CN102696479A

CN102696479A - Method for propagating stonegarlic quickly and efficiently

Info

Publication number: CN102696479A
Application number: CN2012101206623A
Authority: CN
Inventors: 汪仁; 贺佳; 夏冰; 彭峰; 江玉梅; 李晓丹
Original assignee: Institute of Botany of CAS
Current assignee: Institute of Botany of CAS
Priority date: 2012-04-24
Filing date: 2012-04-24
Publication date: 2012-10-03

Abstract

The invention provides a high-efficiency rapid propagation method of Lycoris safflower, and the tissue culture steps of Lycoris safflower for large-scale production and propagation to meet market demand include: A. disinfection of explants, B. induction of adventitious buds, C. . Proliferation of adventitious buds, D. Rooting culture, E. Seedling hardening and transplanting. Adopting the method of plant tissue culture to propagate Lycoris safflower is not only not affected by external conditions, but can be carried out in all seasons, saves the land occupied by seedlings, reduces production costs, and can preserve all the excellent traits of the mother body, and the genetic traits are stable. This method can form a large amount of excellent test-tube seedlings in a short period of time, carry out large-scale and factory production, and provide a large amount of raw materials for industries such as ornamental gardening and pharmacy.

Description

A kind of short-tube lycoris of safflower efficiently method for quickly breeding

Technical field

The present invention relates to safflower short-tube lycoris method for quickly breeding efficiently, belong to artificial propagation and the cultivation method technical field of plant.

Background technology

Safflower short-tube lycoris (L.radiata) is an Amaryllidaceae Lycoris herbaceos perennial, and underground have spherical bulb, a membranous squamation of outsourcing crineous.The petal color and luster is scarlet, has another name called " the husky China of graceful pearl ", is important ornamental plants and medicinal plant.The main medicinal ingredient of safflower short-tube lycoris bulb is various lycorines and galanthamine.Lycorine and derivative thereof have certain active anticancer, and ability anti-inflammatory, analgesic, calm and emetic, and be also effective in cure to amoebic dysentery.Galanthamine is the invertibity anticholinesterase, is used to treat senile dementia, the paralysis that central paralysis diseases such as polio cause, myasthenia gravis etc.These compositions all can extract with commercial size at present.

Under field conditions (factors), most short-tube lycoris nature ripening rates are very low, though solid, but receive the restriction of growing environment, can sprout into the few of seedling.Mostly present safflower short-tube lycoris is triploid, is main with vegetative propagation mainly, and annual female bulb is split into 2 daughter bulbs and grows into 2 new plant with " division naturally " method, and the natural propagation coefficient is very low.In recent years, because a large amount of extractions of pharmaceuticals industry, the safflower short-tube lycoris (L.radiata) that former distribution is the widest, reserves are maximum and the wild resource of Lycoris aurea (L.aurea) also sharply reduced.Long-term artificial propagation is the reason that causes safflower short-tube lycoris bulb to be degenerated, and has a strong impact on the resistivity and kind ball quality of safflower short-tube lycoris to adverse circumstance, and has restricted the breeding process of safflower short-tube lycoris.

Summary of the invention

The object of the present invention is to provide a kind of short-tube lycoris of safflower efficiently method for quickly breeding, carry out large-scale production and train the needs of the research work of other subjects that are the basis with safflower short-tube lycoris group.

Technical scheme of the present invention is achieved in that the main feature of this kind short-tube lycoris method for quickly breeding is:

The sterilization of A explant: the bulb of choosing perennial safflower short-tube lycoris is cleaned, and peels off outer scale, and the excision root cuts bulb the first half about 1/2～2/3.The bulb that leaves plateau is divided into 4～8; Amway cleaning solution with 0.1% shakes washing 15～20min on shaking table, behind flowing water flushing 30～60min, again on superclean bench with 75% alcohol rinsing 30s; After the sterile water wash 4 times; Clorox with containing 1% aseptic water washing 4-5 time, blots surface moisture with aseptic filter paper at last;

The B inducing clumping bud: the fritter bulb of the bacterium of will going out is inoculated in the following medium: the MS minimal medium, and the auxin of debita spissitudo, suitably pH value carried out illumination cultivation 30～40 days;

The C adventitious bud proliferation: treat that indefinite bud grows to 2-3cm, the bulb that will have indefinite bud is inoculated in the following medium: MS cultivates basically, the auxin of debita spissitudo, suitably pH value;

D culture of rootage: choose the test-tube plantlet of robust growth, be inoculated in the following medium together with clove: 1/2MS or MS minimal medium, sucrose 60～90g/L, the auxin of debita spissitudo, suitably pH value carried out illumination cultivation 50～60 days.

E acclimatization and transplants: the tissue culture bottle lid is opened, and added a spot of distilled water, carry out the nonsterile conditions domestication; With tweezers tissue cultivating seedling is taken out from bottle, is transplanted in the cultivation matrix of having sterilized, shelter from heat or light the domestication 3 weeks, during the irregular 1/2MS nutrient solution that sprays.

The elongation technology scheme of described safflower short-tube lycoris method for quickly breeding comprises: short-tube lycoris breeds the auxin that relates in the described medium fast among pH value scope among sterilization reagent in the steps A and time, the step B and step B, C, D, the E; Comprise 6-Bian Ji adenine (6-BA), indolebutyric acid (IBA), methyl (NAA), 2; The 4-dichlorphenoxyacetic acid (2,4-D) wherein one or more.

The elongation technology scheme of described safflower short-tube lycoris method for quickly breeding comprises: the seed disinfection in the steps A is shaken washing 15～20min and is contained 1% clorox sterilization, 20～30min with the Amway cleaning solution of reagent 0.1% on shaking table.

The elongation technology scheme of described safflower short-tube lycoris method for quickly breeding comprises: step B, and C, pH value scope is 5.8～7.0 among the D.

The elongation technology scheme of described safflower short-tube lycoris method for quickly breeding comprises: it is characterized in that the involved auxin of step B is 6-Bian Ji adenine (6-BA) 3.0～5.0mg/L, methyl (NAA) 0.1～0.5mg/L.

The elongation technology scheme of described safflower short-tube lycoris method for quickly breeding comprises: auxin involved among the step C does; Auxin 6-Bian Ji adenine (6-BA) 5.0～6.0mg/L; 2; The 4-dichlorphenoxyacetic acid (2,4-D) 0.5～2.0mg/L, methyl (NAA) 0.1～0.5mg/L.

The elongation technology scheme of described safflower short-tube lycoris method for quickly breeding comprises: involved auxin is indolebutyric acid (IBA) 1.0～2.0mg/L among the step D, sucrose 60～90g/L.

Adopt the method breeding safflower short-tube lycoris of Plant Tissue Breeding, not only can break away from the influence of external condition, the four seasons can both produce, and can practice thrift the occupation of land of growing seedlings, and reduce production costs.Plant tissue culture technique is a totipotency of utilizing cell, takes one block organization of cell clique on the plant corpus, through the control of artificial condition, make these tissues form millions upon millions of plant, and preserved whole merits of parent, and stabilization characteristics of genetics.Tissue-culturing quick-propagation is the effective way of meeting the need of market as early as possible at present, also is the basis of quality saving (ultralow temperature preservation), mutation breeding and genetic transformation simultaneously.This method has kept the plant activity of safflower short-tube lycoris, can form a large amount of good test-tube plantlets in a short time, carries out scale, batch production production.

Description of drawings

Fig. 1 is the safflower short-tube lycoris bulb figure that begins to sprout indefinite bud, the indefinite bud of Fig. 2 propagation and growth, and Fig. 3 is the safflower short-tube lycoris plant of strong plantlets and rootage, Fig. 4 is safflower short-tube lycoris clove and the plant before the acclimatization and transplants.

Embodiment

Instance below in conjunction with the quick breeding of safflower short-tube lycoris is explained embodiment of the present invention.

The inducing and cultivating of instance 1 indefinite bud

1. get the underground bulb of safflower short-tube lycoris, peel off outer scale, the excision root cuts bulb the first half about 1/2～2/3.The bulb that leaves plateau is divided into 4～8; Amway cleaning solution with 0.1% shakes washing 15～20min on shaking table, behind the flowing water flushing 60min, again on superclean bench with 75% alcohol rinsing 30s; After the sterile water wash 4 times; With contain 1% liquor natrii hypochloritis, the 20～30min (add several Tween 80s) that sterilizes in the liquor natrii hypochloritis, aseptic water washing 4-5 time blots the surface of the seed moisture with aseptic filter paper at last.

2. the safflower short-tube lycoris bulb fritter that disinfects directly is inoculated in the adventitious bud induction culture base: add 6-Bian Ji adenine (6-BA) 3.0mg/L, methyl (NAA) 0.2mg/L, sucrose 30g/L, proper pH value in the MS minimal medium; 28 ± 1 ℃ of cultivation temperature, intensity of illumination 1000～3000lx, light application time 10～16h carries out illumination cultivation.

Instance 2 grow thickly inducing of bud and taking root, transplanting of test-tube plantlet

1. after cultivating 30 days, treat that the bud seedling grows to 2-3cm, the renewed vaccination of bud seedling in adventitious buds proliferation medium of the present invention, is carried out enrichment culture.The inducing clumping bud medium is to be minimal medium with the MS medium, and adds auxin 6-Bian Ji adenine (6-BA) 5.0mg/L, and 2,4 dichlorophenoxyacetic acid (2,4-D) 1.0mg/L, methyl (NAA) 0.5mg/L, sucrose 30g/L carries out enrichment culture.Cultivate more than 30 days, inductivity reaches 98%, and most of bud seedling can induce the bud number of growing thickly and reach 7-8.

2. choose the test-tube plantlet of robust growth, be inoculated into then on the 1/2MS minimal medium, indolebutyric acid (IBA) 1.0mg/L, sucrose 60g/L carries out root induction.Cultivate after 40 days, rooting rate reaches 86%, and the average root number reaches 18.5.

3. after safflower short-tube lycoris tissue cultivating seedling culture of rootage 1.5-2 month, begin to carry out acclimatization and transplants; At first tissue culture bottle is moved into illumination box, carry out alternating temperature and become light acclimation 5-7 days, again the tissue culture bottle lid is opened; And add 10ml distilled water, carry out nonsterile conditions domestication 2-3 days.Then with tissue cultivating seedling be transplanted to the cultivation matrix of having sterilized (mire: perlite: garden mould=1: 1: 5), place in the greenhouse that does not have the light of heating, shelter from heat or light 3 weeks of domestication, during the irregular 1/2MS nutrient solution that sprays, transplant the back survival rate and reach 78%.

The safflower short-tube lycoris tissue-culturing rapid propagation situation of above instance is seen accompanying drawing.

Claims

1. an efficient rapid breeding method for Lycoris safflower, characterized in that:

A. Disinfection of explants: Select the bulbs of perennial Lycoris safflower and wash them, peel off the outer scales, cut off the roots, and cut off about 1/2 to 2/3 of the upper half of the bulbs. Divide the bulbs with bulb discs into 4-8 pieces, shake and wash them with 0.1% Amway washing liquid on a shaker, rinse them with running water for 30-60 minutes, and then rinse them with 75% alcohol for 30 seconds on the ultra-clean workbench. After washing with sterile water for 4 times, rinse with 1% sodium hypochlorite and sterile water for 4 to 5 times, and finally dry the surface moisture with sterile filter paper;

B, tufted bud induction: the sterilized small piece bulb is inoculated in the following medium: MS basal medium, the auxin 6-benzyl adenine (6-BA) 3.0～5.0mg/L of appropriate concentration, Naphthaleneacetic acid (NAA) 0.1-0.5mg/L, appropriate pH value, cultured under light for 30-40 days;

C. Proliferation of adventitious buds: After the adventitious buds grow to 2-3cm, the bulbs with adventitious buds are inoculated into the following medium: MS basic culture, plant growth hormone 6-bianyl adenine (6-BA) 5.0-6.0 mg/L, 2,4-dichlorophenoxyacetic acid (2,4-D) 0.5~2.0mg/L, naphthaleneacetic acid (NAA) 0.1~0.5mg/L, proper pH value;

D, rooting culture: select the test-tube seedlings with strong growth, and inoculate in the following medium together with small bulbs: 1/2MS or MS basic medium, sucrose 60～90g/L, indole butyric acid (IBA) 1.0～2.0mg/L L, appropriate pH value, cultured under light for 50-60 days;

E. Seedling hardening and transplanting: Open the cap of the tissue culture bottle, and add a small amount of distilled water to acclimatize under the condition of bacteria; take out the tissue culture seedlings from the bottle with tweezers, transplant them into the sterilized cultivation medium, and cover them. The yin acclimatization was carried out for 3 weeks, during which the 1/2MS nutrient solution was sprayed irregularly.

2. A kind of high-efficiency Lycoris safflower rapid propagation method according to claim 1, is characterized in that 0.1% Amway washing liquid of the reagent for seed disinfection in step A is shaken and washed on a shaker for 15-20min and sterilized with 1% sodium hypochlorite. Bacteria 20 ~ 30min.

3. A highly efficient rapid propagation method for Lycoris safflower according to claim 1, characterized in that the pH range involved in steps B, C, and D is 5.8 to 7.0.