Agrobacterium-Mediated Delivery of Recombinant Viral Clones into Maize Seedlings for Gene Silencing

Abstract

Source: Beernink, B. M. et al. Direct Agroinoculation of Maize Seedlings by Injection with Recombinant Foxtail Mosaic Virus and Sugarcane Mosaic Virus Infectious Clones. J. Vis. Exp. (2021)

This video demonstrates the Agrobacterium-mediated delivery of recombinant viral clones into maize seedlings for gene silencing. It outlines the process of Agrobacterium infection, systemic viral spread, and RNA interference-driven phenotypic changes.

Protocol

1. Injection
NOTE: Maize seedlings from 4-7 days old can be used for injection. Seedling growth rate is greatly affected by growth conditions, amount of photosynthetically active radiation (PAR) (i.e., higher PAR in the greenhouse than in the growth chamber), and genotype, among other things that can be difficult to control in greenhouse conditions. Plants can be injected as young as 4 days old when they are 2-3 cm tall with no leaves expanded, and as old as 7 days when the lowermost rounded-tip leaf is expanded. The success rate of this inoculation method drops rapidly as plants age beyond 7 days after sowing. The injection site is the same regardle....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 mL syringes	Fisher Scientific	14955450	alternatively, BD 309659
Agrobacterium tumefaciens Strain GV3101			Carries vir plasmid encoding T-DNA transfer machinery, RifR, GmR, from lab stock
Fertilizer			Peters Fertilizer 15-15-15 Concentrate
Large Pots	Kordlok	SQL0550	5x5x4" or bigger. For transplanting seedlings.
Maize Golden Bantam Sweet Corn Seed	American Meadows, West Coast Seeds
Soil Substrate	SunGro Horticulture	SS#1-F1P	Sunshine Mix #1/Fafard-1P, any soil mix that maize grows well in is sufficient