2019-12_18-017 – Establish early-stage field trials for new Huanglongbing-tolerant canker-resistant transgenic scions

Establish early-stage field trials for new Huanglongbing-tolerant canker-resistant transgenic scions

Report Date: 01/20/2020
Project: 18-017   Year: 2019
Category: Plant Improvement
Author: Zhonglin Mou
Sponsor: Citrus Research and Development Foundation, Inc.

The project has five objectives:
(1) Remove the flowering-promoting CTV and the HLB bacterial pathogen in the transgenic plants
(2) Graft CTV- and HLB-free buds onto rootstocks
(3) Generate a large number of vigorous and healthy citrus trees
(4) Plant the citrus trees in the site secured for testing transgenic citrus for HLB responses
(5) Collect the field trial data

In this quarter, the following activities have been performed:

(1) To ensure the quality of the citrus plants for the field trial, protein levels in the propagated plants were analyzed. Leaf tissues were collected for total protein extraction. After extraction, total protein was separated in SDS-PAGE gel. The proteins were transferred onto a nitrocellulose memebrane. The blot was probed with an antibody against the overexpressed protein. It was found that all progeny plants express good amount of the transgenic protein. The plants were carefully managed by regular watering and fertilization. This batch of plants will be transplanted into the field at the end of April or early May of 2020.

(2) Purify antibody against a major citrus defense protein. One boost was conducted to the rabbits. Serum was extracted form both rabbits and purified with protein A beads. The purifed antibody was tested using the citrus protein transiently expressed in Nicotiana benthamiana. It was also tested using total protein extracted from the sweet orange ‘Hamlin’. The titer of the antibody has been improved by the boost. The antibody can now be used to analyze other citrus materials.

(3) Analyze the transgenic plants already planted in the field. The size of the plants in the field at Ft Pierce USDA ARS were measured. Leaf samples were collected from each plant. Total protein and RNA have been extracted. Assays of the transgenic protein levels and CLas titers are ongoing.

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