Progress 03/01/19 to 02/29/24
Outputs
Target Audience:PI have presented research findings related to this project in workshop held in the University of FloridaUF/IFAS Protected Agriculture and Controlled Environment Forum. Students, postdocs, extension agents, and growers attended this meeting. During 01/2024-3/2024, we have trained a Ph.D. student, three undergraduate students, one SkillBridge Veteranintern from Florida Veterans Agriculture program. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?During the year of 2024, three undergraduate students, one Ph.D. one postdoc recieve training. Over this five years of project, we have trained 2 graduate students, 2 postdocs and 13 undergraduate students, 3SkillBridge Veteranintern from Florida Veterans Agriculture program. How have the results been disseminated to communities of interest?In 2024, PI has presented research work in the workshop held in UF"Protected Agriculture and Controlled Environment Forum"March 21 - Friday, March 22, 2024 During this project, we have presented our research findings to various stakeholders (extension agents, growers, research scientists, seed producers, retailers) through various channels such as newsletter, research articles, conference papers, extension events and open house demonstration. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
We have completed RNA sequencing of lsahg3 mutants and lettuce wildtype to compare the effect of loss of function in AHG3 gene on seed development and seed germination. Over this five years of project, we have created 17 transgenic or gene editted lines, we have discovered the different function of AHG3 and AHG1 in lettuce in regulation seed development, seed germinatio and flowering. We have published 10research articles, 1 book chapter, 25 conference abstracts/presentations withwith acholowdegement for this project.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2024
Citation:
Zhaoyuan Lian1,2, Jiang Tao2, Yufei, Liang1, Wanxing, Hu1, Huimin, Peng1, Hanghang, Zhang1?Haijun Gong1, Chunxiang You3, Guiluan Wang2,3* Li Liu4*, Heqiang Huo2*(2024)Auxin Affects Gene Editing Efficiency through Regulating Chromatin Accessibility and Plant Regeneration Process, Plant Physiology (Under review)
- Type:
Journal Articles
Status:
Under Review
Year Published:
2024
Citation:
Tao Jiang1, Fangchen Liu1,2, Keila Rodriguez1, Nicholas Rabanal1, Zhaoyuan Lian3, Mengfei Lin4, Haijun Gong3, Songhu Wang5, Heqiang Huo1,2 (2024), Genome-wide Analysis of the SPL Gene Family in Lettuce (Lactuca sativa) and Its Role in miR156-Mediated Plant Development, and Plant Regeneration, Horticultural Plant Journal (Under Review)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Zhaoyuan Lian1,2, Li Liu3, Haijun Gong2, Kede Liu4, Chunxiang You5, and HEQIANG HUO1 (2024) Characterization of Transposon-induced Snapdragon Mutants with Improved Anthocyanin Biosynthesis and Stress Tolerance. SIVB June 8 to June 12, St Louis, 2024
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Progress 03/01/23 to 02/29/24
Outputs
Target Audience:The target audience include vegetable breeders from private and public sectors, horticultralists, research scientists and professionals from academic and industry. Additionally, gradaute students, undergraduate students, postdoctoral researchers are educational targets, who recieved training from this project. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?On Ph.D. student, 3 undergraduate students, and one postdoc recived training under this project. How have the results been disseminated to communities of interest?Yes, the results from this project have been disseminated to research communities through multiple professional conferences. What do you plan to do during the next reporting period to accomplish the goals?Extension applicaiton will be followed to deeply understand the distinct funciotn of both AHG1 and 3 in controlling flowering and seed dormancy.
Impacts
What was accomplished under these goals?
Creating transgenic lines was an integral part of the project to elucidate the molecular mechanisms underlying lettuce seed germination and flowering time. Through many obstacles such as different flowering time and environmental stress due to Florida's climate, we have finally succeeded in obtaining the necessary lines including overexpressing, silencing, and genetic crosses of microRNA 156 and 172, DOG1, and AHG1/3. These transgenic lines will facilitate in-depth studies exploring the functional interplay between microRNA156/172, DOG1, and AHG1/3, shedding light on their roles in response to environmental cues and developmental processes. Thus, these lines hold promise for enhancing our understanding of plant adaptation to diverse environmental conditions and may contribute to the development of improved lettuce varieties with enhanced stress tolerance and optimized flowering traits.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Jiang TP, Rodriguez EU, Norman D, Luo Y, Sandoya G, Huo HQ (2023) Enhancing Postharvest Quality and Environmental Resilience in Lettuce through Manipulating miR164 and SGR1, Annual meeting of American Society of Horticultural Science, Orlando, FL, July 31-August 4,2023.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Jiang TP, Rodriguez KU, Kopsell D, Huo HQ (2023) Improving Lettuce Organ Size and Postharvest Quality through Editing of OSG1 and OSG2 Genes Annual meeting of American Society of Horticultural Science, Orlando, FL, July 31-August 4, 2023.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2023
Citation:
Jiang TP, Rodriguez EU, Norman D, Luo Y, Sandoya G, Huo HQ (2023) Manipulating miR164 and STAY-GREEN 1 to Mitigate Environmental Effect on Lettuce Postharvest Quality. Annual meeting of Society of In-Vitro Biology, Norfolk, VA, June 10-14, 2023.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2023
Citation:
Jiang TP, Zhao Hg, Kim J, Huo HQ (2023) Auxin transport is required for mir156-induced de novo root regeneration in detached lettuce leaves. Plant Animal Genome, San Diego, CA, January 13-18, 2023.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2023
Citation:
Huo HQ (2023) Application of morphogenic regulators for in planta and in vitro transformation in multiple plant species. Plant Animal Genome, San Diego, CA, January 13-18, 2023.
|
Progress 03/01/22 to 02/28/23
Outputs
Target Audience:PI have presented research findings related to this project in several scientifc conferences including Plant Animal and Genome, American Society of Horcitultural Sciences. The primary target audiences include vegetable breeders from private and public sectors, horticultralists, research scientists and professionals from academic and industry. Changes/Problems:Obstacles and Solutions -Lettuce suffered poor pollination when they were grown in the greenhouse during the summer in Florida. Only a small amount of seeds (~10-20 seeds/plant) were collected and regrown during the winter. à Started seed sowing earlier in September instead of November for the maturation and flowering time to be during the cooler season in Florida to increase seed production. -Although lettuce can flower continuously and their flowering duration can be up to one month, the strong delayed flowering phenotype in some transgenic lines prevented genetic crossing. à Grow the delayed flowering transgenic lines 1 month earlier than the early flowering transgenic lines to align the flowering time. At the same time, the CRISPR mutant seeds were used for plant transformation using vectors MIR172 and STTM156 to acquire complementary lines. -High temperature and high humidity in Florida caused many pest problems (mostly insects and fungus) in the greenhouses and affected plant development and health à Avoid growing plants in the greenhouses during the high-temperatures months and only grow them during the cooler season. -AHG1 and AHG3 each have two homologs in lettuce and CRISPR knockout only happened in one homolog for one line and the other homolog in another line. àTo ensure complete knockout of AHG1 and AHG3, the lines were crossed to get homozygous mutation. What opportunities for training and professional development has the project provided?Two undergraduate students (Keila Rodriguez, Hispanic female, and Nicholas Rabanal male) from University of Central Florida, two undergraduate students (Audrey Tatman, female; Michael Lerer male) from Valencia College, two veteran (Kevin White from U.S. Marine Corps, and David Cepero from U.S. Coast Guard) from SkillBridge Veterans Florida Ag Program, two Ph.D. students (Chi Nguyen and Zhaoyuan Lian) and one postdoc (Dr. Tao Jiang) were trained throught this project. The Ph.D. student Chi Nguyen and the postdoc Dr. Tao Jiang made major contributions to this project. These students and scholars also gained professional experience by presenting their work at national and international meetings. How have the results been disseminated to communities of interest?The primary dissemination of our results has been through publications and presentations at scientific meetings including . In addition, we We have presented research results and developed methods in 2022 ASHS, 2023 PAG Conferences, two invited webinars by Elolife, Ball Horitculture, In addition, we made numerous presentations to visitors and at other institutions to make these stakeholders aware our results.The presentations of our research findings have led to recognication by different research groups. As a result, the PI of this project has been invited by two biotechnology companies such as Elolife, Ball Horitculture, Plant Animal Genome and The Israeli Center for Genome Editing (GE) in Agriculture, Pensivinnia State University. Additionally, the student sponsored by this project has received the following awards 2022 Edgar A. Martin Scholarship 2022 Sweetwater Oaks Scholarship Chair-elect for SSEST Professional Group, American Society for Horticultural Science (ASHS) What do you plan to do during the next reporting period to accomplish the goals?The following are what we will focus next: 1) Advance transgenic lines to T3 generations Cross overexpression/silencing lines with AHG1/AHG3 mutant. Grow double transformation lines in the greenhouses. Record flowering time of all the lines. 2)Collect seeds from transgenic lines including double transformation lines. Prepare space and materials to pot the transgenic lines in cooler maternal environments. 3)Grow various plants under cooler maternal environments to check the responses of seeds and collect samples for small RNA sequencing and RNA sequencing 4) Collect morphological and physiological data of the transgenic lines under the cooler maternal environments. Analyze RNA sequencing data and design experiments for seed germination test 5) Characterization of seed performance under different treatments, measuring the expression of relevant genes 6) Grow various plants under high maternal environments to check the responses of seeds and collect samples for small RNA sequencing and RNA sequencing, data analysis and manuscript preparation 7) Characterization of seed performance under different treatments, measuring the expression of relevant genes, data analysis, and manuscript preparation 8)Sequencing data analysis and manuscript preparation
Impacts
What was accomplished under these goals?
Transgenic plants overview -The transgenic lines and their current status are as followed: Overexpression 1. pgwb423-AHG1-ox (2x35S:LsAHG1)à T3 plants in greenhouse, early flowering phenotype 2. pgwb402-MIR156-ox (35S:LsMIR156) àT3 plants in greenhouse, delayed flowering phenotype 3. pgwb402Ω-MIR172-ox (2x35S:LsMIR172)à T3 seeds germinated Silencing 1.POX135-STTM172 (2x35S:STTM172) à T3 plants in greenhouse, delayed flowering phenotype 2.pGSA1403-STTM156 (3x35S: STTM156)à T3 plants in greenhouse, early flowering phenotype 3. pGSA1165-RNAiDOG1 (35S: RNAiDOG1)à T4 plants in greenhouse, early flowering phenotype CRISPR 1.PHN-C2X-AHG1 (Cas9-AHG1)à Gene edited confirmed with sequencing, T3 plants in greenhouse, no delayed flowering 2.PHN-C2X-AGH3 (Cas9-AGH3)à Gene edited confirmed with sequencing, T3 plants in greenhouse, delayed flowering phenotype Research Data Not all plant materials were available for testing, the following data were obtained using the readily available plant materials to understand the phenotype of the transgenic lines. Seed germination test -Seeds from the transgenic lines listed above were placed on filter paper with water only, and they have the same germination rate as the WT except for CRISPR-AHG3 mutant lines. The AHG3 mutant seeds showed delayed germination on water when compared to the WT, -Although AHG1 mutant did not have delayed germination on water when compared to WT; when treated with 150mM NaCl, AHG1 mutant showed significant delayed in germination compared to WT. These data suggested that AHG3 may play a key role in regulating seed dormancy/germination in lettuce. Seedlings Salinity Stress Test -Four weeks old AHG1 and AHG3 mutants were treated with 1g of NaCl per Liter of soil for 2 weeks to determine their susceptibility to salinity stress. However, after 14 days, there were no significant differences in the mutants and WT based on morphological and electrolyte leakage measurements. Flowering Phenotype in the transgenic plants -Transgenic lines that supposedly have delayed flowering phenotype including AHG1/AHG3 mutant, MIR156-ox, and STTM172 were sowed in September (1 month earlier than other transgenic lines) to match the early flowering lines (STTM156, MIR172, AHG1-ox, and DOG1-RNAi) for genetic crossing between the mutant and the overexpression/silencing lines. However, it was observed that AHG1 mutant did not show a delayed flowering phenotype while the AHG3 mutant showed delayed flowering as well as MIR156-ox and STTM172. Transgenic lines DOG1-RNai, STTM156, AHG1-ox were sowed in October and currently have early signs of bolting and are expected to flower earlier compared to the wild type. Additional phenotypes in the transgenic plants -Besides flowering phenotypes, plant size was noticeably different between transgenic lines and WT, where AHG3 mutant and MIR156-ox were significantly smaller than the WT. Obtaining overexpression/silencing/complementary transgenic lines is an integral part of the project and as we overcome different obstacles along the way, the acquisition of the necessary lines is not far away. There are still many experiments to conduct, data to analyze, and molecular mechanisms to connect. The continuation of this project will uncover a network that is critical for seed germination and flowering in lettuce. Both of these developmental stages are especially sensitive to high temperatures and information is urgently needed to produce lettuce that can combat climate change.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Jiang TP, Zhao Hg, Kim J, Huo HQ (2023) Auxin Transport is Required for miR156-Induced De Novo Root Regeneration in Detached Lettuce Leaves, 2023,Plant Animal Genome, San Diego, California., January 13-18, 2023.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2023
Citation:
Huo HQ (2023) Application of Mophorgenic Regulators for in Planta and in Vitro Transformation in Multiple Plant Species, Plant Animal Genome, San Diego, California., January 13-18, 2023.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Jiang TP, Zhao Hg, Kim J, Huo, HQ (2022) Auxin Transport May be Required for miR156-Induced De Novo Root Regeneration (DNRR) in Detached Lettuce Leaves, Chicago, Illinois, July 30 to August 3, 2022.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Lian ZYG, Huo HQ (2022) Application of Mophorgenic Regulators for in planta and in vitro Transformation in Multiple Plant Species, 9th international Horticulture Research Conference, Wuhan, China, November 20-23, 2022.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Lian ZYG, Nguyen CDG, Wang GP, Chen JJ, Huo HQ (2022) Improvement of In-Planta or In Vitro plant Transformation by Application of Developmental Regulators, Chicago, Illinois, July 30 to August 3, 2022.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Wang GP, Deng Z, Huo HQ (2022) T-DNA-Free Gene Editing through Transient Suppression of the POLQ Gene in Plants, Annual meeting of The Society for In Vitro Biology (SIVB), San Diego, CA, June 4-7, 2022
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Lian ZYG, Nguyen CDG, Wang GP, Chen JJ, Wilson S, Ozias-Akins P, Huo HQ (2022) Application of Developmental Regulators to Improve In-Planta or In Vitro Transformation in Plants, Annual meeting of The Society for In Vitro Biology (SIVB), San Diego, CA, June 4-7, 2022
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Nguyen CDG, Creech M&, Xiang DY&, Sandoya G, Kopsell, D., Huo H (2022) Performance of Different Lettuce Cultivars Grown Hydroponically under Fluorescent and Light-emitting Diode Light Growth Conditions, HortScience, 57(11), 1447-1452
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
7. Lian ZYG, Nguyen CDG, Liu L, Wang GLP, Chen JJ, Wang SH, Yi GJ, Wilson S, Gong H and Huo H (2022) Improved Genetic Transformation by Applying Developmental Regulators through Agrobacterium Injection of Adult Plants or In-vitro Tissue Culture, Plant Biotechnology Journal 20(8),1622-1635
|
Progress 03/01/21 to 02/28/22
Outputs
Target Audience:The target audience for this research includes academic leafy vegetable researchers, public and commercial leafy vegetable breeders, vegetable seed companies, extension personnel, leafy vegetable growers with research interests in environmental effect on seed quality and plant bolting in the southern U.S. and subtropical regions in the world. Changes/Problems:Some research activities were partially suspended due to the the spread of new covid virus in 2021; Despite the great progress made in 2021, implementation of some experiments was delayed. What opportunities for training and professional development has the project provided?As proposed in the grant proposal, the PI provided excellent training opportunities for the postdoctoral associate ( Dr. Tao Jiang, Dr. Grace Wang)and one Ph.D student (Ms. Chi Nguyen)through this project. In 2021, this project also provided opportunities for two undergraduate intern students (Zuriama Hernandez and Matt Davis) from a minority college "Valencia College-Lake Nona", and a master studentFahima Alam from University of South Florida. the Ph.D student supported by this grant has recevied 3 awards in 2021 1. National Association of Plant Breeders (NAPB) Borlaug Scholar, 2021 2. Sidney B. Meadows Scholarship Fund, Arthur A. "Buck" Jones Scholarship, 2021 3. University of Florida's Department of Environmental Horticulture Scholarship, 2021 How have the results been disseminated to communities of interest?The primary dissemination of our results has been through publications and presentations at scientific meetings. We have presented research results and developed methods in 2021 ASHS, 2021Independent Plant Breeders' Conference, 2021 ASHS webinar,2021 Vitrual Garden Club webinar. 2021 Virtual meeting National Association of Plant Breeders (NAPB) (Student presentation); and one extension conference Annual Meeting of The Florida State Horticultural Society. What do you plan to do during the next reporting period to accomplish the goals?We will continue our efforts to 1) perfom phenotypic evaluation of different transgenic lines2) advance the double mutants to homozygous lines;3) test the plant responses to environmental stresses. 4) understand the molecular mechanisms of regulating flowering and seed germination under temperature stress.
Impacts
What was accomplished under these goals?
To understand the genetic and molecular mechanisms underlying the regulation of seed thermoinhibition and early flowering by temperature in lettuce, we have proposed three objectives for this project. Objective 1: Elucidate the Role of DOG1-AHG1/3 in regulating miRNA production; Objective 2: Determine Temperature Effects on Regulation of Seed Maturation and Dormancy by the DOG1-AHG1/3-miRs Module; Objective 3: Determine Temperature Effects on Regulation of Flowering by the DOG1-AHG1/3-miRs Module. In 2021, we have advanced the following transgenic lines Overexpression lines 1. pgwb423-AHG1-ox (2x35S:AHG1) was advanced to T2 generation;2. pgwb402-MIR156-ox (35S:MIR156)was advanced to T2 generation;3. pgwb402Ω-MIR172-ox (2x35S:MIR172) was advanced to T2 generation. Noticeable phentypic differences were observed between overexpression lines and wild type under high temperature greenhouse. Silencing lines: 1.POX135-STTM172 (2x35S:STTM172) was advanced to T2 generation;2.POX135-STTM156 (3x35S: STTM156) was advanced to T2 generation;3. pGSA1165-RNAiDOG1 (35S: RNAiDOG1)was advanced to T4generation. CRISPR Genome Editing lines 1.PHN-C2X-AHG1 (Cas9-AHG1)was adavanced to Cas9 Free T2 plants;2.PHN-C2X-AHG3 (Cas9-AGH3) was advanced to Cas9 Free T2 plants Besides the main objectives that I proposed, additional projects were implemented with part supports from this award.' Project 1: Anther Regeneration Test comparison between Salinas WT and miR156 Overexpression Lines Goal: To use lettuce anther to develop instant homozygous inbred lines and avoid lengthy breeding process. Objectives: 1.Evaluate different media compositions for anther regeneration in vitro. 2.Determine the correct flowering stage to use for anther regeneration. 3.Compare anther regenerative capabilities between Salinas WT and miR156 overexpression lines. Objective 1 and 2: Tested 3 different medium and 2 stages of lettuce flower bud to determine the best media for anther regeneration protocol Protocol: Stage 3 and 4 of lettuce flower bud were cut in the morning, sterilized with bleach and tween20, washed with sterilized water, placed on ice while cutting the anthers out of the buds, placed anthers on one of the media below (MS1, MS2, or MS3) , and placed plates at 35C for 7 days in the dark, then placed plates under light for 5 more days, then moved to regeneration media (R) until callus and new shoots come out then place on rooting media (MS) only. Set 1,2, and 3 are anthers at stage 4, long bud with tight closed end. I believe the buds with open end are past the uninucleate stage and would be too late to use for regeneration. -Observation: the anthers after 35C treatment became soft and flaccid and maintained it shape, this was different from the preliminary trial (maybe because media was too wet when the anthers were placed on them?). The preliminary trial anthers were also soft but not watery and the anthers seemed to split apart. The callus for this stage is mostly white callus surrounding the anthers with some green callus now and then. Set 4,5, and 6 are anthers at stage 3, the buds that started to get long and big enough to cut to get the anthers out. Noticed the colors are half green, half yellow. Really young buds have completely green anthers and older buds have completely yellow anthers. This is the stage where they are making a transition? But the anthers are slightly yellow with the tip being green. -Observation: The anthers after 35C treatment maintained it shape and harden the tissue (can feel the hardness when touched it with a tweezer). The callus seemed to "pop" out of the anther and it is fully green callus condensed into a ball. Noticed watery tissue on callus, will move them to Shoot elongation media like lettuce transformation regeneration media (0.05mg/L NAA and 0.01mg/L BAP) to see it can help with shooting as well as watery callus. Set 7 and 8 are anthers at stage 3, but they are not heat treated at 35C. They were placed in the dark at 25C for 7 days and put in light for 4 days then moved to R media like the rest of the set before. Result: Stage 3 and 4 lettuce buds were chosen for media test due to their size and found that at stage 3 on MS1 media, green calluses were formed while stage 4 on all media turned pale and did not develop any further. Conclusion: Stage 3 lettuce buds will be used on MS1 media for anther regeneration test Objective 3: Comparing stage 3 WT lettuce buds and miR156-ox on MS1 media After putting on R media, the callus formed nicely. However, long incubation on R media prevented shooting and caused watery callus (most likely due to high concentration of BAP). Therefore, when the callus is big enough, the callus need to be moved to MSS media with lower concentration of BAP and some NAA for shooting and shoot elongation. Result: Although green calluses and tiny shoots were seen on both WT and miR156-ox anthers on regeneration media MSS, they were not able to grow any further and no whole plants were regenerated. Project 2 : miR156 involved auxin-Induced de novo root regeneration (DNRR) via mediating polar auxin transport (PAT) in lettuce miR156 is required for auxin-induced DNRR in plants, however, fewstudies examine whethermiR156 affectsthe auxin biosynthesis or transport or both. we have used the transgenic plant materials to dissect the mechanism of miR156-induced root regeneration. Results of this project : The adventitious rooting ability dramatically increased in miR156OX (miR156 over-expression lines) of lettuce leaf compared to the wide type, while decreased in STTM-miR156 (miR156 suppressed lines), which are confirmed by histological analysis. Interestingly, miR156-induced rooting could be inhibited by application of N-1-naphthylphthalamic acid (NPA), a most widely used auxin transport inhibitor, whereas application of L-Kynurenine (Kyn), one auxin biosynthesis inhibitor, has no such inhibitory effect. Project 3:miR156 promotes plant regeneration through in-vitro tissue culture in lettuce As reported, two phytohormones, auxin and cytokinin, play critical roles in the process of plants regeneration using different types of explants. Although plants aretotipotent and competent, and de-novo shoot could regenerate from these in-vitro culture explants, the regenerative capacity decreases with developmental age of explants, andthe molecular basis for this phenomenon remains elusive. Results of this project : overexpression of miR156 significantly promote shoot regeneration when lower concentration of auxin and cytokinin was applied to tissue culture. In contrast, suppression of miR156 decreased the regeneration capaicity of miR156-STTM silencing line relative to the wild type. Addtionally, expressing 156 can minimize the negative effect of aging on de-novo shoot regeneration. We have observed that aged leaf explants from miR156 overexpression lines but not wild type and miR156-STTM silencing line exhibited strong shoot regeneration capacity.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Nguyen CD, Li JC, Mou BQ, Gong HJ, Huo HQ (2021)A case study of using an efficient CRISPR/Cas9 system to develop variegated lettuce, Vegetable Research 1 (1), 1-10
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Nguyen CD, Chen JJ, Clark D, Perez H, Huo HQ (2021) Effects of Maternal Environment on Seed Germination and Seedling Vigor of Petunia� hybrida under Different Abiotic Stresses. Plants 10 (3), 581
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Nguyen CD, Chen JJ, Clark D, Perez H, Huo HQ (2021) Effects of Maternal Environment on Seed Germination and Seedling Vigor of Petunia� hybrida under Different Abiotic Stresses. Plants 10 (3), 581. 2021 Annual ASHS Meeting of American Society of Horticultural Science, Denver, Colorado August 5-9, 2021.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Nguyen CD, Li JCv, Mou BQ, Gong HJ, Huo HQ (2021)A case study of using an efficient CRISPR/Cas9 system to develop variegated lettuce, 2021 Annual ASHS Meeting of American Society of Horticultural Science, Denver, Colorado August 5-9, 2021.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Huo HQ (2021) Efficiently manipulating genes for plant genetic improvement, 2021 Independent Plant Breeders Conference, Gainesville, Florida.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Huo HQ, Development of Stress-Resilient Horticultural Plants using Advanced Breeding Tools 2021 Annual ASHS Meeting of American Society of Horticultural Science, Denver, Colorado August 5-9, 2021
- Type:
Other
Status:
Published
Year Published:
2021
Citation:
Huo HQ, (2021) Breeding of Horticultural Plants with Biotechnology Tools, ASHS Webinar, September 22, 2021.
- Type:
Other
Status:
Published
Year Published:
2021
Citation:
Huo HQ, (2021) Breeding of Horticultural Plants with Biotechnology Tools, Florida Master Garden Club, October 13th, 2021.
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Progress 03/01/20 to 02/28/21
Outputs
Target Audience:The target audience for this research includes academic researchers, public and private leafy vegetable breeders, vegetable seed companies, extension personnel, leafy vegetable growers who are intrested in envrionmental effect on plant development and growth. The PI and one postdoc supported by this project participated in 2020 annual conference for American Society of Horticultural Sciences ; Approximately 1500 scientists, growers, and seed company representatives attended the 2020 ASHS vitro meetings. The graduate student Chi Nguyen sponsored by this project received UF-CALS graduate fellowship award and the National Horticultural Foundation Fellowship award. Changes/Problems:The development and evaluation of plant materials are highly dependent on seasonal climate, thus the pandemic outbreak in 2020 has significnatly delayed the implmentation of this project. What opportunities for training and professional development has the project provided?As proposed in the grant proposal, the PI provided excellent training opportunities for the postdoctoral associate and one Ph.D student through this project. In addition, this project also provided opportunities for three undergraduate intern students (Roni Phillips,Tristan Dunzelman, Alexander Moritz-Long) from the underrepresented local college "Valencia College-Lake Nona". The undergraduate students were trained in PI's lab on agricultural biotechnology such as tissue culture, genetic transformation, maintaining genetic plants, physilogical and mrophylogical measurements, etc. After recevied training from PI's team, Alexander Moritz-Long is inspired to pursue higher degree in plant science. How have the results been disseminated to communities of interest?The primary dissemination of our results has been through publications and presentations at scientific meetings. For examples, we have presented research results and developed methods in 2020 ASHS, and FSHS (Florida State Horticultural Society) and EPAF (Extension Professional Associations of Florida). In addition, PI was invited to give vitual lecture on plant breeding with advanced agricultural biotechnonogy to National Gardener Club and Vallencia College. What do you plan to do during the next reporting period to accomplish the goals?We will continue our efforts to 1) Genotype and phyentopye the genetic plant materials that we have developed, 2) to generate double mutants through crossing and perform functional characterization;3) test the plant responses to environmental stresses; 4) perform transcriptome analysis to understand the regulatory pathway.
Impacts
What was accomplished under these goals?
1) we have construct the following plasmid DNA as proposed in our proposal, Overexpression: 1). pgwb423-AHG1-ox (2x35S:AHG1), 2). pgwb402-MIR156-ox (35S:MIR156); 3) pgwb402Ω-MIR172-ox (2x35S:MIR172); 4) pgwb414-DOG1-ox (35S:DOG1). Silencing: 1).POX135-STTM172 (2x35S:STTM172); 2).pGSA1403-STTM156 (3x35S: STTM156); 3). pGSA1165-RNAiDOG1 (35S: RNAiDOG1).CRISPR: 1).PHN-C2X-AHG1 (Cas9-AHG1); 2).PHN-C2X-AHG3 (Cas9-AGH3); 3). AHG1+AHG3. The T0 plants are currently flowering in the greenhouse. 2) Overexpression of MIR156 and Silencing of MIR172 showed strong delayed flowering phenotype in T0 plant while other lines are flowering normally. 3) Overexpression of LsMIR156 resulted in enhanced rooting in lettuce. We observed that overexpression of LsMIR156 promotes rooting in lettuce. Increasing root amount and early rooting were observed from lettuce cuttings. By contrast, suppression of miR156 lines affect root generation, resulting in fewer roots and late rooting. Auxin-related genes (LsAUXs, LsPINs, LsIAAs, LsARFs, LsGH3s, LsTOR) were hypothesized to be invovled in the enhanced rooting of miR156ox lettuce, and will be further examined in the miR156ox and MIR156-STTM lettuce. 4) Organ size is regulated by both genetic and environmental factors in plants. We observed that there was an increase in both seed size by 31.4% and seed weight by 43.8%, respectively, in miR156ox line; while LsmiR156 suppression lines have smaller seeds. Interestingly, the leaf size in Sal-LsMIR156ox shrink significantly with more branches and leaves. Another microRNA, miR172, has an inverse effect in seed size regulation, with a 42.1% decrease in seed size of LsMIR172 overexpressed lines. 5) additional genetic lines such as gene editing of DA1 and EOD1 are under delopmentment to understand whether there is any link between these size regulator and miR156.
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2020
Citation:
1. Sadhukhan AP, Huo HQ (2020) Improvement of floriculture crops using CRISPR/Cas genome editing techniques, in CRISPR/Cas Genome Editing - Strategies And Potential For Crop Improvement , pp69-90, Eds Anjanabha Bhattacharya, Vilas Parkhi and Bharat Char
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Baz Hu, Creech M, Chen JJf, Bradford KJ, Huo HQ, (2020) Water-Soluble Carbon Nanoparticles Improve Lettuce Seed Germination under Salinity Stress, Agronomy 10(8):1192
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Lian ZYG, Wilson Sf, Chen JJf, Huo HQ, (2020) Efficient Plant Regeneration and Agrobacterium-Mediated Genetic Transformation of Antirrhinum majus, Plant Cell, Tissue and Organ Culture,142(3):527-536
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
6. Xiang DYP, Nguyen CG, Fletcher Lf., Clark Df, Huo HQ (2020) Effects of preharvest LED light, melatonin and AVG treatments on the quality and vase life of postharvest cut snapdragons, Flower and Landscaping Journal 6:14-19
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Xiang DYP, Nguyen CG, Clark Df., Huo HQ (2020) Effects of Melatonin and AVG in alleviating dark-induced plant deterioration of three Pilea species, The Horticulture Journal 89(5):609-618
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
1. Jiang TP, Nguyen CG, Michelmore R. Bradford K, Huo HQ (2020), miR156 Plays Crucial Roles in Temperature-Dependent Regulation of Seed Thermoinhibition and Flowering in Lettuce (Lactuca sativa. L), 2020 Annual ASHS Meeting of American Society of Horticultural Science, Orlando, Florida, August 9-13, 2020.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
4. Lian ZYG, Wilson S. Chen JJ, Huo HQ (2020) Efficient Plant Regeneration and Agrobacterium-Mediated Genetic Transformation of Antirrhinum majus, 2020 Annual ASHS Meeting of American Society of Horticultural Science, Orlando, Florida, August 9-13, 2020.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
5. Baz Hu, Bradford KJ, Chen JJf, Creech M, Huo HQ, (2020) Water-Soluble Carbon Nanoparticles Improve Lettuce Seed Germination under Salinity Stress, 2020 Annual ASHS Meeting of American Society of Horticultural Science, Orlando, Florida, August 9-13, 2020.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
6. Creech M., Baz Hu, Plontke Au and Huo HQ (2020) An Improved Protocol for Efficient In-Vitro Micro-propagation of Two Blueberry Cultivars. 2020 Annual ASHS Meeting of American Society of Horticultural Science, Orlando, Florida, August 9-13, 2020.
- Type:
Other
Status:
Published
Year Published:
2020
Citation:
Invited Extension Talk in 2020
Huo, HQ, Breeding Garden Plants with Hybridization, National Gardener Club, October 27 2020 (59 audience)
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Progress 03/01/19 to 02/29/20
Outputs
Target Audience:The target audience for this research includes academic leafy vegetable researchers, public and commercial leafy vegetable breeders, vegetable seed companies, extension personnel, leafy vegetable growers with research interests in environmental effect on seed quality and plant bolting in the southern U.S. and subtropical regions in the world. The PI and one graduate student (Chi Nguyen)supported by this project participated in two research conferences; one is annual conference for American Society of Horticultural Sciences held at Las Vegas, Nevada, July 21-25 2019; another one is annual conference for Society of In Vitro Biology held in Tampa, Florida, June 8-12, 2019. Approximately 2500 and 400 scientists, growers, and seed company representatives attended 2019 ASHS and 2019 SIVB meetings, respectively. The graduate student Chi Nguyen developed an efficient genome editing method for lettuce was awarded for travel grant from ASHS and SIVB. Her work on lettuce genome editing sponsored by this USDA-NIFA project also earned her a 3rd place for her oral presentation competition from SIVB. In addition, a lab technician Matthew Creech gave an oral presentation at 131st Annual Meeting of The Florida State Horticultural Society, Orlando, FL June 9-11, 2019. Changes/Problems:I was challenged by the retaining the senior research personnel last year. One talented postdoc Dr. Ayan Sadhukhan was initially successfully recruited from India, however he had to quit and went back India in the end of last October due to his severe illness. However, during his stay with us, Dr. Ayan Sadhukhan and I drafted a book chapter which is in press. After his leaving, there was a 3-month work gap between October 2019 and February, 2020. Moreover, development of some transgenic plant materials have been greatly delayed due to Coronavirus pandemic and lockdown of our institutions, although some progresses like plasmid DNA constructions and tissue culture preparation have been made. Given this circumstance and depending on the progress of our project, we may request a year of non-cost-extension in the future. What opportunities for training and professional development has the project provided?As proposed in the grant proposal, the PI provided excellent training opportunities for the postdoctoral associate and one Ph.D student through this project. In addition, this project also provided opportunities for two undergraduate intern students (Hanna Baz and Abigail Plontke) from a minority college "Valencia College-Lake Nona". One of them will soon publish her first paper about lettuce seed germination. How have the results been disseminated to communities of interest?The primary dissemination of our results has been through publications and presentations at scientific meetings. For examples, we have presented research results and developed methods in 2019 ASHS, 2019 SIVB and one extension conference Annual Meeting of The Florida State Horticultural Society. In addition, we have showcased more than 220 visitors from local high school, minority colleges, vegetable growers and nurseries in year of 2019. For example, I showcased to educate >150 local extension agents and growers in our plant health seminar held in our center on December 3rd, 2019. What do you plan to do during the next reporting period to accomplish the goals?We will continue our efforts to 1) develop transgenic materials that are essential for accomplishing this project, 2) crossing transgenic or mutants to develop double genetic lines 3) collect transcrit expression data 4) evaluate phenotypes if possible.
Impacts
What was accomplished under these goals?
To understand the genetic and molecular mechanisms underlying the regulation of seed thermoinhibition and early flowering by temperature in lettuce, we have proposed three objectives for this project. Objective 1: Elucidate the Role of DOG1-AHG1/3 in regulating miRNA production; Objective 2: Determine Temperature Effects on Regulation of Seed Maturation and Dormancy by the DOG1-AHG1/3-miRs Module; Objective 3: Determine Temperature Effects on Regulation of Flowering by the DOG1-AHG1/3-miRs Module. In our grant proposal, we have proposed to implement the majority of objective 1 in the year 1 and another two objectives rely on the development of transgenic materials that will be developed in year 1 and year 2. Progress: We have performed RACE to clone the AHG1 gene from lettuce cv "Salinas". The AHG1 was inserted and fused into the Gateway vector with PGWB417 or PGWB517 and fused with a 35S promoter and myc tag at 3' terminal end. The myc tag will enable us to perform immunoprecipitation pull-down assay. These two overexpression constructs have been transformed into Salinas lettuce and Arabidopsis dog1-5-Ls156 mutants, but no transgenic plants have been generated. We have created two CRISPR-Cas9 constructs to knock out the LsAHG1 and LsAHG3. We have identified two copies of LsAHG1 and two copies of LsAHG3. We picked up guided RNA sequences to target all copies of LsAHG1 or LsAHG3 separately. AHG3 is presumably involved in the both DOG1 and ABA signaling pathways, while AHG1 is predominately involved in the DOG1 signaling pathway. Single mutants LsAHG1 or LsAHG3 will allow us to elucidate the mechanism easily. Double mutants will be generated through crossing. LsMIR172-STTM: we have generated a construct to suppress LsMIR172 in the Salinas lettuce. A LsMIR172-STTM PI lettuce line has been previously generated, interestingly, the preliminary data showed that more branching but not early flowering was oberserved in this PI-LsMIR172-STTM line. Unlike the "Salinas" lettuce, the PI line exhibited photoperiod-insensitive flowering type, which may explain that suppression of miR172 in this lettuce did not greatly promote flowering. A duplicated experiment is ongoing to confirm our observation. LsMIR172ox: we have generated LsMIR172-overexpression Salinas lettuce, our preliminary data suggested that these plants exhibit early flowering phenotype and better germination at high temperature. Genome editing of miR172: we have completed a construct to knock out the miR172 using genome editing. Transformation will be performed soon.
Publications
- Type:
Book Chapters
Status:
Awaiting Publication
Year Published:
2020
Citation:
Ayan Sadhukhan and Heqiang Huo, Chapter 4: Improvement of floriculture crops using CRISPR/Cas and genome editing techniques in CRISPR/Cas Genome Editing - Strategies And Potential For Crop Improvement, editor: Anjanabha Bhattacharya, Vilas Parkhi and Bharat Char, Springer, In press.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2020
Citation:
Zhaoyuan Lian, Chi D Nguyen,Sandra Wilson, Jianjun Chen, Haijun Gong,Heqiang Huo,An Efficient Protocol for Agrobacterium-Mediated Genetic Transformation of Antirrhinum majus, Plant Tissue Cell and Organ Culture, under review
- Type:
Journal Articles
Status:
Submitted
Year Published:
2020
Citation:
Water-Soluble Carbon Nanoparticles Improve Lettuce Seed Germination under Salinity Stress
Baz,H.,Creech M., Liu, L., Chen, JJ, Bradford, K. Huo,HQ, Journal of American Society of Horticultural Sciences, submitted
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Nguyen LD, Li JC, Mou BQ, Huo HQ,Development of Variegated Lettuce Using CRISPR/Cas9 Technology, annual conference for American Society of Horticultural Sciences held at Las Vegas, Nevada, July 21-25 2019
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