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: 11/07/2019
Project: 18-017   Year: 2019
Category: Plant Improvement
Author: Zhonglin Mou
Sponsor: Citrus Research and Development Foundation

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, we have conducted the following activities:

(1) Nurture small grafted transgenic plants in the greenhouse. The plants are regularly watered and fertilized to ensure vigorous growth. We still keep propagating the transgenic lines using budwoods free of CTV and CLas. We have generated enough progeny plants for this project. These plants will be transplanted in the Spring of 2020.

(2) Analyze rabbit serum against a major citrus defanse protein. The protein was purified and submitted for antibody development. The first batch of rabbit serum has been tested. Briefly, the citrus protein was transiently expressed in Nicotiana benthamiana. Total protein was extracted and separated in SDS-PAGE gel. After transferring the proteins onto a nitrocellulose membrane, the blot was probed with the rabbit serum. A strong specific band was detected in the sample from plants expressing the citrus defense protein, but not from plants transformed with an empty vector, indictaing that the antobody development is successful. Total protein was also extracted from the sweet orange ‘Hamlin’ and analyzed with the serum. The signal is rather weak. A boost has been requested to enhance the titer of the serum.

(3) Observe the transgenic plants already planted in the field. The 69 plants (49 transgenic plants and 20 controls) in the field at Ft Pierce USDA ARS were taken care of by the crew under the supervion of Dr. Ed Stover. The PI regularly communicate with Dr. Stover on the growth of the plants. We will measure the plants in the fourth quarter of this year.

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