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INVESTIGATOR: Meng, Y.

PERFORMING INSTITUTION:
ALCORN STATE UNIVERSITY
ALCORN STATE, MISSISSIPPI 39096

BIOTECHNOLOGICAL APPROACHES FOR IMPROVING SWEETPOTATO CROP WITH MULTIPLE VIRUS RESISTANCE

NON-TECHNICAL SUMMARY: Sweetpotato (Ipomoea batatas (L.) Lam.; Convolvulaceae), a plant widely grown in all tropical and subtropical areas, is among the 10 most important food crops worldwide. As a crop produced by vegetative propagation, "cultivar decline" due to viral infections significantly reduces sweetpotato yield and storage root quality. In the United States, Sweet potato feathery mottle virus (SPFMV) and Sweet potato leaf curl virus (SPLCV) are two of the most prevalent sweetpotato viruses that cause devastating diseases and yield reductions. Even though resistance to viral infections remains the most cost effective way of disease management, to date, immune resistance to these viruses has not been identified in I. batatas. In this study, we will explore a novel biotechnological method to develop transgenic sweetpotato plants with resistance to these two viruses. The overall goal of this project is to develop and manage sweetpotato viral disease resistance through biotechnology approaches in US, aiming at increasing the sweetpotato yield, quality and investment return for farmers. Specifically, we propose the following objectives: 1) To develop a plant transformation and regeneration protocol for the production of value-added sweetpotato lines using popular production lines; 2) To create transgenic virus resistance lines in sweetpotato to multiple important epidemic viruses; 3) To conduct biological evaluation of the transgenic lines for their resistance to multiple viruses under diverse growth conditions and agricultural practices. Successful execution of this project will significantly boost quality of life and environment in the underserved communities and enhance research and education capacities of Alcorn State University.

OBJECTIVES: The overall goal of this project is to develop and manage sweetpotato viral disease resistance through biotechnology approaches in US, aiming at increasing the sweetpotato yield, quality and investment return for farmers. Specifically, we propose the following objectives: 1) To develop a plant transformation and regeneration protocol for the production of value-added sweetpotato lines using popular production lines; 2) To create transgenic virus resistance lines in sweetpotato to multiple important epidemic viruses; 3) To conduct biological evaluation of the transgenic lines for their resistance to multiple viruses under diverse growth conditions and agricultural practices.

APPROACH: Objective 1: To develop a plant transformation and regeneration protocol for the production of value-added sweetpotato lines using popular production lines.Sweet potato varieties 'Beauregard' and 'Jewel', and ASU breeding line "Excel 2", will be used for transformation. All the plants material will be subcultured every 4 weeks onto a fresh MS medium (Murashige and Skoog, 1962) supplemented with 3% sucrose. Cultures are maintained in a temperature controlled chamber at 26°C with a 16-h photoperiod, with light intensity ranging from 400 to 1,000 μmol m-2 s-1. Dissected leaf disks, petiole and stem segments from the in vitro manipulated stock will be used as explants to gene transformation.In the United States, SPFMV and SPLCV are two of the most prevalent sweetpotato viruses that cause devastating diseases and yield reductions. In this study, we will explore novel biotechnological approaches to develop the PDR in transgenic sweetpotato plants with resistance to these two viruses. The SPFMV and SPLCV genomes were cloned from the MS field isolates by Dr. Chunquan Zhang (Co-PI) (Zhang et. al., 2015, Akrong et. al., 2016). Dr. Zhang further engineered part of coat protein (CP) gene of SPFMV and the replication origin region of SPLCV into a binary vector pCAMBIA1303 for Agrobacterium-mediated or direct biolistic delivery-based genetic transformation for transgene expression. SPFMV CP partial gene sequence and SPLCV replication origin sequence were designed in antisense for the optimal induction of RNA silencing in sweetpotato. Likewise, the SPFMV CP partial gene sequence and SPLCV replication origin sequence were blasted with the available isolates in US, and the conserved regions were selected, thus the transgenic sweetpotato is expected to have SPFMV and SPLCV resistance in and beyond Mississippi. With a two-year of pre-experiment, a high efficient transformation and plant regeneration system has been optimized for sweetpotato cultivars 'Jewel' in our lab (Meng et. al, 2016).Objective 2: To create transgenic viral resistance lines in sweetpotato to multiple important epidemic viruses.To evaluate the transgenic events generated in objective 1, we will conduct molecular characterization by doing PCR and Southern blot analysis. Total genomic DNA will be isolated from leaf tissue of putative transgenic and untransformed (control) plants using the ZR Plant/Seed DNA MiniprepTM kit (Zymo Research, SA). PCR analysis will be used for initial detection of putative transgenic plants. Primers complementary to the individual transgene segments of SPFMV and SPLCV will be used to detect the integrity of the transgene construct in plants. Amplification will be performed in an automated thermal cycler. For Southern blot analysis, total plant DNA (20 μg) from putative transgenic plants will be digested overnight at 37 °C with restriction endonuclease HindIII. DNA fragments will be separated by electrophoresis at 40 V for 3 h in a 1.2 % agarose gel and blotted onto positively charged nylon membranes (Roche, Germany) using the VacuGene XL Vacuum blotting System (GE Healthcare Bio-Sciences, Sweden). DNA fragments will be fixed to the membrane by UV crosslinking for 2-3 min (Stratalinker, Stratagene, CA, USA). The hybridization probe corresponding to the SPFMV segment will be used for the detection of the transgene and generated and labeled with alkali-labile digoxigenin (DIG-dUTP) in PCR using the PCR DIG Probe Synthesis kit (Roche, Germany). Hybridization of the probe to membrane and subsequent chemiluminescence detection by enzyme immunoassay will be performed using the DIG High Prime DNA Labeling and Detection Starter Kit II (Roche, Germany) according to manufacturer's instructions.Objective 3: To conduct biological evaluation on the transgenic lines for their resistance to multiple viruses under diverse growth conditions and agricultural practices.Transgenic plants will be assayed for virus resistance according to the method described by Okada et al. (2002). Vine samples that were NCM-ELISA positive for infection with isolates of SPFMV and SPLCV will be graft-inoculated onto 3 week-old indicator plants, Ipomoea setosa Ker Gawl. Two to three weeks post inoculation, typical virus induced symptoms will be observed on growing leaves of indicator plants. The vine cuttings from these virus-infected I. setosa plants served as scions (inoculum) in grafting experiments.

PROGRESS: 2020/03 TO 2021/03
Target Audience:The graduate and undergraduate students, lab technician, extension agents, farmers are the terget audience for this project. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During the execution of this project, the graduate and undergraduate students, lab technician, extension agents were trained with better skills and knowledge related with plant tissue culture techniques, crop genetic engineering and gene transformation, disease diagnosis techniques and field practices. Three courses related with tissue culture and genetic engineering techniques were developed at undergraduate and graduate class levels. All the personnel mentioned above are benefit for the training and professional development. How have the results been disseminated to communities of interest?The research data were presented to several scientific meetings regionally or nationally. What do you plan to do during the next reporting period to accomplish the goals?Our next goals to be accomplished will be objectives 2 and 3. Especially, to create more transgenic viral resistance lines in sweetpotato to multiple important epidemic viruses and to conduct biological evaluation the transgenic lines for their resistance to multiple viruses under diverse growth conditions and agricultural practices. PI, Co-PIs and collaborators will continue to arrange meetings to discuss plans for execution of the goals.

IMPACT: 2020/03 TO 2021/03
What was accomplished under these goals? We having been focusing objectives 2 and 3 in year three. Plant regeneration protocols for four sweetpotato lines have been optimized. An Agrobacterium-mediated transformation protocol was well-developed in the lab, and 12 transgenic plants were obtained from one sweetpotato line; the transgenic events were confirmed by PCR. We are continuing to generate transgenic sweetpotato lines on another sweetpotato line. The viral resistance of transgenic plants is in investigation under controlled environment, by using biological approach and indicating plant setosa grafting method. The results needs to be further clarified.

PUBLICATIONS (not previously reported): 2020/03 TO 2021/03
Type: Journal Articles Status: Published Year Published: 2020 Citation: Meng Y, Zhang C and Njiti V (2020). Virus elimination in sweetpotato: from meristem-tip culture to storage roots production: a review. Adv Agri Tech Plant Sciences 3(1): 180048