Gene technology for grain legumes: can it contribute to the food challenge in developing countries?

Transfer of genes from heterologous species provides the means of selectively introducing new traits into crop plants and expanding the gene pool beyond what has been available to traditional breeding systems. With the recent advances in genetic engineering of plants, it is now feasible to introduce into crop plants, genes that have previously been inaccessible to the conventional plant breeder, or which did not exist in the crop of interest. This holds a tremendous potential for the genetic enhancement of important food crops. However, the availability of efficient transformation methods to introduce foreign DNA can be a substantial barrier to the application of recombinant DNA methods in some crop plants. Despite significant advances over the past decades, development of efficient transformation methods can take many years of painstaking research. The major components for the development of transgenic plants include the development of reliable tissue culture regeneration systems, preparation of gene constructs and efficient transformation techniques for the introduction of genes into the crop plants, recovery and multiplication of transgenic plants, molecular and genetic characterization of transgenic plants for stable and efficient gene expression, transfer of genes to elite cultivars by conventional breeding methods if required, and the evaluation of transgenic plants for their effectiveness in alleviating the biotic and abiotic stresses without being an environmental biohazard. Amongst these, protocols for the introduction of genes, including the efficient regeneration of shoots in tissue cultures, and transformation methods can be major bottlenecks to the application of genetic transformation technology. Some of the key constraints in transformation procedures and possible solutions for safe development and deployment of transgenic plants for crop improvement are discussed.

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Genetic transformation technology: Status and problems

Abiotic stress is the major limiting factor of plant growth and crop yield which can be improved by osmoprotectants. Proline acts as an osmoprotectant and plays an important role in osmotic balancing, protection of sub-cellular structures, enzymes and in increasing cellular osmolarity that provide the turgor necessary for cell expansion under stress conditions. ∆1-pyrroline-5-carboxylate synthetase (P5CS), a rate-limiting enzyme in proline biosynthesis which is known for conferring enhanced salt and drought stress is subjected to feedback inhibition by proline. Therefore, in the present study, we used a mutagenized version P5CSF129A of wild P5CS which is not subjected to feedback control. Efficient in vitro transformation of embryonic structures of pigeonpea (Cajanus cajan (L.) Millsp.) was obtained using Agrobacterium tumefaciens strain LBA4404 harbouring a modified binary vector pCAMBIA 1301 carrying the hptII gene for resistance to hygromycin sulphate, GUS reporter gene, encoding β-glucuronidase, and the Vigna aconitifolia P5CSF129A genes under a constitutive 35S promoter. Embryonic structures showed blue color when tested for GUS after first cycle of antibiotic selection. Integration of T-DNA into nuclear genome of transformed plants and its sexual transmission to the progeny of the transgenic plants are confirmed by PCR amplification of 340 bp hptII, 800 bp P5CSF129A fragments and Southern blot hybridization analysis. The resultant primary transgenic plants showed more proline accumulation than their non-transformed plants. Levels of proline were also elevated in T1 transgenic plants when grown in the presence of 200 mM NaCl. In addition to their enhanced growth performance, more chlorophyll and relative water content under high salinity, these plants also had lower levels of lipid peroxidation. This suggests that overproduction of proline might play an important role against salt shock and cellular integrity.

Expression of the Vigna aconitifolia P5CSF129A gene in transgenic pigeonpea enhances proline accumulation and salt tolerance

Summary Conventional breeding methods have not been very successful in producing pest-resistant genotypes of pigeonpea, due to the limited genetic variation in cultivated germplasm. We have developed an efficient method to produce transgenic plants of pigeonpea by incorporating the cry1Ab gene of Bacillus thuringiensis through Agrobacterium tumefaciens-mediated genetic transformation. The novel tissue culture protocol is based on the direct regeneration of adventitious shoot buds in the axillary bud region ofinvitro germinating seedlings by suppressing the axillary and primary shoot buds on a medium containing a high concentration ofN 6 -benzyladenine (22.0mM). The tissue with potential to produce adventitious shoot buds can be explanted and used for co-cultivation with A. tumefaciens carrying the synthetic cry1Ab on a binary vector and driven by a CaMV 35S promoter. Following this protocol, over 75 independently transformed transgenic events of pigeonpea were produced and advanced to T2 generation. Amongst the recovered primary putative transformation events, 60% showed positive gene integration based on initial polymerase chain reaction (PCR) screening. PCR analysis of the progenies from independent transformants followed gene inheritance in a Mendelian ratio and 65% of the transformants showed the presence of singlecopy inserts of the introduced genes. Reverse transcription– polymerase chain reaction analysis showed that the transcripts of the introduced genes were normally transcribed and resulted in the expression of Cry1Ab protein in the tested T2 generation plants. Interestingly, the content of Cry1Ab protein as a percent of total soluble protein varied in different tissues of the whole plant, showing the highest expression in flowers (0.1%) and least in the leaves (0.025%) as estimated by enzyme-linked immunosorbent assay. The transgenic plants produced in this study offer immense potential for the improvement of this important legume of the semi-arid tropics for resistance to insect pests.

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Agrobacterium-mediated production of transgenic pigeonpea (cajanus cajan l. millsp.) expressing the synthetic bt cry1ab gene

Age and orientation of the cotyledonary leaf explants determine the efficiency of de novo plant regeneration and Agrobacterium tumefaciens-mediated transformation in Jatropha curcas L.

A protocol for efficient plant regeneration from leaf explants of pigeonpea [Cajanus cajan (L.) Millsp.] was developed for the production of transgenic plants. Leaf explants from 4- to 5-day-old in vitro raised seedlings were most efficient in producing multiple adventitious shoots in 90% of the explants on shoot induction medium [Murashige and Skoog (MS) medium +5.0 μM benzyladenine +5.0 μM kinetin]. Shoot buds originated from the petiolar cut end of the explants and elongated rapidly on medium containing 0.58 μM gibberellic acid. Over 80% of the elongated shoots rooted well on MS medium containing 11.42 μM indole-3-acetic acid and were transplanted with 100% success. The procedure reported here is very simple, efficient and reproducible, and is applicable across diverse genotypes of pigeonpea. The usefulness of this system for further studies on the genetic transformation of pigeonpea has been demonstrated in biolistics-mediated gene transfer by using nptII and uidA as marker genes, where 50% of the selected plants showed gene integration and expression.

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S. Dayal

Papers

An efficient protocol for shoot regeneration and genetic transformation of pigeonpea [ Cajanus cajan (L) Millsp] using leaf explants.