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Method for establishing sterile leaf source for inducing gingko callus

A callus induction and establishment method technology, applied in the field of plant tissue culture, can solve the problems of injury, browning death, poor callus growth ability, etc., and achieves the effects of high efficiency, simple technical operation, and strong popularization and application value.

Active Publication Date: 2019-10-01
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current method of callus induction is to inoculate and cultivate the leaves directly from the seedlings after disinfection. Since the leaves are directly treated with disinfectants and are injured, most of them will brown and die after inoculation. Even if the callus induced after survival Poor ability, it will brown and die after several generations

Method used

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  • Method for establishing sterile leaf source for inducing gingko callus
  • Method for establishing sterile leaf source for inducing gingko callus
  • Method for establishing sterile leaf source for inducing gingko callus

Examples

Experimental program
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Effect test

Embodiment 1

[0030] A method for establishing a sterile leaf source for ginkgo callus induction, comprising the following steps:

[0031] (1) From the first ten days of April to the first ten days of May, cut the young shoots of adult female and male plants that were born in the same year, and measure the content of flavonoids and lactones in the leaves of the shoots to be inoculated, and screen the shoots with high content as the initial inoculation s material. Remove the leaves, wash the young shoots with detergent to remove surface dirt, wash them in running water for 30 minutes, then soak them in 70% ethanol for 30 seconds, wash them with sterile water for 3 to 4 times, and wash them in 0.3% KMnO 4 Soak in the solution for about 25 minutes, rinse with sterile water 4 to 5 times until the branches are free of KMnO 4 color.

[0032] (2) Cut young shoots (not lignified) into 2-5cm stem segments with 1-3 buds on the ultra-clean bench, inoculate in MS+0.1mg / LNAA+0.5mg / L 6-BA+0.5 % active...

Embodiment 2

[0036] A method for establishing a sterile leaf source for ginkgo callus induction, comprising the following steps:

[0037] (1) From late March to late April, cut the young shoots of the 3-5 year old ginkgo seedlings that were born in the same year, and measure the flavonoid and lactone content of the leaves of the shoots to be inoculated, and screen high-content shoots as Material for initial inoculation. Remove the leaves, wash the young shoots with detergent to remove surface dirt, wash them in running water for 30 minutes, then soak them in 70% ethanol for 30 seconds, wash them with sterile water for 3 to 4 times, and wash them in 0.3% KMnO 4 Soak in the solution for about 25 minutes, rinse with sterile water 4 to 5 times until the branches are free of KMnO 4 color.

[0038](2) Cut the young shoots into 2-5cm stem segments with 1-3 buds on the ultra-clean bench, and inoculate them in the axillary buds (or terminal buds) of MS+0.1mg / L NAA+0.5mg / L 6-BA ) on the induction...

Embodiment 3

[0042] A method for establishing a sterile leaf source for ginkgo callus induction, comprising the following steps:

[0043] (1) From the first ten days of December to the first ten days of May, take the ginkgo seed cores stored for more than 4 months, peel off the bony middle testa, wash the seed embryos with detergent to remove surface dirt, soak them in water for 24 hours, and soak them in 70% ethanol in turn 2min, washed 3-4 times with sterile water, in 0.3% KMnO 4 Soak in the solution for about 25 minutes, rinse with sterile water 4 to 5 times, until there is no KMnO on the surface of the seeds 4 color.

[0044] (2) On the ultra-clean bench, the embryos were taken out under aseptic conditions, inoculated on the embryo induction medium of MS+0.1mg / LNAA+0.5mg / L6-BA, cultured for 40 days, and the mature embryos grew into embryo seedlings, The seedling height is 2-3cm, and the leaves are 2-3 pieces. The medium contains 3.0% sucrose, 0.65% agar, and the pH is about 5.8. Th...

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Abstract

The invention discloses a method for establishing a sterile leaf source for inducing a gingko callus, and belongs to the technical field of plant tissue culture. The method comprises the following steps: taking young branches or mature embryos of ginkgo as materials, and culturing the young branches or mature embryos of the ginkgo on a bud induction medium or an embryo induction medium for 40 daysto obtain plantlets or embryo seedlings, cutting sterile leaves of the plantlets, and beveling the bases of the original stem sections of the plantlets; or cutting off the roots of the embryo seedlings, and keeping subculture for 40 days; after the sterile leaves of the plantlets or embryo seedlings are cut again, cutting long sterile shoots without leaves into stem sections with buds with the size of 2-3 cm, and further culturing on a subculture medium for 40 days; and then repeatedly carrying out stem bud induction, leaf extraction, leaf harvesting and stem continued culture to obtain a sterile leaf source for inducing the gingko callus. The method has the advantages of high yield, high efficiency, no seasonal limitation, continuous stability, and steady flow. The obtained sterile leaves can be directly used for callus induction and have quite high popularization and application value.

Description

technical field [0001] The invention relates to the technical field of plant tissue culture, in particular to a method for establishing a sterile leaf source for ginkgo callus induction. Background technique [0002] Ginkgo biloba L. is a unique tree species in my country. Its leaves contain two important flavonoids and terpene lactones, which have anti-oxidant and therapeutic effects on cardiovascular diseases, so they have important medicinal value. At present, ginkgo flavonoids and terpene lactones are mainly extracted from ginkgo leaves, but the content is low and decreases year by year with the growth of ginkgo tree age. In production, a large number of ginkgo young forests need to be planted to produce ginkgo leaves, which takes up a lot of farmland. The content of ginkgo leaf inclusions planted in the field is greatly affected by the external environment, but it is difficult to control the change of the environment artificially. The Ginkgo callus and cell culture tec...

Claims

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Application Information

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IPC IPC(8): A01H4/00
CPCA01H4/005
Inventor 陈颖王瑞敏冯凯沈瑒刘瑞陈燕琼张涛
Owner NANJING FORESTRY UNIV
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