Genetic Transformation Lab: Research

The research in Dr. Zeng-yu Wang's laboratory currently focuses on developing reliable plant regeneration and genetic transformation systems for different cool season forage grasses and cloning important agronomic genes. The aim of our tissue culture and transformation program in the Forage Improvement Division is to generate novel plant material by direct introduction of agronomical genes into important forage crops, and thus to assist or complement the forage breeding program.

Figure 1.

(A) PDS/1000 biolistic device used for microprojectile bombardment.
(B) Suspension cells of tall fescue plated on filter paper before microprojectile bombardment.
(C) Hygromycin resistant calli obtained after selection.
(D), (E) Transgenic plantlets regenerated from the hygromycin resistant calli.
(F) Transgenic tall fescue plants growing in the greenhouse.

The main aspects of the tissue culture and transformation program include: establishment of efficient plant regeneration and genetic transformation systems for different forage species; cloning of potentially useful agronomical genes and promoters; and generation and characterization of transgenic forage plants with improved agronomic characteristics. The cool-season forage grasses we are currently working on are tall fescue (Festuca arundinacea), Russian wildrye (Psathyrostachys juncea) and tall wheatgrass (Thinopyrum ponticum). It is known that these outcrossing monocot forage species are among the most recalcitrant plants to genetically manipulate in vitro. We are also using the available genomics information of Medicago truncatula to identify genes for improvement of the important forage legume alfalfa.

The establishment of an efficient tissue culture system is the basis for genetic manipulation of monocot grass species. We have established an efficient plant regeneration system for tall fescue, Russian wildrye and tall wheatgrass. The protocol is based on large-scale genotype screening for the induction of embryogenic callus, and the subsequent establishment of single genotype-derived embryogenic suspension cultures. Green plants were regenerated from the established suspension cultures at high frequency and were transferred to the greenhouse and the field. The embryogenic cell clusters from the established suspension cultures are ideal targets for biolistic transformation. This is especially true for outcrossing grass species, since the use of single genotype-derived cell suspensions allows the generation of transformants from the same genotype. The suspension cells of tall fescue and Russian wildrye have been used as direct targets for microprojectile bombardment to generate transgenic plants. A chimeric hygromycin phosphotransferase (hph) gene, which renders transformed cells resistant to hygromycin, was used as a selectable marker. Hygromycin resistant calli were obtained after microprojectile bombardment of suspension cells and subsequent selection in the presence of hygromycin (Figure 1A-C). Transgenic tall fescue and transgenic Russian wildrye plants were regenerated from the hygromycin resistant calli (Figure 1D-E). Molecular analyses confirmed the transgenic nature of these regenerated plants. Transgenic tall fescue and transgenic Russian wildrye plants have been transferred to the greenhouse and the field. The development of the transformation technology in grasses provides opportunities to generate value added material by the direct introduction of chimeric genes.

Projects aiming at improving agronomic traits of forage grass and legume are in progress. Genetic improvement of forage quality, drought tolerance and phosphate uptake are the main targets of the tissue culture and transformation program.

Forage digestibility, especially when plant matures, is a limiting factor for animal production. It is known that lignification of plant cell walls is largely responsible for lowering digestibility of forage tissues. We analyzed lignin deposition at different development stages of tall fescue and cloned cDNAs of two enzymes involved in lignin biosynthesis: caffeic acid O-methyltransferase (COMT) and cinnamyl alcohol dehydrogenase (CAD). For the first time in a major forage grass species, we have obtained transgenic tall fescue plants with reduced enzyme activity, reduced levels of lignin, altered lignin composition and improved digestibility. In order to isolate more genes involved in lignin biosynthesis, a cDNA library has been constructed from stem tissue of tall fescue for EST sequencing.

Drought tolerance is a major target of improvement for cool season perennial grasses in the southern Great Plains. A project aimed at isolating genes involved in regulation of stress inducible genes and generating transgenic grass plants was initiated. We have isolated a number of regulatory genes involved in stress regulation in M. truncatula and generated a large number of transgenic alfalfa plants using these genes. We have also constructed a cDNA library from drought stressed tall fescue and taken the EST approach to isolate regulatory genes in this species.

Improving plant phosphate uptake may have an impact on forage production, since phosphate is immobile in soil and very often deficient. Jointly with Dr. Maria Harrison, we have a project aimed at improving plant phosphate acquisition by expressing phytase and/or acid phosphatase. Root specific promoters have been generated and characterized, a large number of transgenic white clover plants have been generated and being tested. The potentially useful transgenic materials identified will be incorporated into the breeding programs of cool-season forages in the Forage Improvement Division.