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Plant Physiol & Biochem, Vol. 23 (1) , 67-72, Janu ary, 1996

Studies on Dif ferentiation in in vitro Grown Shoot tips of Chickpea

Vijay Paul, C handra R., Sangccta K hcta r pa l and Raghuvecr Polisetty Plant Tissue Culture Laboratory. Di vision or Plant Physiology, Indian Agricultural Research Institute, New Delhi-IIO 012

T he major objective of the pre sen t st udy was to observe changes in physiolog ical and bioch emi cal parameters associated wi th growth and di fferentiation of shoot tips (ST) g r ow n in either p lant ind uction medium (PI M : 1\1S + N/\,\ 2m g + IRA 2mgl I) or ca llus induction medium (C IM : I\IS + 2, 4-H 2m g + K in 0. 25mg/l). Fo r th is, shoo t tips of chic kpea (Cicer arietlnum L.) var. BG -256 fr om 7 d old, a scep ticnlly grow n chickpea were used as experi menta l material. S hoot tips differentiated to plants with multiple shoots in PIL\I, whereas they remained as callus in CII\I. Differenti ation of ST in I'IM accompanied by significantly more g r ow t h as obse r ved by increa sed dry weights, more sucrose a nd soluble suga r s, more nitrate reductase activity. The n it ro gen a nd protein was sign ifica ntly more in ST grow n in C IM after IS DA I till 30 HA l.

Key words Cice r arietinum 1.., ca llus. ShOOI tip, sucrose, soluble sugars, nitrate reductase

INTRODUCTION study were obtained from Division of Plant Physiology, Indian Agricultural Research Institute. Seeds were sur­face sterilized with 0,1% mercuric chloride for 3 min For maximum utilization of tissue culture {or crop im­followed by repealed washings with double distilled water. provement, regeneration from cells is a prerequi site, In

pulses, regeneration on routine basis and with higher fre­About 5-10 mrn length, shoot tips from 7 d old sterile quency is yet to be achieved, Ce ll or tissue under cultural seedling were used as explant material. Explants wereconditions undergoes several physiological and biochemi ­inoculated on either PIM, which is MS media supple­cal changes. Some of these change s are directly related mented with 2 mg/L NAA and IBA each; or on CIM with the process of regenerat ion. Plant growth regulat ors which is MS media supplemented with 2 mg/L 2,4-0 andplay a major role in inducing different iation. A few studies 0.25 mg/L Kin. Cultures were maintained at temperature indicating the relation ship between biochemical changes of 25±2C; and light intensity was kept at 900 lux.with process ofdifferenti ation arc: stimulation of efficient

shoot organo genesis in Glyc ine n ULl:, with low ammonium In this laboratory, using different cxplants of chickpea,nitrate concentration (Shetty et 01. 1992) ; increased induc­a reprod ucibl e rege nerat ion meth od was established tion of regeneration and protein synthesis in alfa alfa lines (Chandra et of. 1993) and a few physiological and bio­with both nitrate and ammonium (Skokut et a l. 1985) ; chemica l parameters were studied as a function of process of dedifferentiation protein s speci fic to morpho­different explants and days after inoculation (Arijali et genic callus lines in various genotypes of sugarcane spe­01. 1995 ). The present work is done with the major cies (Rarngopal 1994); nitrate reduct ion in Pin us pinaster objective to study changes in growth. nitrogen (N), pro­(Faye et 01. 1986). tein (P) sucrose, total soluble sugar (TSS) content, and nitrate reductase activity (NRA) in shoot tips induced

MATERIALS AND l\IF.TIIO DS either by plant induction or ca llus induction media.

Seeds of chickpea variety I3G-256, used in the present Morphological, physiological and biochemical observations were taken on day one and later at weekly

Ab breviations : CIM-eallu5 Induction Media; Pl~ I-Plan l l n d lic t ill ll Media; intervals up to 30 days. Uninocul ared ST, served asN-Nilmgen Content; P-Protein Content; TSS-Tolal Soluble Sugars: NI{i\­

control. Fresh and dry weights of ST were recorded at Nitrate Reductase Activity; DW-DI)' Weights; NAA-n-Naphthalcne Acetic Acid; lI3A· lndolc Butyric Acid; 2 .~ -D - 2, -1 -D ic h lmop he ll o\ y acetic acid. all sampling dates and were expressed as mglexplant.

68 Paul, Chandra, Khetorpal and Polisetty

Reduced N was estimated using N-autoanalyser, using the procedure detailed in Technicon Monograph I, 1974. N was expressed as percent (%). Total protein content was arrived by the formula %P = %Nx6.25 .

Nitrate reductase activity (NRA) was determined by using the method given by Hageman and Ilucklesby (1971) and was expressed as n moles N0

1 g' fr wt h'. For estimation

of total soluble sugars (TSS), Dubios et al. (1956) method was used and TSS was expressed as rng glucose equiva­lents g! d wt Sucrose estimation was done using Van Handel (1968) method and was expressed as rng g-I d WI

RESULTS ANI> DISCUSSION

Morphological observation

Shoot tips (ST) induced by PIM, differentiated to plants, and differentiation was morphologically observed from 4 days after inoculation (DAI). By end of growth period i.e. 30 DAI, multiple shoots were formed with profuse rooting. In CIM, callus was initiated and by 30 DAI, the entire ST was converted to callus mass, thus non-differ­entiated.

Dry weights (mg explant')

Upto 7 DAI, the increase in DW was similar in both the treatments (Table 1). Differences in OW were observed from 7 DAJ. Beyond 7 OAf, rapid dry matter accumu­

lation was noticed in rIM, whereas the corresponding increase was slow in CIM. Total OW increase by 30 DAI was 26.4 fold in PlM and 12 fold in CIM (when com­pared with respective control). At 30 OAI, ST induced by PIM recorded 100% more OW in comparison to elM (Table f).

Significant changes In DWs in ST grown in PIM and CIM was visible only from 15 OAJ. This implies that the changes in OW due to induction by plant growth regu­lators was visible only after 15 DAJ. After 15 days, rIM recorded significantly more OW. This increased OW may be related to differentiation of S1' in PIM. Thus, not only type of explant (Anjali et at. 1995), but also type of medium (Nee lam et at. ]I)!loa) and hormonal supple­ment in the medium contribute to the difference in OW and their culmination into differentiation.

Nitrate reductase activity (I\'RA)

At all the observation days, NRA in ST was found to be significantly more in rIM than in CIr-.·1. Both the treatments recorded highest NRA on I DAI and 15 OAI. At initial stages (1-7 OAI), NRA in PIM was 11-12% more, whereas at later stages (21 and 30 OAI) NRA in ST in riM was 34 to 35% above those grown in CIM (Table 2). Increased NRA in both (lIM and CIM over the control was observed . In the present study MS media which contained nitrogen in both NO\ and Nil: forms might have contributed to the increase. Requirement of both NO; and Nil', for expression of NRA was reported by Thorpe et al ( 1989) and Saloncn and Simola (1989).

Table I. Cumulati ve increase in dry weight (rug/explant) of shant tip ofchickpea, variety lI(i-256 as a function ofdays alter inoculation A and 13 represent per ccnt l/D: increase (i)/dccrcase (!-) 0\ er control (kept at IUO%) and /\is + 2.4-1) + KIN treatment (kept at 100%) on the same day respectively

Treatments

o

MS+NAA+ II3A 1.9

A IO()%

13

MS + 2,4-0 + KIN 1.9

A 100%

Standard Error Critical di/Terence at 1%

Treatments 0.3669 1.3367

Intervals IUI1l9 1.890~

Treatment x interval O.R999 3.2743

69 / 11 vitro g r o ll' lI shoot tips ill chickpea

Nitrogen (N) and protein (P) content

Nitrogen content was higher at all DAI 111 ST induced by CIM than in PIM. Maximum N conten t in both PIM and CIM was recorded at 7 DAI (Table 3). There was increase by 19 and 27% nitrogen conten t of ST in PIM and CIM respectively.

More N content was seen in di ff erentiating ST than in non-differentiating hypocotyl when induced by PIM (Chandra et 01. 1993) , Present study indicates that hor­mone supplement in the media will exert influence on N content of the tissue. Thus ST grown in CIM recorded more N and protein content than those grown in PIM (Tabl e 3). From the study it could be inferred that a few specific polypeptides rather than total protein decide the differe ntiation in S1' as seen in Brassica by Crouch and

Sussex (1 981) or in Icttucc by Mac Issac and Sawhney ( 1990),

As protein content (%) is calcul ated from the corre­sponding nitrogen content, changes in protein content exactly coincided with changes in N content (Table 4).

Sucrose content and total soluble sugar content (mg/ g d wt)

Sucrose content was significantly higher in ST induced by rIM than in CIM. Sucrose content increased in both the treatments from I DAI to 30 DAJ. But the increase was 9.5 fold in PIM, whereas it was only 3.3 fold in CIM at 30 DAI when compared to the initial sucrose content (Tab le 5).

T ab le 2. N itrate reduc tase ac tiv it), (n mulcs Nt ) ,- / g f\\ t/ h ) of'shoo t tip o f ch ickpea, var iety BG-256 as a fun cti on o f days a fter inocul ation . A and B rep resent per cent liD : inc rea se (I) decrease (J. ) o ver co ntro l (ke pi a t 100%) and MS + 2. 4-[) + KIN treatment (kept at 100%) on the sa me d ay respect ive ly

T reatments Days afte r in ocul ati on mean

0 7 15 22 30

MS + NAA + lil A 10.88 1904 1534 1835 11 38 1225 1454

A I O()% 75%i 40.9%i 68.7%i 4.6%i 12.6%i 33.6%i

13 II.3 %i 12.9%i 24.8%i 34.oi 35.oi 18.2%i

MS + 2,4-0 + KIN 1088 1710 1359 1470 849 907 1230

A 100% 57. I%i 24.9%i 35% i 11.9%1­ 16.6%.1­ l3%i

Standard E rro r C ritica l difference a t 1%

T re a tmen ts 69.24.t9 252.26

Intervals 48.9636 178.374

T reatment x inte rva l 119.9359 436.93

T ab le 3. Pe r ce nt ni tro gen (N ) ofshoot t ip (It! chickpea ,var iety B(j -256 as a func tio n o f da ys a fter inocul ati on . A and B represent per cent (/D : increase ( i)1decrease ( .l.) m e r con tro l (kept <I I IOlJ%) and MS + 2,4-D + K IN treatment (kept a t 100%) on the sa me day respe ctively

70 Paul, Chandra. Kl ietarpa l and Polisetty

Table 4. Per cent proteins of shoot tip of Chickpea, variety IlG-156 as a functin of days after noculation, A and 13 represent per cent II 0 : increase (i)/ decrease (.1.) over control (kept at 100%) and ~l S + 1,4 -D -t- KIN treatment (kept at 100%) on the same day respectively

Table 5. Sucrose content (mg/g d wt .) of shoot tip od Chickpea, variety I3G-256 as a function f days after inoculation. A and B represent per cent I/O: increase (i) /dccrcasc (t) over control (kept at 100%) and MS + 2,4-0 + KIN treatment (Kept at 100%) on the same day respectively

Treatments Days after inoculation mean

o 7 15 22 30

MS +NAA + lOA 9.6 50.9 51 76.7 100.9 57.8

A 100% 43 foldI 4.3 foldi 6.9 fold I 9.5 foldi 5 foldI

n 2.0 foldi 3.3 foldi 1.5 foldi 1.4 fold i 1.6 foldi

MS + 2,4-0 + KIN 9.6 16.6 II.R 30 .9 41.6 22.\

A 100% 72.9%i 22.9Q "i 2.2 !'lIIJi 3.3 foldi 1.3 foldi

Standard Error Critical differen ce at 1%

Treatments 0.6907 2.90

Intervals 0.8917 3.75

Treatment x interval 1.5444 6.50

Total soluble sugar content was significantly higher in ST induced by PIM than by CIM, at all stages of growth (Table 6). In PIM, ST recorded very high sucrose and sugar content (Table 5 and 6). This study indicates that sucrose uptake is influenced by hormones in the media. Lower TSS in CIM could be due to lower invertase activity in presence of 2, 4-0 in CIM. Low sucrose uptake in sugarbeet discs treated with 2, 4-0 and low alkaline invertase activity in Lemna minor was reported by Saftner et al (1983) and Frick (1991) respectively. On the other hand increased sucrose and glucose content in differentiated shoot tips were reported by Brown et al (1979) in tobacco and Chaturvedi et al (1974) in Citrus

grandis .

CONCLUSION

From this study it can be concluded that when ST was induced by PtM, it differentiated to multiple shoots and plants and when induced by CIM, only callus was developed, part of which, turned embryogenic after 15­21 DAl.lncreased dry weights, increased nitrate reductase activity, increased sucrose and TSS in ST are characteristic of differentiating ST induced PlM. As differentiation was morphologically observed at 4 DAI, all the biochemical changes before 4 OAI might have led to differentiation . On the other hand non-differentiating ST induced by CIM recorded high N and protein content than thos e induced by Pl M. Thus induction of

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71 In vitro grown shoo t tips in chickpea

Table 6. Sugar content [mg (gluco se equivalcnu/g d \\1.) of shoot tip of chickpea. variety BG-256 as a function of days after inoculat ion. A and B represent per cent I/O: increase (i)/deerease (.1.. ) over control (kept at 100%) and MS + 2.4-0 + KIN treatment (kept at 100%) on the same day respectively

Treatments Days after inocula tion mean

o 7 15 22 30

MS +NAA+ inA 42.9 48 .9 65.5 78.6 95.2 120.7 75.3

A 100% 13.9%i 52.7%i 83.2%i 1.2 fold 'T 1.8 fold'] 75.5%i

13 11.8%i 2.89 foldi 1.2 foldi 1.9 fold T 2.4 fold'] 1.2 fold'I

MS + 2,4-0 + KIN 42.9 44 16.8 36.3 32 35.2 34.5

A \00 % 2.6%i 60.8%.1. 15.4%.1. 25.4% .1. 17.9%.1. 19.5%.1.

Standard Error Critical ditTerence at 1%

Treatments 2.8 192 11.55

Intervals 3.9869 16.34

Treatment x interval 6.9056 28.30

differentiation by plant growth regulators is accompained by changes in biochem ical processes, some of which are studied at present.

ACKNOWLEDG El\IENTS

Authors thank Dr. S.C Bhargava, present l leatl, Dr. Y.P. Abrol, ex-Head of division of Plant Physiology for the co-operation and help rendered durin g the course of investigation. They thank Dr. I3. Baldev, Emeritus Sci­entist for providing facilities. Authors are grateful for the IARI fellowship (to VI') and the financial help of the Council of Scientific and Industrial Research, New Delh i.

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