987. 88)23.24 Singh & Chaturvedi (1999)48 Barlass & Skene (1982)33

n :c » -l c ::0 < en S2 ~

~ n =i ::0 C en j CIl CIl C en n c r -l C ::0 en

..-o 00 '-0

Scion

Citrus spp.

C. uurUlltifuliu C. aurantifolia C. auranti/olia

C. auranti/olia C. clementina C. limon C. limun C. maxima (c. grandis) C. medica C. paradisi C. paradisi C. reticulata C. reticulata

C. sinensis C. sinensis C. sinensis C. sinensis

C. sinensis C. rericulara x C. sinensis

Table 5-Micrografting of shoot meristems of Citms scion species ranging from 200 ~m to I rom on various rootstocks - Ex vitro plants

Common name Rootstock Reference

'Temple'tangor, 'Eureka' lemon, 'Washington 'navel orange, 'Satsuma' mandarin, 'Marsh ' grapefruit, 'Cuban ' Shaddock Lime Lime Lime

Lime Clementine Lemon Lemon Shaddock (meristem ex nucellars) Citron Grapefruit Grapefruit Mandarin Mandarin

Sweet orange Sweet or.ange Sweet orange Sweet orange

Sweet ora\1ge 'Temple'tangor

P. tri/oliata x C. sinensis (Troyer citrange) & P. trifoliata (trifoliate orange)

C. jambhiri (rough lemon) C. sinensis x P. trifoliata (Carrizo citrange) C. jambhiri (rough lemon), C. kama (Kama khatta) & c. limonia (Rangpur lime) Rooted shoots of C. limonia (Rangpur lime) P. tri/oliata x C. sinensis (Troyer citrange) C. jambhiri (rough lemon) P. tri/oliata x C. sinensis (Troyer citrange) P. trifoliata x C. sinensis (Troyer citrange)

C.jambhiri (rough lemon) P. tri/oliata x C. sinensis (Troyer citrange) C. sinensis x P. tri/oliata (Carrizo citrange) P. trifoliata x C. sinensis (Troyer eitrange) C. auratium (sour orange), c. jambhiri (rough lemon) & c. limonia (Rangpur lime) P. trifoliata x C. sine~is (Troyer citrange) C. sinensis x P. trifoliata (Carrizo eitrange) C. Sinlllsis x P. trifoliata (Carrizo eitrange) C. jambhiri (rough lemon), C. kama (Kama khatta) & c. limonia (Rangpur l4,ne) _

- Rooted shoots of C~limQ'lia (Rangpur lime) P. tri/oliata x C. sinensis (Troyer cjtrange)

Murashige et al. (1972 /"

Navarro et aJ. (1975)58 Edriss and Burger (1984)w Singh & Chaturvedi (2000)60; Singh et al. (2000)61

Singh & Chaturvedi (unpublished data) Starrantino & Caruso (1988)62 Navarro et a1. (1975)58 Starrantino & Caruso (1988)62 Huang et ul. (1988 )63

Navarro et al. (1975)58 Navarro et al. (1975i8

Edriss & Burger (1984)59 Navarro et al. (1975)58 Mukhopadhayay et al. (1997)1>4

Navarro e: al. (1975)58 Edriss & Burger (1984)59 Starrantino & Caruso (1988/2

Singh & Chaturvedi (2000) 60; Singh et al. (2000)61

Singh & Chaturvedi (unpublished data) Navarro et aI. (1975 )58

o \0 o

z S2 » z tTl >< "t:I ttl (5 ..r z o < tTl 3::: ttl tTl ;:0

N o o

)

CHATURVEDI el at.: CITRUS TISSUE CULTURE 109 1

The crux of the strategy followed for regenerating

ohwt meristems as small in size as 200 jlm in length

laid in three factors. One, u e of filter papel' b1idge with liquid medium during initial culture of small explants, which prevented their desiccation as well as facilitated ready availability of constituents of growth medium. Secondly, use of a balance of three main growth hormones, namely, cytokinin, auxin and gibberillin to give initial boost to growth of meristems to develop into young shoots so they become competent to sustain growth under culture conditions. And third, establishment of aseptic cultures of Citrus species by inducing axillary shoots in nodal stem segments taken from the fresh growth of mature trees, comprising the source of meristems. In this way, shoot meristems of C. aurantifolia, C. sinensis and C. limonia were grown subsequently on nutrient agar as leafy shoots, which could be induced to proliferate at an enormous rate mainly under the influence of a cytokinin and an auxin (Fig 7). Such cultures of proliferating shoots raised from shoot meristems are free from viruses and other Rathogens and constitute a continuous source of clean and genetically uniform scion and rootstock plant material for micrografting as well as macrografting. A major advantage for micrografting in this process is that even bigger explants of shoot apices can be used for grafting, resulting into a better percentage of success that too obtained with a far less difficulty. Moreover, in case of scion species, like, C. aurantifolia, the developed isolated shoots raised from meristem culture were rooted resulting in the formation of complete plants to be grown in field on their own root system. Similarly, clean and . cloned plants of a rootstock, namely, C. limonia, could De produced by rooting the isolated shoots origihate<1 from its meristem culture, which grew vigor�uslt�nder field conditions.

The shoot meristem-raised plants of C. aurantifolia came . to flowering within just one year of their transplantation to potted soil while growing under glasshouse conditions. Thus, the mafurity of mother plants was carried over even through a shoot meristem barely measuring 200 Jlm and was not lost in vitro during several cycles of vegetati ve multiplication as subcultures. Till very recently, it has not been sure, whether a change from juvenile phase to mature phase of a plant, which is a gradual process, was directly under genetic control or not? But now it has been demonstrated that such a process is actually under the genetic control and in a plant ·like Arabidopsis thaliana this is under control of a single

gene . named as . �QUINT (SQNJ, gene encoding a

?rotem� cyclophIlm 40 (CyP40) . It is indeed very JElterestmg and revealing that the maturity factor, a substance of proteinacious nature has been found common between Arabidopsis and Citrus, which has been elegantly demonstrated by incorporating Arabidopsis LEAFY (LFY) or APETALA 1 (AP 1 ) gene, which i s a flower promotion gene of Arabidopsis. In this way, the transgenic zygotic seedlings as well as nucellar plantlets have been induced to flower within 1 2 to 20 months. Induction of precocious onset of maturation phase in Citrus is very significant in view of its usual prolonged juvenility from 6 to 20 years67.

Rooting of Isolated Shoots Since most of the work in Citrus tissue culture has

been concerned with embryogenesis, be it from nucellar tissue or ovular tissue, the role of type of rooting deciding ex vitro survival of in vitro-raised plantlets has not been considered a problem as they have been grown on their own root system. Otherwise also, Citrus is an easy-to-root genus except species, like, C. sinensis and certain other rootstocks, namely, C. jambhiri and C. kama, as seen in this Laboratory. Other workers have also e�rienced the same rooting problem in C. sinensii5• • Whilst in many of the species, rooting is induced by IAA, in others it is produced in combinations of IAA with IBA or NAA, or, of NAA with IBA. But with difficult-to-root species, like, C. sinensis, incorporation of a phenolic acid along with a combination of auxins was necessary for root induction, that too with a pulse treatment strategy. However, rooting of even well­developed microshoots invariably preceded with intervening callusing, which hampered their ex vitro transplant success, as first experienced in C. grandis, C. aurantifolia and C. sinensis21 • Through an innovative method, rooting of microshoots regenerated from somatic callus could be tailored from several thin roots preceded by callusing to a tap root-like system with no intervening callusing through reducing concentrations of certain inorganic salts of the basal medium, namely, NH4N03, KN03, CaCh and MgS04, which resulted in 1 00% transplant success of the in vitro-raised plantlets of C. grandis, C. aurantifolia and C. sinensiil (Figs 9, 10). The in vitro-raised Citrus plants grew normally under field conditions and attained reproductive phase in due course of time, albeit it varied with the three Citrus species, i.e., ca. 5 years for C. aurantifolia, ca. 1 8

1092 INDIAN J EXP BIOL, NOVEMBER 2001

years for C. sinensis and ca. 6 years for C. grandis. Whilst the somatic callus-regenerated plants of C. aurantifolia were diploid, those of C. sinensis showed variation in their chromosome number, one of which an aneuploid produced seedless fruits of characteristic flavour and taste. A new variety of C. sinensis thus created, could be established through in vitro clonal multiplication for plains of Uttar Pradesh, where C. sinensis otherwise does not flourish.

Unsuccessful transplantation due to undesirable rooting of microshoots has been reported by some other workers also and for circumventing thi s problem, micrografting has been resorted t028. Mortality of rooted microshoots with intervening callusing on ex vitro transplantation to soil is mainly due to non-alignment of vascular tissues of shoot and root and secondly, due to infirm attachment of root system with shoots, which could be solved in the present case of seedless C. sinensis by tailoring the root system of callus regenerated microshoots. Similarly, the microshoots having a tap root-like system, obtained from cultures of proliferating shoots raised from nodal stem segments of mature trees of C. aurantifolia, C. sinensis, C. kama, C. jambhiri and C. limonia and those raised from meristems of C. aurantifolia, C. sinensis and C. limonia, on transplantation to soil, survived 100% and grew normally under field conditions.

Germplasm Preservation Citrus being a heterozygous and open pollinated

tree, its germplasm cannot be preserved through seed storage. As a conventional practice, therefore, preservation of Citrus genotypes is done through maintenance of field collections of specimen plants, which, however, is again not advisable since it is attacked by a number of pathogens. Thus, germplasm preservation can effectively be done through long­term culture of proliferating shoots under normal in vitro growth conditions. Shoots regenerated from somatic callus of C. grandis as early as 1970 were being maintained for the past more than 31 years as regenerative shoot cultures, observed so far68 (Fig 11). Similarly, cultures of proliferating shoots raised from nodal stem explants of mature trees of C. aurantifolia, C. sinensis, C. jambhiri, C. kama and C. limonia as well as those raised from shoot meristem culture of C. aurantifolia, C. sinensis and C. limonia were being maintained as normal growth cultures without any decline in their regeneration potentiality for the past 7 years, observed so far. Cultures of proliferating shoots

raised from meristems are ideal , as the germplasm preserved will be both true-to-type in genetic make-up and free from all pathogens. Such cultures constitute a true Tissue Bank or Gene Bank, which will promote also free exchange of germplasm across the phytosanitary boundaries in the plant quarantine system.

Cryopreservation of embryogenic callus and somatic embryos69-71 does not" merit to be a germplasm preservation method sensu stricto because of chances of genetic infidelity72. 73. Similarly, a lone attempt to preserve C. aurantifolia through its regenerative excised root culture may hardly merit to be a practicable method at least on two counts: First, it has been demonstrated for only a period of 3 years and second, a very low frequency of shoot

. 74 regeneratIOn .

Prospects In view of the ever-increasing world demand of

citrus fruit, it is imperative that citrus production in India should commensurately be increased, for which enough scope exists. For example, the orchard area of selected industrially important Citrus species can be extended. The area of sweet orange has a potential to be increased in Maharashtra, Andhra Pradesh, Bihar, Uttar Pradesh and Karnataka, that of limes in Gujarat, Maharashtra, Bihar, Uttar Pradesh and Andhra Pradesh, while of mandarins in Maharashtra , Karnataka, Madhya Pradesh , Rajasthan and North­East region . In respect of Uttar Pradesh, where the sweet orange does not normally grow has a potential for cultivation in alluvial plains of Agra and lower foot-hills of Western Uttar Pradesh . Similarly, there is enough scope to extend lime and lemon orchards in South-Western semi-arid zone of Uttar Pradesh, which includes the areas, like, Aligarh, Etah, Mainpuri , Mathura and Agra.

Citrus fruit production per tree in India is very low, because of largely infected orchards, if not the lowest amongst the important citrus producing countries like, U.S .A., Brazil , Spain, South Africa, Japan and Israel despite the availability of proper agroclimate in respective areas of its cultivation. That is why, India has no place in Citrus World Trade in spite of the fact that it is the seventh largest producer of citrus fruits.

Main reasons for the di smal position of India in Citrus Industry is the non-application of Biotechnology for improvement of citrus orchards. With the utilization of recent biotechnological achievements in Citrus, namely, shoot meristem

. .....

1

CHATURVEDI et al. : CITRUS TISSUE CULTURE 1093

culture, improved micrografting procedure and rapid production of cloned plants by employing apical and axillary shoots from mature elite citrus trees besides creation of transgenic plants resistant to virus diseases. Such recently developed invaluable techniques will ensure production of healthy (pathogen-free) plants leading to increased production of quality fruits per plant acceptable in the Citrus W orId Trade.

Rapid clonal production of disease-free trees of C. reticulata CNagpur' mandarin) and C. nobilis Lour. x C. deliciosa Tenora CKinnow' mandarin), which are the most promising candidates for export75, require immediate experimentation, particularly for their meristem culture besides genetic transformation for increased shelf-life.

To realize above-mentioned prospects holding great promise to boost Citrus Industry and improve National health, the planned improvement of citrus orchards throughout the country appears to be obligatory at government level even by enacting relevant legislation if need be so.

Acknowledgement The authors are thankful to the Department of

Biotechnology, New Delhi, for granting the project titled "Biotechnological investigations in mango and citrus for their improvement and increased production", the results emanated from which have also been incorporated in the text. The authors also thank the Director, NBRI, Lucknow, for the facilities provided.

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