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Establishment of High Frequency Regeneration System and Genetic Transformation by Particle Bombardment of Perennial Ryegrass

Author: HeJinGang
Tutor: XuZiQin
School: Northwestern University
Course: Cell Biology
Keywords: Perennial ryegrass Mature embryos Plant regeneration bar gene Biolistic transformation
CLC: S543.6;S688.4
Type: Master's thesis
Year: 2007
Downloads: 85
Quote: 1
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Abstract


Ryegrass Gramineae ryegrass species , is widely used as a lawn grass and pasture , using genetic engineering techniques to the study of genetic improvement has been much attention . The experiments establish the perennial ryegrass efficient in vitro regeneration system based on bar gene transfer by particle bombardment , transgenic plants of perennial ryegrass , new ways for the ryegrass varieties improvements provided . , Ryegrass after comparative tests from the establishment of the regeneration system to mature embryos as explants successfully established ryegrass regeneration system regeneration medium formulations suitable for ryegrass mature embryos screened . The experimental results show that the callus induction MS medium 5mg / L 2,4-D as a the best embryogenic callus induction frequency of 23.8 % ; differentiation medium was MS basal medium 1mg / L 6 - BA, the differentiation frequency can reach 33.3% . Second , the establishment of perennial ryegrass genetic transformation system of callus the herbicide Bialaphos sensitivity tests to determine the best choice for screening callus pressure of 2 mg / L , established on the basis of a perennial ryegrass embryogenic more tissue injury Biolistic genetic transformation system . Course of the experiment , the first containing hypertonic mannitol and sorbitol 0.25mol / L MS medium for 5 to 12 hours of vigorous growth of embryogenic callus processing , then the implementation of the gene gun bombardment . Bombardment when the degree of vacuum of 28 inches of mercury , and the helium gas pressure of 1350psi per gun the powder is used in an amount of 500μg / L . Bombardment After 16 hours , the callus is transferred to the medium of the additional Bialaphos Selection culture obtained resistant calli after three ( 10 days) . The resistant calli transferred to differentiation medium , about 20 days after having regenerated seedlings grow . Jiangzai seedlings transferred to rooting medium to grow strong root transplanting . The PCR results confirmed the bar gene has been integrated into the ryegrass genome was also carried out of gus gene expression analysis . 37 transgenic plants , bar genetic conversion rate of 1.54% . The herbicide applicator experiments showed integrate bar gene ryegrass have some resistance to Basta .

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CLC: > Agricultural Sciences > Gardening > Ornamental Horticulture ( flowers and ornamental trees) > Garden Plant Cultivation and application technology > Lawns and ground cover plants
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