Study on efficacy of rnai-snv-rp gene expression against sunflower necrosis disease in sunflower (helianthus annus L.)

Shruthi B

Study on efficacy of rnai-snv-rp gene expression against sunflower necrosis disease in sunflower (helianthus annus L.)

Date

2018-05-30

Authors

Shruthi B

Publisher

Vasantrao Naik Marathwada Krishi Vidyapeeth, Parbhani

Abstract

Sunflower (Helianthus annuus) is an important edible oilseed crop of the world and it belongs to family Asteraceae and genus Helianthus. Sunflower necrosis disease is a major limiting factor for cultivation of sunflower in major sunflower growing areas of India. RNAi mediated Transgenics with replicase protein gene would be a reliable option for the development of resistance against the sunflower necrosis virus.Sunflower transgenics for resistance against sunflower necrosis virus (SNV) using RNAi-SNV-RP gene via Agrobacterium-mediated in-planta transformation protocol was developed.Sunflower seeds of cv. Morden were used as explant for optimization of in-planta transformation method. The in-plantatransformation method was optimized by testing different variables among which bacterial O.D600 1.5 and 200µM acetosyringone was found optimum. It revealed highest i.e. 27.11% transformation efficiency. The putative sunflower transformants were confirmed at molecular level by amplifying 150 bp amplicon of RP gene. Further the efficacy of RNAi-SNV-RP gene expression against sunflower necrosis virus was assessed bychallenging of both transformed as well as wild sunflower plants with SNV. Total RNA was extracted from infected sunflower plants (transformed and wild) by using sodium acetate based extraction buffer. The yield of extracted RNA was 1.1-1.5 µg RNA/µl. The cDNA 0.5µg/µl synthesized from extracted mRNA utilized for semi q-RT-PCR analysis. The semi-qRT-PCR revealed the, decrease in quantity of SNV-RP gene transcript among transformed plants compared to wild one. It reports the restriction on the multiplication of SNV in transformed sunflower plants whereas, abundance of SNV transcript was observed inwild/non transformed sunflower plants. It concluded the effectiveness of RNAi-SNV-RP gene construct towards the suppression of SNV in transformed sunflower and enabled to induce resistance in sunflower against sunflower necrosis virus.