CN1311738C - Aesculus hippocastanum somatic embryogenesis and strain regenerating method - Google Patents

Abstract

Aesculus hippocastanums have extremely high ornamental and medical value. A traditional seeding method for breeding Aesculus hippocastanums has the defect of low speed and consumes a large number of labor resources, material resources and financial resources, and the need for Aesculus hippocastanums in the market can not be satisfied. The present invention provides a tissue culture method for generating somatic embryos and regenerating plants by the seed germs of Aesculus hippocastanums or new buds germinated from plants in the spring, blades cultured from the seed germs, and the cotyledons of somatic embryos growing for half a month. The present invention provides a seedling culturing method with the advantages of short period, high reproduction rate, production steps tightly connected, and low cost for the large-scale planting of Aesculus hippocastanums in the production of forestry.

Description

The European horse-chestnut somatic embryo takes place and plant regeneration method

One, technical field

The invention belongs to the generation of the cell stage in the forest tissue culture technique and clone's plant regeneration in the forest-science

Technical field.

Two, background technology

Hippocastanaceae (Hippocastanaceae) world existing 2 belongs to, and kind surplus wherein Aesculus (Aesculus L) has 30 approximately mainly is distributed in the north temperate zone.China has 10 kinds approximately, mainly is distributed in the subtropical zone in the west and south, and they all are tall and big deciduous trees, and the height of tree is 20～30m usually, can reach 45m.Horse chestnut wherein, long handle horse chestnut, Zhejiang Buckeye, Yunnan horse chestnut, Heavenly Teacher's chestnut, Aesculus turbinata etc., very wide in the region that China distributes, Shaanxi, Henan, Jiangsu, zhejiang and other places all have cultivation, remain the one tree ancient tree in more than 300 year age in east Gansu.Because of climatic variation, China moves in horse chestnut distributing line south now, and only there is the wild horse chestnut of natural distribution in height above sea level mountain region below 700 meters in area, the Qinling Mountains.

European horse-chestnut (Aesculus hippocastanum) is a kind of of Hippocastanaceae Aesculus, and European horse-chestnut originates in northern Greece and Albanian mountain area, and nineteen twenty-six Kosanin has found European horse-chestnut in the Macedonia southeast.European horse-chestnut has been represented the remaining species of the 3rd century plant community, and the sediment deposit humus clay of silt-laden river and temperate climate condition are its best suitable immature soil earth and environment, also can successfully grow in nice and cool gentle climatic zone.Cities such as Beijing of China, Shanghai, Qingdao have introducing and planting.

The tall and big grandness of European horse-chestnut, form is perfectly straight, branches and leaves are luxuriant and well-spaced, hat is as canopy, leaf greatly and shape U.S., the effect of shading is fine.White inflorescence very large when bloom early summer rises sheer from the leafage, and like magnificent candelabra, It is a luxuriant and wonderful spectacle, is one of the world's 5 big famous ornamental tree species, also is one of the world four big street trees (plane tree, lime tree, elm, horse chestnut).Extensively plant in the Europe, the United States various countries and to do street tree and garden is viewed and admired tree, and many horticultural varieties are arranged, before building to plant, roadside avenue system or isolated planting, group planting in the lawn, the hillside is all very suitable.As making setoff, then more grand with other seeds.The famous literature and art Luo Qing of man in Taiwan once ran after fame with " horse chestnut ", published a collection of poems, the fire of lighting art with beauty and the vigorous vitality of horse chestnut, the calling people are to the yearning and the pursuit of natural beauty and artistic beauty, the artistic visual field of developing diversity is in the human art ground understanding world, aesthetically experience in the process in the world and brought into play positive role.European horse-chestnut timber look white, the little Huang of face, matter softness are careful, though corrosion resistant is not rotten, can and make fancy goods and furniture etc. for papermaking.

European horse-chestnut has very high medical medical value, and the horse chestnut that belongs to together is used for medicine, and is early on the books in the ancient books of China.In China's traditional medicine, aesculus seed is used as medicine with " buckeye ", puts down in writing in the Compendium of Material Medica: " buckeye is during sweet, warm, sharp gas is wide; and stomach and alleviating pain ", be used for " stomachache due to emotional depression and the hyperactive liver-qi attacking the stomach, abdominal fullness and distention, premenstrual abdominal pain; mammary swelling; infantile malnutrition due to digestive disturbances or intestinalparasites worm pain, dysentery ", in " logical refined ", supplementary Amplifications of the Compendium of Materia Medica, also be documented.In recent years, German, Japanese scientist has carried out detailed research to the medical value of European horse-chestnut, and medicines such as the injection of Development and Production, tablet, suppository, capsule have been widely used in clinical.Effect is quite satisfied, has good market prospects.

Present domestic various places generally adopt traditional seeding method to breed in the nursery stock production of horse chestnut, and growth rate is very slow, and annual nursery stock only can grow to 20～40cm.The Aesculus seed is stupid stubborn type seed, not anti-dehydration and low temperature.Relevant studies have shown that goes pericarp seed natural air drying will lose germinating capacity because of drying out in 15 days, so should not hide for a long time.Because the mature period of Aesculus seed in September, directly behind the insemination and emergence, need be taked freeze prevention measure when seedling survives the winter.Moreover the seed of horse chestnut is big (the heavily about 16g of average particle), needs more labour during sowing, adopts the wet husky preserving process of band pericarp, also needs running check to ted, in case done, go rotten or the plague of rats.In addition, the solid biennial bearing phenomenon of Aesculus is obvious, and the general cycle is 3～4 years.All these brings great difficulty and hard work amount for the nursery stock production of horse chestnut.

At present domestic to European horse-chestnut research and introducing and planting all seldom, limited document record only limits in the research of seeding and seedling raising, cottage propagation and ecological habit of horse chestnut the tissue culture technique research of European horse-chestnut not seen that as yet report is arranged.External to the research of European horse-chestnut more (seeing Table 1), according to the data record, Radojevic once used the pollen of European horse-chestnut (Aesculus hippocastanum) to cultivate embryoid and obtained complete pollen plant in 1978; Used the prematurity embryo to induce somatic embryo and obtained whole plant in 1989.Profumo et al uses the explants such as plumular axis, cotyledon of Horsechestnut seed successfully to induce somatic embryo respectively at, nineteen ninety in 1989; Dameri et al used blade to induce somatic embryo in 1986, but did not form large-scale production as yet at present.

Table 1 European horse-chestnut foreign study present situation

Explant	The result	Author and age
			Pollen	The body embryo, plant	Radojevic，1978
The cotyledon of mature seed	Callus has root	Profumo et al.，1976，1980
			Plumular axis	Embryo callus, the body embryo	Profumo et al.，1989
The cotyledon of mature seed	Embryo callus subculture, body embryo, secondary body embryo	Profumo et al.，1990，1991
			Leaf	Non-embryonic callus, embryo callus subculture, body embryo	Dameri et al.，1986
The prematurity embryo	Non-embryonic callus, embryo callus subculture, body embryo	Radojevic，1988
				Organ takes place, the embryo of sprouting
Filigree	Non-embryonic callus, embryo callus subculture, body embryo	Jorgensen，1989，1991
			The prematurity ovule	Embryo callus subculture, the body embryo	Radojevic，Marinkovic，1993
Body embryo apical meristem	Leafage, bud breeding, root, plant	Radojevic et al.，1988

In view of the huge medicinal and ornamental value of European horse-chestnut, the domestic market is increasing to the demand of European horse-chestnut, and the introducing and planting of present domestic European horse-chestnut is limited, its modes of reproduction only limits to sowing, reproduction speed is extremely slow, extreme labor intensive, material resources and financial resources can not satisfy the needs of market to European horse-chestnut far away again.

Three, summary of the invention

The goal of the invention of this method is to carry out somatic embryo engineering of European horse-chestnut (Aesculus hippocastanum) and the Study of Regeneration Technique of clone plant, searching is applicable to the European horse-chestnut cell engineering mature technology system of breeding fast, to satisfy the needs of market to European horse-chestnut.

For various reasons, the at present domestic extremely difficult seed that obtains European horse-chestnut, used experiment material progressively obtains by following process: adopt the plumule of seed to breed aseptic seedling, adopt the blade of aseptic seedling or the blade of seed germination seedling to induce indefinite bud and embryoid more earlier, and adopt the cotyledon of embryoid and the blade of sprouting body embryo to induce indefinite bud and embryoid once more.Overcome the difficulty of provenance deficiency, experiment material is many, can accelerate reproduction speed.

Technical solution of the present invention is carried out tissue culture for the seed plumule that adopts European horse-chestnut or the sprouting that spring, plant sprouted, blade and the cotyledon behind the body embryonic development two weeks that the seed plumule is turned out, and its major technique feature is as follows:

A. the plumule with seed carries out tissue culture, plumule growth and the used minimal medium of evoking adventive bud are MS medium (Murashing and Skoog medium, hereinafter to be referred as MS), additional plant hormone is 6-benzylaminopurine (benzylaminopurine, be designated hereinafter simply as BA), 6-chaff aminopurine (6-Furfurylaminopurine, be also referred to as Kinetin, i.e. " kinetin ", be designated hereinafter simply as KT), zeatin (zeatin, be designated hereinafter simply as ZT), methyl (naphthylene acetic acid is designated hereinafter simply as NAA) is removed BA, ZT concentration is lower than the plant hormone combination of NAA;

B. the blade of turning out with the sprouting or the seed plumule of plant sprouting in spring carries out tissue culture, the direct organ generation of excised leaf produces indefinite bud, indefinite bud takes place to produce indirect organ and the used minimal medium of body embryo generation is MS, additional plant hormone is NAA, 2,4-dichlorphenoxyacetic acid (2,4-dichlorophenoxyacetic acid, be designated hereinafter simply as 2,4-D), BA, ZT, KT, heteroauxin (indole-3-acetic acid, be designated hereinafter simply as IAA), indolebutyric acid (indolebutyric acid is hereinafter to be referred as IBA);

C. carry out tissue culture with the cotyledon behind the body embryonic development two weeks, used minimal medium is MS, and additional hormone is NAA, IAA, KT, BA, ZT and TDZ (thidiazuron is the thiadiazole phenylurea) etc.

The following two kinds of minimal mediums of main use in the tissue culture overall process, its standard recipe is as follows respectively:

1.MS medium (Murashing and Skoog medium)

NH ₄NO ₃ 1650mg/l

KNO ₃ 1900mg/l

CaCl ₂·2H ₂O 440mg/l

MgSO ₄·7H ₂O 370mg/l

KH ₂PO ₄ 170mg/l

KI 990mg/l

H ₃BO ₃ 6.2mg/l

MgSO ₄·H ₂O 22.3mg/l

ZnSO ₄·7H ₂O 8.6mg/l

Na ₂MoO ₄·2H ₂O 0.25mg/l

CuSO ₄·5H ₂O 0.025mg/l

CoCl ₂·6H ₂O 0.025mg/l

FeSO ₄·7H ₂O 27.8mg/l

Na ₂-EDTA 37.3mg/l

Inositol 100mg/l

Nicotinic acid O.5mg/l

Thiamine hydrochloride 0.1mg/l

Puridoxine hydrochloride 0.5mg/l

Glycine 2mg/l

2.WPM (Woody Plant Medium) is the woody plant tissure medium

NH ₄NO ₃ 400mg/l

Ca(NO ₃) ₂·4H ₂O556mg/l

CaCl ₂·2H ₂O 96mg/l

MgSO ₄·7H ₂O 370mg/l

KH ₂PO ₄ 170mg/l

K2SO ₄ 990mg/l

Na ₂-EDTA 37.3mg/l

FeSO ₄·7H ₂O 27.8mg/l

MgSO ₄·H ₂O 22.3mg/l

ZnSO ₄·7H ₂O 8.6mg/l

H ₃BO ₃ 6.2mg/l

Na ₂MoO ₄·2H ₂O 0.25mg/l

CuSO ₄·5H ₂O 0.25mg/l

Inositol 100mg/l

Nicotinic acid 0.5mg/l

Thiamine hydrochloride 1mg/l

Glycine 2mg/l

Four, embodiment

Embodiment 1: adopt the plumule of seed to carry out tissue culture

Horsechestnut seed kind skin is hard thick smooth, and very male incision can make its sprouting earlier.Earlier outstanding kind of the skin of radicle during sprouting, the part of exposing kind of skin behind the 5d can reach 4cm, this partial interior is surrounded by plumule, selects not expose the breaking off with the fingers and thumb of plumule under, running water washed 2 hours, on superclean bench with 75% alcohol-pickled 30s, 0.1%HgCl ₂Soak 8～12min, aseptic water washing 5～6 times, aseptic filter paper blots surface moisture, and excision plumule outsourcing thing takes out plumule, is inoculated among the MS and grows a period of time.Since plant the skin densification, cotyledons thick, plumule is not subject to outside contamination, and the practician also can not use the disinfectant sterilization, and the outsourcing thing of directly dialling degerming takes out the plumule inoculation, avoids the pollution of disinfectant to environment.

Plumule growth and the used minimal medium of bud differentiation and proliferation are the MS medium, additional plant hormone is BA, KT, ZT, NAA etc., sucrose 30g/L, agar powder 6.5g/L (pH5.8), the pH value transfers to 5.8 before the sterilization, 121 ℃ of high temperature, autoclaving 18min, cultivation temperature is 24～26 ℃, and illumination (2000lx) 16h/d cultivates down.Because the chemical composition of European horse-chestnut leaf, seed is mainly saponin(e, flavonoids, esculiw etc., so brownization very easily takes place when cultivating, stop seedling to draw nutriment, influence is grown even is caused death, so also can add the vitamin C of 5mg/l in the medium.The low light level is cultivated the illumination cultivation down that 5d changes 2000lx again over to down, and in time cuts the part of browning, and subculture is in fresh medium.

Higher (on 0.8～1mg/L) the medium, induced bud speed is fast, the time of sprouting is short, but the bud passing in time that differentiation is come out gradates and is vitrifying bud transparent, many water at BA or ZT concentration.When being reduced to 0.6mg/L, can prevent the generation of vitrifying bud.

Cultivate about 15d, form macroscopic indefinite bud, can obtain the test-tube plantlet of inducing of horse chestnut after one month at the base portion of seedling.

Different hormones and different hormone combinations to the influence of bud propagation referring to table 2.

Different hormones of table 2 and different hormonal readiness are to the influence of bud propagation

Additional hormone (Ms)	The inoculation number	Inductivity (%)	Average induced bud number	The callus degree
					BA0.4	30	56.7	13.8	-
BA0.6	30	100	21.2	-
					BA0.6+NAA0.1	30	100	35.7	+
BA0.6+NAA0.2	30	50	8.5	++
					BA0.8	30	100	67	-
KT0.2	30	6.67	0.1	-
					KT0.4	30	10	0.3	-
KT0.4+NAA0.2	30	13.3	0.4	++
					KT0.8	30	13.3	0.33	-
ZT0.2	30	53	33.8	-
					ZT0.4	30	100	34	-

ZT0.6	30	100	45	-
					ZT0.8	30	100	77	-

Annotate :-no callus+a small amount of callus ++ more callus

Embodiment 2: adopt the blade of the indefinite bud that branch has just been extracted out or induced by plumule in spring to carry out tissue culture

In the present embodiment, can there be dual mode in the explant source, and the one, get blade on the aseptic seedling that in embodiment 1, obtains, be inoculated on the inducing culture, can save the step of sterilization; The 2nd, draw materials from the branch that just sprouted spring, the good young leaflet tablet of bud green growth conditions (related petiole) gets colors, liquid detergent soaks 2h, flowing water flushing 2h, on superclean bench, sterilize, can put into 4～6 blades during sterilization in each triangular flask, 75% absolute ethyl alcohol 1min, 0.1% 84 thimerosal 8min, aseptic water washing 5～6 times, aseptic filter paper is washed dried surface moisture, cut off the blade edge part, be cut into the blockage of 5mm * 10mm, and at middle arteries and veins portion crosscut 3 cuttves of blade, then with the back side towards the underlying bed on ready medium, be positioned under 25 ℃ the dark condition.Blade is bred in three kinds of modes: directly indefinite bud takes place to produce in organ, indefinite bud takes place to produce organ indirectly, the body embryo takes place to produce organ indirectly, and existing division is as follows:

1. directly organ takes place to produce indefinite bud: used minimal medium is MS, additional plant hormone has NAA, IAA, IBA, BA, ZT etc., move under the 2000lx light after cultivating 10d under the 25 ℃ of conditions in dark place, other condition of culture is with embodiment 1, and blade is cultivated and can be obtained a test-tube plantlet in month.

Different hormones and different hormone combinations to the influence of excised leaf differentiation referring to table 3.

Different hormones of table 3 and different hormonal readiness are to the influence of blade bud differentiation

Additional hormone (MS)	By kind of a number	Inductivity (%)	Average induced bud number
				BA3+KT0.2+NAA0.2	30	23	2.2
BA3+KT0.2+IAA0.2	30	21	1.7
				BA3+KT0.2+IBA0.2	30	19	2.1
BA3+KT0.2+NAA0.4	30	32	4.7
				ZT2+NAA0.2	30	75	8

As can be seen from Table 3, in each is handled, be optimum with ZT2mg/L+NAA0.2mg/L.On this medium, inductivity is 75%, and on average each blade can be induced 8 indefinite buds.

2. organ takes place to produce indefinite bud indirectly: used minimal medium is MS, additional hormone has 2,4-D, NAA, KT, BA, ZT etc., with blade inoculation in MS+2,4-D2+KT0.2 medium on, be positioned under the 25 ℃ of conditions in dark place and cultivate, behind the 20d at vein and edge and incision produce the milky callus, callus is changed on the higher medium of cytokinin concentration, move under the light and cultivate, approximately behind the 10d, the callus edge engenders green projection, gradually grow into macroscopic bud point, along with the prolongation of incubation time, the bud point gradually grows up and is the indefinite bud of green, treats that indefinite bud grows to 2cm when high, can cut down and be inoculated among the MS, or be inoculated in the medium of embodiment 1 design and breed.

Different hormones and different hormone combinations make a difference very big to the indirect organ of blade, referring to table 4.

Different hormones of table 4 and different hormonal readiness are to the influence of blade bud differentiation

Additional hormone (MS)	The inoculation number	Inductivity (%)	Average induced bud number
				BA1+NAA0.2	30	11	1.2
BA3+NAA0.2	30	46	3.6
				KT2+NAA0.2	30	15	0.9
ZT2+NAA0.2	30	59	5.6

As seen MS+BA3+NAA0.2 and ZT2+NAA0.2 medium induce effect relatively good, and inductivity is respectively 46 and 59, and every callus produces 3.6 and 5.6 buds respectively.

3. somatic embryo (being called for short the body embryo) takes place to produce in organ indirectly: under appropriate condition, a large amount of body embryos can appear on the callus of inducing by the European horse-chestnut blade, and the ability that the body embryo breaks up secondary embryo is stronger, can produce a large amount of high quality seedlings at short notice.Blade inoculation in MS+2, on the medium of 4-D2+KT0.2, is positioned under the 25 ℃ of conditions in dark place and cultivates, and beginning gauffer occurs at blade edge and incision behind the 5d, begins to produce callus behind the 10d, and during to 20d, 100% blade has all formed callus.The callus that induces has three types, and a class is a milky, densification, and growth rate is very fast, is granular deposit type, can produce the body embryo through inducing; One class is milk yellow, and is soft, and microscopically is observed, and its cytoplasm is thin, and vacuole is bigger, can produce a small amount of body embryo through further inducing; The 3rd class is a flakes white callus, and the surface has fine hair, and is withered, can not induce the body embryo.The three is easy to difference on mode of appearance, can reject the 3rd class callus, reduces the unnecessary workload in back.

Blade is containing 2, behind the cultivation 20d, will change on the higher medium of cytokinin concentration on the medium of 4-D, induces embryo callus subculture.Must remove 2 this moment, 4-D, otherwise 2, under the effect of 4-D, the dedifferentiation cell constantly divides, and makes cell be in continuous splitting status, can't break up and morphogenesis again.The variable concentrations proportioning of the basic element of cell division and growth hormone, to embryo callus subculture induce influence very big, by table 5 as seen, when BA concentration 5～10 the time, all can induce embryo callus subculture, wherein MS+BA8+NAA1 induce effect best, inductivity reaches 70%.Phenols content height in the European horse-chestnut blade, callus be brown stain easily under light, and be withered dead, and after the embryo callus subculture that induces in the dark moved under the light, also browning was lost body embryo generating ability gradually, must cultivate in the dark.After cultivating 15d embryo callus subculture is inoculated on the differential medium, during to 3d, microscopically can be observed globular embryo, be easy to separate with callus on every side, in ensuing 5d, after globular embryo passed through torpedo embryo, heart-shape embryo successively, the idiosome upper end developed into cotyledon gradually.When 10d, naked eyes can be observed on the callus surface and form numerous leucoplast embryos, and color and luster is sparkling and crystal-clear, and is smooth exquisitely carved, 2 symmetrical cotyledons of tool and the radicle that expands.In this stage, the kind of hormone and concentration are very important to the growth and the maturation of body embryo, see Table 6, comprehensive induction frequency, on average go out three aspects of embryo number and average generation deformity embryo number, and is better with the concentration proportioning of MS+BA5+NAA0.2 and MS+ZT2+NAA0.2.When treating that the body embryo grows to 5～10cm, in time transfer to the body embryo among the 1/2MS that does not add any hormone, it is sprouted, or move in the further medium that reduces of hormone concentration, make it produce secondary embryo.

The different hormone concentrations of table 5 are to inducing the influence of embryo callus subculture

Additional hormone (MS)	The inoculation number	The embryo callus subculture number	Inductivity (%)
				BA5+NAA0.5 BA5+NAA1 BA5+NAA2 BA8+NAA0.5 BA8+NAA1 BA10+NAA1	50 50 50 50 50 50	13 21 15 23 35 28	26 42 30 46 70 56

Different hormones of table 6 and hormone concentration are to the influence of body embryonal induction

Additional hormone (MS)	The inoculation number	Go out embryo callus number	Inductivity (%)	On average go out the embryo number	Average lopsided embryo number
						BA3+NAA0.2 BA5+NAA0.2 BA6+NAA0.2 ZT1+NAA0.2 ZT2+NAA0.2 ZT3+NAA0.2	30 30 30 30 30 30	1 11 8 13 21 14	3.3 36.7 6.7 43.3 70 46.7	4 7 5 8 14 7	1 0.73 0.325 0.15 0.06 0.24

European horse-chestnut is many body embryo to occur at the brownization position of embryo callus subculture or the callus base portion of close medium, after this brownization of callus increase the weight of last death, this brings difficulty for the propagation of body embryo, find in the research to use cotyledonary embryos to induce secondary embryo ratio to be easier to, radicle position at the body embryo produces secondary embryo, is connected closely with parent when initial, begins to separate with parent after growing up gradually, break away from parent at last voluntarily, can independently absorb nutritional development.In the multiplicative stage, it is very important to keep the in-house hormonal balance of body embryo, see Table 7, the MS+KT0.1+NAA0.01 of low concentration and MS+ZT0.1+NAA0.01 can make the body embryo quantity of new generation reach maximum, the propagation frequency is respectively 214,256, when excessive concentration or ratio were inappropriate, the rate of increase descended on the contrary, and lopsided embryo also increases.

Different hormones of table 7 and hormone concentration are to the influence of secondary embryonal induction

Additional hormone (MS)	The inoculation number	Subculture body embryo number	The propagation number	Propagation frequency %
					MS0 KT0.05+NAA0.01 KT0.1+NAA0.01 KT0.5+NAA0.05 ZT0.1+NAA0.01 ZT0.5+NAA0.05	50 50 50 50 50 50	73 113 157 93 178 107	23 63 107 43 128 57	46 126 214 86 256 114

The body embryo that 5～8cm is long changes under the illumination and sprouts after moving into 1/2MS.Stretch out between the cotyledon that 10d left and right sides visible growth o'clock is expanded from two milkys, behind the 15d, launch first pair of compound leaf, germination rate is 98%, and radicle germinates during 20d.Cotyledon becomes big and cracking, produces and much incises, and can cut the test material that is used as embodiment 3.Need 50d approximately, the body embryonic development is to have 2 pairs of compound leaves, very large cracking cotyledon, butt physically well develops, the leaf look bud green plantlet, one week of uncork hardening, after in the greenhouse, taming 15d, can transplant outdoorly, rich water quality management has in addition obtained to originate from the complete body embryo plant of blade.

Embodiment 3: carry out tissue culture with the cotyledon behind the body embryonic development two weeks

The body embryo of European horse-chestnut is more special, and cotyledon can not resemble to grow the body scutellum of other plant and be blade, but in the process of body embryonic development cracking gradually, very plump, be to carry out the adventitious bud inducing and the good material of body embryo generation once more.In this embodiment, MS is a minimal medium, and used hormone is ZT, BA, KT, NAA, IAA and has tried out novel cell mitogen TDZ.Directly cut the cotyledon of body embryo, be inoculated on the ready medium, find that cotyledon produces indefinite bud with direct organ generation form, with direct and indirect organ two kinds of forms take place and produce the body embryo.Different hormone combinations to the direct organ of cotyledon indefinite bud takes place to produce, and still to produce the influence of body embryo very big, sees Table 8.

1. indirectly the body embryo takes place to produce organ: this process is that the growth and the propagation of the inducing of callus and embryo callus subculture, body embryo is identical with the method that produces among the embodiment 2, and the result is similar.

2. direct organ generation evoking adventive bud: the medium of additional separately BA, KT or TDZ is remarkable for the evoking adventive bud effect, and it is faint for inductor embryo effect, BA will get well than KT evoking adventive bud effect, and the MS+BA2+NAA0.2 inductivity is 88, and average induced bud number is 25.6.Novel basic element of cell division TDZ is extremely obvious for the evoking adventive bud effect, and when concentration transferred to 0.005, inductivity reached 100, and the number that on average sprouts is 37, and seedling is in good shape, and growth rapidly; Concentration is 0.01 o'clock, every cotyledon sprouts too much, on average can reach 78, because the restriction of space and nutrition, can not grow and be effective bud, and because One's name is legion, breathe vigorous, make temperature and humidity rising in the blake bottle, the seedling vitrifying is serious, is suitable inducing culture with MS+TDZ0.005+NAA0.2 therefore.

3. directly organ takes place to produce the body embryo: ZT is for the critical function of cotyledon inductor embryo, and listed medium inductivity all reaches 80% in the table, on average goes out the embryo number all above 25.Wherein best with the MS+ZT2+NAA0.2 effect, inductivity and on average go out the embryo number and be respectively 88 and 38, when concentration reduces to 0.5 and attached during with BA1, inductivity and on average go out the embryo number and slightly reduce, be respectively 82 and 30, because the ZT price than other reagent price height, from saving cost consideration, determines with MS+ZT0.5+BA1+NAA0.2 to be the suitable culture medium of inductor embryo.Body embryo propagation mode is with identical among the embodiment 2.

Different hormones of table 8 and hormone concentration are induced the influence of differentiation to cotyledon

Additional hormone (MS)	The inoculation number	Go out bud induction rate %	Number on average sprouts	Go out embryonal induction rate %	On average go out the embryo number
						BA2+NAA0.2 KT2+NAA0.2 ZT2+NAA0.2 ZT1+BA1+NAA0.2 ZT0.5+BA1+NAA0.2 ZT1+KT1+NAA0.2 TDZ0.001+NAA0.2 TDZ0.005+NAA0.2 TDZ0.01+NAA0.2	50 50 50 50 50 50 50 50 50	88 74 38 46 36 56 74 100 100	25.6 17.2 7 7.5 7.2 8.9 21 37 78	2 10 88 86 82 82 4 6 4	1 1.6 38 32 30 25 1.5 2 3

The indefinite bud that differentiates by all means on the 1/2MS or WPM medium of additional 0.4～0.6NAA, produces root system about 2 weeks, and rooting rate is 75%.The embryoid that induces by blade and cotyledon begins about a week to sprout on the 1/2MS medium, begins behind the 20d to take root, and developmental condition is fine.

Adopt the packaged technology of European horse-chestnut tissue-culturing quick-propagation provided by the present invention to carry out the exploitation of European horse-chestnut tissue culture and little propagating technology, it is a kind of effectively approach, in case after obtaining aseptic seedling, can utilize aseptic seedling to breed indefinite bud, also can utilize the blade evoking adventive bud and the embryoid of aseptic seedling, utilize the cotyledon evoking adventive bud and the embryoid of embryoid again, form a good production cycle system, increase reproduction coefficient.Direct organogenetic mode is because without the callus stage, can reduce the generation of variation plant, for keeping select tree in fast numerous, keeping the good characteristic of germ plasm resource to provide safeguard, and the indefinite bud and the embryoid that produce take place by indirect organ, may produce variation plant, this provides the machine of taking along for breed improvement and the seed selection new varieties of forest again.The present invention provides a kind of cycle short for implant mass in production of forestry, reproduction rate height, seedling-cultivating method with low cost.

Claims (7)

1. the cell stage of an European horse-chestnut takes place and the method for plant regeneration, it is characterized in that:

A. the plumule with seed carries out tissue culture, plumule growth and the used minimal medium of evoking adventive bud are the MS medium, additional plant hormone is BA 0.4～0.8mg/L+NAA 0～0.2mg/L, or KT 0.2～0.8mg/L+NAA 0～0.2mg/L, or ZT 0.2～0.4mg/L;

B. the blade of turning out with the sprouting and the seed plumule of plant sprouting in spring carries out tissue culture, indefinite bud takes place to produce for the direct organ generation of excised leaf generation indefinite bud, indirect organ and the used minimal medium of indirect organ generation generation body embryo is MS, wherein:

A. indefinite bud takes place to produce in the direct organ of excised leaf, the plant hormone of medium supplemented is BA 3mg/L+KT 0.2mg/L+NAA 0.2～0.4mg/L, or BA 3mg/L+KT 0.2mg/L+IAA 0.2mg/L, or BA 3mg/L+KT 0.2mg/L+IBA0.2mg/L, or ZT2mg/L+NAA 0.2mg/L;

B. organ takes place to produce indefinite bud indirectly, and the plant hormone of medium supplemented is BA 1～3mg/L+NAA 0.2mg/L, or KT 2mg/L+NAA 0.2mg/L, or ZT 2mg/L+NAA 0.2mg/L;

C. organ takes place to produce the body embryo indirectly, and the additional hormone of inducing the embryo callus subculture stage is BA 5～10mg/L+NAA 0.5～2mg/L; The body embryonal induction stage, additional hormone was BA 3～6mg/L+NAA 0.2mg/L, or ZT 1～3mg/L+NAA 0.2mg/L; The secondary embryonal induction stage, additional hormone was KT 0.05～0.5mg/L+NAA 0.01～0.05mg/L, or ZT 0.1～0.5mg/L+NAA 0.01～0.05mg/L;

C. carry out tissue culture with the cotyledon behind the body embryonic development two weeks, used minimal medium is MS, additional hormone is BA 2mg/L+NAA 0.2mg/L, or KT 2mg/L+NAA 0.2mg/L, or ZT 0.5～2mg/L+BA 0～1mg/L+NAA 0.2mg/L, or ZT 1mg/L+KT 1mg/L+NAA 0.2mg/L, or TDZ 0.001～0.01mg/L+NAA 0.2mg/L.

2. cell stage as claimed in claim 1 takes place and plant regeneration method, it is characterized in that: when cultivating with the plumule of Horsechestnut seed, the concentration of additional hormone BA or ZT is 0.6mg/L.

3. cell stage as claimed in claim 1 takes place and plant regeneration method, it is characterized in that: when the direct organ of blade that sprouting that sprouts with European horse-chestnut plant in spring and seed plumule are turned out took place to produce indefinite bud, additional hormone combinations and concentration value were ZT 2mg/L+NAA 0.2mg/L.

4. European horse-chestnut cell stage as claimed in claim 1 takes place and plant regeneration method, it is characterized in that: when the indirect organ of blade that sprouting that sprouts with European horse-chestnut plant in spring and seed plumule are turned out took place to produce indefinite bud, additional hormone combinations and concentration value were BA 3mg/L+NAA 0.2mg/L or ZT 2mg/L+NAA 0.2mg/L.

5. cell stage as claimed in claim 1 takes place and plant regeneration method, it is characterized in that: when the indirect organ of blade that sprouting that sprouts with European horse-chestnut plant in spring and seed plumule are turned out takes place to produce the body embryo, inducing the additional hormone combinations and the concentration value in embryo callus subculture stage is BA 8mg/L+NAA 1mg/L, the additional hormone combinations in body embryonal induction stage and concentration value are BA 5mg/L+NAA 0.2mg/L or ZT 2mg/L+NAA 0.2mg/L, and the additional hormone combinations in secondary embryonal induction stage and concentration value are KT 0.1mg/L+NAA0.01mg/L or ZT 0.1mg/L+NAA 0.01mg/L.

6. cell stage as claimed in claim 1 takes place and plant regeneration method, it is characterized in that: during with the direct organ generation of the cotyledon evoking adventive bud behind the European horse-chestnut body embryonic development two weeks, additional hormone combinations and concentration value are BA 2mg/L+NAA 0.2mg/L or TDZ 0.005mg/L+NAA 0.2mg/L.

7. cell stage as claimed in claim 1 takes place and plant regeneration method, it is characterized in that: when taking place to produce somatic embryo with the direct organ of cotyledon behind the European horse-chestnut body embryonic development two weeks, additional hormone combinations and concentration value are ZT 0.5mg/L+BA 1mg/L+NAA 0.2mg/L.