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Screening of mungbean and ricebean entries for Mungbean Yellow Mosaic Virus Resistance through Agroinfection M. Sudha, Research Scholar Department of Plant Molecular Biology and Biotechnology, Centre for Plant Molecular Biology, Tamil Nadu Agricultural University, Coimbatore. - Dr. Norman Borlaug Yellow mosaic disease in pulses White flies Agroinfection Agroinfection is an effective method by which infectious viral clones are introduced into plants using Agrobacterium tumefaciens A partial tandem repeat of a virus is constructed and placed in the T-DNA of a binary vector and introduced into A. tumefaciens Upon inoculation of plants with the Agrobacterium strains and transfer of the T-DNA, the viral genome is released in the plant cells from the partial tandem repeat of the virus Studies on Agroinfection in pulses Infectivity analysis of two variable DNA B components of Mungbean yellow mosaic virus-Vigna in Vigna mungo and Vigna radiata Mungbean yellow mosaic virus-Vi Agroinfection by codelivery of DNA A and DNA B from one Agrobacterium Strain Analysis of an isolate of Mungbean yellow mosaic virus (MYMV) with a highly variable DNA B component Infectivity analysis of a soybean isolate of Mungbean yellow mosaic India virus by agroinoculation Agrobacterium strains VA221 - (KA 22 DNA B + KA30 DNA A) VA239 - (KA 27DNA B + KA30 DNA A) These strains cloned from infected black gram were obtained from Dr. K. Veluthambi (Madurai Kamaraj University) MYMV Symptom expression in Co 5 black gram for VA 221 and VA 239 strains Susceptible reaction to MYMV through agroinfection in mungbean LM 122 (field susceptible) Field screening of germplasm entries against MYMV S.No Germplasm Mungbean Number of entries screened 413 Number of resistant entries 28 1. 2. Ricebean 24 24 Agroinfection screening of mungbean entries against MYMV Reaction Resistant to VA221 Number of entries 3 Resistant to VA 239 Susceptible Total 1 24 28 List of field resistant mungbean entries subjected to agroinfection S.No Entries S.No Entries S.No Entries 1 KMG 149 11 ML 131 21 PDM 12-1 2 PUSA 9071 12 ML 1108 22 COGG 917 3 PUSA 9030-BOK 13 EC 27087 23 KMG 189 4 SEL 2 14 ZKM 26 24 SP 84 5 PLS 3087 15 VGG 24 25 SU 4-147 6 PLS 303-B 16 SU 4-145 26 VGG 148 7 VBM-92-3-B 17 SU4-139 27 VGG 04-006 8 NL 5 18 SU4-148 28 ML 1165 9 PDM-84-1464 19 VGG04-013 10 COGG-924 20 ML 818 Resistant to VA221 strain Resistant to VA239 strain MYMV Symptom(s) expression in mungbean – ML 1108 (VA221 resistant ) MYMV Symptom(s) expression in mungbean - ML 818 (VA239 resistant ) PCR amplification in MYMV agroinoculated mungbean entries FOR VA221 Strain Positive control 100bp 1 2 3 4 5 6 7 8 9 10 11 12 13 14151617181920 100bp 1 2 703 bp FOR VA239 Strain 100bp 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17181920 1-20 : Agroinoculated entries Primer used – Coat protein region of DNA A 1. CO 5 blackgram-VA221 2. CO5 blackgram-VA239 Agroinfection screening of ricebean entries against MYMV Reaction Resistant Susceptible Number of entries 24 0 Total 24 List of Rice bean entries subjected to agroinfection S.No Entries S.No Entries 1 V. umbellata TNAU red 13 LRB 89 green 2 LRB 41 14 LRB 11 3 LRB 297 15 LRB 29 l.green mottled 4 LRB 110 Yellow 16 LRB 35 5 LRB 113 original 17 TNAU Yellow 6 LRB 23 18 LRB 111 7 V. Umbellata TNAU green 19 LRB 104 8 LRB 113 black 20 LRB 282 9 LRB mottled green 21 LRB 5 mottled 10 V. Umbellata dark green 22 LRB 55 11 LRB tendrills 23 LRB 109 green 12 LRB 15 green 24 LRB 15 green Resistance reaction to MYMV through agroinfection in ricebean LRB 297 Resistant symptom expression in Ricebean TNAU RED along with positive check blackgram Co 5 through agroinfection PCR amplification of agroinoculated ricebean TNAU RED for VA221 strain 1000bp 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 800 bp 703 bp 700 bp Lane1 : Uninoculated Control TNAU red Lane 2 : First day Lane 3 : Second day Lane 4 : Nineth day Lane 5 : Tenth day Lane 6 : Twelth day Lane 7 : Fifteenth day Lane 8 : Seventeenth day Lane 9 :Twenty-second day Lane10 : Immediately after inoculation( embryos alone) Lane11 : Third day ( embryos alone) Lane12 : Fourth day (embryos alone) Lane13 : Nineth day Lane14 : Tenth day Lane15 :Twelth day Lane16 : Fifteenth day Lane17 : Seventeenth day Lane 18 : Eighteenth day Lane19 : Twentith day Lane 20 : Twenty-first day PCR amplification of agroinoculated ricebean TNAU RED VA221 strain at different growth stages 100bp 1 2 3 4 5 800 bp 703 bp 700 bp Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 : 2 month old lower leaves : 1 month old upper leaves : 1 month old lower leaves : Twenty four days old leaves : Fifteenth days old leaves Ongoing works… Real-Time PCR assays for detection and quantification of MYMV viruses for the identified resistant mungbean entries which were strain specific Identification of resistant proven donors for MYMV resistance through agroinfection which can be used as a genetic material