Development of Resistant Lines against Leaf Blight Disease of Withania somnifera (L.) Dunal. Caused by Alternaria alternata through In vitro Selection
A. Jhankare
Horticultural Biotechnology Laboratory ,Biotechnology Centre, J. N. Agricultural University, Jabalpur 482 004, India.
G. Tiwari
Department of Medicinal and Aromatic Plants, Biotechnology Centre, J. N. Agricultural University, Jabalpur 482 004, India.
M.K. Tripathi
Horticultural Biotechnology Laboratory ,Biotechnology Centre, J. N. Agricultural University, Jabalpur 482 004, India.
G.N. Pandey
Department of Plant Pathology,Biotechnology Centre, J. N. Agricultural University, Jabalpur 482 004, India.
R.P. Patel
Department of Plant Pathology,Biotechnology Centre, J. N. Agricultural University, Jabalpur 482 004, India.
S. Tiwari
Division of Tissue Culture and Transgenics, Biotechnology Centre, J. N. Agricultural University, Jabalpur 482 004, India.
B. S. Baghel
KNK-College of Horticulture, Mandsaur 458 001; Biotechnology Centre, J. N. Agricultural University, Jabalpur 482 004, India.
*Author to whom correspondence should be addressed.
Abstract
Disease tolerant/resistant Withania (Withania somnifera (L.) cv JA-20 and MWS-100) cell lines were selected against leaf blight disease caused by Alternaria alternata. For this purpose callus and cell suspension cultures derived from mature embryos and hypocotyl explants were exposed to purified toxic culture filtrate produced by the fungus supplemented with MS culture medium. Two selection methods were used: a continuous method in which four cycles of selection were performed on toxic medium whereas during discontinuous method, a pause was given after the second and third cycle of selection using non-toxic medium. Approximately 310 calli of JA-20 and 340 of MWS-100 obtained from mature embryo and hypocotyl explants cultures and 675 of JA-20 and 725 of MWS-100 cell clumps/embryoids acquired from cell suspension cultures were exposed to media with effective concentration (10.0 ml/l) of phytotoxin for selection. The discontinuous method proved to be superior as it allowed the calli to regain their regeneration capability. Continuous exposure with toxic culture filtrate resulted up to 77% mortality. In vivo pathogenesity test of regenerated plants from the surviving tolerant/resistant cell lines revealed non-sensitive against pathogen toxin. A total 2 lines of JA-20 and one of MWS-100 were documented resistant/tolerant amongst an array of putative resistant/tolerant lines during S1 generation.
Keywords: In vitro selection, embryogenic calli, cell clumps, cell suspension culture, toxic culture filtrate