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Standardization of Agrobacterium-mediated Genetic Transformation of Pigeonpea [Cajanus cajan (L.) Millsp.] Using Tissue Culture and Non Tissue Culture Based Methods

KrishiKosh

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Title Standardization of Agrobacterium-mediated Genetic Transformation of Pigeonpea [Cajanus cajan (L.) Millsp.] Using Tissue Culture and Non Tissue Culture Based Methods
 
Creator AJITHA, V
 
Contributor YAMINI, K. N
 
Subject sowing, inorganic compounds, physical control, storage, grain, maize, storage structures, germinability, pesticides, insecticides
 
Description Pigeonpea (Cajanus cajan (L.) Millsp.) is an important high protein grain legume of the semi-arid tropics and caters to the protein requirement of the population in Indian sub-continent. The productivity of pigeonpea is constrained by several biotic and abiotic stresses. Since, pigeonpea is a self pollinated crop with narrow genetic base, conventional breeding has no conspicuous effects on the genetic improvement. Genetic engineering offers an attractive alternative at this juncture for several traits which cannot be improved through conventional breeding. Among the various approaches for genetic transformation of plants, Agrobacterium mediated method is most popular and studies on Agrobacterium tumefaciens have provided the basis that has made this soil bacterium the dominant customer for plant transformation. Development of transgenics in pigeonpea remains dogged by poor plant regeneration in vitro with low frequency of transformation. Hence, there is a need for more focused and consistent efforts to develop protocols for obtaining stable in vitro regeneration and genetic transformation. In the present study, attempts were made to standardize regeneration as well as transformation protocols for the both tissue culture based and non-tissue culture i.e., in planta based methods for Agrobacterium mediated transformation of pigeonpea cultivar, Asha (ICPL 87119).
Regeneration from axillary meristem explants (AMEs) of pigeonpea cultivar Asha was attempted. Among the AMEs isolated from 7 day, 9 day and 11 day old seedlings, the 9th day old AMEs were most suitable for regeneration. These were inoculated on to four combinations of shoot induction media (SIM) of which MS with 0.5 mg l-1 BAP (SIM1) was found to be the best for maximum shoot bud induction after two sub-cultures in the same media. Elongation of multiple shoots was tested on MS media with three concentrations of gibberellic acid (GA3) and 0.3 mg l-1 (GA3) gave response to elongation. Only six shoots
elongated and these were used for rooting on half strength MS medium supplemented with 0.2 mg l-1 IBA, however, only one shoot rooted, but could not survive upon transfer to sterile vermiculite for acclimatization.
The factors for tissue culture based Agrobacterium mediated transformation were standardized. Ninth day old AMEs were found to be appropriate for Agrobacterium mediated transformation. Out of the various concentrations (0, 50, 100, 150 and 200 mg l-1) of kanamycin tested, SIM 1 supplemented with 150 mg l-1 kanamycin, was identified as most ideal concentration for selecting the transformants. Among the infection times tested, of few seconds, 15 min and 30 min, for co-cultivation with Agrobacterium, it was revealed that 30 min with the culture having optical density of 0.6 (OD600nm) (among 0.3, 0.5 and 0.6 tested) was optimum with maximum response for transformation (9.75%). A co-cultivation period of two days was found to be optimum as beyond this period, bacterial overgrowth was observed. Also addition of acetosyringone (100 μM), enhanced the response to transformation.
The parameters for in planta based Agrobacterium mediated transformation were standardized. The germinated seedlings from pigeonpea cultivar Asha were observed from 30 h to 70 h after sowing and the germinated seedlings at 44 h to 48 h old (2 days) stage were identified as most optimum for in planta transformation. Agrobacterium culture with OD600nm values of 1.2 and 1.5 were used for infection of pricked seedlings at just emerging plumule for in planta method of transformation. Infection time of Agrobacterium culture 1 h was kept constant for in planta transformation.
Attempts were made to develop transgenic plants through tissue culture based Agrobacterium mediated transformation, using EHA105::pBinAR-cry1Aabc with the standardized parameters. However, shoots were obtained at a low transformation frequency of 1.5%. The shoots developed, did not respond to rooting and hence transgenic plants could not be realized using this method in the present study, which emphasizes the need for more efforts with large number of explants in future with this method.
Through the in planta method of Agrobacterium mediated transformation, 60 T0 plants were obtained and raised in the net house. A preliminary PCR analysis of the T0 plants, indicated that four T0 plants were positive for nptII gene, however these results were not consistent in T1 generation, possibly due to the chimeric nature of T0 plants. In the successive generation, 185 (T1) plants were raised and the 120 of these which survived, were screened for presence of transgenes (Cry1Aabc and nptII) by PCR analysis. However, none of T1 plants were found to be positive for both the transgenes.
In conclusion, it can be said that the present study is a preliminary effort towards standardization of protocols for tissue culture based and in planta method of Agrobacterium mediated transformation for the cultivar Asha of pigeonpea. In future, more efforts need to be made to use a large number of explants for the tissue culture based method and also a large scale experiment (may be thousands of plants) needs to be attempted for in planta transformation. Such attempts will, in future lead to realization of positive transgenic pigeonpea plants carrying Cry1Aabc, which may confer resistance to insect pests such as pod borer.
 
Date 2017-01-03T10:48:31Z
2017-01-03T10:48:31Z
2016
 
Type Thesis
 
Identifier http://krishikosh.egranth.ac.in/handle/1/94087
 
Language en
 
Relation D10018;
 
Format application/pdf
 
Publisher PROFESSOR JAYASHANKAR TELANGANA STATE AGRICULTURAL UNIVERSITY RAJENDRANAGAR, HYDERABAD