Plant Cell Reports

Plant Cell Reports (1996) 16:32-37

© Springer-Verlag1996

Efficient transgenic plant regeneration through Agrobacterium-mediated transformation of Chickpea (Cicer arietinum L.) Sanchayita Kar *, Tony M. Johnson **, Pritilata Nayak, and S.K. Sen Plant Molecular & Cellular Genetics & Centre for Plant Molecular Biology,Bose Institute, P1/12, C.I.T. SchemeVII-M, Calcutta - 700 054, India * Present address." Department of Biochemistry& Molecular Biology,Box 9650, Mississipi State University, MS 39762, USA ** Present address: Department of Biochemistry,Collegeof Biological Sciences, 140 Gortner Laboratory, 1479 Gortner Avenue, St. Paul, MN 55108-1022, USA Received 6 July 1995/Revised version received 24 March 1996 - Communicatedby W. A. Parrott

ABSTRACT: Three genotypes of chickpea ICCV-1, ICCV-6 and a Desi (local) variety were tested for plant regeneration through multiple shoot production. The embryo axis was removed from mature seeds, the root meristem and the shoot apex were discarded. These explants were cultured on medium containing MS macro salts, 4X MS micro salts, B5 vitamins, 3.0 mg/1 BAP, 0.004 mg/1 NAA, 3% (w/v) sucrose and incubated at 260C. The explants were transformed with Agrobacterium tumefaciens strain LBA4404 with binary vector pBI121 containing the uidA and nptlI genes. Multiple shoots were repeatedly selected with kanamycin. The selected kanamycin resistant shoots were rooted on MS medium supplemented with 0.05 mg/1 IBA. The presumptive transformants histochemically stained positive for GUS. Additionally, nptlI assay confirmed the expression of n p t H in kanamycin resistant plants. Transgenic plants were transferred to soil and grown in the green house. Abbreviations: BAP- 6-benzylamino purine; 2,4-D - 2,4-

dichlorophenoxy acetic acid; IAA- Indole acetic acid; IBA-Indole butaric acid; NAA-Naphthalene acetic acid ~TRODUCTION Chickpea (Cicer arietinum L.) is an important grain legume which has worldwide acceptance as a major source of protein for human as well as animal consumption. Much of its grain productivity is adversely affected by damages caused by a lepidopteran pest, the pod borer (Heliothis sp.). Raising insect resistant chickpea lines through conventional breeding is not feasible due to the nonavailability of any germplasm showing resistance against the damage caused by this insect (van der Have, 1970). Thus, establishment of insect resistant transgenic lines of chickpea is considered as a potential improvement strategy. Correspondence to." S. K. Sen

One of the prerequisites for successful gene transfer to plants is the availability of a suitable protocol for transformation which is compatible with in vitro plant regeneration methods of the target plant species. In vitro plant regeneration in chickpea has been reported through organogenesis from shoot meristems (Bajaj and Dhanju, 1979; Sharma et al., 1979; Kartha et al., 1981), immatta'e cotyledons (Shri and Davis, 1992) and through embryogenesis from immature cotyledons (Sagare et at., 1993) and leaflet callus (Bama and Wakhlu, 1993, 1994; Kumar et al., 1994). Furthermore, Agrobacterium mediated transformation in a number of legumes has been previously reported (Mariotti et aL, 1984; Owens and Cress, 1985; Jensen et aL, 1986; Bercetche et at., 1987; Hussey et aL, 1989). There are reports on transgenic plant production of other grain legumes (Vigna aconitifolia , Eapen et aL, 1987; GIycine m a x , Hinchee et al., 1988; P h a s e o l u s , Mariotti et al., 1989; Pisum sativum, DeKathen and Jacobsen, 1990; Puonti-Kaerlas et al., 1990, 1992; Schroeder et al., 1993; Arachis hypogea, McKently et al., 1995). Recently, Fontana et al. (1993) have reported successful transformation of chickpea through A g r o b a c t e r i u m mediated genetic transformation. That report dealt with only one local variety. Though Islam et al (1994) have tested susceptibility of chickpea to A g r o b a c t e r i u m infection, reproducible protocols crossing the genotype barrier for gene transfer in chickpea have yet to be designed. In this communication, we report transgenic plant production of three genotypes of chickpea using the Agrobacterium - mediated gene transfer technique. This has been achieved through development of an efficient method of plant regeneration through multiple shoot formation. We believe that the reported method of plant regeneration in chickpea would be effective for other gene delivery methods, including the particle gun delivery system.

33 MATERIALS AND METHODS

Plant materials and Bacterial strains ICCV-1, ICCV-6 and a Desi (local) genotype were used as the source of plant material. Genotypes ICCV-1 and ICCV-6 were obtained from International Crops Research Institute for the Semi Arid Tropics (ICRISAT), Hyderabad, India and the Desi (local) variety was collected locally. The Agrobacterium strain used was LBA4404 (Hoekama et al., 1983) containing pBI121 (Jefferson et al., 1987) which has a nptll(neomycin phosphotransj%rase H ) gene and a uidA gene driven by nos and CaMV 35S promoter, respectively and nopaline synthetase terminators in both the cases. Dried mature seeds were soaked with 0.1% Tween 20 for 20 rain followed by vigorous washing with water. The seeds were treated with 0.1% HgC12 for 15 min, washed three times with sterile distilled water, then submerged in sterile water and imbibed overnight under sterile conditions. On the following day, seeds were opened, embryo axes removed and the root meristem and shoot apex discarded. The remaining part containing a portion of the hypocotyl as well as epicotyl was used as the experimental material (Fig-lA).

Plant regeneration through multiple shoot formation Fifteen explants per 90 mm Petri dishes from each genotype were cultured in medium containing MS (Murashige and Skoog, 1962) macrosalts, 1X or 4x MS micro salts, B5 vitamins (Gamborg et al., 1968), different concentrations of growth regulators, 3% (w/v) sucrose, pH 5.8 and solidified with 0.8% Difco Bactoagar (Table-l). Cultures were placed at 240C and 16-h/8-h photoperiod under -20 I.tmol photons.m2.s -1 white fluoroscent light. From the intial observations (Table-I) a combination of salts and growth regulators (MS macro salts, 4X MS micro salts, B5 vitamins, 3.0 rag/1 BAP, 0.004 mg/1 NAA (SR medium), which induced maximum number of shoots per explant, was adopted for further experiments. Three weeks after culture, explants producing multiple shoots were transferred to fresh SR medium for shoot elongation. After two weeks, the individual shoots were separated fi'om the explants and cultured for another two weeks before transferring to root induction medium (MS salts, MS vitamins, 0.05 mg/1 IBA, Table-2). Shoots with roots were transferred to half-strength MS medium containing 2% (w/v) sucrose, without exogenous growth regulators. Plants with well developed roots were transferred to soil and grown in the green house.

Transformation and Plant regeneration Agrobactenum strain LBA4404 was grown at 280C overnight in LB (Sambrook et al , 1989) liquid medium containing 100 rag/1 kanamycin. The bacterial cells were collected by centrifugation and resuspended in SR medium to a concentration of lx108 cells/ml. The explants were cultured for 24 h on SR medium prior to cocultivation with bacteria. Fifty explants were cocultivated with l0 ml bacterial suspension for 24 h at 280C on a gyratory shaker at 40 rpm. The explants were washed thoroughly with sterile liquid SR medium, blotted on filter papers and cultured in semi-solid SR medium for 72 h and then transferred to same medium supplemented with 500 rag/1 cefotaxime and 25 rag/1 kanamycin; and incubated at 250C with a 16-h photoperind. After three weeks, the growing shoots were dissected from the primary explant and subcultured in fresh SR medium along with the primary explant in presence of 50 mgfl kanamycin. The green, surviving shoots were subjected to 2-3 more passages of selection by repeated excision of

branches and their exposure to selection medium. The green shoots were transferred to rooting medium without kanamycin for root induction.

Data Analysis All experiments were carried out in five replicates and the standard error of the mean were calculated. The experiments were repeated three times, keeping all the parameters unchanged.

DNA isolation and Southern hybridisation DNA was isolated from young shoots following the method of Dellaporta et al. (1983). Five gg of DNA from each sample were digested with Hind IlI, fractionated in 0.8% (w/v) agarose gel, transferred to Hybond + nylon membrane (Amersham), UV cross-linked. The Pst I cleaved DNA fragment of pBI121 (1.9 kb) containing nptH gene was gel eluted, radiolabelled by random primer method with 32p (Sambrook et al., 1989) and used as probe. The blot was hybridised at 420C, washed two times at 650C with 1X SSC-0.1% SDS, three times with 0.1X SSC-1% SDS. The blot was exposed to Kodak X-Omat film with intensifying screen at -800C for 6 days.

Enzyme Assay Shoots growing in kanamycin supplemented medium were tested for the intracellular presence of Agrobacterium prior to enzyme assay. Shoot segments were cultured in LB medium without antibiotics for 48 h. The medium was then screened microscopically for the presence of bacteria. The nptlI assay of the transgenic plants was carried out as described by Scott et al. (1988). The histochemical GUS assay was carried out according to Jefferson (1987) and the fluorometric assay was performed according to Scott et al. (1988). RESULTS

Plant regeneration through multiple shoot formation A m o n g the g en o t y p es tested, the Desi variety gave greatest n u m b e r o f shoots per explant (Fig. 2). When MS salts without modification were used for shoot Table 1: Effect of medium composition on shoot inductiona MS micro salts

NAA BAP No. of shoots generated/explant (rag/l) (rag/l) ICCV-1 ICCV-6 Desi

1X

1

3

nil

nil

nil

1X

0.5

3

nil

nil

nil

4X

1

3

nil

nil

2.95 + 0.88

4X

1

5

nil

nil

3.18 +0.78

4X

0.5

3

1.72 + 0.45 1.38 +_0.48

5.09+0.79

4X

0.5

5

1.54 + 0.5

1.63+ 0.44

4.99+0.5

4X

0.1

3

6.09 + 0.75 5.99 + 0.79.

11+ 0.96

4X

0.05

3

10.85+0.86

4X

0.04

3

11.2+__0.8 12.7_+1.35 22.51+ 1.51

12.48+1.4 20.21+2.12

a MS macro salts and B5 vitamins were used for all experiments.

34 induction, generation of multiple shoots were not observed. Few explants produced fleshy, abnormal shoots. The use of 4X MS micro salts along with 3.0 mg/l BAP and 0.04 to 0.05 mg/l NAA prompted Table 2: Effect of auxins on root induction in Desi genotypea. MS Basal medium

IAA

Auxins (rag/l) NAA IBA

Frequency of Root Induction (%)

MS

0.0

1/2 MS

0.0

MS

1

0.0

MS

2

0.0

I/2 MS

1

0.0

MS

1

0.0

MS

2

0.0

MS

1

1

49.2+ 0.45

MS

l

53.4+_.1.3

MS

0.5

92.85 + 0.65

Fig. 3: S o u t h e r n blot analysis o f t r a n s g e n i c plants for the p r e s e n c e o f nptll gene. L a n e 1, 2, 4, 5, 6, 7 - g e n o m i c D N A f r o m t r a n s g e n i c p l a n t s ; lane 3 - g e n o m i c D N A f r o m untransformed plant. Table 3: Frequency of transformationa

a In each case, 60 shoots were ufilised for each treatment line.

multiple shoot induction (Table-l). After five weeks of culture covering two subcultures, nearly 20-25 shoots per explant could be obtained in Desi genotype (Fig. 1B). The exact site of incision in the embryo axis influenced the efficiency for shoot bud production. When the root axis 100--

i i!i lPi i !i!i J ~ii@iii~!i

~i

60~ iiiiiiiiiiiiiiiill

i:i:iiiii:i:i

?!;!!ili!i)!iil

384

6

1.56

ICCV-6

408

8

1.96

Desi

572

i0

1.74

ICCV- 1

356

5

1.4

lICfV-6

384

6

1.56

602

7

1.16

.................

ICCV- l

405

7

1.72

ICCV-6

415

8

1.9

Desi

618

10

1.61

Experiment 3:

~}ii~i}!i~: i!iiiiiiiiiiiiiii

20~

0

ICCV- 1

Desi :~:~:~:~...... :~:~

40~

Experiment 1:

Experiment 2:

80--

~

Genotype No. of regenerated No. of Kan Transformation tested shootsb resistant shoots frequency (%)

•........

i!ii!ii!iii~!i ~i~i:}~i!i!i!i!i!i~

ICCV-I

ICCV-6

l iiii@!ii}i Desi

Genotypes F i g . 2 : R e s p o n s e o f explants o f the cultivars tested to shoot regeneration in SR m e d i u m .

a

For each experiment 150 explants/were cocultivated with

Agrobacterium in three replications of 50 explants/petridish. b After 3 weeks of culture

was left undisturbed, a maximum of two shoots per explant were formed. Injury inflicted to the cotyledon attachment region resulted in callus intitiation. Taking into consideration the above parameters, an optimal condition could be created where only the explants devoid

35

Fig,l: Transgenic plant regeneration, a) the explant used for infection (arrow); b) multiple shoot formation on shoot induction medium; c) Shoots proliferating on medium containing 50 mg/1 kanamycin; d) a separated shoot containing a green presumptive transformed and a white (arrow) untransformed branch; e) tile detatched green shoot producing only green branches on selection medium; f) a chimeric shoot histochemically assayed for GUS, the green branch stained and the white remained white; g) a selected, kanamycin-resistant shoot in the rooting medium; h) plantlets in root hardening medium before transfer to soil. Bars represent 2 mm in (a); 5 mm in (b), (d), & (e); 1 cm in (c), (g) & (h) and 250 ~m in (f).

Fig. 4: Enzymatic assay of the transgenic plants for the expression of transgenes: a) fluorometdc assay of the extracts from the transgenic plants to detect expression of uidA (arrow indicates the positive band) - lane 1, +ve control (standard E. colifl-glucuronidase ); lane 2, untransformed plant; lane 3-8, transformed plants; b) nptII enzyme assay of the kanamycin resistant transgenic plants - lane 1, untransformed plant; lane 2-9, individual transgenic plants. Arrow indicates the expression of the transferred nptIl gene resulting in synthesis of neomycin phosphotransferase enzyme.

36 of root axis and shoot apex, but with intact cotyledon attachment region (Fig. 1A) produced multiple shoots. Most of such explants of Desi genotype produced 20-25 shoots per explant (Table- 1). Roots were formed after subculturing for two weeks on MS medium supplemented with 0.5 mg/1 IBA (Fig. 1G). No root induction was observed in absence of auxins or when IBA was replaced with IAA and NAA (Table-2). Culturing on the same medium did not result in further development of the roots. The roots proliferated only when transferred to liquid half-strength MS medium. Growing on top of a Whatman No. 1 filter paper accelerated the growth of the roots (Fig. 1H).

Transfornmtion and transgenic plant regeneralion Transformation was carried out in case of three genotypes under study (Table-3). Three weeks after culturing in 25 mg/1 kanamycin, the green shoots along with yellowishwhite shoots were lransfelled to fresh medium with 50 mg/l kanamycin. After two weeks of culture in this medium most of the shoots became chlorotic, suggesting the elimination of untransformed tissues (Fig. 1C). Green shoots and chimeric shoots with partial white branches (Fig. 1D) were separated and cultured in fresh medium containing 50 mg/1 kanamycin. At this stage some of the chimeric branches were tested histochemically for GUS expression. Green shoots and branches stained blue, whereas the partially white/yellowish-white branches did not (Fig. 1F). Elimination of all chimeric shoots having untransformed branches became possible by repeated separation and subculturing of green branches and shoots for 4-5 subcultures. Shoots with exclusively green branches were obtained, which on medium containing 100 mg/1 kanamycin developed, producing exclusively green shoots (Fig. 1E). These shoots were cultured in root induction medium (fig. lg), and hardening of the induced roots was carried out before transfoTing to soil (Fig. 1H). The presumptive transgenics were tested for the presence and expression of the transgenes. Southern blot analysis of the presumptive transgenics showed the integration of april gene in multiple sites in the plant genome in most cases (Fig.3). The expression of uidA gene was carried out first by histocllemically staining the leaf and root of the transgenic plant. Unlike that of Fontana et aL (1993), no localized expression of GUS was observed, and blue staining was uniform. Fluorometric illuminescence assay of transgenic lines revealed GUS activity in the plant tissue (Fig. 4A). The nptlI assay was also carried out indicating expression of the tlansgene in a number of transgenic lines (Fig. 4B). The glass house grown transgenic plants flowered and produced seeds. Further analysis of these seeds for the presence of the transgenes are underway. DISCUSSION The rate of multiple shoot production and plant regeneration using three different genotypes has broadened the practical scope for transgenic plant

production of chickpea from desirable genotypes. It also suggests that adopting this methodology and choosing the appropriate combinations of plant growth regulators and basal medium, it is possible to achieve in vitro plant regeneration from a number of genotypes of chickpea. The use of 4X micro salts in the medium was found to be essential in the present case as also reported by Barwale et aI. (1986). The rate of plant regeneration in the present case has been -95% and obtaining plants with well developed root system have also been possible in an efficient and repeatable manner. We took advantage of this plant regeneration system for transformation studies. One of the major disadvantages of obtaining plants through organogenesis is the involvement of more than one cell in the shoot initiation process (Vasil, 1985). When organogenesis is used for transformation and subsequent transgenic plant regeneration, the risk of recovering chimeric plants exists. We observed that application of selection pressure before shoot formation largely inhibits shoot formation by eliminating untransformed cells. Repeated selections of rapidly multiplying shoots eliminated untransformed tissues. Unlike that of Fontana et al. (1993), we tried to maximumize the number of shoots in the primary explant. Lowe et aL, (1995) adopted a similar strategy to enrich transgenic sectors and eliminate chimeras. Schroeder et al. (1993) reported in case of Pisum that presence of growth regulators in the cocultivation medium enhanced transformation frequency. We also observed that the presence of growth regulators used for shoot induction in the cocultivation medium is essential in the present case. Absence of growth regulators in the cocultivation medium, greatly reduced transformation frequency and recovery of transgenic plants was sporadic. We found that nptII is an efficient selectable marker for chickpea. We have recovered no escapes using kanamycin as selection agent. This finding does not agree with Puonti-Kaerlas et al. (1990), who reported that the nptII gene is not a suitable selectable marker gene for legumes. All the three chickpea genotypes showed similar levels of transformation frequency. In conclusion, we report on the development of a suitable method of in vitro plant regeneration and transformation of chickpea. A simple procedure for routine transgenic plant production for more than one genotypes of chickpea could be developed. We hope that this method can be effectively adopted for other genotypes of cl~ickpea. The present report also highlights that the nature of multiple shoot regeneration from a number of genotypes, the rate of recovery of transformed shoots and the reproducibility of the present method can quite likely be adopted to other recalcitrant crops like tea and jute.

Acknowledgements: Financial assistances received from the Department of Biotechnology, Govt. of India, UNDP/FAO (IND/87/017) and the Rockefeller Foundation (RF 89026#39) by the laboratory are acknowledged gratefully.

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