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Embryoid-approach blumea balsamifera tissue culture method

A kind of Ainaxiang group, culture medium technology, applied in horticultural methods, botanical equipment and methods, horticulture and other directions, can solve the problems of low detoxification rate, easy browning, high mutation rate, and achieve rooting rate and transplanting seedlings to survive The effect of high rate, no growth cycle and high survival rate

Active Publication Date: 2016-02-10
TROPICAL CORP STRAIN RESOURCE INST CHINESE ACAD OF TROPICAL AGRI SCI
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  • Summary
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The standardized seedling breeding technology and standardized planting technology of Ainaxiang are important guarantees to meet the market's demand for Ainaxiang extract. However, for a long time, its seedling breeding has always relied on division propagation or seed reproduction, with low reproduction efficiency and poor seed production. The uneven quality of seedlings and serious soil-borne diseases and insect pests have seriously affected the breeding and standardized planting of Ainaxiang seedlings
[0004] Introduced in the prior art the Ainaxiang seedling breeding method, which involves artificial beekeeping pollination, seed tank nutrient solution cultivation to improve seed fertility, seedbed treatment, matrix formula and sowing methods and other seedling breeding methods, but its seedling breeding Restricted by the season, the breeding quantity and seedling quality etc. are all restricted, and can't detoxify, can't solve the soil-borne disease and insect pest problem of Ainaxiang seedling breeding kind; In the prior art, about the Ainaxiang tissue culture seedling production method mainly Including disinfection of explants, induction of clustered seedlings, rooting, hardening and transplanting of seedlings, etc., technically speaking, it all belongs to the Ainaxiang tissue culture detoxification and rapid propagation system. Low toxicity rate, easy browning (proliferation rate <20%), high mutation rate, low reproduction efficiency, limited number of annual breeding seedlings, etc.
[0005] The premise of plant transgenic breeding is to obtain plants that can tolerate transgenes well. Callus is a good transgenic material. In the prior art, there are few Ainaxiang tissue culture methods through the embryoid body approach.

Method used

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Experimental program
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Effect test

Embodiment 1

[0040] Example 1 Ainaxiang explant disinfection program optimization

[0041] 1. Effects of penetrants and protective agents on the browning of Ainaxiang explants

[0042] 1. Experimental method: Divide the shoot tip explants or leaf explants of Ai Naxiang into 10 groups, first treat them with different concentrations of Tween 80 and silver nitrate for 15-30 minutes, and then disinfect them with 70% alcohol for 30 seconds and 0.1 % mercuric chloride for 10 min, rinsed with sterile water for 5 times until pH = 7, treated 20 bottles in each group, repeated 3 times, inoculated on hormone-free MS medium, placed in a constant temperature incubator at 30°C for full light cultivation, and After 10 to 12 days, the pollution rate and proliferation rate were counted; the results were: when the explants of Ai Naxiang were directly sterilized with 70% alcohol and 0.1% mercuric chloride for 10 minutes without pretreatment, most of the explants were polluted or browned, It is speculated th...

Embodiment 2

[0049] Screening and optimization of embodiment 2 basal medium

[0050] Divide the shoot tip explants or leaf explants of Ainaxiang into 20 groups, first disinfect them with 70% alcohol for 30 seconds, wash them with sterile water for 3 to 5 times, and then wash them with 0.5% to 2.0% Tween 80 and 5 ~30mg / L silver nitrate with 0.1% mercury liter for 10min, rinse with sterile water 5 times to pH=7, treat 20 bottles for each group, repeat 3 times, inoculate on modified MS medium with different formulations, place at 30°C The constant temperature incubator was cultured under full light, and the growth status of the explants was counted after 10-12 days.

[0051] In order to find the optimal medium suitable for the tissue culture of Ai Naxiang, by setting a series of salt concentration gradients (MS, 3 / 4MS and 1 / 2MS) and adding 10-30mg / L inositol to the Ainaxiang tissue culture Conditions (explant growth) were optimized, and the results are shown in Table 3 and Table 4. (1) Com...

Embodiment 3

[0053] The screening and optimization of embodiment 3 callus induction medium

[0054] Divide the shoot tip explants or leaf explants of Ainaxiang into 10 groups, first disinfect them with 70% alcohol for 30 seconds, wash them with sterile water for 3 to 5 times, and then wash them with 0.5% to 2.0% Tween 80 and 5 ~30mg / L silver nitrate with 0.1% mercuric chloride for 10min, rinsed with sterile water 5 times to pH=7, inoculated in callus induction culture based on modified MS medium with different concentrations of plant growth regulators added Each group was treated with 20 bottles, repeated 3 times, placed in a constant temperature incubator at 30°C under full light, and the growth status of the explants was counted after 10-12 days.

[0055] Appropriate auxin / cytokinin ratio is conducive to the proliferation of callus. When the ratio is too high, it will easily lead to callus browning; if it is too low, the callus will not proliferate or the proliferation coefficient will...

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Abstract

The invention discloses an embryoid-approach blumea balsamifera tissue culture method. The tissue culture method includes the optimized steps of pretreatment, detoxification method implementing, embryogenic callus induction, callus proliferation, clump bud induction, seedling strengthening culturing, rooting culturing and the like. The tissue culture method is characterized in that the blumea balsamifera tissue culture method is carried out through an embryonic callus approach for the first time, and a characteristic culture medium is an induction culture medium of a blumea balsamifera embryogenic callus and is prepared from a 3 / 4 MS culture medium comprising 10 mg / L to 30 mg / L of inositol, 0.1 mg / L to 2.0 mg / L of 2,4-D, 0.1 mg / L to 2.0 mg / L of NAA, 0.2 mg / L to 0.5 mg / L of 6-BA and 0.1 mg / L to 2.0 mg / L of TDZ. In the blumea balsamifera seedling breeding process, the method has the advantages that the increase rate is high, cost is low, the rooting percentage and the seedling-transplanting survival rate are high, and the growth cycle is not limited by the seasons; in addition, the method can also be used for blumea balsamifera germplasm innovation and new variety breeding such as polyploid induction, mutation induction and transgenosis germplasm innovation.

Description

technical field [0001] The invention relates to the technical field of tissue culture, and more specifically, relates to a tissue culture method of Ainaxiang through the embryoid body pathway. Background technique [0002] Aina Xiang ( Blumeabasamifera (L.)DC) belongs to the Asteraceae Inula family, and is a characteristic authentic medicinal material in Hainan, Guizhou, Yunnan, Guangdong and other provinces and regions of China. Sore throat, sore throat, etc. , the main raw material of national medicine for gastrointestinal diseases, with definite curative effect and good market prospect, with an annual output value of 150 to 200 million yuan; in addition, Ainaxiang is also rich in terpenoids and flavonoids, which not only contains l - borneol, α - pinene, l - Monoterpenoids such as camphor, which also contain β -Sesquiterpene compounds such as caryophyllene, also contain flavonoids, flavonols, dihydroflavonoids and other flavonoids, which are important carriers for st...

Claims

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Application Information

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IPC IPC(8): A01H4/00
Inventor 张影波庞玉新王丹胡璇陈晓鹭于福来官玲亮
Owner TROPICAL CORP STRAIN RESOURCE INST CHINESE ACAD OF TROPICAL AGRI SCI