CN109392713A - A kind of tissue culture and rapid propagation method that Kiwi berry east is red - Google Patents

Abstract

The invention discloses a kind of tissue culture and rapid propagation methods that Kiwi berry east is red, are related to Kiwifruit Cultivars tissue culture and rapid propagation method.This method is: the 1. acquisition of aseptic seedling；2. adventitious shoot regeneration induces；3. adventitious bud proliferation, strong seedling culture；4. culture of rootage；5. transplanting；It is characterized by comprising germination medium, regeneration culture medium, proliferation strong seedling culture base and root medias.The present invention is using four kinds of specific aim culture mediums to the red carry out tissue cultures in east, it can a large amount of red Kiwi berry seedlings in east of fast breeding in a short time, and it can guarantee seedling quality to the maximum extent by tissue cultures, improve seedling breeding speed, it realizes seedling industrialized production, meets in production the needs of to eastern Red Indian race seedling；Direct tissue culture kind seedling, establishes kind cutting orchard, breaks the bottleneck for grafting the amount of scion in traditional propagation by grafiting, sufficiently meets the needs of production.

Description

A kind of tissue culture and rapid propagation method that Kiwi berry east is red

Technical field

The present invention relates to the red tissue-culturing rapid propagation sides of Kiwifruit Cultivars tissue culture and rapid propagation method more particularly to a kind of Kiwi berry east Method.

Background technique

The red kiwifruit fruit elongated cylindrical in east, 70-75 grams of Mean Fruit Weight, fruit tip circle, flat, the green and brown color in fruit face, Glabrous, Neat appearance, pericarp is thick, and fruit dot is rare.Pulp is golden yellow, and core surrounding red is bright-coloured；Fresh & Tender in Texture, juice is medium more, and flavor is dense Sweet tea gives off a strong fragrance, and mineral nutrition is abundant, and especially potassium (2600mg/kg) and calcium (446mg/kg), fruit calcium content height are conducive to Storage.With the quickening of Chinese Rhesus Monkeys peach breeding of new variety speed, for Rapid Popularization new varieties and retain the excellent product of new varieties Matter, carrying out breeding using the tissue culture technique of Kiwi berry is most quickly and efficiently one of method, at present the red Kiwi berry in east Industrialization promotion plantation is in examination implementation phase, and it is mainly grafting, tissue cultures east Red Indian race that large area, which plants source of seedling, The method of seedling has not been reported, and grafting obtains the compatibility of scion and stock height, graft union healing in the approach of seedling The growth potential and yielding ability of graft survival rate and later period kind scion are all directly affected, in addition the limitation of scion quality and quantity As east it is red be widely applied, a bottleneck of industrialization production.

Summary of the invention

In view of the deficiencies in the prior art, the object of the invention is that provide a kind of red tissue culture in Kiwi berry east fast Breeding method directly obtains uniform, the excellent red tissue culture seedling in vitro east of quality by method for tissue culture, and by optimize from Body rapid propagation system realizes the red Kiwi berry high quality seedling in fast breeding east, for ever-increasing seedling demand in production.

The technical solution adopted by the present invention is as follows:

To the east of red annotinous branch water planting rudiment be explant, carry out tissue cultures after disinfection, aseptic seedling obtained, with sterile miaoye Handle is regenerating tissues source, carries out induction differentiation, culture of rootage.Adventitious bud Direct Regeneration rate is up to 91.2%, and value-added coefficient is reachable 4.5.It can be quickly obtained a large amount of fine quality seedlings in a short time, realize the seedling industrialized fast seedling growing of eastern Red Indian race.

Specifically, this method includes the following steps:

1. the acquisition of aseptic seedling

1) annotinous branch full from clip robust growth axillary bud on the red elite stand in east at the end of November, is cut into 30cm long shoot for branch Section, every section of 5-10 bud；

2) the branch section morphology upper end being sealed with sealing film, indoor culture in 2 ‰ aqueous sucrose solutions is impregnated in morphology lower end, 25 ± 2 DEG C of environment temperature of holding, guarantee indoor cleaning, the aqueous solution of replacement in 2-3 days, after two weeks, axillary bud eruption；

3) when axillary bud sprout it is long to 1-2cm when, take young tender shoots body on superclean bench, being placed in sterilized empty bottle, 75% wine Smart surface sterilization 1min, pours out alcohol, and it is that 0.1% mercuric chloride deeply sterilizes 4-6min that mass volume ratio, which is added, mercuric chloride solution with it is external Body fullys shake, and guarantees that disinfection thoroughly, pours out thimerosal, and explant more changes in new sterilizing bottle, and 4-5 is rinsed in sterile water concussion It is secondary, it is dried in sterilizing filter paper；

4) remove the wound of axillary bud sprout base portion and the blade of damage, be inoculated on explant germination medium, in illumination condition Lower culture；

5) after 25-30 days, sprout lamina, robust growth obtains aseptic seedling；

2. adventitious shoot regeneration induces

1) in cutting aseptic seedling petiole on superclean bench, it being cut into 0.5cm segment, is placed on regeneration culture medium, pH is adjusted to 5.8, Carry out the regeneration induction of adventitious bud；

2) inoculation material is placed under illumination condition and cultivates；

3) after 35-45 days, petiole Direct Regeneration adventitious bud.

3. adventitious bud proliferation, strong seedling culture

Regeneration obtains adventitious bud after subculture is primary on best adventitious shoot regeneration culture medium, and adventitious bud is cut into single sprout, It is placed on proliferation strong seedling culture base and carries out proliferation strong seedling culture, pH is adjusted to 5.8；

4. culture of rootage

By the relatively uniform high 2-3cm of growth conditions, the adventitious bud for having 5-6 piece leaf cuts, is inoculated on root media and carries out Culture of rootage, 5-7 days i.e. existing visually visible adventitious roots, short and thick, healthy and strong, root long 2.5-3cm after 25-30 days, 8-10 item root Transplanting；

5. transplanting

1) after culture of rootage 25 days, bottle seedling of taking root is directly taken out, the culture medium in foundation is cleaned with tap water；

2) seedling is planted to the small nutritive cube that Nutrition Soil is housed, and nutritive cube top covered plastic film guarantees locating for new transplanting seedling Ambient humidity be 85-90%, temperature control removes plastic film after 25-30 DEG C, 3-7 days, ambient humidity is suitably reduced to 60-75%；

3) conventional water and fertilizer management after 28-30 days, environment temperature control is at 35 DEG C hereinafter, high temperature summer need to shade and Wet-curtain temperature reducing.

Specific requirement:

The germination medium are as follows:+0.8% agar of MS+6-BA 2.0-5.0mg/L+1%-2% sucrose；

In the acquisition of the aseptic seedling, inoculation material condition of culture are as follows: intensity of illumination 2000-2500Lx, illumination 12h/d, culture 25 ± 2 DEG C of room temperature；

The regeneration culture medium are as follows:+0.8% agar of MS+6-BA 0.1-2.0mg/L+NAA 0.1-1.0mg/L+3% sucrose；

In the adventitious shoot regeneration induction, inoculation material condition of culture are as follows: intensity of illumination 2500-2800Lx, illumination 12h/d, training Support 25 ± 2 DEG C of room temperature；

The proliferation strong seedling culture base are as follows: MS+6-BA 0.01-1.0 mg/L+NAA 0.005-0.1mg/L+3% sucrose+ 0.8% agar；

In the adventitious bud proliferation, strong seedling culture, inoculation material condition of culture are as follows: intensity of illumination 3000-3200Lx, illumination 12h/ D cultivates 25 ± 2 DEG C of room temperature；

The root media are as follows: 1/2MS+IBA 0.1-2.0 mg/L+3% sucrose+0.8-1.5% agar；

In the culture of rootage, inoculation material condition of culture are as follows: intensity of illumination 3400-3500Lx, illumination 12h/d, in culturing room 25 ± 2 DEG C of temperature；

The Nutrition Soil ingredient are as follows: fertile soil: turfy soil: perlite=2:1:1.

Innovative point of the invention: the method that initial stage of the invention uses indoor aqueous sucrose solution culture, to inspire axillary bud sprouting As explant source, evade the pollution sources of direct outdoor sampling, greatly reduces the wind that bacterium, Mycophyta and virus carry Danger, while the mercuric chloride processing time is reduced, reduce the injury to explant histocyte physiologically.To material in tissue culture procedures The intensity of illumination of receiving makes gradient adjustment, and domestication is transplanted to it and plays induction adaptation well.Late stage of culture is taken root Stage increases the usage amount of agar powder, is more advantageous to root of hair, cleaning when also being more conducive to transplant to plant foundation position culture medium, To reduce the damage to root, transplanting survival rate is greatly improved.Seedling is directly transplanted without domestication, saves time and manpower phase Resource is closed, from largely reducing production cost.The control of temperature and humidity in transplanting initial stage certain time, is conducive to seedling The environment after rapidly adapting to transplanting in short term, both shortens breeding time, also greatly improves seedling transplanting survival rate.

The present invention has following advantages and good effect:

1. the present invention using four kinds of specific aim culture mediums to the red carry out tissue cultures in east, can in a short time a large amount of east of fast breeding it is red Kiwi berry seedling, and can guarantee seedling quality to the maximum extent by tissue cultures, seedling breeding speed is improved, realizes seedling Industrialized production meets in production the needs of to eastern Red Indian race seedling；

2. explant uses water planting budding mode during providing, the dirt in aseptic seedling establishment process can be farthest controlled Dye largely improves the breeding speed of seedling, shortens the breeding cycle of seedling, reduce the cost of tissue culture.

3. during seedling fostering, the enhancing of the change of condition of culture, especially intensity of illumination is conducive to seedling accumulation Nutrient growth ability enhances seedling resistance, greatly improves later period tissue culture seedling transplanting survival rate；

4. planting transplantation of seedlings early period, to the strict control of the temperature and humidity of transplanting environment, it can guarantee seedling transplant survival to the greatest extent Rate, to reduce loss, reduce production cost.

5. obtaining eastern Red Indian race seedling relative to previous cuttage, grafting, tissue-culturing rapid propagation nursery more can guarantee seedling quality, seedling Growth is consistent, neat, and can meet production within the shorter time to the quantitative demand of seedling；

6. direct tissue culture kind seedling, establishes kind cutting orchard, breaks the bottleneck for grafting the amount of scion in traditional propagation by grafiting, fill Divide and meets the needs of production.

Specific embodiment

It is described in detail with reference to embodiments.

One, embodiment 1

A kind of tissue culture and rapid propagation method that Kiwi berry east is red, is completed in the steps below:

1, the acquisition of aseptic seedling:

The annotinous branch full from clip robust growth axillary bud on the red elite stand in east at the end of November, is cut into 30cm long shoot section, and every section 10 buds, morphology upper end seal with sealing film, and indoor culture in 2 ‰ aqueous sucrose solutions is impregnated in morphology lower end, keep environment temperature 25 ± 2 DEG C of degree guarantees that indoor cleaning, the aqueous solution of replacement in 2 days, after two weeks, axillary bud eruption take when sprout grows to 1-2cm For young tender shoots body on superclean bench, being placed in sterilized empty bottle, 75% alcohol surface sterilization 1min pours out alcohol, and matter is added Amount volume ratio is that 0.1% mercuric chloride deeply sterilizes 5min, and mercuric chloride solution fullys shake with external body, guarantees that disinfection thoroughly, pours out disinfection Liquid, explant more change in new sterilizing bottle, and sterile water concussion is rinsed 4-5 times, dry in sterilizing filter paper, remove sprout base The wound in portion and the blade of damage are inoculated on+0.8% agar of explant germination medium MS+6-BA3.0 mg/L+2% sucrose, After 26 days, sprout lamina, robust growth obtains aseptic seedling.Inoculation material is cultivated under illumination condition, intensity of illumination 2400Lx, 25 ± 2 DEG C of room temperature of culture, illumination 12h/d.

2, adventitious shoot regeneration induces:

In cutting aseptic seedling petiole on superclean bench, it is cut into 0.5cm segment, is placed in regeneration culture medium MS+BA0.5mg/L+ On+0.8% agar of NAA0.2mg/L+3% sucrose, pH is adjusted to 5.8, carries out the regeneration induction of adventitious bud.Inoculation material is placed in illumination Under the conditions of cultivate.Intensity of illumination 2500Lx cultivates 25 ± 2 DEG C of room temperature, illumination 12h/d.After 35 days, petiole Direct Regeneration is not Normal bud, quantity is more, and is clump bud, and differentiation is fast, and color bright green, adventitious bud Direct Regeneration is up to 91.2%.

3, adventitious bud proliferation, strong seedling culture:

Regeneration obtains adventitious bud after subculture is primary on best adventitious shoot regeneration culture medium, and adventitious bud is cut into single sprout, It is placed on+0.8% agar medium of MS+6-BA0.2mg/L+NAA0.05mg/L+3% sucrose and carries out proliferation strong seedling culture, pH is adjusted to 5.8.Intensity of illumination 3000Lx cultivates 25 ± 2 DEG C of room temperature, illumination 12h/d.

4, culture of rootage:

By the relatively uniform high 2-3cm of growth conditions, the adventitious bud for having 5-6 piece leaf is cut, and is inoculated in 1/2MS+IBA0.5mg/L Culture of rootage is carried out on the root media of+1.2% agar of+3% sucrose, does not have to adjust pH.Intensity of illumination 3500Lx, in culturing room 25 ± 2 DEG C of temperature, illumination 12h/d, 7 days i.e. existing visually visible adventitious roots, short and thick, healthy and strong, rooting rate 98%, root long after 25-30 days 2.5-3cm, 8-10 item root, can transplant.

5, it transplants:

After culture of rootage 25 days, bottle seedling of taking root is directly taken out, cleans the culture medium in foundation with tap water, seedling is planted to equipped with battalion Support the small nutritive cube of soil, Nutrition Soil ingredient are as follows: fertile soil: turfy soil: perlite=2:1:1.Add plastic film above nutritive cube, Guarantee that ambient humidity locating for new transplanting seedling is 85%-90%, plastic film, ring are removed in temperature control after 25-30 DEG C, 7 days Border humidity is suitably reduced to 60%-75%.Conventional water and fertilizer management after 30 days, environment temperature control is at 35 DEG C hereinafter, high temperature summer needs Shading and Wet-curtain temperature reducing.High survival rate is counted after two months up to 95%.

Two, embodiment 2

A kind of tissue culture and rapid propagation method that Kiwi berry east is red, is completed in the steps below:

1, the acquisition of aseptic seedling:

The annotinous branch full from clip robust growth axillary bud on the red elite stand in east at the end of November, is cut into 30cm long shoot section, and every section 10 buds, morphology upper end seal with sealing film, and indoor culture in 2 ‰ aqueous sucrose solutions is impregnated in morphology lower end, keep environment temperature 25 ± 2 DEG C of degree guarantees that indoor cleaning, the aqueous solution of replacement in 2 days, after two weeks, axillary bud eruption take when sprout grows to 1-2cm For young tender shoots body on superclean bench, being placed in sterilized empty bottle, 75% alcohol surface sterilization 1min pours out alcohol, and matter is added Amount volume ratio is that 0.1% mercuric chloride deeply sterilizes 4min, and mercuric chloride solution fullys shake with external body, guarantees that disinfection thoroughly, pours out disinfection Liquid, explant more change in new sterilizing bottle, and sterile water concussion is rinsed 4-5 times, dry in sterilizing filter paper, remove sprout base The wound in portion and the blade of damage are inoculated on+0.8% agar of explant germination medium MS+6-BA2.0 mg/L+2% sucrose, After 28 days, sprout lamina, robust growth obtains aseptic seedling.Inoculation material is cultivated under illumination condition, intensity of illumination 2000Lx, 25 ± 2 DEG C of room temperature of culture, illumination 12h/d.

2, adventitious shoot regeneration induces:

In cutting aseptic seedling petiole on superclean bench, it is cut into 0.5cm segment, is placed in regeneration culture medium MS+BA0.1mg/L+ On+0.8% agar of NAA0.1mg/L+3% sucrose, pH is adjusted to 5.8, carries out the regeneration induction of adventitious bud.Inoculation material is placed in illumination Under the conditions of cultivate.Intensity of illumination 2500Lx cultivates 25 ± 2 DEG C of room temperature, illumination 12h/d.After 40 days, petiole Direct Regeneration is not Normal bud, quantity is more, and is clump bud, and differentiation is fast, color bright green, and adventitious bud Direct Regeneration rate is up to 90.8%.

3, adventitious bud proliferation, strong seedling culture:

Regeneration obtains adventitious bud after subculture is primary on best adventitious shoot regeneration culture medium, and adventitious bud is cut into single sprout, It is placed on+0.8% agar medium of MS+6-BA0.01mg/L+NAA0.005mg/L+3% sucrose and carries out proliferation strong seedling culture, pH tune To 5.8.Intensity of illumination 3000Lx cultivates 25 ± 2 DEG C of room temperature, illumination 12h/d.

4, culture of rootage:

By the relatively uniform high 2-3cm of growth conditions, the adventitious bud for having 5-6 piece leaf is cut, and is inoculated in 1/2MS+IBA0.1mg/L Culture of rootage is carried out on the root media of+0.8% agar of+3% sucrose, pH5.8, intensity of illumination 3400Lx cultivate room temperature 25 ± 2 DEG C, illumination 12h/d, 7 days i.e. existing visually visible adventitious roots, short and thick, healthy and strong, rooting rate 96%, root long 2.5- after 25-30 days 3cm, 8-10 item root, can transplant.

5, it transplants:

After culture of rootage 25 days, bottle seedling of taking root is directly taken out, cleans the culture medium in foundation with tap water, seedling is planted to equipped with battalion Support the small nutritive cube of soil, Nutrition Soil ingredient are as follows: fertile soil: turfy soil: perlite=2:1:1.Add plastic film above nutritive cube, Guarantee that ambient humidity locating for new transplanting seedling is 85%, plastic film, environmental wet are removed in temperature control after 25-30 DEG C, 7 days Degree is suitably reduced to 70%.Conventional water and fertilizer management after 28 days, environment temperature control is at 35 DEG C hereinafter, high temperature summer need to shade and wet Curtain cooling.

High survival rate is counted after two months up to 94.5%.

Three, embodiment 3

A kind of tissue culture and rapid propagation method that Kiwi berry east is red, is completed in the steps below:

1, the acquisition of aseptic seedling:

The annotinous branch full from clip robust growth axillary bud on the red elite stand in east at the end of November, is cut into 30cm long shoot section, and every section 10 buds, morphology upper end seal with sealing film, and indoor culture in 2 ‰ aqueous sucrose solutions is impregnated in morphology lower end, keep environment temperature 25 ± 2 DEG C of degree guarantees that indoor cleaning, the aqueous solution of replacement in 2 days, after two weeks, axillary bud eruption take when sprout grows to 1-2cm For young tender shoots body on superclean bench, being placed in sterilized empty bottle, 75% alcohol surface sterilization 1min pours out alcohol, and matter is added Amount volume ratio is that 0.1% mercuric chloride deeply sterilizes 6min, and mercuric chloride solution fullys shake with external body, guarantees that disinfection thoroughly, pours out disinfection Liquid, explant more change in new sterilizing bottle, and sterile water concussion is rinsed 4-5 times, dry in sterilizing filter paper, remove sprout base The wound in portion and the blade of damage are inoculated in+0.8% agar of explant germination medium MS+6-BA5.0 mg/L+1.5% sucrose On, after 25 days, sprout lamina, robust growth obtains aseptic seedling.Inoculation material is cultivated under illumination condition, intensity of illumination 2500Lx cultivates 25 ± 2 DEG C of room temperature, illumination 12h/d.

2, adventitious shoot regeneration induces:

In cutting aseptic seedling petiole on superclean bench, it is cut into 0.5cm segment, is placed in regeneration culture medium MS+BA2.0mg/L+ On+0.8% agar of NAA1.0mg/L+3% sucrose, pH is adjusted to 5.8, carries out the regeneration induction of adventitious bud.Inoculation material is placed in illumination Under the conditions of cultivate.Intensity of illumination 2800Lx cultivates 25 ± 2 DEG C of room temperature, illumination 12h/d.After 42 days, petiole Direct Regeneration is not Normal bud, quantity is more, and is clump bud, and differentiation is fast, and color bright green, adventitious bud Direct Regeneration is up to 91%.

3, adventitious bud proliferation, strong seedling culture:

Regeneration obtains adventitious bud after subculture is primary on best adventitious shoot regeneration culture medium, and adventitious bud is cut into single sprout, It is placed on+0.8% agar medium of MS+6-BA1.0mg/L+NAA0.1mg/L+3% sucrose and carries out proliferation strong seedling culture, pH is adjusted to 5.8.Intensity of illumination 3200Lx cultivates 25 ± 2 DEG C of room temperature, illumination 12h/d.

4, culture of rootage:

By the relatively uniform high 2-3cm of growth conditions, the adventitious bud for having 5-6 piece leaf is cut, and is inoculated in 1/2MS+IBA2.0mg/L Culture of rootage is carried out on the root media of+1.5% agar of+3% sucrose, does not have to adjust pH, intensity of illumination 3500Lx, in culturing room 25 ± 2 DEG C of temperature, illumination 12h/d, 5 days i.e. existing visually visible adventitious roots, short and thick, healthy and strong, rooting rate 94%, root long after 25-30 days 2.5-3cm, 8-10 item root, can transplant.

5, it transplants:

After culture of rootage 30 days, bottle seedling of taking root is directly taken out, cleans the culture medium in foundation with tap water, seedling is planted to equipped with battalion Support the small nutritive cube of soil, Nutrition Soil ingredient are as follows: fertile soil: turfy soil: perlite=2:1:1.Add plastic film above nutritive cube, Guarantee that ambient humidity locating for new transplanting seedling is 90%, plastic film, environmental wet are removed in temperature control after 25-30 DEG C, 5 days Degree is suitably reduced to 60%.Conventional water and fertilizer management after 30 days, environment temperature control is at 35 DEG C hereinafter, high temperature summer need to shade and wet Curtain cooling.

High survival rate is counted after two months up to 92.8%.

Claims (1)

1. a kind of tissue culture and rapid propagation method that Kiwi berry east is red, comprising the following steps:

1. the acquisition of aseptic seedling

1) annotinous branch full from clip robust growth axillary bud on the red elite stand in east at the end of November, is cut into 30cm long shoot for branch Section, every section of 5-10 bud；

2) the branch section morphology upper end being sealed with sealing film, indoor culture in 2 ‰ aqueous sucrose solutions is impregnated in morphology lower end, 25 ± 2 DEG C of environment temperature of holding, guarantee indoor cleaning, the aqueous solution of replacement in 2-3 days, after two weeks, axillary bud eruption；

3) when axillary bud sprout it is long to 1-2cm when, take young tender shoots body on superclean bench, being placed in sterilized empty bottle, 75% wine Smart surface sterilization 1min, pours out alcohol, and it is that 0.1% mercuric chloride deeply sterilizes 4-6min that mass volume ratio, which is added, mercuric chloride solution with it is external Body fullys shake, and guarantees that disinfection thoroughly, pours out thimerosal, and explant more changes in new sterilizing bottle, and 4-5 is rinsed in sterile water concussion It is secondary, it is dried in sterilizing filter paper；

4) remove the wound of axillary bud sprout base portion and the blade of damage, be inoculated on explant germination medium, in illumination condition Lower culture；

5) after 25-30 days, sprout lamina, robust growth obtains aseptic seedling；

2. adventitious shoot regeneration induces

1) in cutting aseptic seedling petiole on superclean bench, it being cut into 0.5cm segment, is placed on regeneration culture medium, pH is adjusted to 5.8, Carry out the regeneration induction of adventitious bud；

2) inoculation material is placed under illumination condition and cultivates；

3) after 35-45 days, petiole Direct Regeneration adventitious bud；

3. adventitious bud proliferation, strong seedling culture

Regeneration obtains adventitious bud after subculture is primary on best adventitious shoot regeneration culture medium, and adventitious bud is cut into single sprout, It is placed on proliferation strong seedling culture base and carries out proliferation strong seedling culture, pH is adjusted to 5.8；

4. culture of rootage

By the relatively uniform high 2-3cm of growth conditions, the adventitious bud for having 5-6 piece leaf cuts, is inoculated on root media and carries out Culture of rootage, 5-7 days i.e. existing visually visible adventitious roots, short and thick, healthy and strong, root long 2.5-3cm after 25-30 days, 8-10 item root Transplanting；

5. transplanting

1) after culture of rootage 25 days, bottle seedling of taking root is directly taken out, the culture medium in foundation is cleaned with tap water；

2) seedling is planted to the small nutritive cube that Nutrition Soil is housed, and nutritive cube top covered plastic film guarantees locating for new transplanting seedling Ambient humidity be 85-90%, temperature control removes plastic film after 25-30 DEG C, 3-7 days, ambient humidity is suitably reduced to 60-75%；

3) conventional water and fertilizer management after 28-30 days, environment temperature control is at 35 DEG C hereinafter, high temperature summer need to shade and Wet-curtain temperature reducing；

It is characterized by:

The germination medium are as follows:+0.8% agar of MS+6-BA 2.0-5.0mg/L+1%-2% sucrose；

In the acquisition of the aseptic seedling, inoculation material condition of culture are as follows: intensity of illumination 2000-2500Lx, illumination 12h/d, culture 25 ± 2 DEG C of room temperature；

The regeneration culture medium are as follows:+0.8% agar of MS+6-BA 0.1-2.0mg/L+NAA 0.1-1.0mg/L+3% sucrose；

In the adventitious shoot regeneration induction, inoculation material condition of culture are as follows: intensity of illumination 2500-2800Lx, illumination 12h/d, training Support 25 ± 2 DEG C of room temperature；

The proliferation strong seedling culture base are as follows: MS+6-BA 0.01-1.0 mg/L+NAA 0.005-0.1mg/L+3% sucrose+ 0.8% agar；

In the adventitious bud proliferation, strong seedling culture, inoculation material condition of culture are as follows: intensity of illumination 3000-3200Lx, illumination 12h/ D cultivates 25 ± 2 DEG C of room temperature；

The root media are as follows: 1/2MS+IBA 0.1-2.0 mg/L+3% sucrose+0.8-1.5% agar；

In the culture of rootage, inoculation material condition of culture are as follows: intensity of illumination 3400-3500Lx, illumination 12h/d, in culturing room 25 ± 2 DEG C of temperature；

The Nutrition Soil ingredient are as follows: fertile soil: turfy soil: perlite=2:1:1.