CN103535275A

CN103535275A - Tissue-culture rapid multiplication method of nerviliae fordii corm

Info

Publication number: CN103535275A
Application number: CN201210266184.7A
Authority: CN
Inventors: 李守岭; 白燕冰; 高燕
Original assignee: Dehong Tropical Agriculture Institute of Yunnan
Current assignee: Dehong Tropical Agriculture Institute of Yunnan
Priority date: 2012-07-15
Filing date: 2012-07-15
Publication date: 2014-01-29

Abstract

The invention provides a tissue-culture rapid multiplication method of nerviliae fordii corm and relates to the technical field of bioengineering. The tissue-culture rapid multiplication method is realized by the following scheme: the nerviliae fordii corm is used as an explant, sterilized and treated, germination culture is carried out onto the corm, and root stocks are obtained through culture; multiplication culture is carried out onto the root stocks to form a plurality of root stocks; and corm-induced culture is carried onto the root stocks to form a plurality of corm provenances. The method disclosed by the invention can be used for improving corm multiplication speed and proliferation rate of the nerviliae fordii and is beneficial to industrialized production of the nerviliae fordii corm.

Description

Ford nervilia leaf bulb tissue culture quick propagation culturing method

Technical field

The present invention relates to the bulb cultural method of medicinal ford nervilia leaf, relate in particular to a kind of ford nervilia leaf bulb tissue culture quick propagation culturing method.

Background technology

Ford nervilia leaf is a kind of rare Chinese medicine, mainly be distributed in Guangdong, Guangxi, the ground such as Sichuan and Yunnan, it is one of Guangdong real estate " southern medicine ", its medicinal part is blade or complete stool, in recent years, because the market price is better, people disorderly adopt disorderly and dig, mostly be that leaf belt bulb is excavated processing, wild resource is on the verge of exhaustion, in recent years, ford nervilia leaf artificial cultivation one is that the traditional bulb of sampling carries out artificial propagation, but because 1～2 bulb is only bred in the every strain of ford nervilia leaf every year, each bulb can only develop into a strain whole plant, not only reproduction rate is low, and the cycle is longer, the 2nd, utilize tissue culture technique to breed ford nervilia leaf seedling, but sapling multiplication technology is also immature, reproduction coefficient is low.

Summary of the invention

In order to overcome the deficiency in existing ford nervilia leaf sapling multiplication method, the invention provides a kind of ford nervilia leaf bulb tissue culture quick propagation culturing method, the method can improve propagation by corm speed and the propagation multiple of ford nervilia leaf, is conducive to the batch production of ford nervilia leaf bulb and produces.

The technical solution adopted for the present invention to solve the technical problems is: take ford nervilia leaf bulb as explant, sterile-processed after, bulb is sprouted to cultivation, by cultivation, obtain root-like stock; Then root-like stock is bred to cultivation, form a large amount of root-like stocks; Again root-like stock is carried out to bulb induction and cultivate, can form a large amount of bulb provenances.

Explant is chosen and is sterilized: the bulb of choosing the anosis worm of robust growth, first use detergent immersion, with toothbrush, the earth on bulb is washed, then with 75% alcohol-pickled 30s, be placed on the 10～15min that sterilizes in 0.1% mercuric chloride solution, then use aseptic water washing 5～6 times.

Sprout and cultivate: bulb is inoculated on germination medium, germination medium is mg/litre+NAA0.5～1.0, MS+BA2.0～4.0 mg/litre+KT0.5～1.0 mg/litre+LH (lactoalbumin hydrolysate) 1.0 grams per liters+sucrose 30 grams per liters, germination medium pH5.8～6.2,25～27 ℃ of cultivation temperature, intensity of illumination 1500～2000Lx, illumination every day 12 hours, incubation time 30～45 days, induces root-like stock.

Root-like stock propagation is cultivated: root-like stock is inoculated on proliferated culture medium, proliferated culture medium is mg/litre+NAA2.0～4.0, MS+BA2.0～4.0 mg/litre+GA3 2.0～4.0 mg/litre+10～20% coconut milk+sucrose 30 grams per liters, proliferated culture medium pH5.8～6.2,25～27 ℃ of cultivation temperature, intensity of illumination 1500～2000Lx, illumination every day 12 hours, incubation time 30～45 days, obtains a large amount of root-like stocks.

Bulb induction is cultivated: root-like stock is inoculated on inducing culture, inducing culture is 1/2MS+BA0.1～0.5 milligram+NAA0.1～0.5 mg/litre+10～20% coconut milk+0.1% charcoal, proliferated culture medium pH5.8～6.2,25～27 ℃ of cultivation temperature, dark culturing, incubation time 50～60 days, obtains a large amount of bulb provenances, is directly used in cultivation and produces.

The invention has the beneficial effects as follows, can improve ford nervilia leaf sapling multiplication speed and increment multiple, shorten the sapling multiplication cycle, be conducive to carry out batch production production.

Embodiment

The bulb of choosing the anosis worm of robust growth is explant, concrete grammar comprises the steps: that (1) explant chooses and sterilize: the bulb of choosing the anosis worm of robust growth, first use detergent immersion, with toothbrush, the earth on bulb is washed, then with 75% alcohol-pickled 30s, be placed on the 10～15min that sterilizes in 0.1% mercuric chloride solution, then use aseptic water washing 5～6 times; (2) sprout and cultivate: bulb is inoculated on germination medium, germination medium is mg/litre+KT0.5～1.0, mg/litre+NAA0.5～1.0, MS+BA2.0～4.0 milligram+LH (lactoalbumin hydrolysate) 1.0 grams per liters+sucrose 30 grams per liters, germination medium pH5.8～6.2,25～27 ℃ of cultivation temperature, intensity of illumination 1500～2000Lx, illumination every day 12 hours, incubation time 30～45 days, induces root-like stock; (3) root-like stock propagation is cultivated: root-like stock is inoculated on proliferated culture medium, and proliferated culture medium is mg/litre+NAA2.0～4.0, MS+BA2.0～4.0 mg/litre+GA ₃2.0～4.0 mg/litre+10～20% coconut milk+sucrose 30 grams per liters, proliferated culture medium pH5.8～6.2,25～27 ℃ of cultivation temperature, intensity of illumination 1500～2000Lx, illumination every day 12 hours, incubation time 30～45 days, obtains a large amount of root-like stocks; (4) bulb induction is cultivated: root-like stock is inoculated on inducing culture, inducing culture is 1/2MS+BA0.1～0.5 milligram+NAA0.1～0.5 mg/litre+10～20% coconut milk+0.1% charcoal, proliferated culture medium pH5.8～6.2,25～27 ℃ of cultivation temperature, dark culturing, incubation time 50～60 days, obtains a large amount of bulb provenances.

Claims

1. a ford nervilia leaf bulb tissue culture quick propagation culturing method, is characterized in that: take ford nervilia leaf bulb as explant, sterile-processed after, bulb is sprouted to cultivation, by cultivation, obtain root-like stock; Then root-like stock is bred to cultivation, form a large amount of root-like stocks; Again root-like stock is carried out to bulb induction and cultivate, can form a large amount of bulb provenances; Concrete steps are as follows:

(1) explant is chosen and is sterilized: the bulb of choosing the anosis worm of robust growth, first use detergent immersion, with toothbrush, the earth on bulb is washed, then with 75% alcohol-pickled 30s, be placed on the 10～15min that sterilizes in 0.1% mercuric chloride solution, then use aseptic water washing 5～6 times;

(2) sprout and cultivate: bulb is inoculated on germination medium, germination medium is mg/litre+KT0.5～1.0, mg/litre+NAA0.5～1.0, MS+BA2.0～4.0 milligram+LH (lactoalbumin hydrolysate) 1.0 grams per liters+sucrose 30 grams per liters, germination medium pH5.8～6.2,25～27 ℃ of cultivation temperature, intensity of illumination 1500～2000Lx, illumination every day 12 hours, incubation time 30～45 days, induces root-like stock;

(3) root-like stock propagation is cultivated: root-like stock is inoculated on proliferated culture medium, and proliferated culture medium is mg/litre+NAA2.0～4.0, MS+BA2.0～4.0 mg/litre+GA ₃2.0～4.0 mg/litre+10～20% coconut milk+sucrose 30 grams per liters, proliferated culture medium pH5.8～6.2,25～27 ℃ of cultivation temperature, intensity of illumination 1500～2000Lx, illumination every day 12 hours, incubation time 30～45 days, obtains a large amount of root-like stocks;

(4) bulb induction is cultivated: root-like stock is inoculated on inducing culture, inducing culture is 1/2MS+BA0.1～0.5 milligram+NAA0.1～0.5 mg/litre+10～20% coconut milk+0.1% charcoal, proliferated culture medium pH5.8～6.2,25～27 ℃ of cultivation temperature, dark culturing, incubation time 50～60 days, obtains a large amount of bulb provenances.