In planta transformation of pigeon pea: a method to overcome recalcitrancy of the crop to regeneration in vitro

Abstract

Development of transgenics in pigeon pea remains dogged by poor plant regeneration in vitro from transformed tissues and low frequency transformation protocols. This article presents a non-tissue culture-based method of generating transgenic pigeon pea (Cajanus cajan (L.) Millisp.) plants using Agrobacterium-Ti plasmid-mediated transformation system. The protocol involves raising of whole plant transformants (T0 plants) directly from Agrobacterium-infected young seedlings. The plumular and intercotyledonary meristems of the seedling axes are targeted for transformation. The transformation conditions optimized were, pricking of the apical and intercotyledonary region of the seedling axes of two-day old germinating seedlings with a sewing needle, infection with Agrobacterium (LBA4404/pKIWI105 carrying uid A and npt II genes) in Winans' AB medium that was added with wounded tobacco leaf extract, co-cultivation in the same medium for 1h and transfer of seedlings to soilrite for further growth and hardening and subsequent transfer of seedlings to soil in pots in the greenhouse. Out of the 22-25 primary transformants that survived infection-hardening treatments from each of the three experiments, 15 plants on the average established on the soil under greenhouse conditions, showed slow growth initially, nevertheless grew as normal plants, and flowered and set seed eventually. Of the several seeds harvested from all the T0 plants, six hundred were sown to obtain progeny (T1) plants and 350 of these were randomly analysed to determine their transgenic nature. PCR was performed for both gus (uid A) and npt II genes. Forty eight of the 350 T1 plants amplified both transgenes. Southern blot analysis substantiated the integration and transmission of these genes. The protocol ensured generation of pigeon pea transgenic plants with considerable ease in a short time and is applicable across different genotypes/cultivars of the crop and offers immense potential as a supplemental or an alternative protocol for generating transgenic plants of difficult-to-regenerate pigeon pea. Further, the protocol offers the option of doing away with a selection step in the procedure and so facilitates transformation, which is free of marker genes.

Expression of Cry2Aa, a Bacillus thuringiensis insecticidal protein in transgenic pigeon pea confers resistance to gram pod borer, Helicoverpa armigera

“…Primary transformants of pigeon pea cv. Pusa 992 were developed using the apical meristem-targeted Agrobacterium -mediated in planta transformation strategy 19 . About 50 seedlings were subjected to in planta transformation of which 28 individual T 0 plants (primary transformants) established in the green house that grew normally, flowered and set seeds.…”

“…The recalcitrance of pigeon pea to tissue culture and reduced ability in the development of transformants has resulted in researchers adopting non-conventional approaches for the development of transformants in this crop 15 , 16 , 18 , 19 . In the present study, a tissue culture independent apical meristem-targeted in planta transformation that was standardized earlier by our group was used to develop transformants in pigeon pea 19 . The strategy mainly deals with the in planta inoculation of the shoot apical meristem and allowing them to grow ex vitro .…”

“…armigera in pigeon pea. In addition, utilizing a non-tissue culture-based in planta approach 19 for the development of stable and effective events can be an added advantage due to the recalcitrance of pigeon pea to tissue culture. Earlier, cry2Aa has been tested and proven effective against H .…”

Conventional and Molecular Approaches towards Genetic Improvement in Pigeonpea for Insects Resistance

“…Recently, the protocol was also proved to be feasible in pigeonpea [91]. At ICRI-SAT, seedling-derived leaf explants have also been employed successfully to obtain high frequency of genetic transformation in pigeonpea through biolistics [82].…”

In planta Transformed Cumin (Cuminum cyminum L.) Plants, Overexpressing the SbNHX1 Gene Showed Enhanced Salt Endurance

“…Although the in vitro method of plant regeneration-transformation is applicable to variety plants species [ 36 – 38 ] but the method is laborious and not promising because of the requirement of sterile conditions [ 39 ] and also somaclonal variations or morphological abnormalities as a result of the long phase of regeneration [ 40 – 42 ]. Also, most of the plant species are highly recalcitrant to in vitro regeneration [ 39 , 43 ]. To overcome the constraints mentioned above, the in vivo method of Agrobacterium -mediated transformation, known as in planta are gaining more importance.…”