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Method for genetic transformation and regeneration of eucalyptus urophylla

A technology of genetic transformation and Eucalyptus urophylla, applied in the field of genetic transformation and regeneration of Eucalyptus urophylla

Inactive Publication Date: 2011-08-31
ZHANJIANG NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] To sum up, at present, there are no examples at home and abroad of using the hypocotyls of sterile seedlings of Eucalyptus urophylla as explants for genetic transformation and achieving success

Method used

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  • Method for genetic transformation and regeneration of eucalyptus urophylla
  • Method for genetic transformation and regeneration of eucalyptus urophylla
  • Method for genetic transformation and regeneration of eucalyptus urophylla

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Experimental program
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Embodiment example

[0048] Select 1g of mature and plump Eucalyptus urophylla seeds, soak in warm water at 50-55°C and cool naturally for 4-6 hours, rinse twice with sterile water on an ultra-clean workbench, sterilize with 70% ethanol for 1 minute, and rinse with sterile water for 2 minutes. After the first time, add 15% (volume ratio) NaClO (3.75% relative to chlorine) solution, adopt the secondary disinfection method, the disinfection time is 9+9min, rinse with sterile water 5-6 times, and inoculate to the sterilized environment without hormones. 1 / 2 MS medium (supplemented with 0.7% agar, 3% sucrose, pH 5.8-6.0). After 7 days of dark cultivation at 25°C, transfer to a temperature of 25±2°C, light for 12 hours / day, and light intensity of 2000-2500 lx for 6 days, and then grow aseptic seedlings of Eucalyptus urophylla with a seedling age of 13 days.

[0049] Take 13-day-old seedlings (containing only 1 pair of cotyledons), remove the terminal buds and roots, cut the hypocotyls into 0.5-1.0 cm l...

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Abstract

The invention belongs to a method for genetic transformation and regeneration of a eucalyptus, and particularly relates to the method for genetic transformation and regeneration by taking hypocotyledonary axis of an aseptic seedling of a eucalyptus urophylla as an explant. The hypocotyledonary axis of the seedling of the eucalyptus urophylla is taken as the explant, calluses are pre-cultured and induced, co-culture transformation is performed with agrobacterium tumefactions carrying exogenous genes, kanamycin is used for selection, and adventitious bud induction, bud proliferation, adventitious bud growth, rootage and transplantation are performed for getting a plant to be transformed. PCR (polymerase chain reaction) detection and RT-PCR (reverse transcription-polymerase chain reaction) detection prove that, as for the obtained plant of the eucalyptus urophylla to be transformed, the exogenous genes are introduced into a genome of the eucalyptus urophylla and expressed; the transformed plant having better resistance to phytophthora nicotianae is found in the anti-disease detection of in vitro blades of the eucalyptus urophylla of a reverse radish-anti-fungal protein (RS-AFP2); and the relevant enzyme activity determination result of the resistance shows that, after inoculation of the transformed plant, the activities of L-phenylalanin ammo-nialyase (PAL), peroxidase (POD) and polyphenol oxidase (PPO) are enhanced.

Description

technical field [0001] The invention belongs to the genetic transformation and regeneration method of eucalyptus, in particular to the genetic transformation and regeneration of Eucalyptus urophylla. Background technique [0002] Eucalyptus (Eucalyptus) belongs to Mytaceae (Mytacea), Eucalyptus (Eucalyptus), native to Australia, there are more than 1,000 species and subspecies. Eucalyptus has many remarkable characteristics: ①The material is hard, and the economic value of wood, bark, leaves, and flowers is very high. It is not only a timber forest, an economic forest, a shelter forest, a scenic forest, but also a good energy tree species; Especially in the young forest stage, the growth is fast, which greatly shortens the production cycle and can obtain higher economic benefits; ③ There are many types and strong stress resistance, including both heat-resistant and cold-resistant tree species, less pests and diseases, and more resistant to thinness. Planted in different cli...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H4/00
Inventor 曾富华
Owner ZHANJIANG NORMAL UNIV
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