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Professor Jayashankar Telangana State Agricultural University, Hyderabad (Telangana State)

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20172
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Thesis Type: Ph.D × Subject: Biotechnology and Molecular Biology ×
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    ThesisItemOpen Access
    STRATEGIES TO DEVELOP TRANSGENIC CASTOR (RICINUS COMMUNIS L.) TOLERANT TO NECROTROPHIC FUNGI
    (Professor Jayashankar Telangana State Agricultural University, 2017) LAKSHMIDEVI, G; DINESH KUMAR, V
    Castor is one of the important commercial non-edible oilseed crops of India. India is the leading producer of this crop in the world. Grey mold disease caused by the necrotrophic fungus Botryotinia ricini, which predominantly affects the inflorescence, results in yield losses of more than 80% under favourable conditions offsetting the achievements made through crop improvement approaches. Various approaches adopted for control of this disease have not been successful making it the major biotic stress factor in reducing the productivity of this crop. And also, limited genetic variability in the germplasm of castor with respect to the resistance/tolerance against grey mold necessitates the adoption of alternate strategies, such as biotechnological tools, for overcoming the huge losses inflicted by this fungus. In the most of the previous studies with single transgene approach in different crops, only partial resistance has been reported against necrotrophic fungi such as Botrytis and it has been opined that simultaneous expression of multiple transgenes might lead to better tolerance. Therefore, to develop transgenic castor tolerant to grey mold, three genes viz., AtEBP (Arabidopsis Ethylene Responsive Element Binding Protein), an antiapoptotic gene, BIK1 (Botrytis Induced Kinase1), a receptor kinase and ERF1 (Ethylene Response Factor1), a regulator of ethylene response were chosen for their simultaneous expression. Each of these three genes is reported to impart varying degrees of resistance in Arabidopsis against Botrytis cinerea through different modes of action. At IIOR, appropriate gene constructs had been developed using these three genes and three promoters AtACS4, AtACS5 and AtACS7 that are known to drive elevated expression in inflorescence parts. As an effort to validate these constructs, transgenic tobacco plants expressing these constructs had been realized and characterized. These transgenic plants had shown better tolerance against necrotrophic fungi like Alternaria and Botrytis. During the present investigation, to assess the cumulative effect of expressing the three genes (AtEBP1, BIK1 and ERF1) simultaneously in a plant in imparting resistance against fungi, the gene cassettes were pyramided by crossing the plants carrying single gene cassettes. At each step of stacking the genes, the resultant progeny plants were confirmed for the presence and expression of the transgenes using PCR and RT-PCR techniques. Thus transgenic tobacco plants carrying and expressing one, two or all the three gene cassettes were realized. These plants were subjected to disease bioassays using the necrotrophic fungus Alternaria alternata as well as hemi-biotrophic fungus Phytophthora parasitica pv. nicotianae. All the transgenic plants, irrespective of whether they carried single or two or three transgenes, when compared to the control or untransformed plants, showed better tolerance to both the tested fungi albeit with differential degree of tolerance. Combination of AtEBP1+BIK1 showed better tolerance against Alternaria while BIK1+ERF1 showed highest level of tolerance against Phytophthora. Thus, the present study demonstrated the advantage of expressing more than one transgene in imparting fungal tolerance. Efforts were made for developing a tissue culture based transformation protocol in castor. Even though shoots were obtained, they did not develop further to yield rooted plantlets. Meristem based transformation approaches also did not yield any transgenic shoots in castor. As castor is a recalcitrant crop with less amenability for transformation using tissue culture, in planta method of transformation as reported earlier was tried though with limited success. Therefore, in planta transformation method was further modified and using this protocol, a total of 37 putative transgenic castor plants, carrying different gene cassettes were identified in the T1 progeny analysis. These plants will be further confirmed for the stable inheritance and expression of the transgene(s) and they will be studied for their disease tolerance. Thus, in the present investigation, proof-of-concept was established with respect to the advantage of simultaneous expression of more than one transgene in imparting fungal tolerance using tobacco as a model system. Putative transgenic castor plants with different gene cassettes were realized using a modified in planta transformation protocol.
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    ThesisItemOpen Access
    MARKER ASSISTED SELECTION IN ADVANCED BACKCROSS POPULATION OF RICE VARIETY, MTU1010 FOR BACTERIAL BLIGHT AND BLAST RESISTANCE
    (Professor Jayashankar Telangana State Agricultural University, 2017) SRAVANTHI PENDEKANTI; DURGA RANI, CH.V
    Biotic stresses, such as Bacterial leaf blight (BB) and Blast diseases account for significant yield losses annually in rice. The major resistance genes viz., xa13 and Xa21 for BB and Pi54 and Pi1 for blast offer resistance to these diseases. MTU1010 (Cottondora Sannalu) is a mega rice variety of India released from Andhra Pradesh Rice Research Institute (APRRI), Maruteru. It is a short duration, high yielding; long slender, BPH tolerant variety occupied maximum area in India particularly during Rabi season. It is susceptible to bacterial blight (BB) and blast diseases, which are endemic to many rice growing areas in India. M-16-59 is a four gene pyramided line developed from the ongoing DBTDBSRR sub project IV funded by DBT, Govt of India at Institute of Biotechnology. M-16-59 possessed two BB resistance genes (xa13 and Xa21) and two blast resistance genes (Pi54 and Pi1). This line was developed by intercrossing of two BC2F1 plants of MTU1010 / GPP2 and MTU1010 / NLR 145. M-16-59 carrying four genes with 92% recurrent parent genome (RPG, MTU1010) and was identified from the ICF2 segregating population. The present investigation was attempted to further improve the recovery of RPG along with four target genes. Resistance genes linked/gene specific molecular markers were used for foreground selection. M-16-59 was validated for the presence of target genes by using xa13-prom (functional marker) for xa13 gene, pTA248 (STS marker) for Xa21 gene, Pi54 MAS (functional marker) for Pi54 gene and RM224 (gene linked marker) for Pi1 gene and found that the resistance alleles were present in accordance with earlier reports. Recurrent (MTU1010) and (M-16-59) donor parents showed polymorphism for the selected target genes by using the above said markers. Polymorphic primer pairs that are spread all over rice genome were used for background selection to carry out MAS. Parental polymorphic screening was carried out between two donors and recurrent parent (MTU1010) with 354 SSR markers. Out of 354 SSR markers, 83 markers showed distinct polymorphism between two donors and recurrent parent. 64 markers showed distinct polymorphism between MTU1010 and GPP2 whereas, 54 SSR markers showed distinct polymorphism between MTU1010 and NLR 145. In the earlier study of Aruna Kumari, 2013 out of 616 SSR markers tested, xx 108 showed polymorphism between recurrent parent (MTU1010) and the two donor parents (GPP2 and NLR 145) during the development of M-16-59. Among the 108 polymorphic markers, 9 markers were not recovered in M-16-59 (the plant with highest RPG 92%). In addition to the above mentioned 83 polymorphic markers, 9 non recovered polymorphic markers from earlier study were included. Thus a total of 191 (99 markers from earlier study i.e., till M-16-59 development and 92 from F1 to BC1F2 of present study) polymorphic markers were used for tracking the advanced pyramided lines carrying 4, 3 and 2 target gene combinations. In the present study, F1, BC1F1, BC1F2 and BC1F3 materials were generated. F1 and BC1F1 generations are equal to BC3F1 and BC4F1 generations, respectively as two backcrosses were completed earlier. In each generation foreground selection with genes linked/gene specific molecular markers was carried out, while background selection was carried out in F1, BC1F1, and BC1F2 generations. RPG was varied from 96.0% to 97.8% in BC1F2 population. Out of 120 F1 plants, nine plants having required four gene combination, viz., xa13, Xa21, Pi54 and Pi1 in heterozygous condition were found. These nine F1 plants were screened for the recovery percent of recurrent parent genome by using parental polymorphic markers. F1-27th plant with highest recurrent parent genome (95.6%) was selected and BC1F1 plants were generated. Eight SSR markers were not recovered in F1 progeny when analysed for RPG recovery. The BC1F1-198th plant (96.8%) was selfed to produce BC1F2 population. A total of 1060 BC1F2 plants were screened and homozygous plants possessing four genes, three genes and two genes were identified. Phenotyping for BB was carried out with highly virulent IIRR isolate, DX-020. As compared to MTU1010, the plants having both xa13 and Xa21 genes exhibited very small lesion lengths indicating a very high level of resistance followed by the plants with Xa21 gene alone and xa13 gene alone. The BC1F2 plants (possessing xa13, Xa21, Pi54 and Pi1 in homozygous condition) were screened with six parental polymorphic SSR markers for selecting those “positive” plants possessing maximum recurrent parent genome. Highest RPG was recorded in BC1F2-198-52nd (97.8%) plant. The BC1F2 plants were also evaluated for Days of 50 % flowering, Plant height and Grain type in comparison with MTU1010. Twenty five plants were advanced to BC1F3 generation based on BB resistance and above mentioned phenotypic characters. The selected BC1F3 progenies were screened for blast and BB resistance at IIRR, Rajendranagar and Agriculture Research Institute, Rajendranagar, Hyderabad during wet season, 2016. Four BC1F3 progenies with four target genes (xa13xa13Xa21Xa21Pi54Pi54Pi1Pi1) showed very high level of resistance to both the diseases. These families along with other resistant progenies possessing three gene and two gene combinations (total 25 lines) were analysed for yield and yield attributes including DUS characters along with MTU1010. BC1F2-198-52nd line found similar to MTU1010 with respect to yield and yield related characters besides showing resistance to both BB and blast. The three gene and two gene improved lines (BC1F3-198-30, BC1F3-198-494, BC1F3-198-843, BC1F3-198-702 and BC1F3-198-511) were on par with MTU1010 and have been forwarded for further multiplication. Improved lines of MTU1010 can be advanced for multi-location testing under All India Coordinated Rice Improvement Project (AICRIP) for their evaluation and possible release for the benefit of rice farmers. The recommendation and adoption of durable biotic stress resistant varieties not only saves the input cost but also saves the environment by reducing the number of chemical sprays. Finally the farmer can earn more income from unit land area. Key words: Bacterial leaf Blight, Blast, molecular markers, foreground selection, background selection, recurrent parent genome, molecular breeding, MTU1010, xa13, Xa21, Pi54 and Pi1 genes.