Skip to Main Content
 

Global Search Box

 
 
 
 

Files

File List


Mingde Liu final thesis.pdf (62.23 MB)

ETD Abstract Container

Abstract Header

Gene Modification of Rubber Producing Dandelions

Liu, Mingde
http://rave.ohiolink.edu/etdc/view?acc_num=osu1566222527954184

Abstract Details

2019, Master of Science, Ohio State University, Food, Agricultural and Biological Engineering.
Natural rubber (cis-1,4-polyisoprene) is an important and irreplaceable biopolymer widely used in industry all over the world. However, the present sole natural rubber resource, Hevea brasiliensis, has several problems, including narrow genetic diversity, pathogen attack, restricted growth area, competition with other crops and high labor consumption of latex collection, which make this current rubber production system unsustainable. Taraxacum kok-saghyz (TK, rubber dandelion) and Taraxacum brevicorniculatum (TB) are dandelion species with wide environmental adaption and short life cycle, and their roots contains high quality rubber. T. kok-saghyz is being developed as a new potential natural rubber producing crop, while T. brevicorniculatum is a good model for research on rubber biosynthesis and dandelion gene-phenotype interaction. To domesticate and commercialize rubber producing dandelions, increased rubber yield and reduced ecological risk are two main goals. With the rapid development of precise gene manipulation methods and highly efficient transformation systems, improvement of T. kok-saghyz and T. brevicorniculatum is accelerated. Thanks to the capability of shooting out from hairy root, TK and TB may be rapidly transformed by Agrobacterium rhizogenes. Moreover, hairy roots phenotype could also be used as a visible marker. Combining with CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/ CRISPR associated protein 9), genome modification research was done in my master`s project. As a representative example of wind dispersal species, the seed of dandelion dispersed by the wind and convectional updraft, Taraxacum officinale is one of the major weed species in North America. Although T. kok-saghyz is not vigorous enough to be a weed now, it is also stereotyped as having high ecological risk, which will impede the extension and commercialization of T. kok-saghyz. Pappus is a cluster of fibrous hairs connected to the achene, which maintains stability of the seeds in the wind and maximizes descent time to help achieve long distance dispersal. Two ESTs that four-fold upregulated in the pappus of Gerbera hybrida were aligned to the T. kok-saghyz genome using BLAST. The aligned pappus-related genes in T. kok-saghyz were knocked out using CRISPR/Cas9 system and confirmed in regenerated plantlets by Sanger sequencing. The mutant showed a late flowering phenotype. However, more replication is needed to validate this result. The structure and function of pappus both need to be observed after the mutant flowering. This research may disrupt pappus structure and reduce ecological risk and limit the seeds lost. To my knowledge, this project is the first study manipulating pappus-related genes in T. kok-saghyz. In addition to reducing ecological risk, increasing rubber yield is a significant goal in rubber producing dandelion domestication. The gene encoding an N-terminal truncated 3-hydroxy-3-mthylglutaryl-coenzyme A reductase (HMGR), which is the rate-limiting enzyme in the mevalonate pathway (MVA pathway) which produces IPP, the monomer of the rubber polymer, was introduced into TB. As an apomictic species, progeny was generated as clones from two independent confirmed HMGR transgenic T. brevicorniculatum lines. Exogenous HMGR expression was confirmed in both HMGRoe2 TB and HMGRoe6 TB lines. HMGRoe TBs contained higher rubber and resin concentration compared to wild type TB, seemingly at the expense of soluble carbohydrates. Furthermore, HMGR overexpression improved the cold tolerance of T. brevicorniculatum. Cold environment also enhanced root growth in wild type TB. This research suggests that HMGR overexpression increases rubber biosynthesis in T. brevicorniculatum via enhanced IPP production via the MVA pathway and might also improve cold tolerance to help extend the production region or growth season duration. Future studies of Taraxacum kok-saghyz after removal of the Ri gene will provide a clearer indication of the effect HMGR overexpression on rubber biosynthesis. Natural rubber is an end product stored in roots of T. kok-saghyz. Roots also store inulin, a polyfructan carbohydrate. As an antagonistic photosynthetic carbon sink of natural rubber, suppressing inulin biosynthesis might increase the flux of photpsynthetic carbon to the MVA pathway. Fructan:fructan 1-fructosyltransferase (1-FFT) is a key enzyme in inulin biosynthesis, and directly catalyzes inulin polymerization. The two 1-FFT CRISPR/Cas9 gene knock T. kok-saghyz lines were crossed with wild type high rubber yielding clones to segregate out the knockout mutants from the hairy root gene. Restriction enzyme digestion site loss method and Sanger sequencing were used to screen for the mutants in progeny. The hairy root phenotype showed a 1:1 segregation ratio, as expected. However, the 1-FFT mutant were lost after crossing, which might because of a chimeric problem in transformation or that not all copies of 1-FFT were mutagenized during the CRISPR knockout process. A better way to remove the hairy root phenotype without losing mutagenized genes and a faster method to screen large numbers of mutated plants need to be figured out in the future.
Katrina Cornish (Advisor)
John Cardina (Committee Member)
144 p.
Agricultural Engineering; Horticulture

Recommended Citations

Citations

  • Liu, M. (2019). Gene Modification of Rubber Producing Dandelions [Master's thesis, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1566222527954184

    APA Style (7th edition)

  • Liu, Mingde. Gene Modification of Rubber Producing Dandelions. 2019. Ohio State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1566222527954184.

    MLA Style (8th edition)

  • Liu, Mingde. "Gene Modification of Rubber Producing Dandelions." Master's thesis, Ohio State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu1566222527954184

    Chicago Manual of Style (17th edition)

osu1566222527954184
89
© 2019, some rights reserved.Creative Commons License Gene Modification of Rubber Producing Dandelions by Mingde Liu is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. Based on a work at etd.ohiolink.edu.
This open access ETD is published by The Ohio State University and OhioLINK.