CN1320110C - Method for establishing buffalo grass regeneration system through somatic embryogenesis way - Google Patents

Abstract

The present invention relates to a method for establishing a regeneration system of buffalo grass by using buffalo grass mature embryos as materials. The method comprises four culture stages: (1) explants are directly cultured to induce calluses; (2) the calluses are subcultured; (3) the calluses are induced to obtain somatic embryos; (4) the somatic embryos regenerate into plantlets. Culture mediums used by the former three stages in the culture process are MS culture mediums containing 2, 4-D and 6-BA of different mixture ratios, and the somatic embryos are developed into complete plantlets on an MS culture medium without hormone in the fourth stage. The calluses are dried for some time before embryoids are induced, which markedly improves the inducing rate of the somatic embryos.

Description

Set up the method for buffalo grass regeneration system by body embryogenesis path

Technical field

The present invention relates to the method that buffalograss isolated culture regeneration system is set up.Specifically relating to buffalograss seed maturity embryo is explant, sets up the method for its regeneration system by somatic embryo generation approach.Belong to the plant cell engineering field.

Background technology

Buffalograss [Buchloe dactyloides (Nutt.)] is the Gramineae buffalo grass, has another name called the buffalo grass.Originate in temperate zone, middle part, North America and semiarid zone, subtropics.Be a kind of poor growth, the perennial short turfgrass of the season type cold-resistant, heat-resisting, that drought resistance is strong.

The lawn is the basis and the important component part of urban afforestation as the artificial vegetation under the human intervention, plays protection in the human lives, improve and beautifies the environment, and is one of important symbol of weighing a national civilization degree and urban modernization.Since the nineties in last century, developed rapidly along with socioeconomic, China's Lawn Industry has entered a new development climax, and Lawn Industry has become extremely important integral part in the modern garden construction.As park green land, greenery patches, residential quarters, both sides of highway and bank protection greenery patches, various playgrounds greenery patches etc., constantly build in each city of China on the lawn of various difference in functionality purposes.Large-area turf establishment has promoted the fast development of China's Lawn Industry, thereby cause the demand of turf grass species is sharply increased, the China that acts on lawn more and more is subject to people's attention, the variation of essence has also taken place in its value, status and effect, and it changes the industry that integrates economy, ecology and social benefit into from single economic functions.But China is except that subtropical zone and Tibet, and the grass seeds that the various places lawn planting is selected for use is import cold-season-type grass seeds more than 95%, as annual bluegrass, rye grass, Festuca Arundinacea etc.Cool-season grasses requires fertile soil, and water requirement is relatively large, enters semidormancy season at the summer high temperature high humidity, disease and pest takes place easily, and a large amount of applying pesticides and chemical fertilizer cause environmental pollution, the spring and autumn growth is vigorous in addition, pruning frequency height, the overhead charges on lawn are high.If miscarriage, about 5 years, will degenerate in the lawn, causes the situation of " afford to plant, can not support " in some lacks of water of China and economically underdeveloped area.The scarcely anti-trample of cold-season turfgrass, people can only view and admire and can not enter.Therefore, the area of planting is big more, and the space that can lie fallow is just more little.And in order to bring into play its garden landscape effect, big flood pouring green grassland that will be frequent, and in recent years, the situation of China's shortage of water resources is serious day by day, this has aggravated the contradiction of China's water resources anxiety undoubtedly.In order to solve these contradiction, in recent years, introduced the theory in non-irrigated scape gardens gradually abroad, warm season turf begins to be subject to people's attention in the garden landscape construction with characteristics such as its stronger resistance and low water consumption.

The forties in 20th century, buffalograss is introduced China as the soil conservation plant, at first plants experimentally in Gansu province, and the back all has big area successfully to introduce a fine variety on China North China, northwest and northeast and other places as turfgrass.One of feature that buffalograss is the most outstanding is to have drought resistance, can keep fine quality low the grade under the maintenance level.It enters dormant state when extreme drought, in case suitable the recovery rapidly again of condition grown.The prosperity of buffalograss stolon also has rhizome to take place sometimes.The fine and close characteristic of the aggressiveness of its stolon, habit and turf makes it have stronger soil conservation ability.Studies confirm that, in the buffalograss adaptation zone, its day evapotranspiration have only 6mm, all lower than cold-season-type or other warm season turf.Domestic scholars needs to have done number of research projects aspect the water characteristic buffalograss in recent years.(Beijing Forestry University's journal, 2003,11,25 (6): studies show that 39～44) the year evapotranspiration that buffalograss is watered under the water condition in abundance is 758.66mm, and the year evapotranspiration under the limitation water-feeding condition is 624.28mm such as ZhaoBing Xiang.It is 424mm to 527mm that buffalograss needs the water scope in the year of Beijing area.Buffalograss can enter dormant state rapidly under drought stress, again can very fast recovery growth in case condition is suitable.This is because the characteristic that crawl energy for growth and the drought stress lower blade of its powerful fibrous root system, tool aggressiveness can curl to a great extent.Under the natural rainfall condition of Beijing area, but the buffalograss normal growth need not to irrigate, thereby has the people to be called " rain is supported type " turfgrass.In recent years, being applied to by the motorway more and more, the place of low-maintenance such as airfield runway, golf course and some playgrounds, is a kind of warm season turf that has development potentiality.But buffalograss also comes with some shortcomings as turfgrass, and as having only a growth season in 1 year, the green phase is shorter, and leaf color is light, and this has influenced it to a certain extent and has viewed and admired and practical value.Although by cradling and measures such as water and fertilizer management, can the green phase of proper extension, can not reach ideal effect.Therefore cultivating green phase long new China of buffalograss by breeding technique is humid euphorbia platymiscium center of distribution, and China just has 10 kinds in 15 kinds of the whole world, almost the distribution of humid euphorbia is all arranged the each province according to Chinese Plants will China, and kind is the most basic solution route.

The seed selection work to the buffalograss new variety abroad starts from 1936 the earliest, and its breeding direction mainly is to cultivate the costly new variety in level ground such as anti-low pruning, length of green phase, leaf color depth, disease and insect resistance.So far, means such as overseas utilization conventional breeding method such as ploidy breeding and cross-breeding have been cultivated many buffalograss new variety that are applicable to different functions such as golf course, highway slope protection greening.Twentieth century eighties, USGA associating University of California and University of Nebraska emphasis have been cultivated some and have been applicable to the new variety of golf course and other ornamental lawn planting, nineteen ninety-five, University of Nebraska selects the buffalograss new variety of first seminal propagation, and making becomes possibility with seed sowing mode planting buffalograss.

For a long time, cultivating long new variety of green phase is one of breeding emphasis of people, but regrettably, up to the present, the buffalograss new variety that the green phase obviously prolongs are not cultivated success yet.Tracing it to its cause, is because there is limitation in traditional breeding way to the improvement of this proterties.Therefore, it is imperative to utilize modern biotechnology that this proterties is improved.And set up high frequency, stable plant regeneration system is to utilize modern biotechnology to carry out the prerequisite of breed improvement.With buffalograss is the warm season turf of representative, and its regeneration system is set up quite difficulty.Therefore, buffalo grass regeneration system is created as key for biotechnology breeding.

Buffalograss isolated culture and plant regeneration research only have indivedual reports.(Hort.Sci.Abs, 1991,26,150) such as Hughes etc. (AgronAbs, Amer.soc.Agron.Madison, WI, 1991,177) and Moore have induced callus from seed and the tender cauline leaf tissue of children of buffalograss, but fail to obtain regeneration plant.The button friend people wait (Hangzhou Botanical Garden's communication, 1996,13 (2), 45～46) to utilize buffalograss " magnificent No. one " stem section also to induce callus, but finally do not obtain regeneration plant.Only Fei S etc. (Plant Cell Org.andCult., 2000,, 60,197～203; Plant Cell.Org.Cult., 2001,70,275～279, InternationalTurfgrass Society Research, 1997,8,283～289) be explant with buffalograss prematurity inflorescence, containing 9uM (2mg/L) 2,4-D, Mu of 5～15mg/L AgNO3 and 5～15g/LCH draws in Bouguer and the Si Kuge substratum (MS substratum, composition sees Table 1) and induces callus, behind the succeeding transfer culture 30d, containing 2.25～9 μ M2,4-D, 0.44 induce the bud of growing thickly among～1.32 μ M 6-BA, containing 4.5～9 μ M2, induce embryo callus on the substratum of 4-D, and obtained regeneration plant.Though set up the regeneration system of buffalograss by this method, because drawing materials, the prematurity inflorescence is subject to seasonal restrictions greatly, therefore be greatly limited in actual applications.In the various explants of buffalograss isolated culture, its seed has easily collecting, storage tolerance than other explants, draws materials advantage and mature embryo Endogenous Hormone Contents in Vitro height in the seed such as not to be subject to seasonal restrictions, cell has potential vigorous splitting ability, is the good material that is used for isolated culture.

Summary of the invention

The objective of the invention is with the buffalograss mature embryo is the regeneration system that cultivated material is set up buffalograss.

The present invention is to be that explant is cultivated with buffalograss Texoka mature embryo, sets up the method for buffalo grass regeneration system with this.

The present invention has set up regeneration system by isolated culture buffalograss mature embryo, and the regeneration plant proterties homogeneous that is obtained, stable is easy to genetic engineering procedure.

The present invention sets up the method for buffalo grass regeneration system, with buffalograss Texoka mature embryo is explant, cultivate, described method comprises four cultivation stages: (1) explant is directly cultivated evoked callus (2) callus succeeding transfer culture (3) callus induction somatic embryo (4) somatic embryo regeneration plant, wherein (1) (2) are dark culturing, (3) be the low light level cultivation of 200～600 luxs, (4) be the light cultivation of 1500～3000 luxs, whole culturing process, culture temperature is 20 ℃～30 ℃, every cultivation renewed bright substratum in 20 days, wherein the minimum medium of four-stage is the MS substratum, carbon source is for adding sucrose and each 15g/L of glucose in the substratum, used hormone is in the substratum in (1) stage: 0.1～5mg/L2,4-D (2, the 4-dichlorphenoxyacetic acid) and 0.1～3mg/L6-BA (6-benzylamino VITAMIN B4), used hormone is in the substratum in (2) stage: 0.1～5mg/L2,4-D (2, the 4-dichlorphenoxyacetic acid) and 0.1～3mg/L6-BA (6-benzylamino VITAMIN B4), used hormone is in the substratum in (3) stage: 0.05～5mg/L2,4-D (2, the 4-dichlorphenoxyacetic acid) and 0.01～3mg/L6-BA (6-benzylamino VITAMIN B4), substratum in (4) stage is the MS substratum that does not use hormone, wherein the substratum in (1) stage also adds the STS (silver thiosulfate) of 1～20mg/L, the substratum in (2) stage also adds 500～2000mg/L caseinhydrolysate, it is characterized in that before (3) the stage callus induction embryoid callus being carried out in gnotobasis 12～96 hours, temperature is 20 ℃～50 ℃ a drying treatment.

The present invention sets up the method for buffalo grass regeneration system, preferred scheme may further comprise the steps (1) explant and is inoculated in interpolation 1.5-2.5mg/L 2, the substratum of the STS (silver thiosulfate) of 4-D (2,4 dichlorophenoxyacetic acid), 0.05～0.2mg/L6-BA (6-benzylamino VITAMIN B4) and 5～10mg/L; (2) induce the callus that obtains to be inoculated in and add 2.0～3.0mg/L2,4-D (2,4 dichlorophenoxyacetic acid), the substratum of 0.1～0.3mg/L 6-BA (6-benzylamino VITAMIN B4) and 800～1200mg/L caseinhydrolysate; (3) to carry out 36～60 hours, temperature in gnotobasis be 20 ℃～30 ℃ drying treatment to the callus of subculture; (4) callus crossed of drying treatment is inoculated in and adds 0.1-1mg/L 2, the substratum of 4-D (2,4 dichlorophenoxyacetic acid) and 0.05～0.3mg/L mg/L 6-BA (6-benzylamino VITAMIN B4); (5) induce the somatic embryo that obtains to be inoculated in the substratum that does not add plant-growth regulator, above-mentioned substratum other compositions except that hormone are identical with the MS substratum, carbon source is for adding sucrose and each 15g/L of glucose, wherein (1) (2) are dark culturing, (3) be the low light level cultivation of 200～600 luxs, (5) are that the light of 1500～3000 luxs is cultivated whole culturing process, culture temperature is 20 ℃～30 ℃, and fresh culture was changed in every cultivation in 20 days one time.

The present invention is with matured seeds of buffalograss (Buchloe dactyloides (Nutt.) Texoka), making breaking dormancy handles, after seed directly sterilized, be inoculated in the substratum in (1) stage, cultivate after 3 days, the mature embryo of rudiment is taken out in aseptic technique, continues to place former substratum to cultivate perpendicular to after several sections of the plumular axis cuttings.Under the substratum in above-mentioned (1) stage and culture condition, cultivated 20 days, callus induction rate reaches 86.67%, change the substratum in (2) stage over to, increment volume of per 20 days subcultures increases 1.3-2.0 doubly approximately, choose wherein fine and close white or the faint yellow callus of structure, drying is handled the culture medium culturing 20 days in (3) stage that changed over to, and the somatic embryo inducement rate is 16.83%, and drying treatment improves 9.97% before the embryoid induction rate is handled.Change the body embryo substratum of (4) stage over to, shoot regeneration frequency is 36.2%, and transplanting survival rate reaches 95%.

The present invention is easy to induce callus from the buffalograss mature embryo, and kept the good quality of callus, institute's inductive somatic embryo can produce the regeneration plant of high quantity, stable, homogeneous, so this system can be used for genetically engineered or cell engineering breeding.

The present invention is when initial cultivation, because the existence of buffalograss seed endogenetic fungus makes to obtain quite difficulty of mature embryo sterile culture thing, the inventor is by experiment repeatedly in a large number, the sterilising method that obtains suiting (Qian Yongqiang, Practaculture Science, 2005,22 (2), 101-105).After seed handled 60s with 70% alcohol, with 70%NaClO original solution (available chlorine is 7～10%) immersion 25～30 minutes, the rate of depolluting reached more than 95%.Its principle may be because it is close with the osmotic pressure of bacterium, constantly infiltrates to thalline is inner gradually before the sex change at bacterium surface albumen, tropina is dewatered, solidify sex change and reach the sterilization purpose.The sterilising effect of clorox is better than mercuric chloride, and hypochlorous oxygenizement is its topmost sterilization mechanism in the clorox.Form hypochlorous acid in water, after having an effect with cell walls, and because of molecule is little, neutral so can invade in the cell with protein generation oxygenizement or destroy its phosphate dehydrogenase, makes the carbohydrate metabolism imbalance and causes necrocytosis.Find among the present invention that buffalograss carries disease germs to plant NaClO is had high susceptibility.

Embodiment

Further illustrate the present invention in the following embodiments, this does not limit the scope of the invention.

Embodiment 1

Matured seeds of buffalograss (Buchloe dactyloides (Nutt.) Texoka) is purchased in the Chinese Academy of Agricultural Sciences, and seed is made breaking dormancy and handled, and the test determination percentage of germination is 90%; Behind the washings thorough washing, soak 24h～48h in the mobile tap water, seed is taken out from its bud grain husk, with 70% alcohol to seed treatment 60s after, soak 30min with 70%NaClO original solution (available chlorine is 7～10%), be inoculated in callus inducing medium, add 1.7mg/L 2,4-D (2, the 4-dichlorphenoxyacetic acid), 0.09mg/L6-BA (6-benzylamino VITAMIN B4), the STS of 9mg/L (silver thiosulfate).Wherein, the preparation of substratum is that hormone is directly made an addition in other compositions of Ms substratum, and autoclaving makes then.After the dark culturing 3 days, aseptic technique is taken out, and with the mature embryo of rudiment, continues to place former substratum to cultivate perpendicular to after several sections of the plumular axis cuttings.Continue dark culturing.

Used plant and instrument is equipment such as the known ultra-clean working machine of plant tissue culture, automatic high pressure Autoclave.Cultivate in the whole process at whole regeneration system, the medium pH value is 5.8, and temperature is 25 ± 1 ℃.

To induce the callus that obtains, add 2.2mg/L2,4-D (2,4 dichlorophenoxyacetic acid), 0.28mg/L 6-BA (6-benzylamino VITAMIN B4), 1200mg/L caseinhydrolysate at the MS substratum.Dark culturing.

Choose wherein fine and close white or the faint yellow callus of structure in the callus of succeeding transfer culture, on Bechtop, aseptic technique places the sterile petri dish that is covered with dry filter paper, temperature is 25 ℃, and be 40 hours storage period, changes over to immediately to contain 0.5mg/L 2,4-D (2, the 4-dichlorphenoxyacetic acid), in the MS substratum of 0.27mg/L 6-BA (6-benzylamino VITAMIN B4), low light level cultivation (light intensity is 500～1000 Luxs),

The inductive somatic embryo is changed on the MS substratum that does not add plant-growth regulator, make it grow up to whole plant.In light intensity is the illumination cultivation of 1500～3000 Luxs.Light application time is at 10～14h/d, and somatic embryo grows up to the complete plant of root, stem and leaf through 20 days cultivation, transplants in vermiculite hardening 15 days, can move into outdoor big Tanaka.

The regeneration system that aforesaid method is set up, callus induction rate reaches 86.67%, and the embryoid induction rate reaches 16.83%, and wherein drying treatment improves 9.97% before the embryoid induction rate is handled.Regeneration rate reaches 36.2%, and transplanting survival rate reaches 95%.

Embodiment 2

The cultivation program is all undertaken by embodiment 1, and institute's difference is to add 2 in the callus inducing medium, and 4-D is 2.0mg/L, 6-BA is 0.13mg/L, STS is 10mg/L, and adding hormone in the callus subculture medium is 2, and 4-D is 2.5mg/L, 6-BA is 0.13mg/L, caseinhydrolysate is 1100mg/L, and the drying treatment time is 48 hours, and adding hormone in the somatocyte training inducing culture is 2,4-D is 0.13mg/L, and 6-BA is 0.09mg/L.

Embodiment 3

The cultivation program is all undertaken by embodiment 1, and institute's difference is to add 2 in the callus inducing medium, and 4-D is 2.0mg/L, 6-BA is 0.07mg/L, STS is 8mg/L, and adding hormone in the callus subculture medium is 2, and 4-D is 3.0mg/L, 6-BA is 0.3mg/L, caseinhydrolysate is 800mg/L, and the drying treatment time is 60 hours, and adding hormone in the somatocyte training inducing culture is 2,4-D is 0.8mg/L, and 6-BA is 1.0mg/L.

Embodiment 4

The cultivation program is all undertaken by embodiment 1, and institute's difference is to add 2 in the callus inducing medium, and 4-D is 2.5mg/L, 6-BA is 0.18mg/L, STS is 5mg/L, and adding hormone in the callus subculture medium is 2, and 4-D is 2.0mg/L, 6-BA is 0.2mg/L, caseinhydrolysate is 1000mg/L, and the drying treatment time is 36 hours, and adding hormone in the somatocyte training inducing culture is 2,4-D is 0.8mg/L, and 6-BA is 0.15mg/L.

Embodiment 5

The cultivation program is all undertaken by embodiment 1, and institute's difference is to add 2 in the callus inducing medium, and 4-D is 1.5mg/L, 6-BA is 0.05mg/L, STS is 7mg/L, and adding hormone in the callus subculture medium is 2, and 4-D is 3.0mg/L, 6-BA is 0.3mg/L, caseinhydrolysate is 950mg/L, and the drying treatment time is 36 hours, and adding hormone in the somatocyte training inducing culture is 2,4-D is 1.0mg/L, and 6-BA is 0.3mg/L.

Embodiment 6

The cultivation program is all undertaken by embodiment 1, institute's difference is to add 2 in the callus inducing medium, and 4-D is 1.8mg/L, and 6-BA is 7mg/L for 0.11mg/L STS, adding hormone in the callus subculture medium is 2,4-D is 2.3mg/L, and 6-BA is 0.1mg/L, and caseinhydrolysate is 1000mg/L, the drying treatment time is 60 hours, adding hormone in the somatocyte training inducing culture is 2, and 4-D is 1.0mg/L, and 6-BA is 0.1mg/L.

Embodiment 7

The cultivation program is all undertaken by embodiment 1, and institute's difference is to add 2 in the callus inducing medium, and 4-D is 2.0mg/L, 6-BA is 0.2mg/L, STS is 7mg/L, and adding hormone in the callus subculture medium is 2, and 4-D is 2.5mg/L, 6-BA is 0.10mg/L, caseinhydrolysate is 900mg/L, and the drying treatment time is 45 hours, and adding hormone in the somatocyte training inducing culture is 2,4-D is 0.10mg/L, and 6-BA is 0.2mg/L.

Table one: MS minimum medium (Murashige ﹠amp; Skoog, 1962) composition

Component	Composition	Content (mg/L)
			Macroelement	Ammonium nitrate	1650
Saltpetre	1900
		Potassium primary phosphate		170
Bitter salt	370
		Two hydration calcium chloride		440
Trace element	Four anhydrous manganeses				22.3
		Zinc vitriol	8.6
	Boric acid			6.2
		Potassiumiodide	0.83
	Two molybdic acid hydrate sodium			0.25
		Cobalt chloride hexahydrate	0.025
	Salzburg vitriol			0.025
		Organic element	Inositol		100
Nicotinic acid	0.5
			Vitamin	0..1
Pyridoxine hydrochloride	0.5
			Glycine	2.0
Molysite	Ferrous sulfate				27.8
		Disodium ethylene diamine tetraacetate	37.3

Table two: B5 minimum medium (Gamborg etc., 1968) composition

Component	Composition	Content (mg/L)
			Macroelement	Ammonium sulfate	134
Saltpetre	3000
		Potassium primary phosphate		150
Bitter salt	500
		Two hydration calcium chloride		150
Trace element	Four anhydrous manganeses				10
		Zinc vitriol	2
	Boric acid			3
		Potassiumiodide	0.75
	Two molybdic acid hydrate sodium			0.25
		Cobalt chloride hexahydrate	0.025
	Salzburg vitriol			0.025
		Organic element	Inositol		100
Nicotinic acid	1
			Vitamin	1
Pyridoxine hydrochloride	10
			Molysite	Ferrous sulfate	27.8
Disodium ethylene diamine tetraacetate	37.3

Table three: N6 minimum medium (Zhu Zhiqing, 1974)

Component	Composition	Content (mg/L)
			Macroelement	Ammonium sulfate	460
Saltpetre	2830
		Potassium primary phosphate		400
Bitter salt	185
		Two hydration calcium chloride		166
Trace element	Four anhydrous manganeses				10
		Zinc vitriol	1.5
	Boric acid			1.6
		Potassiumiodide	0.8
	Organic element			Glycine	2.0
Nicotinic acid		0.5
			Vitamin	1
Pyridoxine hydrochloride		0.5
			Molysite	Ferrous sulfate	27.8
Disodium ethylene diamine tetraacetate	37.3

Table four: DPD minimum medium composition (Durand etc., 1973)

Component	Composition	Content (mg/L)
			Macroelement	Ammonium nitrate	270
Saltpetre	1480
		Potassium primary phosphate		80
Bitter salt	340
		Two hydration calcium chloride		570
Trace element	Four anhydrous manganeses				5.0
		Zinc vitriol	2.0
	Boric acid			2.0
		Potassiumiodide	0.25
	Two molybdic acid hydrate sodium			0.1
		Cobalt chloride hexahydrate	0.01
	Salzburg vitriol			0.015
		Organic element	Inositol		100
Nicotinic acid	4.0
			Vitamin	4.0
Pyridoxine hydrochloride	0.7
			Glycine	1.4
Folic acid	0.4
			Vitamin H	0.04
N.F,USP MANNITOL	54.0
			Molysite	Ferrous sulfate	27.8
Disodium ethylene diamine tetraacetate	37.3

Claims (2)

1. method of setting up buffalo grass regeneration system, it is characterized in that with buffalograss Texoka mature embryo be explant, cultivate, described method comprises four cultivation stages: (1) explant is directly cultivated evoked callus (2) callus succeeding transfer culture (3) callus induction somatic embryo (4) somatic embryo regeneration plant, wherein (1) (2) are dark culturing, (3) be the low light level cultivation of 200～600 luxs, (4) be the light cultivation of 1500～3000 luxs, whole culturing process, culture temperature is 20 ℃～30 ℃, every cultivation renewed bright substratum in 20 days, wherein the minimum medium of four-stage is the MS substratum, carbon source is for adding sucrose and each 15g/L of glucose in the substratum, used hormone is in the substratum in (1) stage: 0.1～5mg/L2,4-dichlorphenoxyacetic acid 2,4-D and 0.1～3mg/L6-benzylamino VITAMIN B4 6-BA, used hormone is in the substratum in (2) stage: 0.1～5mg/L2,4-dichlorphenoxyacetic acid 2,4-D and 0.1～3mg/L6-benzylamino VITAMIN B4 6-BA, used hormone is in the substratum in (3) stage: 0.05～5mg/L2,4-dichlorphenoxyacetic acid 2,4-D and 0.01～3mg/L6-benzylamino VITAMIN B4 6-BA, substratum in (4) stage is the MS substratum that does not use hormone, wherein the substratum in (1) stage also adds the silver thiosulfate STS of 1～20mg/L, the substratum in (2) stage also adds 500～2000mg/L caseinhydrolysate, it is characterized in that before (3) the stage callus induction embryoid callus being carried out in gnotobasis 12～96 hours, temperature is 20 ℃～50 ℃ a drying treatment.

2. in accordance with the method for claim 1, it is characterized in that this method may further comprise the steps (1) explant and is inoculated in interpolation 1.5-2.5mg/L 2,4-dichlorphenoxyacetic acid 2, the substratum of the silver thiosulfate STS of 4-D, 0.05～0.2mg/L6-benzylamino VITAMIN B4 6-BA and 5～10mg/L; (2) induce the callus that obtains to be inoculated in and add 2.0～3.0mg/L2,4-dichlorphenoxyacetic acid 2,4-D, the substratum of 0.1～0.3mg/L 6-benzylamino VITAMIN B4 6-BA and 800～1200mg/L caseinhydrolysate; (3) to carry out 36～60 hours, temperature in gnotobasis be 20 ℃～30 ℃ drying treatment to the callus of subculture; (4) callus crossed of drying treatment is inoculated in and adds 0.1-1mg/L 2,4 dichlorophenoxyacetic acid 2, the substratum of 4-D and 0.05～0.3mg/L mg/L 6-benzylamino VITAMIN B4 6-BA; (5) induce the somatic embryo that obtains to be inoculated in the substratum that does not add plant-growth regulator, above-mentioned substratum other compositions except that hormone are identical with the MS substratum, carbon source is for adding sucrose and each 15g/L of glucose, wherein (1) (2) are dark culturing, (3) be the low light level cultivation of 200～600 luxs, (5) are that the light of 1500～3000 luxs is cultivated whole culturing process, culture temperature is 20 ℃～30 ℃, and fresh culture was changed in every cultivation in 20 days one time.