Optimization of in vitro transformation protocol and RNAi based gene silencing for viral (Cucumber Mosaic Virus) resistance in Bhut jolokia (Capsicum chinense Jacq.)
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Date
2021
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Abstract
Bhut jolokia (Capsicum chinense Jacq.), one of the most popular and hottest chillies
in the world, is widely cultivated in Assam and other North Eastern regions in India. A
member of the Solanaceae family, Bhut jolokia is characterized by very high pungency due
to the presence of high amount of phenolic alkaloid ‘Capsaicinoids’. It is an ideal chilli
variety for extraction of oleoresin and capsaicin, which have high market demand due to
their industrial uses and medicinal properties. Bhut jolokia production is challenged by
several biotic constraints, particularly viral diseases, which affect its quality and yield.
Among the viruses, Cucumber Mosaic Virus (CMV) causes severe crop damage, leading to
low productivity. Current control measures for CMV are mainly preventive through vector
management strategies, which are not adequate in controlling the disease. An effective way
to control the disease is the use of biotechnological tools such as RNA interference (RNAi)
technology to engineer resistance against the virus. Plants expressing a copy of a viral gene
in sense and/or antisense orientation have shown resistance upon infection with the virus via
post-transcriptional gene silencing. In the present investigation, an in vitro regenerationtransformation
strategy has been optimized for Bhut jolokia and, a hairpin RNA (hpRNA)
based gene silencing construct has been developed using the Replicase gene from CMV.
The study was initiated by callus induction from Bhut jolokia leaf segments in MS
basal medium. Very good quality callus were induced in MS medium supplemented with
0.5 mg/l or 1 mg/l 2,4-D. Multiple shoot induction and regeneration from callus were
obtained in MS medium supplemented with 8.5 mg/l KIN and 0.5 mg/l TDZ along with 5
mg/l AgNO3 with maximum shoot initiation frequency of 95% and regeneration frequency
of 90%. Root regeneration was found to be optimum in half strength MS medium
supplemented with 1.5 mg/l NAA within 4 weeks of culture with maximum rooting
frequency of 70%. For standardization of an Agrobacterium-mediated genetic
transformation system, the strain LBA4404 carrying pCAMBIA1301 binary vector
construct with gusA as the reporter gene and hptII and nptII as selection marker genes was
used. Transformation was carried out using 45 days old callus and also with intact Bhut
jolokia seeds as explants. Hygromycin concentration of 9 mg/l was found to be optimum for
efficient selection of putative transformants. From a total of 30 nos. of callus infected by
Agrobacterium, 9 numbers of putative transformed shoots were regenerated in presence of
selection agent. Finally, only 2 (6.66%) fully rooted plants survived out of which, only 1
plant finally survived during hardening in the green house. Moreover, out of 30 nos. of
infected seeds, a total of 7 numbers of putative transformed seedlings were developed.
Finally, only 1 (3.33%) seedling survived, which was transferred to the green house for
hardening. Thus, both callus and seeds could be used as explants for transformation in Bhut
jolokia, although the frequency of putative transformants obtained using callus explants was
higher than that in seed transformation. The putative transformants were confirmed by GUS
histochemical assay and PCR analysis. For developing the RNAi construct, a 323 bp
Replicase gene sequence was cloned into pHANNIBAL vector both in sense and anti-sense
orientations. The construct was then transferred to pBI121 binary vector, which was
electroporated into Agrobacterium strain LBA4404 for plant transformation. Functional
validation of the CMV Replicase hp-RNA construct was done through bioassay in model
plant Nicotiana benthamiana by Agro-infiltration. Transgene expression in N. benthamiana
was confirmed by RT-PCR analysis. The bioassay results indicated suppression of CMV
infection in Agro-infiltrated N. benthamiana plants when mechanically inoculated with
CMV sap. Further, DAS-ELISA established the functional efficiency of the hpRNA
construct in providing considerable level of resistance against CMV infection. The in vitro
regeneration-transformation strategy and the hpRNA based gene silencing construct,
developed through this study would serve as a foundation towards future studies on
engineering resistance against CMV in Bhut jolokia.