CN104396750A - Tissue culture propagation method for clematis lanuginosa - Google Patents

Abstract

The invention discloses a tissue culture propagation method for clematis lanuginosa. The excellent effects of high multiplication rate, stable hereditary feature, high reproduction coefficient and vigorous test-tube plantlet growth are achieved through the steps of primary culture, multiplication culture, rooting culture, seedling hardening and transplanting mainly, the germinating rate of the callus of the clematis lanuginosa is up to 98%, the multiplication multiple is up to 5.7 times, the rooting rate is up to 100%, and the final survival rate is up to 96%.

Description

The tissue culture propagation method of a kind of mao of leaf clematis

Technical field

What the present invention relates to is a kind ofly be applicable to a mao tissue culture propagation method for leaf clematis.

Background technology

Hair leaf clematis climbs up by holding on to liana, Chang Weidan leaf to life, extremely rare trifoliolate leaf; Dan Huading is raw; The sharp point in top, base portion is gradually narrow, and inner face is without hair, and base goes out the raw net arteries and veins of three straight middle arteries and veins and side can be seen, during outside is straight along three, arteries and veins forms a lance-shaped band, and by the bent pubescence of yellow, outside is by the shallow fine hair be close to, and edge is bordering on without hair; In June at florescence, really July phase is to August.Originate in northeast, Zhejiang.Often be born in the mountain valley between height above sea level 100-400 rice, small stream limit shrubbery.

At present, not yet someone studies to utilize tissue culture technology to breed a mao technology for leaf clematis, and tissue culturing system not yet sets up, and popularizing of improved seeds not yet completes, and nursery stock disparities between supply and demand are increasingly sharpened, and has had a strong impact on Landscape Application and the popularization of hair leaf clematis.

Summary of the invention

The present invention is directed to the tissue culture propagation method that existing deficiency provides a kind of mao of leaf clematis.

The present invention realizes above-mentioned purpose by the following method.

A tissue culture propagation method for mao leaf clematis, the method comprises the following steps:

(1) selection of explant: take the hair leaf clematis tender stem segments of robust growth to make explant 4 ~ July;

(2) material processing method: first brush away gently with the dust of banister brush by hair leaf clematis surface, ultra violet lamp 10min is used after 10 minutes with running water, proceed to superclean bench, superclean bench is first used the alcohol immersion 10 seconds of 70%, and then proceed to dripped 2 ~ 3 POLYSORBATE 80s 0.1% mercuric chloride solution in soak 2-3min, use aseptic water washing again 3 ~ 4 times, finally aseptically separating materials, it is long that stem section cuts into 0.4 ~ 0.6cm;

(3) Initial culture: under sterile conditions the explant that step (2) obtains is seeded on Initial culture base, this medium with the addition of 2 of 2.5mg/L in 3/4MS conventional medium, the GA of NAA, 0.6mg/L of 4-D, 0.7mg/L, PH 6.5, cultivation temperature is 21 DEG C, periodicity of illumination 13h/d, luminous intensity is 2100-2300lux, until cultivation after about 14 days, when sprouting grows to about 2.0cm, namely start the next stage;

(4) Multiplying culture: the explant sprouting of Initial culture is cut and is seeded on subculture medium, this medium with the addition of 2 of 2.5mg/L in 1/2MS conventional medium, the GA of NAA, 0.6mg/L of 4-D, 0.7mg/L, PH 6.5, cultivation temperature is 21 DEG C, periodicity of illumination 13h/d, and luminous intensity is 2100-2300lux, when sprouting grows to 2.0cm, namely start the next stage;

(5) culture of rootage: the explant sprouting of squamous subculture is cut and is seeded on root media, this medium is the NAA of IAA, the 0.8mg/L that with the addition of 2.0mg/L in MS conventional medium, the KT of 1.2mg/L, PH 6.5, cultivation temperature is 24 DEG C, periodicity of illumination 13h/d, and luminous intensity is 2000lux, when new talent grows to 2.0cm, namely start the next stage;

(6) acclimatization and transplants: first blake bottle is moved to greenhouse, natural conditions lower refining seedling 7d, then sealed membrane is untied, hardening 3d, suitable shading during hardening, intensity of illumination is made to be 40% of natural daylight, after hardening completes, taking-up seedling cleans the medium of root attachment, be transplanted to the weight ratio rice chaff ash through sterilization: perlite: vermiculite=1: in the mixed-matrix of 2: 2, temperature controls at 25 DEG C, front 5d humid control, about 80%, reduces humidity later gradually, until under transplanted seedling is placed in natural conditions.

Beneficial effect: the present invention adopts tissue culture technique to hair leaf clematis, with tender stem section for explant, through explant sterilization, Initial culture, Multiplying culture, culture of rootage and acclimatization and transplants step, the nutritional need of test-tube plantlet in research incubation, the regulation and control that hormone breaks up seedling growth, solve the microbody fast breeding of hair leaf clematis, the important technological problems of root induction, establish perfect Mao Ye clematis tissue culturing system, reach efficient explant differentiation and proliferation and the high-quality of inducing and ensure the object that seedling survives, and save whole merits of parent, stabilization characteristics of genetics.This method can form a large amount of excellent test-tube plantlet in a short time, carries out scale, factorial praluction.

Embodiment

Below in conjunction with embodiment, the present invention is further illustrated, and following embodiment, only for illustration of the present invention, does not limit the present invention.

The tissue culture propagation method of hair leaf clematis, the method comprises the following steps:

(1) selection of explant: take the hair leaf clematis tender stem segments of robust growth to make explant 4 ~ July;

(2) material processing method: first brush away gently with the dust of banister brush by hair leaf clematis surface, ultra violet lamp 10min is used after 10 minutes with running water, proceed to superclean bench, superclean bench is first used the alcohol immersion 10 seconds of 70%, and then proceed to dripped 2 ~ 3 POLYSORBATE 80s 0.1% mercuric chloride solution in soak 2-3min, use aseptic water washing again 3 ~ 4 times, finally aseptically separating materials, it is long that stem section cuts into 0.4 ~ 0.6cm;

(3) Initial culture: under sterile conditions the explant that step (2) obtains is seeded on Initial culture base, this medium with the addition of 2 of 2.5mg/L in 3/4MS conventional medium, the GA of NAA, 0.6mg/L of 4-D, 0.7mg/L, PH 6.5, cultivation temperature is 21 DEG C, periodicity of illumination 13h/d, luminous intensity is 2100-2300lux, until cultivation after about 14 days, when sprouting grows to about 2.0cm, namely start the next stage;

(4) Multiplying culture: the explant sprouting of Initial culture is cut and is seeded on subculture medium, this medium with the addition of 2 of 2.5mg/L in 1/2MS conventional medium, the GA of NAA, 0.6mg/L of 4-D, 0.7mg/L, PH 6.5, cultivation temperature is 21 DEG C, periodicity of illumination 13h/d, and luminous intensity is 2100-2300lux, when sprouting grows to 2.0cm, namely start the next stage;

(5) culture of rootage: the explant sprouting of squamous subculture is cut and is seeded on root media, this medium is the NAA of IAA, the 0.8mg/L that with the addition of 2.0mg/L in MS conventional medium, the KT of 1.2mg/L, PH 6.5, cultivation temperature is 24 DEG C, periodicity of illumination 13h/d, and luminous intensity is 2000lux, when new talent grows to 2.0cm, namely start the next stage;

(6) acclimatization and transplants: first blake bottle is moved to greenhouse, natural conditions lower refining seedling 7d, then sealed membrane is untied, hardening 3d, suitable shading during hardening, intensity of illumination is made to be 40% of natural daylight, after hardening completes, taking-up seedling cleans the medium of root attachment, be transplanted to the weight ratio rice chaff ash through sterilization: perlite: vermiculite=1: in the mixed-matrix of 2: 2, temperature controls at 25 DEG C, front 5d humid control, about 80%, reduces humidity later gradually, until under transplanted seedling is placed in natural conditions.

Through the cultivation of above-mentioned steps, the rate of sprouting of the callus of hair leaf clematis reaches 98%, and proliferation times reaches 5.7 times, and rooting rate reaches 100%, and final survival rate reaches 96%.

Claims (1)

1. a tissue culture propagation method for hair leaf clematis, is characterized in that the method comprises the following steps:

(1) selection of explant: take the hair leaf clematis tender stem segments of robust growth to make explant 4 ~ July;

(2) material processing method: first brush away gently with the dust of banister brush by hair leaf clematis surface, ultra violet lamp 10min is used after 10 minutes with running water, proceed to superclean bench, superclean bench is first used the alcohol immersion 10 seconds of 70%, and then proceed to dripped 2 ~ 3 POLYSORBATE 80s 0.1% mercuric chloride solution in soak 2-3min, use aseptic water washing again 3 ~ 4 times, finally aseptically separating materials, it is long that stem section cuts into 0.4 ~ 0.6cm;

(3) Initial culture: under sterile conditions the explant that step (2) obtains is seeded on Initial culture base, this medium with the addition of 2 of 2.5mg/L in 3/4MS conventional medium, the GA of NAA, 0.6mg/L of 4-D, 0.7mg/L, PH 6.5, cultivation temperature is 21 DEG C, periodicity of illumination 13h/d, luminous intensity is 2100-2300lux, until cultivation after about 14 days, when sprouting grows to about 2.0cm, namely start the next stage;

(4) Multiplying culture: the explant sprouting of Initial culture is cut and is seeded on subculture medium, this medium with the addition of 2 of 2.5mg/L in 1/2MS conventional medium, the GA of NAA, 0.6mg/L of 4-D, 0.7mg/L, PH 6.5, cultivation temperature is 21 DEG C, periodicity of illumination 13h/d, and luminous intensity is 2100-2300lux, when sprouting grows to 2.0cm, namely start the next stage;

(5) culture of rootage: the explant sprouting of squamous subculture is cut and is seeded on root media, this medium is the NAA of IAA, the 0.8mg/L that with the addition of 2.0mg/L in MS conventional medium, the KT of 1.2mg/L, PH 6.5, cultivation temperature is 24 DEG C, periodicity of illumination 13h/d, and luminous intensity is 2000lux, when new talent grows to 2.0cm, namely start the next stage;

(6) acclimatization and transplants: first blake bottle is moved to greenhouse, natural conditions lower refining seedling 7d, then sealed membrane is untied, hardening 3d, suitable shading during hardening, intensity of illumination is made to be 40% of natural daylight, after hardening completes, taking-up seedling cleans the medium of root attachment, be transplanted to the weight ratio rice chaff ash through sterilization: perlite: vermiculite=1: in the mixed-matrix of 2: 2, temperature controls at 25 DEG C, front 5d humid control, about 80%, reduces humidity later gradually, until under transplanted seedling is placed in natural conditions.