The development of transformation technologies provides opportunities to generate value-added materials through the direct introduction of potentially useful agronomic genes. The agronomic gene can be either cloned into the plasmid containing the marker gene or remain independent of the marker gene and introduced into plants by co-transformation. When an agronomic gene and the selectable marker gene reside in the same plasmid, they will be transferred as a single entity, and most of the transgenics will contain the agronomic gene after selection. In the case of co-transformation, the genes reside in different plasmids, and when introduced into plant cells at the same time, transgenics containing the agronomic gene can be recovered at a reasonable frequency after selection.

The candidate transgenes can be from either tall fescue or unrelated species. Transgenic tall fescue plants have been produced with the objective of improving forage digestibility, nutritional value (see Chapter 11), disease resistance (see Chapter 8 and Chapter 24), and abiotic stress tolerance (see Chapter 4). Although much of the work has been for concept testing, the results are promising and justify further efforts in refining the systems for future use in cultivar development.

Animal productivity is closely related to in vitro dry matter digestibility of forages (see Chapter 16), and lignification of plant cell walls is largely responsible for lowering digestibility of forage tissues. Lignin in grasses comprises guaiacyl (G) units derived from coniferyl alcohol, syringyl (S) units derived from sinapyl alcohol, and p-hydroxyphenyl (H) units derived from p-coumaryl alcohol (Baucher et al., 1998). Both concentration and composition of lignin are important factors influencing cell wall degradability of forages (Vogel and Jung, 2001). Genetic modification of lignin has been effective in improving digestibility/degradability in dicot species such as tobacco (Nicotiana tabacum L.), alfalfa (Medicago sativa L.), poplar (Populus spp.), and Arabidopsis thaliana (L.) Heynh. (Baucher et al., 1998; Boudet et al., 2003; Dixon et al., 2001; Humphreys and Chapple, 2002; Reddy et al., 2005).

Many genes and enzymes are involved in the lignin biosynthetic pathway (Dixon et al., 2001; Reddy et al., 2005). Two lignin biosynthetic genes, cinnamyl alcohol dehydrogenase (CAD) and caffeic acid O-methyltransferase (COMT) were cloned and characterized in tall fescue (Chen et al., 2002). The enzyme CAD catalyzes the reduction of cinnamaldehydes to cinnamyl alcohols, which is the last step in the biosynthesis of lignin precursors (Baucher et al., 1998). The COMT is a multi-specific enzyme involved in the 3-O-methylation of monolignol precursors at the aldehyde or alcohol levels (Dixon et al., 2001; Humphreys and Chapple, 2002). Transgenic tall fescue plants were produced by biolistic transformation using sense and antisense CAD and COMT gene constructs. Severely reduced mRNA levels and significantly decreased enzymatic activities were found in some transgenic lines. These transgenic tall fescue plants had reduced lignin concentration, altered lignin composition, and increased dry matter digestibility (7.2-10.5%) (Chen et al., 2003, 2004). The results demonstrated that down-regulation of endogenous genes is an effective approach for improving forage digestibility of grasses. In addition to CAD and COMT, down-regulation of other lignin genes, such as caffeoyl CoA 3-O-methyltransferase (CCOMT), p-coumarate 3-hydroxylase (C3H), hydroxycinnamoyl-coenzyme A shikimate/quinate hydroxycinnamoyltransferase (HCT), 4-coumarate:CoA ligase (4CL), and cinnamyl CoA reductase (CCR), may lead to reduced lignin and improved dry matter digestibility.

Under normal grazing conditions, sulfur-rich amino acids (methionine and cysteine) are among the most limiting essential amino acids for wool growth in sheep (Ørskov and Chen, 1990; Reis and Schinckel, 1963). Direct infusion into the abomasum (by-passing the rumen) of cysteine alone or its precursor methionine can increase wool growth by up to 100% (Reis, 1989). To improve protein quality of tall fescue, transgenic plants were produced with the sunflower seed albumin 8 gene encoding a rumen-stable protein rich in sulfur-containing amino acids (Wang et al., 2001). The transgene driven by either the CaMV35S promoter or the light inducible cab promoter was stably integrated in the plant genome. The expected transcript was produced and the corresponding sulfur-rich SFA8 protein accumulated at levels of up to 0.2% of total soluble protein in leaves of individual transgenic plants (Wang et al., 2001). It would be necessary to achieve an expression level of the SFA8 to about 2% of total leaf protein to make a significant impact on ruminant diets (Wang et al., 2001). Strategies for increasing the accumulation levels of foreign proteins in forage plants may include the use of stronger promoters or the development of a chloroplast transformation system to produce transplastomic lines. Chloroplast transformation offers unique advantages in plant biotechnology, including high levels of foreign protein expression and transgene containment due to the lack of gene transmission through pollen (Ruf et al., 2001).

Turfgrass quality (see Chapter 26) can be enhanced by genetic transformation. The Agrobacterium tumefaciens ipt gene encodes the enzyme isopentenyl transferase, which catalyses the rate-limiting step in cytokinin biosynthesis (Chou et al., 1998). Introduction of the ipt gene into turf-type tall fescue by biolistic transformation resulted in enhanced tillering ability, increased levels of chlorophylls a and b, and improved cold tolerance (Hu et al., 2005). The transgenic tall fescue plants were more vigorous and stayed green longer than nontransgenic plants under lower temperature conditions (Hu et al., 2005).

Fungal diseases limit maintenance of tall fescue lawns. Two major fungal diseases of tall fescue are brown patch, caused by Rhizoctonia solani Kühn, and gray leaf spot, caused by Magnaporthe grisea (T. T. Hebert) Yaegashi & Udagawa (Dong et al., 2008). Transgenes from various sources were introduced into tall fescue through Agrobacterium-mediated transformation. The alfalfa b-1,3-glucanase AGLU1 gene, a truncated frog dermaseptin SI gene and the bacteriophage T4 lysozyme gene conferred resistance to both diseases (Dong et al., 2007; Dong et al., 2008). The introduction of rice Pi9 gene conferred resistance against gray leaf spot (Dong et al., 2007). The disease resistance experiments were performed in the greenhouse. Field tests and inoculation with other fungal pathogens are needed to confirm the function of these genes under natural environments.

Progress in transgenic research with related species provides useful information for tall fescue. Delayed flowering in red fescue (Festuca rubra L.) and down-regulation of pollen allergens in ryegrass are two such examples. Delayed flowering of forages can be beneficial for herbage quality. By introducing a strong floral repressor TERMINAL FLOWER1 (LpTFL1) isolated from perennial ryegrass into red fescue, floral development in transgenic red fescue plants was delayed or arrested (Jensen et al., 2004, 2001). Transgenic lines showing low to intermediate expression of LpTFL1 flowered approximately 2 wk later than the controls, and transgenic lines showing very high LpTFL1 expression levels remained nonflowering after exposure to natural vernalization conditions in two successive years (Jensen et al., 2004). The development of hypoallergenic ryegrass is another interesting approach. Grass pollen is a widespread source of airborne allergens and is a major cause of hay fever and seasonal allergic asthma (Petrovska et al., 2004). Genes encoding major pollen allergenic proteins, Lol p 1 and Lol p 2, were cloned from perennial ryegrass. Transgenic perennial ryegrass and Italian ryegrass were produced using antisense Lol p 1 and Lol p 2 transgenes under the control of a pollen-specific promoter. The accumulation levels of Lol p 1 and Lol p 2 allergens in pollen were significantly reduced in the transgenic ryegrass plants (Petrovska et al., 2004). The development of hypoallergenic grass cultivars is expected to have major benefits for public health.


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