Department of Plant Molecular Biology and Biotechnology,

1. 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.

2. Pulses - slow runners ? - Dr. Norman Borlaug

3. Yellow mosaic disease in pulses White flies

4. 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

5. Studies on Agroinfection in pulses • Infectivity analysis of two variable DNA B components of Mungbean yellow mosaic virus-Vignain Vignamungoand Vignaradiata • Mungbean yellow mosaic virus-ViAgroinfection by codelivery of DNA A and DNA B from one AgrobacteriumStrain • 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

6. 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)

7. MYMV Symptom expression in Co 5 black gram for VA 221 and VA 239 strains

8. Susceptible reaction to MYMV through agroinfection in mungbean LM 122 (field susceptible)

9. Field screening of germplasm entries against MYMV

10. Agroinfection screening of mungbean entries against MYMV

11. List of field resistant mungbean entries subjected to agroinfection Resistant to VA239 strain Resistant to VA221 strain

12. MYMV Symptom(s) expression in mungbean – ML 1108 (VA221 resistant )

13. MYMV Symptom(s) expression in mungbean - ML 818 (VA239 resistant )

14. PCR amplification in MYMV agroinoculated mungbean entries FOR VA221Strain 100bp 1 2 3 4 5 6 7 8 9 10 11 12 13 14151617181920 Positive control 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 • CO 5 blackgram-VA221 • CO5 blackgram-VA239 1-20 : Agroinoculated entries Primer used – Coat protein region of DNA A

15. Agroinfection screening of ricebean entries against MYMV

16. List of Rice bean entries subjected to agroinfection

17. Resistance reaction to MYMV through agroinfection in ricebean LRB 297

18. Resistant symptom expression in Ricebean TNAU RED along with positive check blackgram Co 5 through agroinfection

19. PCR amplification of agroinoculatedricebean TNAU RED for VA221 strain 800 bp 1000bp 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 700 bp 703 bp 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 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

20. 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 : 2 month old lower leaves Lane 2 : 1 month old upper leaves Lane 3 : 1 month old lower leaves Lane 4 : Twenty four days old leaves Lane 5 : Fifteenth days old leaves

21. 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

22. Thank you