Screening of mungbean and ricebean entries for Mungbean Yellow Mosaic Virus Resistance through Agroinfection

Department of Plant Molecular Biology and Biotechnology,Centre for Plant Molecular Biology, Tamil Nadu Agricultural University,

Coimbatore.

M. Sudha,Research Scholar

- Dr. Norman Borlaug

White flies

Yellow mosaic disease in pulses

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

AgroinfectionAgroinfection

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 Indiavirus by agroinoculation

Studies on Agroinfection in pulses

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 Number of entries

screened

Number of resistant entries

1. Mungbean 413 28

2. Ricebean 24 24

Agroinfection screening of mungbean entries against MYMVAgroinfection screening of mungbean entries against MYMV

ReactionReaction Number of entriesNumber of entries

Resistant to VA221 3Resistant to VA 239 1

Susceptible 24Total 28

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

List of field resistant mungbean entries subjected to agroinfectionList of field resistant mungbean entries subjected to agroinfection

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

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

FOR VA221 Strain

703 bp

PCR amplification in MYMV agroinoculated mungbean entries

FOR VA239 Strain

Positive control

1-20 : Agroinoculated entriesPrimer used – Coat protein region of DNA A

100bp 1 2

1. CO 5 blackgram-VA2212. CO5 blackgram-VA239

100bp 1 2 3 4 5 6 7 8 9 10 11 12 13 14151617181920

100bp 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17181920

Agroinfection screening of ricebean entries against MYMV

Reaction Number of entries

Resistant 24Susceptible 0

Total 24

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

List of Rice bean entries subjected to agroinfection

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

1000bp 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

703 bp

Lane1 : Uninoculated Control TNAU red Lane 2 : First dayLane 3 : Second dayLane 4 : Nineth dayLane 5 : Tenth dayLane 6 : Twelth dayLane 7 : Fifteenth day Lane 8 : Seventeenth dayLane 9 :Twenty-second day

Lane10 : Immediately after inoculation( embryos alone)Lane11 : Third day ( embryos alone)Lane12 : Fourth day (embryos alone)Lane13 : Nineth dayLane14 : Tenth dayLane15 :Twelth dayLane16 : Fifteenth day Lane17 : Seventeenth dayLane 18 : Eighteenth dayLane19 : Twentith dayLane 20 : Twenty-first day

PCR amplification of agroinoculated ricebean TNAU RED for PCR amplification of agroinoculated ricebean TNAU RED for VA221 strainVA221 strain

700 bp

800 bp

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

100bp 1 2 3 4 5

703 bp700 bp

800 bp

PCR amplification of agroinoculated ricebean TNAU RED VA221 strain at different growth stages

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