PhD

Okra (Abelmoschus esculentus L. Moench, Malvaceae) is one of the leading
vegetable crops in hot and humid tropics. Unfortunately, this climate is conducive for
many of the pests and diseases. Okra is susceptible to viruses such as Yellow vein
mosaic virus (YVMV) and Enation leaf curl virus (ELCV), belonging to the genus
Begomovirus (family Geminiviridae). Because of the favourable conditions prevailing
in the coastal region, the losses in Kerala state are 60-100%, depending upon the stage
of plant growth and the severity of infection. RNAi is one of the promising molecular
biology approach against the viral diseases. Keeping the above facts in view, the
present study “RNA mediated resistance to Yellow vein mosaic virus in okra” was
taken up at the Centre for Plant Biotechnology and Molecular Biology, CoA, Thrissur
from September 2017 to May 2021.
The high yielding and YVMV susceptible popular okra cv. Salkeerthi was
selected for the development of resistance using RNAi mechanism. Total DNA was
isolated from the YVMV infected plant and part of the βC1 gene (187 bp) of the virus
was amplified using primers VβC1F and VβC1R. Sequence information of PCR
product has revealed that the gene is 90-95% identical with the Indian isolates. The
βC1 gene sequence was analysed using IDT software and 10 siRNAs were found at
three different position (19-44, 34-59, 99-124 bp). Through Restriction Mapper, it
was confirmed that the sequence selected for the preparation of sense and antisense
strand, do not possess recognition sites for SmaI, HindIII and MauBI restriction
enzymes which are present in the pRNAiLIC vector. The output of VSupPred
revealed that the fragment does not contain any Viral Suppressor Regions (VSRs),
with a high prediction score (0.625).
The hairpin RNAi construct harbouring the region of βC1 gene of β satellite of
Begomovirus of okra was generated using pRNAi-LIC (CD3-1285) vector. The SmaI
digested plasmid produced three fragments, vector backbone (9842 bp), Pdk intron
(1641 bp) and ccdB gene (614 bp) and the digested plasmid was treated with dTTP.
Product-1 was PCR amplified (215 bp) with VLIC1 and VLIC2 primers, using the
DNA from YVMV infected plant as template. Product-2 was PCR amplified (243 bp)
with VLIC3 and VLIC4 primers using product-1 as template. Product-1 and product-2
were eluted from the gel and treated with dATP. The dATP treated PCR products and
dTTP treated SmaI digested plasmid were mixed together and ligated by incubation at
65ºC for 5 min. followed by 22ºC for 15 min. Ligated product was successfully
transformed in competent cells of E. coli (DH5α) and incubated on LB medium
containing Kanamycin and Chloramphenicol. Colony PCR was performed, the
transformation efficiency was found to be 80%. Plasmid was isolated from the
positive DH5α colony and sequenced using the primers VLIC5 and VLIC6. The
sequence data had shown that both sense and antisense strands are at right position
and direction. Plasmid containing ihpRNA-βC1 cassette was successfully transformed
into the competent cells of Agrobacterium (GV3101) and incubated on LB medium
containing Kanamycin, Chloramphenicol and Rifampicin. Colony PCR was
performed, the transformation efficiency was found to be 100%. Plasmid was isolated
from the positive GV3101 colony and sequenced using the primers VLIC5 and
VLIC6. Sequence data has further confirmed that both sense and antisense strands are
at right position and direction.
The ihpRNA-βC1 cassette was successfully transformed into okra cv.
Salkeerthi using in planta method of Agrobacterium mediated transformation. The
transformation efficiency observed was 11.42% and the transformation was confirmed
by the amplification of sense strand using the primers VLIC1 and VLIC5. cDNA was
prepared from the total RNA isolated from transformed and control plants. siRNA
synthesis was confirmed using the primers VLIC1 and VLIC5 (400bp) and Ubiquitin
gene was confirmed using the primer UBQ7 (187 bp). Silencing potential of the RNA
interference of βC1 gene and the development of resistance was evaluated by keeping
the 15-day old transformed and control plants along with YVMV infected plants
inside containment facility, with whiteflies released into insect cage for infection. All
the control plants and one transgenic plant have shown the YVMV symptoms after 10
days. Three transgenic plants were healthy with no symptoms. The present
investigation was successful in the development of YVMV resistant okra plants
carrying ihpRNA-βC1 using pRNAi-LIC (CD3-1285) plasmid vector. The further
evaluation is needed in the coming generations for the identification of stable
transgenic lines.