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The Regeneration System Establishment and the Genetic Transformation Study of the Purple Majesty

Author: HuangLi
Tutor: LiMingYang
School: Southwestern University
Course: Floriculture
Keywords: ornamental grass Purple Majesty the Regeneration System Genetic the transforma-tion System the means of agrobacterium-mediated genetic transformation GUS gene instantan-eous expression detection
CLC: S682.36
Type: Master's thesis
Year: 2010
Downloads: 20
Quote: 0
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Abstract


Purple Majesty is belong to the Pennisetum of poaceae. It is a kind of new ornamental grass.Because of the graceful form,kinds of colours,good adaptability, Purple Majesty is gradually into landscape designer’s view as the development of landscaping in the world.The blade,stem and inflorescence of Purple Majesty are all purple.And its inflorescence is cattail-like. Purple Majesty is used in landscape with glared all day or shading partly.lts seedling is gree and its stems and main vein are turned purple when the eight leaves grow up. Purple Majesty has strong drought and high tempreture resistanse.However, Purple Majesty can’t resist hypothermia and it will die nearly below sixteen degree.So it was limited in landscaping bacause of the tempreture factor. On the basis of the regeneration of Purple Majesty, its resistance of low tempreture will be increased by genetic transformation to the callus.Thus it will be the base of using more extensive of Purple Majesty.1 the regeneration system establishment of Purple MajestyThe study was focused on the regeneration system of Purple Majesty with it’s mature seeds and N6 medium. The surface sterilization measure of Purple Majesty seeds was 75% alcohol 1 minute and then 0.1% mercuric chloride 13 minutes. The best callus induction rate was one hundred percent when N6 supplemented 2.0 mg/L 2,4-D,0.3mg/L6-BA,690mg/L Proline,500mg/L Hydrolyzation casein,3% cane sugar and 6 g/L Agar. It can restrain the tissue browning supplementing 100 mg/L Vc into the callus induction medium. The best differentiation rate was nity percent when N6 supplemented 0.1 mg/L 6-BA and 0.2% activated carbon. The root development medium was 1/2 MS supplemented with 3% cane sugar and 6g/L Agar.2 the genetic transformation system study of Purple MajestyWhen Chimonanthus praecox (L.) Link cold adaptation protein gene was shifted into callus by means of agrobacterium-mediated genetic transformation by use of callus as explants, the genetic transformation system of Purple Majesty was preliminarily studied. By detecting the transient expression of GUS gene, this study probed into different factors effected on Purple Majesty transformation, which were pre-culture, different types of infectious liquid, concentration of agrobacterium, time of infection, time of co-culture and addition of AS. The results showed that these were important factors in Purple Majesty’transformation. The optimal method of transformation was as follows:the callus was pre-cultured in the medium N6+2.0 mg/L 2,4-D+0.3 mg/L 6-BA +100 mg/L Vc+690mg/L Proline+500mg/L Hydrolyzation casein for 3～5 days. After the bacterial liquid whose OD600 was 0.1 by the second activation was centrifuged, thalli were collected. The OD600 of bacterial liquid should be about 0.7 after thalli was resuspended in liquid MS+AS100 um/L.Then the callus that had been pre-cultured was put into the resuspended bacterial liquid to be infected for 15 minites in the shaking bed. These callus were transferred into the medium N6+2.0 mg/L 2,4-D+ 0.3 mg/L 6-BA+100 mg/L Vc+690mg/L Proline+500mg/L Hydrolyzation casein in the darkness for 3 days. Later, it was time to wash bacteria off on the surface of callus in the liquid MS+Cef 500 mg/L for 30 minites.And next, after bacterium on the surface of callus was soaked up by aseptic water, which had been washed by aseptic water more than 5 times, the callus was selectively cultured in the medium N6+ 0.1 mg/L 6-BA+ 100 mg/L Vc +0.2% activated carbon+690mg/LProline+500 mg/L hydrolyzation casein+30mg/LG418+200mg/L Cef. Under these optimum conditions,the rate of transient expression of GUS gene in Purple Majesty could be 40%.

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CLC: > Agricultural Sciences > Gardening > Ornamental Horticulture ( flowers and ornamental trees) > Perennial Flowers > Other Flowers > Foliage plants
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