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Study on Dissociation and Culture of Protoplasts of Medicago Sativa L.

Author: WangJuan
Tutor: ShiShangLi
School: Gansu Agricultural University
Course: Grassland
Keywords: Alfalfa Cotyledons Callus Protoplast Separate Culture
CLC: S541.9
Type: Master's thesis
Year: 2010
Downloads: 107
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Abstract


Alfalfa (Medicago sativa) cultivation earliest and most widely distributed, nutrient-rich, arid and semi-arid areas of improving soil nitrogen fixation indispensable perennial quality legume forage, known as the \But in terms of yield, quality and resistance need to be further improved, and the existing pasture can not meet the growing needs of the animal husbandry, cultivating strong resistance, high yield and quality of alfalfa varieties, has become many breeders the main objective. Different flowers due to alfalfa the complex features pollination, using conventional breeding methods to cultivate new varieties has been greatly restricted. Development of plant cell engineering and genetic engineering technology to provide a new way to improve the stress resistance of alfalfa. The trial four alfalfa varieties Shimizu alfalfa (Medicago sativa L.cv.Qingshui) the Algonquin (of Medicago stativa L.cv. Algonguin), Gannon No. 1 the (the Medicago stativa L.cv.Gannong No. 1) tourists (Medicago stativa L.cv.Euroke) for the materials, to establish alfalfa callus regeneration system as a basis for the establishment of alfalfa protoplast isolation and culture. 1. The alfalfa varieties Shimizu alfalfa, Algonquin, Gannon, visitors hypocotyls explants, plant regeneration research. The results showed that: (1) four alfalfa varieties under hypocotyl out more rate order Algonquin 93.7% gt; Gannon, 92.6% gt; Shimizu alfalfa 92.2% gt; visitors to 87.7%. (2) four alfalfa varieties induce embryonic callus optimum medium MS 2.0 mg/L2 ,4-D 1.0 mg / L NAA 2.5% sucrose, 0.6% agar callus highest rate; induction rate is as follows: Algonquin 91.7%, visitors to 89.6% and 88.3% of the fresh water alfalfa, Gannon, 87.3%. (3) of the four varieties of alfalfa callus accurately preserving the best medium of MS 0.5 mg / L KT, 0.3 mg / L NAA 2.5% sucrose, 0.6% agar highest differentiation rate, differentiation rate as follows: Gannon 1 93.0 %, Shimizu alfalfa 89.7% of visitors to 86.5% and 84.5% of Algonquin. (4) four varieties of alfalfa roots in the rooting medium 1/2MS 0.1mg/LNAA 1% sucrose, 0.6% agar, the best, The rooting rate as follows: Gannon No. 1, 96.0%, 94.6% of Algonquin tourists 93.3%, 92.5% of Shimizu alfalfa. 2 soft embryogenic callus induction of alfalfa varieties Algonquin and sterile cotyledons material enzymatically dissociated protoplasts, protoplast culture. The study of different enzyme solution, hydrolysis time, callus following generation time and osmotic pressure on alfalfa free protoplast and callus protoplasts split in KM8P medium, the results show that: ( 1) embryogenic callus culture 12 days following the material under 2% cellulase 0.5% pectin 1% hemicellulose enzymes conditions for 12 h, enzymatic mannitol concentration of the enzyme solution was 0.55 mol / L, obtained yield of 2.56 * 106 g 1 and vitality for 88% of the protoplasts. Protoplast cultured for 4 d in KM8P 2.0 mg/L2 ,4-D 1.0 mg / LNAA medium first cell division, the division frequency of up to 50.2%; 2 to 3 weeks after the formation of small cell groups, and then to mannitol The concentration of 0.1mol / L ~ 0.2mol / L medium 2 or 3 generations, is conducive to small cell groups continued to divide and form callus. (2) the Algonquin alfalfa Cotyledons material, 2% cellulase 0.5% pectin 1% hemicellulose enzymes conditions, enzymatic 8 h, the enzyme solution mannitol concentration of 0.55 mol / L available yield of 2.19 * 106 g 1 vitality for 87.6% of the protoplast.

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CLC: > Agricultural Sciences > Crop > Fodder crops,pasture > Perennial legume > Other
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