DEVELOPING SWEETPOTATO GERMPLASMS WITH INCREASED PROTEIN LEVELS AND IMPROVED VIRAL DISEASE RESISTANCE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: ACTIVE
• Funding Source: OTHER GRANTS
• Reporting Frequency: Annual
• Accession No.: 1028610
• Grant No.: 2022-38821-37359
• Cumulative Award Amt.: $499,685.00
• Proposal No.: 2021-12830
• Project Start Date: May 1, 2022
• Project End Date: Apr 30, 2026
• Grant Year: 2022
• Program Code: [EQ]- Research Project

Recipient Organization
ALCORN STATE UNIVERSITY
(N/A)
ALCORN STATE,MS 39096

Performing Department
Agriculture

Non Technical Summary
Food security and nutrition security exist as the major concerns in many countries of the world. 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. However, sweetpotato contains lower protein content compared to other staple foods, e.g., maize, rice, wheat and soybean. Moreover, as a crop produced by vegetative propagation, "cultivar decline" due to viral infections significantly reduces sweetpotato yield and storage root quality. In this study, we will apply novel biotechnological methods to develop sweetpotato plants with improved nutrition and viral resistance, thus, sweetpotato lines with higher nutritional value and increased yield production will be available for Mississippi sweetpotato seed industry and farmers. Specifically, our objectives are 1). To generate Arabidopsis QQS and sweetpotato NF-YC4 transgenic sweetpotato to increase protein levels and disease resistance in sweetpotato roots via Agrobacterium-mediated transformation method; 2). To generate sweetpotato via CRISPR/Cas9 targeted mutagenesis for overexpression of sweetpotato NF-YC4 to increase protein levels and disease resistance in sweetpotato roots; 3). To test the composition of protein and starch for the new sweetpotato varieties generated in objectives 1 and 2; 4). To test viral disease resistance for the new sweetpotato varieties generated in objectives 1 and 2. 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.

Animal Health Component

0%

Research Effort Categories

Basic

100%

Applied

0%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
202	1450	1081	100%

Knowledge Area
202 - Plant Genetic Resources;

Subject Of Investigation
1450 - Sweet potato;

Field Of Science
1081 - Breeding;

Keywords

sweetpotato

molecular breeding

protein levels

viral resistance

Goals / Objectives
To generate sweetpotato lines with higher nutritional value and increased yield, our objectives are 1). To generate Arabidopsis QQS and sweetpotato NF-YC4 transgenic sweetpotato to increase protein levels and disease resistance in sweetpotato roots via Agrobacterium-mediated transformation method; 2). To generate sweetpotato via CRISPR/Cas9 targeted mutagenesis for overexpression of sweetpotato NF-YC4 to increase protein levels and disease resistance in sweetpotato roots; 3). To test the composition of protein and starch for the new sweetpotato varieties generated in objectives 1 and 2; 4). To test viral disease resistance for the new sweetpotato varieties generated in objectives 1 and 2. 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.

Project Methods
Objective 1: To generate Arabidopsis QQS and sweetpotato NF-YC4 transgenic sweetpotato to increase protein levels and disease resistance in sweetpotato roots via Agrobacterium-mediated transformation method.Plant materialsSweetpotato varieties PI318846 and 'Jewel' cultivars PI 566638 and PI566648 will be used for transformation.Vector constructionConstruction of QQS expression and Sweetpotato NF-YC4 over-expression vectorsQQS was cloned from Arabidopsis thaliana ecotype Columbia-0 (Col-0) and sweetpotato NF-YC4 was cloned from Ipomoea batatas varieties listed above. A 35S::AtQQS fusion construct was made by cloning the amplified full-length QQS coding sequence (with primers: QQSCDS.F and QQSCDS.R; AtQQS sequence is underlined), and a 35S::IbNF-YC4 fusion construct was made by cloning the full-length sweetpotato IbNF-YC4 coding sequence (with primers: NF-YC4CDS.F and NF-YC4CDS.R; IbNF-YC4 sequence is underlined) into a binary vector pB2GW7 (Karimi et al., 2002) (Table 1). The full-length QQS coding sequence was also cloned into another binary vector pK2GW7 (Karimi et al., 2002) for co-expression of AtQQS and IbNF-YC4 in sweetpotato. AtQQS or IbNF-YC4 would be expressed under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter as previously tested. The 35S::QQS-pB2GW7 or -pK2GW7 and 35S::IbNF-YC4-pB2GW7 vectors have been constructed in Li lab, and have been transformed into Agrobacterium tumefaciens strain EHA105 in Meng lab for sweetpotatoPlant transformation and regenerationA high-efficient transformation and plant regeneration system in sweetpotato has been well developed in Meng lab.1). Agrobacterium-mediated transformation. A single bacterial colony of the A. tumefaciens strain EHA105 harboring pB2GW7- or pK2GW7-based constructs will be used to inoculate 5 mL of standard LB liquid media with 100 mg/L spectinomycin or 50 mg/L kanamycin in a shaker (200 rpm) at 28°C for overnight growth. A 50-µL aliquot of this overnight bacterial culture will be transferred into a 250-mL Erlenmeyer flask with 50 mL LB media in 100 mg/L Spectinomycin and 200 µM acetosyringone in a shaker (200 rpm) at 28°C until the optical density of culture reaches 0.4 - 0.6 at 600 nm. Bacterial cells are centrifuged at 6,000 rpm for 10 min at 25°C and re-suspended in same volume of bacterial infection media (MS salt, 40 mg/L thiamine, 100 mg/L myoinositol, 30 g/L sucrose, 6.49 µm 4FA and 200 µM acytosyringone, pH 5.5).Surface sterilized explants will be transferred into a Petri dish (20 x 100 mm) containing about 25 mL of agrobacterium culture, and kept in the dark at 28°C without agitation for 15 min. Then the explants will be cultured with co-cultivation medium (MS salt, 40 mg/L thiamine, 100 mg/L myoinositol, 30 g/L sucrose, 6.49 µM 4FA and 200 µM acytosyringone, 0.3 % phytogel, pH 5.5), and incubated at 28°C in the dark for 4 days.2). Selection and plant regeneration. Following this co-culture step, the explants will be blotted onto sterile filter paper, then transferred onto selective regeneration media (optimized medium contains 200 mg/L carbenicillin and 0.5 mg/L glufosinate), and sealed and incubated in the culture chamber (16 h photoperiod, 3000 lux and 28/26°C). After selection for 5-6 days, the explants will be transferred to callus and shoot induction medium (optimized medium contains 200 mg/L carbenicillin and 0.5 mg/L glufosinate) for callus and shoot induction and selection. The explants will be subcultured onto the fresh medium every two-three weeks. Friable and glufosinate-resistant calli should form on media plates after 6 weeks. Excise and transfer regenerated shoots to the rooting medium (optimized medium contains 200 mg/L carbenicillin and 0.5 mg/L glufosinate) in Megenta boxes. Transfer well-rooted putative transformants to pots, and cover them with transparent plastic cups for a week or so to let them acclimate.3). Transgenic events confirmation. Transgenic plants derived with Agrobacterium-mediated transformation will be confirmed by PCR and PCR product sequencing.Objective 2: To generate novel sweetpotato lines via CRISPR/Cas9 targeted mutagenesis for overexpression of sweetpotato NF-YC4 to increase protein levels and disease resistance in sweetpotato roots.Vector selection and guide RNA construction: pDicAID_nCas9-PmCDA_NptII_Della (Addgene) will be used as the vector for Cas9 and guide RNA expression in planta. The plasmid pDicAID_nCas9-PmCDA_NptII_Della contains a modified version of Cas9 with a point mutation that leads to deficient nicknase activity. Petromyzon marinus cytidine deaminase (PmCDA1) is fused to nCas9 to direct the cytidine to thymine mutation on target DNA. IbCas9 codon on this vector has been optimized for dicot applications.CRISPR/Cas9 targets have been identified using on-line guide RNA design software E-CRISP for sweetpotato NF-YC4 gene amplified and sequenced in Li lab from three sweetpotato lines. The sweetpotato CRISPR/Cas9 constructs will be prepared initially in Meng lab. As soon as the additional CRISPR target sequences of NF-YC4 genes are identified, the CRISPR/Cas9 constructs will be transferred to A. tumefaciens strain EHA105, and positive clones harboring the constructs will be used to transform sweetpotato. Leaf/stem explants will be regenerated and rooted on selection media. Screening for genome edited lines will be conducted at the plantlet stage.About 100 transformants from each of the above transformation design will be screened. To speed up positive selection, total RNA will be extracted at the callus stage, and reverse-transcribed into cDNA using Oligo (dT)20 as primer. The cDNA will be used with NF-YC4 specific primer pairs to amplify sweetpotato NF-YC4 mRNA gene and the PCR products will be sequenced to look into changes in the gene sequence to identify transformants with putative NF-YC4 mutations.Objective 3: To test the composition of protein and starch for the new sweetpotato varieties generated in objectives 1 and 2.This experiment would be performed in Li Lab. Starch quantification and protein quantification are routinely performed in Li Lab. Li Lab will optimize the protocols for sweetpotato and do the starch assay and protein assay to quantify the protein and starch levels in the new sweetpotato varieties generated in this project. At least three biological replicates will be used.Novel sweetpotato varieties with high protein content would be identified.Objective 4: To test the viral disease resistance for the new sweetpotato varieties generated in objectives 1 and 2. This objective will be conducted in Meng lab in year 3. Novel sweetpotato lines generated from objective 1 and 2 will be assayed for virus resistance according to the method described by Okada et al. (2002). Vine samples that are PCR or RT-PCR positive for infection with isolates of SPFMV, SPLCV, SPVC, SPVG and SPV2 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. All plants will be kept at 25°C in an insect proof growth room in the Controlled Environment Research Unit (CERU) at ASU, under 80% relative humidity, and be fertilized weekly with 3:1:3 (nitrogen: phosphate: potassium). Approximately 4 weeks after planting, transgenic plants and controls will be graft-inoculated with scions of virus-infected I. setosa. Plants will be maintained in the growth room for a period of 12 weeks to allow for viruses to translocate through the graft and progress into the transgenic sweet potato plant. Twelve weeks post inoculation, scions from inoculated transgenic and control plants will be graft inoculated back onto 3-week-old I. setosa plants.

Progress 05/01/23 to 04/30/24

Outputs
Target Audience:The graduate and undergraduate students, post-doctorate research scientist, lab technician, extension agents, farmers are the target 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, post-doctorate, 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 well 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, including the annual Mississippi Academy of Sciences Meeting at Hattiesburg, MS, the 18th 1890 Association of Research Directors (ARD) Research Symposium at Nashville, TN, and the 10th annual Center of Research Symposium at Alcorn State University 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 test transgenic lines in sweetpotato with enhanced protein levels and viral resistance. CRISPR/Cas9 targeted mutagenesis for overexpression of sweetpotato NF-YC4 will be developed to increase protein levels and disease resistance in sweetpotato roots. PI, Co-PIs and collaborators will continue to arrange meetings to discuss plans for execution of the goals.

Impacts
What was accomplished under these goals? We have been focusing on objective 1 in year 2: to generate Arabidopsis QQS and sweetpotato NF-YC4 transgenic sweetpotato to increase protein levels and disease resistance in sweetpotato roots via Agrobacterium-mediated transformation method. Arabidopsis QQS expressing vectors were constructed. QQS was cloned from Arabidopsis thaliana ecotype Columbia 0 (Col-0). The vector was transformed into Agrobacterium tumefaciens strain EHA105. A high-efficient sweetpotato regeneration system and Agrobacterium-mediated gene transformation system for sweetpotato 'Red Jewel" cultivar was well-developed. Preliminary results showed that expression of foreign genes in kanamycin resistant plants was achieved using A. tumefaciens strain EHA105 harboring the expression cassette. The PCR result showed that the foreign QQS gene has been transformed into the sweetpotato line PI566648 (Red Jewel).

Publications

• Type: Journal Articles Status: Published Year Published: 2024 Citation: 1. Justin George, Gadi V.P. Reddy, Philip Wadl, William Rutter, Julianna Culbreath, Lau Pierre, Tahir Rashid, Matthew C. Allan, Suzanne D. Johaningsmeier, Amanda Nelson, Ming Li Wang, Augustine Gubba, Kai-Shu Ling, Yan Meng, Daniel Collins, Sathish Ponniah, Prasanna H. Gowda (2024). Sustainable Sweetpotato Production in the United States: Current Status, Challenges, and Solutions. Agronomy Journal. P.1-31 DOI: 10.1002/agj2.21539
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: 1. Alpha Jones, Chunquan Zhang and Yan Meng (2024). Developing efficient biotechnological approaches for sweetpotato virus detection and removal. Association of 1890 Research Directors Research Symposium 2024, Nashville, Tennessee
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: 2. Tatyana Hollingbird, Chunquan Zhang, Lei Wang, Ling Li and Yan Meng (2024). Developing sweetpotato germplasm with enhanced performances by using Agrobacterium-mediated transformation. Association of 1890 Research Directors Research Symposium 2024, Nashville, Tennessee
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: 3. Alpha Jones, Chunquan Zhang and Yan Meng (2024). Developing efficient biotechnological approaches for sweetpotato virus detection and removal. 10th Annual Center for Research Excellence Symposium, Alcorn State University, Lorman, Mississippi
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: 4. Alpha Jones, Chunquan Zhang and Yan Meng (2024). Developing efficient biotechnological approaches for sweetpotato virus detection and removal. 88th Annual Mississippi Academy of Sciences Meeting, Hattiesburg, Mississippi
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: 5. Tatyana Hollingbird, Chunquan Zhang, Lei Wang, Ling Li and Yan Meng (2024). Developing sweetpotato germplasm with enhanced performances by using Agrobacterium-mediated transformation. 88th Annual Mississippi Academy of Sciences Meeting, Hattiesburg, Mississippi
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: 6. Alpha Jones, Chunquan Zhang and Yan Meng (2024). Developing efficient biotechnological approaches for sweetpotato virus detection and removal. 88th Annual Mississippi Academy of Sciences Meeting, Hattiesburg, Mississippi
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: 7. Alpha Jones, Chunquan Zhang and Yan Meng (2024). Developing efficient biotechnological approaches for sweetpotato virus detection and removal. 10th Annual Center for Research Excellence Symposium, Alcorn State University, Lorman, Mississippi
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: 8. Tatyana Hollingbird, Chunquan Zhang, Lei Wang, Ling Li and Yan Meng (2024). Developing sweetpotato germplasm with enhanced performances by using Agrobacterium-mediated transformation. 10th Annual Center for Research Excellence Symposium, Alcorn State University, Lorman, Mississippi
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: 9. Alpha Jones, Chunquan Zhang and Yan Meng (2024). Developing efficient biotechnological approaches for sweetpotato virus detection and removal. 10th Annual Center for Research Excellence Symposium, Alcorn State University, Lorman, Mississippi

Progress 05/01/22 to 04/30/23

Outputs
Target Audience:The graduate and undergraduate students, post-doctorate research scientist, lab technician, extension agents, farmers are the target 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, post-doctorate, 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 well 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, including the annual Mississippi Academy of Sciences Meeting at Biloxi, MS and the 9th annual Center of Research Symposium at Alcorn State University. 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 transgenic lines in sweetpotato with enhanced protein levels and viral resistance. CRISPR/Cas9 targeted mutagenesis for overexpression of sweetpotato NF-YC4 will be developed to increase protein levels and disease resistance in sweetpotato roots. PI, Co-PIs and collaborators will continue to arrange meetings to discuss plans for execution of the goals

Impacts
What was accomplished under these goals? We have been focusing on objective 1 in year 1: to generate Arabidopsis QQS and sweetpotato NF-YC4 transgenic sweetpotato to increase protein levels and disease resistance in sweetpotato roots via Agrobacterium-mediated transformation method. The constructs containing Arabidopsis QQS and sweetpotato NF-YC4 genes were constructed. An Agrobacterium-mediated transformation protocol was well-developed in the lab; an optimized regeneration protocol for line PI566648 was also developed. Selection antibiotic-resistant calli were obtained from sweetpotato line PI566648, we are inducing the regenerated transgenic plants now.

Publications

• Type: Journal Articles Status: Published Year Published: 2023 Citation: Lei Wang, Andrew J. Tonsager, Wenguang Zheng, Yingjun Wang, Dan Stessman, Wei Fang, Kenna E. Stenback, Alexis Campbell, Rezwan Tanvir, Jinjiang Zhang, Samuel, Cothron, Dongli Wan, Yan Meng, Martin H. Spalding, Basil J. Nikolau, Ling Li (2023). Single-cell genetic models to evaluate orphan gene function: the case of QQS regulating carbon and nitrogen allocation. Frontiers. Frontiers in Plant Science. 14:1126139. doi: 10.3389/fpls.2023.1126139
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Favour Afolabi, Yan Meng and Ling Li (2022). Utilizing novel biotechnology to develop new sweetpotato varieties with improved protein levels and broad-spectrum disease resistance. 8th Annual Center for Research Excellence Symposium, Lorman, Mississippi. Alcorn State University, Lorman, Mississippi
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Tymesha Nabors, Chunquan Zhang and Yan Meng (2022). Developing Agrobacterium-mediated transformation approach to improve the viral resistance in sweetpotato ((Ipomoea batatas L.). 8th Annual Center for Research Excellence Symposium, Alcorn State University, Lorman, Mississippi
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Yan Meng, Tymesha Nabors, Favour Afolabi and Chunquan Zhang (2022). Developing Agrobacterium-mediated transformation approach to improve the viral resistance and protein levels in sweetpotato (Ipomoea batatas L.). 86th Annual Mississippi Academy of Sciences Meeting, Biloxi, Mississippi
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Tatyana Hollingbird, Chunquan Zhang and Yan Meng (2023). Developing sweetpotato germplasms with increased protein levels and improved viral disease resistance. 87th Annual Mississippi Academy of Sciences Meeting, Biloxi, Mississippi
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Jacob Piazza and Yan Meng (2023). Optimization of plant regeneration protocol for selective elite lines in sweetpotato (ipomoea batatas (l.) Lam). 87th Annual Mississippi Academy of Sciences Meeting, Biloxi, Mississippi
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Alpha Jones, Chunquan Zhang and Yan Meng (2023). Using meristem-tip tissue culture technique to remove viruses from sweetpotato. 87th Annual Mississippi Academy of Sciences Meeting, Biloxi, Mississippi