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 regardless of the age of the seedlings.

1. Wearing safety goggles, inject the bacterial suspension into the seedlings 2-3 mm above the coleoptilar node using a 25G x 5/8" needle attached to a 1 mL disposable syringe.
   NOTE: The coleoptilar node is where crown roots will eventually form. This is the lowest node on the plant. Typically, there will be a color change from green to white at and below the node. The injection location is just above the meristem. Dissecting a few seedlings at this stage may help with visualizing the location of the meristem and consequently the proper injection site.
2. Apply gentle pressure to the syringe until the suspension fills up the coleoptile or is visible in the whorl, depending on the growth stage of the plants. This is approximately 100-200 µL of suspension.
   NOTE: If it is difficult to inject the suspension into the seedling, the injection site may be too low. Moderate pressure is all that should be needed to inject the suspension.
3. Inject all seedlings, changing syringes and needles for each construct.

2. Continued plant care

1. Transplant the injected seedlings to 13 x 13 x 15 cm or larger pots when they are 7-8 days old.
2. Maintain growth conditions (16 h photoperiod and fertilizing once per week).

3. Confirmation of infection (Phenotypically)

1. Phenotypically score plants between 14-21 days post inoculation (DPI). Lesions from silencing the control genes lesion mimic 22 or phytoene desaturase can easily be seen on the leaves and are distinct from FoMV symptoms.

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