Legume Res., 29 (3) : 163-168, 2006 ESTABLISHMENT OF AN IN VITRO REGENERATION SYSTEM SUITABLE FOR AGROBACTERIUM MEDIATED TRANSFORMATION OF KABULI TYPE CHICKPEA (CICER ARIETINUM L.) P. Das and B.K. Sarmah* Department of Agricultural Biotechnology, Assam Agricultural University. Jorhat - 785 013, India ABSTRACT An in vitro regeneration system using cotyledonary explants with half embryonic axes was developed for kabuli type chickpea. Murashige and Skoog s (MS) medium was supplemented with different concentrations of two cytokinins (BAP and Kinetin) and an auxin (NAA) in order to determine the optimum concentration of hormones required for regeneration of large number of multiple shoots. Healthy multiple shoots were also used for direct rooting in tissue culture and also grafted in vitro. Shoots were also regenerated following the same protocol after the explants were infected with an Agrobacterium strain AGL1. Thus, this regeneration protocol will be useful for Kabuli type chickpea transformation using Agrobacterium tumifaciens as vector. INTRODUCTION Among the legume crops, chickpea (Cicer arietinum L.) provides high quality and quantity of proteins. Two major types of chickpea cultivars, namely, kabuli and desi are cultivated worldwide on 10.2 m ha of land with a production of 7.9 m tons (FAO, 1994). In spite of being such an important crop, the yield of chickpea is not stable due to its susceptibility to a number of biotic and abiotic stresses (Singh, 1992). Conventional breeding methods for the future improvement of chickpea, particularly, for the development of insect and disease resistant varieties are found to be limiting due to narrow genetic base and sexual incompatibility that exists with wild relatives. Biotechnological approach such as gene technology can give new dimension towards achieving this goal. Reports on gene technology for the development of transgenic chickpeas are confined to desi type only (Fontana et al., 1993; Kar et al., 1996; Krisnamurthy et al., 2000; Das et al., 2002). So far there is no such report of in vitro regeneration and transformation of kabuli type chickpea. Before initiating transformation work, it is worth studying the regeneration * Author for correspondence: email: bksarmah @ aau.ac.in ability of a plant species using explants without and with Agrobacterium infection. Thus the objective of the present study was to develop an efficient in vitro regeneration system in kabuli type chickpea, before and after infecting the explants with Agrobacterium tumifaciens, so that the system could be used for future genetic improvement purpose. MATERIAL AND METHODS Plant regeneration: In the present investigation, market collected kabuli type chickpea seeds were surface sterilized with 70 per cent ethanol for 1.5-2.0 min, followed by 1 per cent sodium hypochloride (Nice chemicals, Cochin, 5 per cent available chloride v/v) for 20 minutes. The seeds were than rinsed with sterile distilled water for 3-4 times and soaked over night in sterilized water supplemented with Plant Preservative Mixture (PPM, Astratec. Inc., Australia.) @ 1mg/l. Explants (Fig. 1a) containing half embryonic axes were prepared using a sharp sterilized blade (Surgeon No. 10) by splitting the seeds longitudinally along the embryonic axes. Seed coats were removed and explants were inoculated in the sterile petridishes (20 x 100 cm) containing first regeneration medium (RI).
164 LEGUME RESEARCH Fig. 1a. Cotyledonary explants with half embryonic axis in co-cultivation medium (B5) Murashige and Skoog s medium (MS) supplemented five different combinations of phytohormones (RIa - RIe) (Table 1) were tested with different concentration of two cytokinins (BAP and Kinetin) and one Auxin (NAA) in order to determine the optimum concentration of hormones required for regeneration of large number of multiple shoots. Fifty explants were inoculated for each treatment (R1 medium). After 14 days, the explants producing shoots in the first regeneration media were subcultured to second regeneration media (RIIa-e) (Table1) containing only cytokinins for induction of multiple shoots. Cotyledons and roots if any were removed from the explants before subculturing into RII media. Observations were recorded after 14 days for number of multiple shoots per explant for each of the five media combinations. Explants containing multiple shoots were then transferred to third regeneration medium (RIII) (MS + 0.1mg/l BAP + 0.1mg/l Kinetin) for production of healthy shoots. After 14 days in RIII, number of explants producing healthy shoots that were used for direct rooting or grafting was recorded in order to determine percent shoot regeneration. All explants were cultured at 25±2 0 C temperature with a light regime of 3000-4000 lux for 14-16 hours a day. Plant regeneration using explants infected with Agrobacterium: Agrobacterium strain AGL1 was cultured in MGL medium (Grafinkel, 1980) supplemented with 20mg/l rifampacin for selection of Agrobacterium strain. Seeds were surface sterilized and kept in Plant Preservative Mixture (PPM) solution (1mg/l) for overnight. Cotyledonary explants, with half embryonic axes were prepared, after rinsing the seeds 3-4 times with sterile distilled water. The explants were left in MSO (MS basal) for 30 minutes to 1 hr. Pipette off the MS0 and the explants were infected with the overnight grown Agrobacterium (0.6-1.0 OD) for 45 minutes. Embryogenic axes were dipped in the Agrobacterium culture. Explants were then transferred to the co-cultivation medium (B5 + 1 mg/l BAP + 1 mg/l NAA + 100 um Acetosyringone) for 3 days. Explants were then washed with sterile water for 3-4 times and blotted with sterile filter paper for about 30 minutes before they were transferred into regeneration medium RI (MS + BAP 0.5 mg/ l + Kin 0.5 mg/l + NAA 0.05 mg/l + timentin 150 mg/l). Antibiotic timentin (125mg/L) was added to check the growth of Agrobacterium during regeneration. In all 50 explants were cultured in RI medium. Five explants were cultured per petridish. After 14 days explants were subcultured to RII (MS + BAP 0.5 mg/l + Kin 0.5 mg/l + timentin 150 mg/l) as described above. Mean numbers of multiple shoots produced per explant was recorded. Direct rooting in tissue culture: Shoots were cultured in B 5 medium (Gamborg, 1968) supplemented with 2% sucrose after dipping in IBA solution (1 mg/l) for 15-20 seconds. Observation was recorded for induction of roots every 2-3 days for a period of about one month. In vitro grafting: Seeds of desi type cultivar (Vijay) were used for the preparation of rootstocks (In our lab we found Vijay as the best rootstock out of number of cultivars tested including kabuli type). Seeds were germinated on waters agar medium (0.8%) and 4-5 day old seedlings were used for preparing
Vol. 29, No. 3, 2006 165 Table 1. Composition of modified MS media for multiple shoot induction in kabuli type chickpea from cotyledonary explants Regeneration stage Medium Growth regulators (mg/l) BAP Kinetin NAA RIa 0.25 0.5 0.05 RIb 0.5 0.25 0.05 RI RIc 0.5 0.5 0.05 RId 1.0 0.5 0.05 RIe 0.5 1.0 0.05 RIIa 0.25 0.5 RII RIIb 0.5 0.25 RIIc 0.5 0.5 RIId 1.0 0.5 RIIe 0.5 1.0 rootstocks. After inserting the scion (healthy regenerated shoot) into the stock. A silicon ring (0.5 mm diameter) was used to hold the scion and stock tightly. Grafting was performed under the laminar hood. After every two days, any axillary shoots emerging from the rootstock were cut off. Grafted plants were kept in culture room at 25± 2 0 C temperature and light regime of 3000-4000 lux for 6-7 days, and eventually transferred to earthen pots (18cm. in diameter) containing soil and compost in 4:1 ratio in the plastic house. For hardening the plants were covered with a plastic pot and kept in the shade for 4-5 days with minimal exposure to light every morning for removal of moisture from the cover and providing fresh air. After 5 days the cover was removed gradually in the subsequent 7-8 days. Statistical analysis: To evaluate the best medium for regeneration of kabuli-type chickpea complete-randomized design (CRD) was followed. Fisher s-t test was performed to examine the significance of difference, wherever required, between the average values observed across different groups. RESULTS AND DISCUSSION Induction of multiple shoots: The shoots were regenerated (Fig. 1b) and multiple shoot were induced (Fig. 1c) in different combinations of MS medium using cotyledonary explants. The frequency of shoot Fig. 1b. Initiation of shoot regeneration from an explant in the first regeneration medium after 14 days of culture Fig. 1c. Multiple shoots developed from a single explant in the second regeneration medium after 14 days of culture initiation and multiple shoot inductions are presented in Table 2. Highest (89.9%) shoot
166 LEGUME RESEARCH Table 2. Induction of multiple shoot from cotyledonary explant in modified MS media after one month in culture Medium* Explants Explants % Explants Multiple in RI cultured with shoots shoot in First Second to RII in RI RII (Mean) regeneration regeneration (RI) (RII) RIa RIIa 50 43 86.9 6.06 RIb RIIb 50 38 75.0 7.61 RIc RIIc 50 42 84.2 8.13 RId RIId 50 41 82.4 9.64 RIe RIIe 50 39 78.9 6.63 Total no of explants inoculated = 50; * Composition of media given in Table 1. Table 3. Frequency of rootable/graftable shoots in third regeneration medium (RIII) using the explants producing multiple shoots Sequence of media Explants with Explants with % explants multiple shoots shoots for with shoots RI RII in RIII grafting/ for rooting/ rooting grafting RIa RIIa 43 (50) 13 30.2 RIb RIIb 38 (50) 16 42.8 RIc RIIc 42 (50) 28 66.6 RId RIId 41 (50) 32 78.0 RIe RIIe 39 (50) 18 46.1 Figure in parenthesis indicates the initial number of explants inoculated in RI. initiation was observed in the medium RIa followed by 84.2% in RIc. The frequency of shoot initiation was the lowest (75.0%) in RIb. Observations for mean numbers of multiple shoots in different media combinations were recorded after 14 days of culture in RII media. Mean number of shoots was observed to be highest (9.64) in regeneration medium RIId, followed by a mean of 8.13 shoots per explant in RIIc. The mean numbers of multiple shoot was lowest (6.06) in RIIa. Statistical analysis revealed that mean value of multiple shoot induction in RIId was not significant to that obtained in RIIc but showed significant difference with the lowest mean (6.06) observed in RIIa. The explants with multiple shoots in all five media combination were further subcultured into a third regeneration medium RIII (MS + 0.1mg/l BAP + 0.1mg/l Kinetin for production of healthy elongated shoots. It was observed that after 14 days of subculture in RIII medium, percentage of explant with graftable or rootable shoots was found to be highest (78.0%) (Table 3), when explants were inoculated first in RId for initiation of shoot regeneration, followed by subculturing in RIId for multiple shoot induction and eventually into RIII for generation of healthy graftable/rootable shoots. The results of shoot induction using cotyledonary explant with half embryonic axes in Kabuli type chickpea on media supplemented with two cytokinins (BAP and Kinetin) concur with those obtained by Kartha et al. (1981), Bradt and Hess (1993) and Kar et al., (1996). Cytokinins are involved in the regulation of shoot formation such as in axillary bud development by antagonizing apical dominance and
adventitive organogenesis (Skoog and Miller, 1957 Wang et al., 1991, Stefaans et al., 1990). However, in the second subculture we removed the roots if any, from the explants and also removed the cotyledons and cultured in the auxin free media to enhance the multiple shoot induction frequency. The multiple shoots obtained in RII were subcultured to third regeneration medium (RIII) wherein the cytokinin concentration was further reduced to 0.1 mg/l. Sarmah and Higgins (1999) observed that reduced cytokinin concentration in second and subsequent subculture induced better shoot growth as they slowed down the cells to undergo further regeneration. Induction of multiple shoots using cotyledonary explants infected with Agrobacterium: As it was found statistically that the explants produced highest frequency of multiple shoots in the medium RIIc, we inoculated explants in this medium after the explants were infected by the Agrobacterium strain AGL1 in order to observe if explants produced almost equal frequency of multiple shoots. It was observed that after 14 days in RIIc, the mean number of shoots produced by the explants was 9.78. The t- value for the two mean i.e., mean numbers of multiple shoots in RIIc, with and without (Table 2) Agrobacterium infection was found to be 1.97 (critical value = 2.45). It was observed that the mean number of shoots in second regeneration medium (RIIc) did not show any significant difference with the mean number of shoots obtained upon infection with Agrobacterium strain AGL1. Thus, this protocol for the induction of multiple shoots and eventual regeneration of plants in kabuli type chickpeas will not be altered with the Agrobacterium infection. In vitro rooting: For in vitro rooting in culture, 20 shoots were grown in B 5 medium after dipping in IBA (1mg/l) for 20 sec. It was observed that even after one month in culture, no roots were produced. Shoots became Vol. 29, No. 3, 2006 167 vitrified and tips were necrotic without any roots. Our results suggest that direct rooting in Kabuli type chickpeas compares unfavourably with those of desi type chickpea. It was observed that when the healthy shoots were hardened to rooting medium, no roots were induced even after one month in culture. Besides, the shoot tips became necrotic after 25 days of culture. However Fontana et al., (1993) obtained roots from regenerated shoots in MS medium supplemented with 0.5 mg/l IAA and 0.05 mg/l kinetin and 1% w/v sucrose in desi type chickpea. Kar et al. (1996) obtained about 95% rooting in desi type chickpea using MS medium supplemented with 0.05 mg/l IBA. Sarmah and Higgins (1999) reported poor rooting efficiency (23%) in Australian desi type chickpea and recommended grafting as an alternative in chickpea to establish the putative transformants. In vitro grafting: Following the protocol described in the Material and Method, total 14 shoots were grafted in vitro (Fig. 1d) and eventually transferred into the polyhouse for their establishment as plants. Though all of them established only 9 plants grew up to maturity and produced seeds (Fig. 1e). Thus percent regenerated plants grew up to the maturity was 64.2. The seeds were harvested and stored for their future use as negative controls in transformation research. In vitro grafting was performed on desi type chickpea rootstocks.the regenerated shoots were successfully established (65%) through grafting. However the percent establishment can be further increased considerably with time as it depends upon the skill of the worker. Sarmah and Higgins (1999) obtained 100 per cent success in plant establishment through in vitro grafting. Moreover, Krishnamurthy et al. (2000) successfully attempted in vitro grafting as a mean of rooting in desi type chickpea. From the above study, it can be inferred that in kabuli type chickpea, multiple
168 LEGUME RESEARCH Fig. 1d. A kabuli type regenerated shoot grafted on desi type (Vijay) rootstock Fig. 1e. A complete regenerated kabuli type chickpea plant attaining maturuty shoots can be produced in higher number in the media combination in which first regeneration medium RIc is followed by two subsequent subculturing in second regeneration medium RIIc and third regeneration medium RIII at 14 days interval. But since both frequency of graftable/rootable shoots as well as frequency of shoot establishment were highest in RIId, so in transformation experiment where not only the quantity, but quality of the shoot is also important for successful establishment of the putative transformants, we recommend the media sequence where RId is followed by two successive subcultures in RIId and RIII at 14 days interval. Once multiple shoots are induced after infection with Agrobacterium, it would not be difficult to establish the healthy shoots through grafting. Thus this regeneration protocol can be used for efficient regeneration of kabuli type chickpea in genetic transformation experiments using Agrobacterium as vector. ACKNOWLEDGEMENT The author is grateful to the McKnight foundation, USA for providing fund and T.J. Higgins of Plant Industry, CSIRO, Australia for the Agrobacterium strain and Mr. Sanjay Borthakur for assisting in statistical analysis. REFERENCES Brandt, E.B. et al. (1994). In Vitro Cell Dev. Biol., 30: 75-80. Das, A. et al. (2002). Fourth National Symposium on Biochemical Engineering and Biotechnology, New Delhi, pp. 174-176. FAO (1994). In: Food and Agricultural Organization of the United Nations : Production Yearbook. FAO, Kave, Italy. Fontana G.S. et al. (1993). Plant Cell Rep., 12: 194-198. Gamborg, O.l. (1968). Exp. Cell Res., 50: 151-158. Grafinkel, J. (1980). J. Bacteriol., 144: 732-743. Kartha, K.K. et al. (1981). Canadian J. Bot., 1671-1679. Kar, S. et al. (1996). Plant Cell Report, 16: 32-37. Krishanamurthy, K.V. et al. (2000). Plant Cell Report., 19: 235-240. Skoog and Miller, C.O. (1957). Symp. Soc. Exptl. Biol., 11: 118-131. Sarmah, B.K. and Higgins, T.J. (1999). In: Annual Report, The Mc. Knight Foundation, Minneapolis, Minneapolis, USA, 15-22. Singh, K.B. (1992). Field Crop Res., 53: 161-170. Stefaans, P.O. et al. (1990). In: Plant Cell Culture : A Practical Approach. (Dixon, R.A. and Gonzales, R.A. ed.), pp. 127-134. Wang, P.J. and Charles, A. (1991). In: Biotechnology in Agriculture and Forestry Vol. 17, (Bajaj, Y.P.S. ed.), Springer-Verlag, N.Y., pp. 41-49.
Fig. 1a. Cotyledonary explants with half embryonic axis in co-cultivation medium (B5) Fig. 1b. Initiation of shoot regeneration from an explant in the first regeneration medium after 14 days of culture Fig. 1c. Multiple shoots developed from a single explant in the second regeneration medium after 14 days of culture Fig. 1d. A kabuli type regenerated shoot grafted on desi type (Vijay) rootstock Fig. 1e. A complete regenerated kabuli type chickpea plant attaining maturuty