| Lucerne (Medicago sativa) is one of the best pasture grass species in the world and it has been cultivated for more than2000years in China. Lucerne is named as ’King of Pasture Grass’ due to some of its unique characteristics such as high yield, high protein content and good palatability. Since lucerne plays important roles in animal husbandry, the improvement of its stress resistance and forage quality with genetic engineering technology would benefit the animal husbandry in China.Siberian wildrye(Elymus sibiricus) and Elymus nutans are perennial grass species in genus Elymus, family Poaceae. These grasses can be grown in varieties soil types and are tolerant to drought, cold and saline-alkaline.The above-mentioned plants E. sibiricus and E. nutans are also highly economic forage grasses. In order to improve the animal husbandry industry in China and to meet different manufacture requirements it is crucial to breed forage grasses with high quality using genetic engineering technology. However, the knowledge regarding the tissue culture of these two forage grasses, especially the plant tissue regeneration system is lacking. In this study, a regeneration system for these two grasses was developed based on previous research on perennial ryegrass(Lolium perenne), tall fescue (Festuca arundinacea) and Chinese wildrye (Leymus chinensis).1. The establishment of regeneration system and GUS gene transformation of lucerneIn this study, we developed a tissue culture regeneration system of lucerne using the cotyledon of cultivar ’Zhongmu No.1’as an explant vector, which provided a foundation for the further transgenetic work of lucerne. By using this regeneration system, a reporter gene GUS was transferred ino the vector using agrobacterium-mediated transformation technique and putative Kan resistant plants were obtained as a result. Further details are as below:1) Vector and medium. To select a proper explant vector, both cotyledon and hypocotyl axis were subjected in tissue culture. Results showed that both tissues had high callus induction rates; however, cotyledon had a better differentiating capacity and was a better vector compared to hypocotyl axis. Three media, UM+2.0mg/L2,4-D+0.25mg/L KT (M1), UM+2.0mg/L KT (M2) and1/2MS0(M3) were developed in this study. Media M1, M2and M3were callus induction, callus differentiation and root induction, respectively.2) The agrobacterium-mediated transformation technique was used to transfer GUS gene to cultivar ’Zhongmu No.1’. The cotyledons of6-day old lucerne seedlings were pre-cultivated for3days after tissues had beenimmerged in Agrobacterium solution (OD600=0.5) for10minutes. Degerming culture processes were conducted by transferring tissues to medium which contained500mg/L Cef antibiotics.The concentration of antibiotics was reduced by100mg/L in each generation. Selection culture was then obtained by transferring the induced callus to medium that contained50mg/L Kan. Root induction was obtained by transferring plant tissues to1/2MS0medium.3) Lucerne transformation and PCR identification. After selection,26Kan resistant plants were obtained. PCR identification results showed that all the studied plants were false positive.2. Studies on the tissue culture and plant regeneration of E. sibiricus and E. nutansThe dissection tissues (2-3mm) of E. sibiricus and E. nutans were used as the explant vectors. Results showed media contained MS+2.5mg/L2,4-D+30g/L sucrose+7.0g/L agar and MS+3.0mg/L2,4-D+30g/L sucorse+7.0g/L agar provided a better callus for E. sibiricus and E. nutans, respectively. The best callus differentiation was obtained for both the grass species using media contained MS+1.0mg/L KT+1.0mg/L6-BA+30g/L sucrose+7.0g/L agar.1/2MS was the most effective medium for root induction. By using these media, we regenerated10E. sibiricus plants and2E. nutans plants. |
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