Background

The importance of forage (particularly greenfeed, Zea mays ssp. mexicana L. to modern agricultural production and livestock breeding has been described in Chapter 1. In order to meet the needs of agriculture and consumers the creation, selection and fixation of the requirements for plant phenotypes using plant breeding for improving plant materials is the primary task (Moose & Mumm 2008). Traditional breeding projects are focused on increasing productivity and quality and disease resistance. With the development of molecular biology it has been shown that traditional breeding methods through phenotypic selection cannot alone satisfy all the requirements of breeding. Thus, modern cultivars have limited tolerance of abiotic stresses, which greatly affect the huge productivity requirements in the world and in China. In order to resolve these problem in the shortest possible time, the introgression of abiotic stress tolerance into modern cultivars is essential work for the future (Dunwell 2014). Until now, phenotype selection, marker assisted selection (MAS), and genetic engineering have been the main methods used to produce low temperature tolerant cereals. In addition, exploring available germplasm resources in wild relative species is becoming a high value activity. In plant, crop and forage studies, it has been shown that wild relatives have a rich genetic diversity with characteristics of wildness, aggressiveness and resistance. Therefore they are usually used as valuable original materials for genetics, breeding and natural gene banks for locating and adding new diversity to cultivated cultivars, which resolves the problem of limited genes available in traditional gene pooling for traditional genetic breeding (Inoue, Fujimori & Cai 2007). Cold tolerance is an important ability of plants to defend against temperature change and is an irreplaceable trait in worldwide production of many crops. In different districts, plants need to develop lots of strategies to adjust to cold stress for living by promoting a series of complex processes of physiological rearrangement (Mishra, Heyer & Mishra 2014). The low temperature injury of plants includes chilling injury (>0 °C) and freezing

165 injury (<0 °C) (Verslues et al. 2006). These two stresses can greatly reduce plant growth and development (Ensminger, Busch & Huner 2006). Low temperature can lead to formation of ice crystals in the cell, which affects the structure of cell membranes and makes cells lose function. Cells can reduce the damage of chilling injury or freezing injury by improving sugar content, increasing antioxidants, taking mechanical action and inducing molecular chaperone chemicals (Leyva et al. 1995). Meanwhile, metabolic processes of cell membrane can also be changed by low temperature, such as photosynthesis and respiration (Ryan 1991). The reaction center and light-harvesting antennas of the basic photosynthesis membrane system lie on chloroplast thylakoid phospholipid bilayer-membranes. These attend the process of transforming light energy into biochemical usable chemical potential energy (ATP) and redox potential energy (NADPH) (Ensminger, Busch & Huner 2006). Under low temperature stress, the structural stability of the lipid bilayeris reduced. Especially the photosynthesis PSII reaction center can be harmed under this stress (Huner et al. 1993).

In addition, low temperature can also change the activity and the reaction speed of enzymes and metabolism. When plants detect a low temperature signal, different kinds of defensive pathway are activated. These include physical structure adaptation, changing the content of the cell membrane and the arrangement of the cell cytoskeleton; osmoregulation leading to osmotic chemical concentration increase, with soluble sugar, betaine, proline, free amino acids, and ions; antioxidant composition and reactions increase and changes of a number of kinases (Kaplan et al. 2004; Levitt 1980). According to previous research results, more than 70% of 400 types of metabolite products can be induced by low temperature (Cook et al. 2004b). Soluble proteins, soluble sugars and proline are the most important metabolites for osmoregulation. These methods and vitamin E changes also can reduce plant injury under low temperature (Maeda et al. 2006).

In order to better understand cold or drought tolerance in plants and use it for tolerant crops breeding, many genes have been isolated from maize which then rely on the results

166 from model plant Arabidopsis and tobacco to ascertain their functions. Such genes includes ZmMKK4, ZmCPK4, ZmLEA5C, ZmNAC55, ZmDBP4, ZmDBP3, ZmCLC-d, ZmMPK4 and ZmDBF3 (Jiang et al. 2013; Kong et al. 2011; Liu et al. 2014; Mao et al. 2016; Wang & Dong 2009; Wang et al. 2015b; Zhou et al. 2016; Zhou et al. 2012), which have all been implicated in improving cold or drought tolerance of target plants significantly. However, no tolerance related genes have been isolated from teosinte except a tb1 (teosinte branched 1) gene, which has been extensively studies (Clark et al. 2006; Vann et al. 2015). Actually, the creation of pasture germplasm resources with molecular sequencing technology has seldom been reported for forage studies. Combining pasture germplasm resources and new generation genetics technologies (Transcriptome, Gene expression profiles analysis, Functional gene analysis) will offer genetics related reference information for the creation of pasture breeding information and improving breeding efficiency. At the transcriptional level, use of transcriptome sequencing and digital gene expression profile analysis of plant resistance gene expression will benefit researchers through constructing related tolerance gene resources banks, which will provide important reference information. Meanwhile, gene functional research will be expanded from Arabidopsis to include Zea mays ssp. mexicana L. Until now, few molecular biological studies have been done related to this plant. So these researches will improve the level of knowledge and offer molecular evidence for future breeding strategies of Zea mays ssp. mexicana L.