DECIPHERING THE GENETIC MECHANISMS UNDERLYING GENOTYPE-DEPENDENT SOMATIC EMBRYOGENESIS AND PLANT REGENERATION RESPONSE IN MAIZE (ZEA MAYS L.)

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1020442
• Project Start Date: Oct 1, 2019
• Project End Date: Sep 30, 2023
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218

Performing Department
Agronomy

Non Technical Summary
In order to meet global food, feed, fiber and biofuel needs in the face of climate change and predicted population growth, crop improvement programs may choose to utilize tissue culture-based approaches to clonally propagate and/or genetically enhance crops via genetic engineering or gene editing. When using those approaches, the ability to regenerate plants from tissue culture is critical for success. Many genotypes and varieties of crop plants are unable, however, to form the optimal, embryogenic, regenerable culture type that is ideal for plant regeneration. This results in significant impediment of progress in tissue culture-based crop genetics research and enhancement. Research is being conducted in the locally and globally important crop, maize (corn), to decipher the genetic mechanisms underlying the ability to form embryogenic, regenerable tissue cultures. New knowledge generated from this research will contribute to a better understanding of the basic biological processes involved in formation of somatic (clonal) embryos in plants, and can aid in the development of crop germplasm with improved regeneration ability and the design of enhanced tissue culture systems for crops that can be applied to a wider array of germplasm within those crops. In addition to generation and dissemination of new, valuable information, novel germplasm will be developed as part of this research, including regenerable, transformable forms of maize inbred line, B73, an important line in maize breeding and genetics research, and informative genetic mapping population lines that will be made available for broader utilization in maize genetics research and breeding.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	1510	1080	100%

Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1510 - Corn;

Field Of Science
1080 - Genetics;

Keywords

corn

maize

plant genetic engineering

plant tissue culture

crop biotechnology

crop improvement

somatic embryogenesis

plant genome editing

Goals / Objectives
The major goalof this project isto decipherthe molecular genetic mechanisms controlling in vitrosomatic embryogenesisand plant regeneration response in maize, and to use the knowledge and germplasm generated from the research to aid in the development of efficient, genotype-independent crop propagation, transformation and gene-editing systems. An additional goal of the research is totrain graduate students on the projectto become the next generation of plant breeders, geneticists, and genome engineers/editors in both the public and private sectors.Specificresearch objectives of the project areto genetically dissect a major Quantitative Trait Locus (QTL) associated with high embryogenic, regenerable culture response in maize; to identify and characterize candidate gene(s)/sequences involved in control of tissue culture response;and, to map the genetic control of modifiers of culture response/transformation efficiency using the recently developed morphogenic regulatory gene (Bbm/Wus) expression system. All three specificobjectives will involve the use of fine-mapping assays, phenotypic analysis, genome sequence interrogation, and genetic engineering techniques for candidate sequence testing/confirmation.

Project Methods
This project involves research aimed at deciphering the genetic mechanisms underlying plant somatic embryogenesis and regeneration from tissue culture in the globally important crop, maize. Research will be conducted via genetic fine-mapping of a previously identified quantitative trail locus (QTL), located on the long arm of chromosome 3 in maize. The goal of the experiments is the identification and characterization of candidate genes controlling embryogenesis and regeneration. An additional goal is to identify "modifier" genes or regions of the genome that enhance embryogenic, regenerable tissue culture response via genetic mapping of genes enhancing the response of systems employing expression of morphogenetic regulatory genes (Bbm/Wus2) in segregating populations. General methodology will include development and molecular marker analysis of maize genetic mapping populations, phenotypic analysis of mapping lines for embryogenic culture and plant regeneration responses. Candidate gene(s) will be identified by association of high response with genomic regions/sequences, using genome sequence database and RNA expression analysis comparisons. Candidate gene sequences will be cloned/synthesized into gene expression vectors and tested via transformation for confirmation of candidate gene expression and effect on embryogenic, regenerable response.Efforts-Results from the research will be shared at scientific conferences each year of the grant period, and via publication in scholarly journals at approximately year 3-4 of the grant period. The new knowledge generated through this research is expected to aid in the development of enhanced plant regeneration systems for both public and private plant genome research and crop improvement efforts. Graduate student mentoring and training will occur throughout the entire grant period, as well as training of undergraduate students through participation in research as hourly lab assistants, and in classroom lab exercises.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience: The target audience of the research within the past year included scientists and researchers in the areas of plant genomics and epigenomics, plant biology and plant biotechnology. Additionally, the target audience included undergraduate students, graduate students, and postdoctoral research associates through education and training associated with the research. Changes/Problems:Due to the Covid19 pandemic and related restrictions, overall research progress was slowed (though not stopped) in 2020. What opportunities for training and professional development has the project provided?A graduate student is conducting the research and receiving training and professional development opportunities related to plant genetics and genomics research and application of biotechniques to crop genetic research and improvement. The student also gained training in multidisciplinary collaborative research and had the opportunity to give presentations at research conferences. How have the results been disseminated to communities of interest?Results have been presented at scientific conferences, grower association meetings, through publication, and through press interviews/releases. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, it is planned that the candidate gene vectors will be transformed into maize and transgenic plants and progeny will be assayed for effects on somatic embryogenesis response, somatic embryo development and plant regeneration. It is also planned that progeny lines within the maize "interacting gene(s)" mapping populations will be grown and transformed with the embryogenesis-related morphogenes and assayed for embryogenic response, somatic embryo development and plant regeneration.

Impacts
What was accomplished under these goals? Crop genome engineering and editing systems are critical tools for the advancement of plant functional genomics research and genetic improvement of crop plants for enhanced food, feed, fiber and bioenergy related traits, improved resistance to biotic and abiotic stresses, and development of sustainable farming systems. When using those approaches, the ability to regenerate plants from tissue culture is critical for success. Many varieties and genotypes of crop plants are unable, however, to form the optimal embryogenic tissue culture type that is ideal for plant regeneration, resulting in significant impediment of advancement in crop genetics research and enhancement. Deciphering the genetic mechanisms controlling the development of somatic embryos and embryogenic cultures would enable development of "genotype-independent" crop genome engineering/editing systems, greatly accelerating progress in the genetic study and improvement of crop plants. Research is underway to determine genetic factors involved in somatic embryogenesis in the locally and globally important crop, maize (corn). Through genome mapping techniques, several candidate genes were identified in this research that appear to play a significant role in the process of somatic embryogenesis. Sequences of 3 candidate genes(WOX2A-A188, WOX2A-B73, GRAS23-PH207) were identified and cloned into expression vectors to test whether overexpressing and/or expressing the genes at native levels results in changes in somatic embryogenesis in maize. Genome mapping experiments aimed at detection of genes with important "interactions" (modifying effects) with morphogenic genes (BBM and WUS2) involved in embryogenesis were initiated. To determine the optimal maize population to use to conduct the mapping experiments, 9 inbred parental lines (PHP02, PHK76, PHK56, P39, LH182, LH85, 3IIH6, W606S, andGaspé flint) were screened for embryogenic culture response following transformation of the lines with embryogenesis-related morphogenic genes. Parents with high and low embryogenic responses were identified, and maize populations generated from crosses of high and low parents were planted for use in genome mapping studies. New knowledge being generated through this research will enhance understanding of the genetic mechanisms underlying plant somatic embryo development and is being utilized in the development and optimization of new genotype-independent crop genome engineering/editing systems. Establishment of systems will lead to accelerated advancements in crop functional genomics research and genetic improvement applications.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Lin G., He C., Zheng J., Koo D-H., Le H.,1, Zheng H.,1, Tamang T.M., Lin J., Liu Y., Zhao M., Hao Y., McFraland F., Wang B., Qin Y., Tang H., McCarty D.R., Wei H., Cho M-J., Park S., Kaeppler H., Kaeppler S., Springer N., Liu Y., Schnable P.S., Wang G., White F.F., Liu S. 2020. Chromosome-level Genome Assembly of a Regenerable Maize Inbred Line A188. bioRxiv. https://doi.org/10.1101/2020.09.09.289611