Plant Cell Reports
Plant Cell Reports (1993) 12:521-524
9 Springer-Verlag1993
Somatic embryogenesis and plant regeneration from callus cultures of chickpea (Cicer arietinum L.) K.S. Barna and A. K. Wakhlu Department of Biosciences, University of Jammu, Jammu-180004, India Received November 24, 1992/Revised version received April 26, 1993 - Communicated by G. C. Phillips
ABSTRACT
Somatic embryogenesis and plant regeneration were obtained from immature leaflet callus of chickpea. Numerous globular embryos developed on the surface of callus on Murashige and Skoog's (1962) medium containing 25 ~M 2,4-dichlorophenoxyacetic acid. These globular embryos differentiated into mature somatic embryos upon removal of 2,4-dichlorophenoxyaeetic acid. The maturation of embryos was significantly affected by pH, photoperiod, abscisic acid and genotype. Callus continued to produce somatic embryos for over 8 subcultures at 4 week intervals. Two per cent of the embryos formed plants on medium containing 15 ~M gibberellia acid and 1 ~M indole-3-butyrlc acid. Desiccation of embryos for a period of 3 d increased their rate of conversion into plants from 0.9 to 2.8%. All regenerated plants showed normal morphological characteristics. : 2,4-D: 2,4-diahlorophenoxyaeetic acid; NAA: 1-naphthaleneacetic acid; Kn: Kinetin; 8A: 6-benzyladenine; TDZ: Thidiazuron; GA3: Gibberellic acid; IBA: Indole-3-butyric acid; ABA: AbseiSic acid; AC: Activated charcoal; AgNOq: Silver nitrate; PVP: Polyvinylpyrrolidone-40,O00 mol wt; MS: Murashige and Skoog (1962) medium. Abbreviations
INTRODUCTION
The chickpea (Cieer arietinum L.) is the most important pulse crop of India. Regeneration of chickpea from callus cultures is reported to be difficult (Altaf and Ahmad 1990). Development of in vitro conditions for achieving comple-t-e and reproducible plant regeneration is essential for the exploitation of tissue culture techniques for improvement of this species. Rao and Chopra (1989) recently have obtained somatic embryos but were unable to obtain plants. This
paper
reports
Correspondence to. A. K. Wakhlu
for
the
first
time plant regeneration through the induction of somatic embryos in chickpea. The effects of explant source, growth regulators, pH, photoperiod, genotype, culture age, and partial desiccation treatments are discussed. M A T E R I A L S AND METHODS
Seven chickpea genotypes were used, including ILC-72, Annigeri and JG-62 (provided by Dr. R.P.S. Pundit, International Crops Research Institute for Semi-Arid Tropics, Patencheru), Flip 85-37 (provided by Dr. M.C. Saxena, International Center for Agricultural Research in the Dry Areas, Alepo), BG-267 (provided by Dr. P.N. Bahl~ Indian Agricultural Research Institute, New Delhi), and Gaurav and C-235 (provided by Dr. K.C. Soni, Sher-e-Kashmir University of Agricultural Sciences and Technology, Jammu). Seeds were surface-sterilized with 70% ethanol for 1 min, then in 0.2% mercuric chloride solution for 4 min. They were rinsed with sterile d i s t i l l e d water and germinate~ a~septically under 16 h light (30 9E m- s -• at 25~ on an agar solidified ~-strength MS basal medium. Immature leaflets (3 mm x 1 mm) were excised under aseptic conditions from 1-week-old seedlings. Cotyledons were excised from surface-sterilized seeds after soaking them in sterile distilled water for 24 h. For the initiation of embryogenic callus cultures, the explants were exposed to 2,4-D and NAA (5-50 tiM) s~ngly or in combination with Kn or BA (0.5-2.5 >M). Calli derived in this manner were subcultured using 5 callus pieces (ca 300 mg fresh weight) per treatment, with 3 replications. For maturation of somatic embryos, calli bearing globular embryos were grown on a medium supplemented with BA~ Kn or TDZ (5 tiM). Different pH levels (4.5-7.5), photoperiods (16 h light or 24 h dark) and genotypes were tested. Four replications of embryogenia r (ca 300 mg fresh weight) were used per treatment. The pH
522 of the c o n t r o l m e d i u m was a d j u s t e d to 5.8 prior to a u t o c l a v i n g , and c u l t u r e s were m a i n t a i n e d unde r p h o t o p e r i o d of 16 h light : 8 h d a r k at a t e m p e r a t u r e of 25~ unless otherwise stated. For partial d e s i c c a t i o n , 280 e m b r y o s w e r e s e a l e d in an empty, dry, s t e r i l e 50 ml f l a s k and kept in the light for 3 or 6 d before i n o c u l a t i n g on e m b r y o c o n v e r s i o n medium. Two r e p l i c a t i o n s w e r e used per t r e a t m e n t for c o n v e r s i o n of e m b r y o s into p l a n t l e t s . All experiments were repeated at least once. Observations were recorded a f t e r 4 weeks of c u l t u r e . D a t a on the n u m b e r of c a l l i s h o w i n g e m b r y o g e n e s i s and plantlet formation were subjected to arcsin transformation for proportions b e f o r e a n a l y s i s , and w e r e c o n v e r t e d b a c k to p e r c e n t a g e s for p r e s e n t a t i o n in tables ( S n e d e c o r and C o c h r a n 1968). M e a n s were c o m p a r e d u s i n g D u n c a n ' s new m u l t i p l e r a n g e test ( D u n c a n 1955). For h i s t o l o g i c a l analysis, mature s o m a t i c e m b r y o s w e r e fixed for 24 h in FAA ( f o r m a l i n : g l a c i a l a c e t i c acid : e t h a nol, 1:1:18 v/v), dehydrated in an ethanol series, and e m b e d d e d in p a r a f f i n wax (M P 52~ S e c t i o n s of I0 ~ m t h i c k n e s s were cut on a r o t a r y m i c r o t o m e and s t a i n e d with safranin-fast green combination (Johansen 1940).
Tal~le I. Effect of auxins on formation of embryogenlc cultures from immature leaflet and cotyledon derived calll of Cicer arletlnum cv. C-235 after 4 weeks of culture.
Auxin #M
Number of calll showingembryogenesls%1 Leaflet Cotyledon (Mean) (Mean)
NAA 5 25
Oa 0a
50
2.4 a
2,4-D 5 25 50
b 29.5 71.4~ 16.9
Source
df
Replication Auxin Source (A) Auxin conc (8) Explant source (C) AxB B x~ AXC AxBxC Error
5 I 2 I 2 2 I 2 55
O O 0 MS 78.26 5614.47 303.83 7650.85 676.52 303.82 5614.46 676.53 62.99
F NS 1.24,, 89.13, 4.82** 121.46,, 10.74. 4.82** 89.13,, 10.72
Means with same superscript are not signTfTcantly different from each other at the 5% level byDuncan's new multiplerange test,
NS : Not significant * Significant at 5% level
R E S U L T S AND D I S C U S S I O N **Highly significantat I% level. Friable c r e a m y c a l l u s was p r o d u c e d from the cut ends of immature leaflet and c o t y l e d o n e x p l a n t s on a m e d i u m c o n t a i n i n g 5MM 2,4-D w i t h i n 1"2 w e e k s of e x p l a n t i n g . T h e c a l l u s d e r i v e d from i m m a t u r e l e a f l e t s differentiated numerous globular embryos (Fig I). T h e s e c a l l i w e r e n o d u l a r and w h i t e and c o n s i s t e d of small, t h i n - w a l l e d , and densely cytoplasmic cells with a centrally-placed prominent nucleus. The induction of somatic embryogenesis was s i g n i f i c a n t l y i n f l u e n c e d by a u x i n source, its concentration, explant source, and their i n t e r a c t i o n s ( T a b l e i). 2 , 4 - D was the m o s t s u i t a b l e of the two a u x i n s o u r c e s tested for i n d u c t i o n of s o m a t i c e m b r yos, w h i l e NAA i n d u c e d e m b r y o g e n e s i s in a small n u m b e r of calli. This finding agrees with the r e p o r t of K y s e l y and J a c o b s e n (1990), who reported 2,4-D treatment to be favourable for s o m a t i c embryogenesis in pea c a l l u s c u l t u r e s . No s o m a t i c e m b r y o s were formed on callus derived from cotyledon explants. T h e n u m b e r of c alli b e a r i n g g l o b u l a r e m b r y o s was g r e a t e s t on a medium supplemented with 2,4-D at a c o n c e n t r a t i o n of 25 ~ M . A d d i t i o n of BA or Kn (0.5, 2.5 ~ M each) decreased the f~equency of calli bearing globular embryos (data not presented). Mature s o m a t i c e m b r y o s w e r e f o r m e d only w h e n the c a l li b e a r i n g g l o b u l a r e m b r y o s w e r e put on 2,4-D-free medium. Use of BA, Kn or TDZ (5 ~M) did not modify the embryo m a t u r a t i o n r e s p o n s e (data not p r e s e n t e d ) .
Among various media pH levels tested, a pH of 6.5 was most s u i t a b l e for e m b r y o m a t u r a t i o n (Table 2). L o w e r i n g of the pH to 4.5 c o m p l e t e l y suppressed the development of mature em b r y o s . E m b r y o g e n e s i s has also been r e p o r t e d to be s e n s i t i v e to pH in Other plants. A pH of 6.8 was o p t i m a l to o b t a i n large n u m b e r s of Effect of medlum pH on maturatlen of somai~icembryos in Clcer arieflnumcv. C-235 after 4 weeks of culture 9
Table 2.
pH
2 Number of mature embryos per callus (Mean)
4.5 5.0 5.5 5.8 6.5 7.5
0d 17-5c 18.7c c 23.Ia 41.4b 32.5
IHormone-freeMS medium used. 2Means with same superscript are not significantly different from each other at the 5% level by Duncan's new multiple range test. Each mean represents 8 samples. embryos in potato (Rashid and Reinert 1983)~ whereas 6.0 pH was optimal for c a r r o t ( S m i t h and K r i k o r i a n 1990). The e m b r y o g e n i c calli g r o w n under light g a v e a high frequency of mature embryos per c a l l u s p i e c e (21), w h i l e calli g r o w n in total d a r k n e s s p r o d u c e d a low n u m b e r of
523 response with 25.7 somatic embryos formed per callus piece, and three other genotypes gave comparable responses. ILC-72 was the least responsive genotype. Genotypic differences with respect to the number of somatic embryos formed per explant also have been reported in Medicago falcata (Chen and Marowitch and G lycine max (Komatsuda and Ohyama 1988).
Figs.l-5 Somatic embryogenesis and plant regeneration from callus culture of chickpea. Fig.l Embryogenic callus with globular embryos formed on medium with 25 ~M 2,4-D after 4 weeks of culture(Scale 5 mm). Fig.2 Mature embryo formed from embryogenic callus on hormone- free MS medium after 4 weeks of culture (Scale i mm). Fig.3 Histological section of mature embryo showing shoot primordium (SP), root primordium (RP) and provascular tissue (PV; Scale 0.5 mm). Fig.4 A plantlet regenerated from somatic embryo on a medium with 15 ~ M GA 3 plus i ~M IBA after 5 weeks of culture (Scale I0 mm). Fig.5 A plant derived from somatic embryo growing in a pot containing garden soil (Scale 5 cm). embryos per callus piece (1.6). Addition of ABA significantly increased the response of embryo maturation (Table 3), as has been previously demonstrated in cultures of Picea glauca (Attree et al 1990 ; Hakman and yon Arnold 1988).-- -Table 3. Effect of ABA on maturation of somatic embryos Clcer arletlnum cv. C-235 after 4 weeks of culture I.
ABA (~M) 0 I 5 10
in
Number of mature embryos per callus 2 (Mean) 23.4 c 37.0 a 33.6 ab 28.7 be
IHormone-free MS medium used, 2Means With same superscript are not significantly different from each other at the 5% level by Duncan's new multiple range test, Each mean represents 8 replicates.
The number of mature embryos formed callus piece varied with the genotype (Table 4). Flip 85-37 gave the best
per
The influence of r e p e a t e d subculturing (age of callus) on the frequency of mature somatic embryos formed per callus piece was tested (Table 5). Embryogenic callus continued to produce mature embryos for over 8 subcultures. Long-term maintenance of embryogenesis has been previously observed in hypocotyl-derived calli of Albizia richardiana (Tomar an--~Gupta 1988) and immature zygotic embryos of soybean and peanut (Sellars et al 1990). The mature somatic embryos had two welldeveloped cotyledons and an elongated hypocotyl root axis (Fig 2). Histological studies of mature somatic embryos showed the presence
Table 4, Effec~ of genotype of Clcer arietinum on formation of mature e~bryos and conversion of somatic embryos into plantlets.
Genotype
ILC-72 JG-62 Gaurav Annlgeri BG-267 C-255 Fllp 85-37
Numberof mature embryos per callus 2 (Mean)
Conversiono f somatic embryos Into plantlets Total number of % plantle~ embryos cultured formation (Mean)
9.2 d
148
0a
15.6~c 19.2ab c 21.2 21.2 abc 23.1 ab 25.7 a
210 180 210 195 258 195
0.46 a 0.13 a 0.50 a 0.13 a 0.93 a 0a
IHormone-free MS medium used.
2Means with same superscript within columns are not significantly drfferent from each other at the 5% level by Duncan's new multiple range test. Each mean represents 8 replicates for formation of mature embryos and 4 replicates for embryo conversion Into plantlets.
of shoot and root primordia andprovascular traces (Fig 3). Best conversion of embryos into plantlets (2%) was observed in the presence of 15 ~M GA~ and 1 ~M IBA (Fig 4; Table 6). The ~evelopment of
524 Table 5.
Effect of subculture (age of callus) on maturationof somatic embryos In CIcer arletinum cv. C-235, using 4 week subculture IntervalsI. Subculture
ACKNOWLEDGEMENTS
17o2 b
We wish to thank Head of the D e p a r t m e n t of Biosciences, University of Jammu, for providing laboratory facilities and Drs. K~hmer and Sjut, Schering A k t i e n g e s e l l s c h a f t , B e r l i n for the gift of thidiazuron. KSB g r a t e f u l l y a c k n o w l e d g e s the f i n a n c i a l s u p p o r t of J a m m u U n i v e r s i t y .
17.3 16.1~
REFERENCES
Number of mature embryos per callus3 (Mean)
Ist2 2nd 4th 6th
23.1~
8th
19.0
Altaf
N, A h m a d MS (1990) In: Bajaj Y P S (ed) Biotechnology in A g r i c u l t u r e and F o r e s t r y , Vol i0, L e g u m e s and Oilseed Crops i, Springer-Verlag, B e r l i n H e i d e l b e r g pp 1 0 0 - 1 1 3
2 Embryogenlc callus obtained after Ist sub-culture of primary em~ryogenic callus.
Attree
SM, T a u t o r u s TE~ D u n s t a n Dig F o w k e LC (1990) Can J Bot 6 8 : 2 5 8 3 - 2 5 8 9
3 Means with same superscr{pt are not significantly different from each other at the 5% level by Duncan's new multiple range test. Each mean represents 8 replicates.
Chen
THH, Marowitch J Physiol 128:271-277
IEmbryogenlc callus maintainedon MS+25 ~M 2,4-D+I000mgl-I~ PVP with 4 week-subculture Intervals. For maturation of embryos, embryogenic callus was inoculatedon hormone-freeMS medium.
Duncan Hakman
e m b r y o s into p l a n t s was e n h a n c e d from 0.9 to 2.8% by d e s i c c a t i n g the e m b r y o s for 3 d ( T able 6). A s i m i l a r p r o m o t i v e e f f e ct of partial desiccation has b e e n n o t i c e d in G l y c i n e max ( P a r r o t t et al 1988). The rate ~ c o n v e r s i o n of s'6-maTic e m b r y o s into plantlets was not influenced by the g e n o t y p e ( T a b l e 4). Effect of various media and partial desiccation on conversion of somatic embryos into plantlets In Cicer arletlnum cv. C-235 after 4 weeks of culture. % plantle+I formation (Mean}
Hormone-freeMS BA I ~M + IBA 0.1 ~M GA 15 ~M + IBA 1.0~M AC30.5% AgNO3 10 ~M
258 246 245 216 145
0.90ab 0.92ab 2.0a %11 b 0
280 280
2.8 a 0.42 ab
Desiccation 2
3 d 6 d
I
Means wlth same superscript are no~ significantly different from each other at the 5% level by Duncan's new multiple range test. Each mean represents 4 replicates.
2Hormone-freeMS medium used. T h i r t y p l a n t s were t r a n s f e r r e d to pots c o n t a i n i n g g a r d e n soil (Fig 5) a f t e r h a r d e n i n g in s t e r i l e v e r m i c u l i t e for about a week. All regenerated plants were morphologically normal. T h e p r o c e d u r e of regenerating whole plants described here opens up the prospects of using biotechnological approaches for c h i c k p e a improvement.
Biometrics S
K o m a t s u d a Tg O h y a m a K Genet 75:695-700 Kysely
ii:
Plant
1-42
(1988)
Physiol
Company
(1988)
Theor
Appl
W, J a c o b s e n HJ (1990) Tiss Org Cult 2 0 : 7 - 1 4
Plant
Cell
M u r a s h i g e T, Skoog 15:473-497
F
(1962)
Physiol
Plant
P a r r o t t WAg D r y d e n Gg Vogt S, H i l d e b r a n d DF~ C o l l i n s GB~ W i l l i a m s EG (1988) In V i t r o Cell D e v e l Biol 2 4 : 8 1 7 - 8 2 0 Rao
Media
(1955)
I, yon A r n o l d Plant 7 2 : 5 7 9 - 5 8 7
J
J o h a n s e n DA (1940) Plant M i c r o s T a r a M c G r a w Hill P u b l i s h i n g Ltd, B o m b a y New Delhi
Table 6.
Total number of embryos cultured
DB
(1987)
BG, C h o p r a VL 134:637-638
Rashid
A, R e i n e r t 116:155-160
Sellars RM, (1990) Smith
(1989) J
J Plant
(1983)
Physiol
Protoplasma
Southward GM, Phillips Crop Sci 3 0 : 4 0 8 - 4 1 4
DL, K r i k o r i a n 77:1634-1647
AD
(1990)
Amer
GC
J Bot
Snedecor GW, Cochran WG (1968) Statistical Methods. O x f o r d and IBH Publishing Co Pvt Ltdg New Delhi Bombay Calcutta Tomar
UK, G u p t a SC Reports 7:70-73
(1988)
Plant
Cell
Plant Cell Reports (1993) 12:521-524
9 Springer-Verlag1993
Somatic embryogenesis and plant regeneration from callus cultures of chickpea (Cicer arietinum L.) K.S. Barna and A. K. Wakhlu Department of Biosciences, University of Jammu, Jammu-180004, India Received November 24, 1992/Revised version received April 26, 1993 - Communicated by G. C. Phillips
ABSTRACT
Somatic embryogenesis and plant regeneration were obtained from immature leaflet callus of chickpea. Numerous globular embryos developed on the surface of callus on Murashige and Skoog's (1962) medium containing 25 ~M 2,4-dichlorophenoxyacetic acid. These globular embryos differentiated into mature somatic embryos upon removal of 2,4-dichlorophenoxyaeetic acid. The maturation of embryos was significantly affected by pH, photoperiod, abscisic acid and genotype. Callus continued to produce somatic embryos for over 8 subcultures at 4 week intervals. Two per cent of the embryos formed plants on medium containing 15 ~M gibberellia acid and 1 ~M indole-3-butyrlc acid. Desiccation of embryos for a period of 3 d increased their rate of conversion into plants from 0.9 to 2.8%. All regenerated plants showed normal morphological characteristics. : 2,4-D: 2,4-diahlorophenoxyaeetic acid; NAA: 1-naphthaleneacetic acid; Kn: Kinetin; 8A: 6-benzyladenine; TDZ: Thidiazuron; GA3: Gibberellic acid; IBA: Indole-3-butyric acid; ABA: AbseiSic acid; AC: Activated charcoal; AgNOq: Silver nitrate; PVP: Polyvinylpyrrolidone-40,O00 mol wt; MS: Murashige and Skoog (1962) medium. Abbreviations
INTRODUCTION
The chickpea (Cieer arietinum L.) is the most important pulse crop of India. Regeneration of chickpea from callus cultures is reported to be difficult (Altaf and Ahmad 1990). Development of in vitro conditions for achieving comple-t-e and reproducible plant regeneration is essential for the exploitation of tissue culture techniques for improvement of this species. Rao and Chopra (1989) recently have obtained somatic embryos but were unable to obtain plants. This
paper
reports
Correspondence to. A. K. Wakhlu
for
the
first
time plant regeneration through the induction of somatic embryos in chickpea. The effects of explant source, growth regulators, pH, photoperiod, genotype, culture age, and partial desiccation treatments are discussed. M A T E R I A L S AND METHODS
Seven chickpea genotypes were used, including ILC-72, Annigeri and JG-62 (provided by Dr. R.P.S. Pundit, International Crops Research Institute for Semi-Arid Tropics, Patencheru), Flip 85-37 (provided by Dr. M.C. Saxena, International Center for Agricultural Research in the Dry Areas, Alepo), BG-267 (provided by Dr. P.N. Bahl~ Indian Agricultural Research Institute, New Delhi), and Gaurav and C-235 (provided by Dr. K.C. Soni, Sher-e-Kashmir University of Agricultural Sciences and Technology, Jammu). Seeds were surface-sterilized with 70% ethanol for 1 min, then in 0.2% mercuric chloride solution for 4 min. They were rinsed with sterile d i s t i l l e d water and germinate~ a~septically under 16 h light (30 9E m- s -• at 25~ on an agar solidified ~-strength MS basal medium. Immature leaflets (3 mm x 1 mm) were excised under aseptic conditions from 1-week-old seedlings. Cotyledons were excised from surface-sterilized seeds after soaking them in sterile distilled water for 24 h. For the initiation of embryogenic callus cultures, the explants were exposed to 2,4-D and NAA (5-50 tiM) s~ngly or in combination with Kn or BA (0.5-2.5 >M). Calli derived in this manner were subcultured using 5 callus pieces (ca 300 mg fresh weight) per treatment, with 3 replications. For maturation of somatic embryos, calli bearing globular embryos were grown on a medium supplemented with BA~ Kn or TDZ (5 tiM). Different pH levels (4.5-7.5), photoperiods (16 h light or 24 h dark) and genotypes were tested. Four replications of embryogenia r (ca 300 mg fresh weight) were used per treatment. The pH
522 of the c o n t r o l m e d i u m was a d j u s t e d to 5.8 prior to a u t o c l a v i n g , and c u l t u r e s were m a i n t a i n e d unde r p h o t o p e r i o d of 16 h light : 8 h d a r k at a t e m p e r a t u r e of 25~ unless otherwise stated. For partial d e s i c c a t i o n , 280 e m b r y o s w e r e s e a l e d in an empty, dry, s t e r i l e 50 ml f l a s k and kept in the light for 3 or 6 d before i n o c u l a t i n g on e m b r y o c o n v e r s i o n medium. Two r e p l i c a t i o n s w e r e used per t r e a t m e n t for c o n v e r s i o n of e m b r y o s into p l a n t l e t s . All experiments were repeated at least once. Observations were recorded a f t e r 4 weeks of c u l t u r e . D a t a on the n u m b e r of c a l l i s h o w i n g e m b r y o g e n e s i s and plantlet formation were subjected to arcsin transformation for proportions b e f o r e a n a l y s i s , and w e r e c o n v e r t e d b a c k to p e r c e n t a g e s for p r e s e n t a t i o n in tables ( S n e d e c o r and C o c h r a n 1968). M e a n s were c o m p a r e d u s i n g D u n c a n ' s new m u l t i p l e r a n g e test ( D u n c a n 1955). For h i s t o l o g i c a l analysis, mature s o m a t i c e m b r y o s w e r e fixed for 24 h in FAA ( f o r m a l i n : g l a c i a l a c e t i c acid : e t h a nol, 1:1:18 v/v), dehydrated in an ethanol series, and e m b e d d e d in p a r a f f i n wax (M P 52~ S e c t i o n s of I0 ~ m t h i c k n e s s were cut on a r o t a r y m i c r o t o m e and s t a i n e d with safranin-fast green combination (Johansen 1940).
Tal~le I. Effect of auxins on formation of embryogenlc cultures from immature leaflet and cotyledon derived calll of Cicer arletlnum cv. C-235 after 4 weeks of culture.
Auxin #M
Number of calll showingembryogenesls%1 Leaflet Cotyledon (Mean) (Mean)
NAA 5 25
Oa 0a
50
2.4 a
2,4-D 5 25 50
b 29.5 71.4~ 16.9
Source
df
Replication Auxin Source (A) Auxin conc (8) Explant source (C) AxB B x~ AXC AxBxC Error
5 I 2 I 2 2 I 2 55
O O 0 MS 78.26 5614.47 303.83 7650.85 676.52 303.82 5614.46 676.53 62.99
F NS 1.24,, 89.13, 4.82** 121.46,, 10.74. 4.82** 89.13,, 10.72
Means with same superscript are not signTfTcantly different from each other at the 5% level byDuncan's new multiplerange test,
NS : Not significant * Significant at 5% level
R E S U L T S AND D I S C U S S I O N **Highly significantat I% level. Friable c r e a m y c a l l u s was p r o d u c e d from the cut ends of immature leaflet and c o t y l e d o n e x p l a n t s on a m e d i u m c o n t a i n i n g 5MM 2,4-D w i t h i n 1"2 w e e k s of e x p l a n t i n g . T h e c a l l u s d e r i v e d from i m m a t u r e l e a f l e t s differentiated numerous globular embryos (Fig I). T h e s e c a l l i w e r e n o d u l a r and w h i t e and c o n s i s t e d of small, t h i n - w a l l e d , and densely cytoplasmic cells with a centrally-placed prominent nucleus. The induction of somatic embryogenesis was s i g n i f i c a n t l y i n f l u e n c e d by a u x i n source, its concentration, explant source, and their i n t e r a c t i o n s ( T a b l e i). 2 , 4 - D was the m o s t s u i t a b l e of the two a u x i n s o u r c e s tested for i n d u c t i o n of s o m a t i c e m b r yos, w h i l e NAA i n d u c e d e m b r y o g e n e s i s in a small n u m b e r of calli. This finding agrees with the r e p o r t of K y s e l y and J a c o b s e n (1990), who reported 2,4-D treatment to be favourable for s o m a t i c embryogenesis in pea c a l l u s c u l t u r e s . No s o m a t i c e m b r y o s were formed on callus derived from cotyledon explants. T h e n u m b e r of c alli b e a r i n g g l o b u l a r e m b r y o s was g r e a t e s t on a medium supplemented with 2,4-D at a c o n c e n t r a t i o n of 25 ~ M . A d d i t i o n of BA or Kn (0.5, 2.5 ~ M each) decreased the f~equency of calli bearing globular embryos (data not presented). Mature s o m a t i c e m b r y o s w e r e f o r m e d only w h e n the c a l li b e a r i n g g l o b u l a r e m b r y o s w e r e put on 2,4-D-free medium. Use of BA, Kn or TDZ (5 ~M) did not modify the embryo m a t u r a t i o n r e s p o n s e (data not p r e s e n t e d ) .
Among various media pH levels tested, a pH of 6.5 was most s u i t a b l e for e m b r y o m a t u r a t i o n (Table 2). L o w e r i n g of the pH to 4.5 c o m p l e t e l y suppressed the development of mature em b r y o s . E m b r y o g e n e s i s has also been r e p o r t e d to be s e n s i t i v e to pH in Other plants. A pH of 6.8 was o p t i m a l to o b t a i n large n u m b e r s of Effect of medlum pH on maturatlen of somai~icembryos in Clcer arieflnumcv. C-235 after 4 weeks of culture 9
Table 2.
pH
2 Number of mature embryos per callus (Mean)
4.5 5.0 5.5 5.8 6.5 7.5
0d 17-5c 18.7c c 23.Ia 41.4b 32.5
IHormone-freeMS medium used. 2Means with same superscript are not significantly different from each other at the 5% level by Duncan's new multiple range test. Each mean represents 8 samples. embryos in potato (Rashid and Reinert 1983)~ whereas 6.0 pH was optimal for c a r r o t ( S m i t h and K r i k o r i a n 1990). The e m b r y o g e n i c calli g r o w n under light g a v e a high frequency of mature embryos per c a l l u s p i e c e (21), w h i l e calli g r o w n in total d a r k n e s s p r o d u c e d a low n u m b e r of
523 response with 25.7 somatic embryos formed per callus piece, and three other genotypes gave comparable responses. ILC-72 was the least responsive genotype. Genotypic differences with respect to the number of somatic embryos formed per explant also have been reported in Medicago falcata (Chen and Marowitch and G lycine max (Komatsuda and Ohyama 1988).
Figs.l-5 Somatic embryogenesis and plant regeneration from callus culture of chickpea. Fig.l Embryogenic callus with globular embryos formed on medium with 25 ~M 2,4-D after 4 weeks of culture(Scale 5 mm). Fig.2 Mature embryo formed from embryogenic callus on hormone- free MS medium after 4 weeks of culture (Scale i mm). Fig.3 Histological section of mature embryo showing shoot primordium (SP), root primordium (RP) and provascular tissue (PV; Scale 0.5 mm). Fig.4 A plantlet regenerated from somatic embryo on a medium with 15 ~ M GA 3 plus i ~M IBA after 5 weeks of culture (Scale I0 mm). Fig.5 A plant derived from somatic embryo growing in a pot containing garden soil (Scale 5 cm). embryos per callus piece (1.6). Addition of ABA significantly increased the response of embryo maturation (Table 3), as has been previously demonstrated in cultures of Picea glauca (Attree et al 1990 ; Hakman and yon Arnold 1988).-- -Table 3. Effect of ABA on maturation of somatic embryos Clcer arletlnum cv. C-235 after 4 weeks of culture I.
ABA (~M) 0 I 5 10
in
Number of mature embryos per callus 2 (Mean) 23.4 c 37.0 a 33.6 ab 28.7 be
IHormone-free MS medium used, 2Means With same superscript are not significantly different from each other at the 5% level by Duncan's new multiple range test, Each mean represents 8 replicates.
The number of mature embryos formed callus piece varied with the genotype (Table 4). Flip 85-37 gave the best
per
The influence of r e p e a t e d subculturing (age of callus) on the frequency of mature somatic embryos formed per callus piece was tested (Table 5). Embryogenic callus continued to produce mature embryos for over 8 subcultures. Long-term maintenance of embryogenesis has been previously observed in hypocotyl-derived calli of Albizia richardiana (Tomar an--~Gupta 1988) and immature zygotic embryos of soybean and peanut (Sellars et al 1990). The mature somatic embryos had two welldeveloped cotyledons and an elongated hypocotyl root axis (Fig 2). Histological studies of mature somatic embryos showed the presence
Table 4, Effec~ of genotype of Clcer arietinum on formation of mature e~bryos and conversion of somatic embryos into plantlets.
Genotype
ILC-72 JG-62 Gaurav Annlgeri BG-267 C-255 Fllp 85-37
Numberof mature embryos per callus 2 (Mean)
Conversiono f somatic embryos Into plantlets Total number of % plantle~ embryos cultured formation (Mean)
9.2 d
148
0a
15.6~c 19.2ab c 21.2 21.2 abc 23.1 ab 25.7 a
210 180 210 195 258 195
0.46 a 0.13 a 0.50 a 0.13 a 0.93 a 0a
IHormone-free MS medium used.
2Means with same superscript within columns are not significantly drfferent from each other at the 5% level by Duncan's new multiple range test. Each mean represents 8 replicates for formation of mature embryos and 4 replicates for embryo conversion Into plantlets.
of shoot and root primordia andprovascular traces (Fig 3). Best conversion of embryos into plantlets (2%) was observed in the presence of 15 ~M GA~ and 1 ~M IBA (Fig 4; Table 6). The ~evelopment of
524 Table 5.
Effect of subculture (age of callus) on maturationof somatic embryos In CIcer arletinum cv. C-235, using 4 week subculture IntervalsI. Subculture
ACKNOWLEDGEMENTS
17o2 b
We wish to thank Head of the D e p a r t m e n t of Biosciences, University of Jammu, for providing laboratory facilities and Drs. K~hmer and Sjut, Schering A k t i e n g e s e l l s c h a f t , B e r l i n for the gift of thidiazuron. KSB g r a t e f u l l y a c k n o w l e d g e s the f i n a n c i a l s u p p o r t of J a m m u U n i v e r s i t y .
17.3 16.1~
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Ist2 2nd 4th 6th
23.1~
8th
19.0
Altaf
N, A h m a d MS (1990) In: Bajaj Y P S (ed) Biotechnology in A g r i c u l t u r e and F o r e s t r y , Vol i0, L e g u m e s and Oilseed Crops i, Springer-Verlag, B e r l i n H e i d e l b e r g pp 1 0 0 - 1 1 3
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e m b r y o s into p l a n t s was e n h a n c e d from 0.9 to 2.8% by d e s i c c a t i n g the e m b r y o s for 3 d ( T able 6). A s i m i l a r p r o m o t i v e e f f e ct of partial desiccation has b e e n n o t i c e d in G l y c i n e max ( P a r r o t t et al 1988). The rate ~ c o n v e r s i o n of s'6-maTic e m b r y o s into plantlets was not influenced by the g e n o t y p e ( T a b l e 4). Effect of various media and partial desiccation on conversion of somatic embryos into plantlets In Cicer arletlnum cv. C-235 after 4 weeks of culture. % plantle+I formation (Mean}
Hormone-freeMS BA I ~M + IBA 0.1 ~M GA 15 ~M + IBA 1.0~M AC30.5% AgNO3 10 ~M
258 246 245 216 145
0.90ab 0.92ab 2.0a %11 b 0
280 280
2.8 a 0.42 ab
Desiccation 2
3 d 6 d
I
Means wlth same superscript are no~ significantly different from each other at the 5% level by Duncan's new multiple range test. Each mean represents 4 replicates.
2Hormone-freeMS medium used. T h i r t y p l a n t s were t r a n s f e r r e d to pots c o n t a i n i n g g a r d e n soil (Fig 5) a f t e r h a r d e n i n g in s t e r i l e v e r m i c u l i t e for about a week. All regenerated plants were morphologically normal. T h e p r o c e d u r e of regenerating whole plants described here opens up the prospects of using biotechnological approaches for c h i c k p e a improvement.
Biometrics S
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