CN106879473A

CN106879473A - A kind of method that larch plant efficient occurs in vitro

Info

Publication number: CN106879473A
Application number: CN201710263530.9A
Authority: CN
Inventors: 朱木兰; 卫志明; 郑武林; 刘航
Original assignee: Shanghai Institutes for Biological Sciences SIBS of CAS
Current assignee: Center for Excellence in Molecular Plant Sciences of CAS
Priority date: 2017-04-21
Filing date: 2017-04-21
Publication date: 2017-06-23
Anticipated expiration: 2037-04-21
Also published as: CN106879473B

Abstract

The invention provides a kind of method that larch plant efficient occurs in vitro, specifically, present invention firstly discovers that being initial explant to the hypocotyl of larch plant seedling, bred in vitro, can induce out the callus containing many cells,primordials, and can further efficient evoking adventive bud generation, and by the elongation of adventitious bud, after taking root and transplanting seedling, a large amount of regeneration plant phenotype uniformity can be obtained high, the good larch plant of genetic stability, in addition, it is also used as one of parent of somatic hybridization, carry out cell hydridization, the larch new germ plasm different from former parent can be created.

Description

A kind of method that larch plant efficient occurs in vitro

Technical field

The present invention relates to larch field of plant cultivation, and in particular to the side that a kind of larch plant efficient occurs in vitro Method.

Background technology

Larch (Larix Mill) belongs to Pinaceae (Pinaceae) fallen leaves abieteae (Laricoideae), and natural distributed exists Northern Hemisphere temperate zone mountain area, the Plain of cool temperature zone and alpine climate area.Larch have widely distributed, strong adaptability, pest and disease damage it is few, The features such as material is excellent, its early stage fast-growing character ranks among the best in all of coniferous tree, is that state forest farm and peasant weigh very much Depending on reproducting tree species, artificial afforestation area's mesh benefit expand；It is also the weight in China northeast, northwest, North China and southern Subalpine region area Want pulpwood and building wood species.But the Larch Growth cycle is more long, generative propagation progeny variation is larger, and cutting propagation is taken root Rate is very low.Tissue cultures have very big potentiality as the main path of larch fast asexual propagation.Therefore, fallen leaves are carried out It is significant to there is research in the high-efficiency in-vitro of pine.

Used as basic technical operation platform, larchen Regeneration System has following main application：

(1) seedling provides conventional method production Larch Seed time and effort consuming, and production efficiency is low in the unit interval, seed The seed production climate in garden influences larger with cultivation management mode, and yield is extremely unstable between year, and seed supply is not enough, seedling Regularity is presented normal distribution.Using efficient Regeneration System, asexual propagation is carried out to excellent maternal plant, be Developing Clonal Forestry Breeding provides technical support.

(2) breed improvement at present, China mainly by manually introducing a fine variety, fine-variety breeding, the traditional breeding method mode such as interspecific hybridization Genetic improvement is carried out to larch.Although having achieved the progress for attracting people's attention in this field, however from technological layer for, Due to these traditional breeding mode human and material resources expend it is larger, by effect of natural conditions, breeding cycle be long, genetic manipulation is difficult Degree is big, the need for larch breeding process can not meet modern Forest Tree Genetic Breeding.

Therefore, this area is in the urgent need to developing a kind of new, large-scale plantation that is can realizing larch plant height The in vitro method for occurring of effect.

The content of the invention

It is an object of the invention to provide it is a kind of it is new, larch plant can be realized large-scale plantation it is efficient from The method that body occurs.

First aspect present invention provides a kind of propagation method of larch plant, including step：

A () carries out sprouting treatment to the seed of the larch plant, so as to obtain the seedling through sprouting；

B () is 4-20 to seedling culture n days through sprouting, wherein n, so as to obtain the seedling through culture；

C () cuts to the hypocotyl of the seedling, obtain hypocotyl segment, and the length of the hypocotyl segment is 1-6mm；With

D () regenerates to described hypocotyl segment, so as to obtain larch plant.

In another preference, in step (b), cultivated 4-20 days from same day day of emerging.

In another preference, in the step (b), the seedling is carried out into culture 5-18 days, it is preferred that 6-16 days.

In another preference, in step (d), including following one or more independent sub-steps：

(d1) described hypocotyl segment is cultivated, obtains callus；

(d2) treatment of sprouting is carried out to the callus, so as to obtain original adventitious bud (that is, bud former base)；

(d3) the original adventitious bud is cultivated, so as to obtain the adventitious bud of elongation；

(d4) adventitious bud to the elongation carries out rooting treatment, so as to obtain larch plant.

In another preference, in the step (c), the length of the hypocotyl segment is 1-5mm, it is preferred that 2- 4mm。

In another preference, in the step (b), also including being carried out disinfection to the seedling the step of.

In another preference, in the step (d), the regeneration is in low nitrogen and/or low NH₄ ⁺Culture medium in carry out.

In another preference, described low nitrogen and/or low NH₄ ⁺Culture medium be based on MS culture mediums low nitrogen and/or Low NH₄ ⁺Culture medium.

In another preference, in the low nitrogen and/or low NH₄ ⁺Culture medium in, except nitrogen and/or NH₄ ⁺Outside its His composition and content it is essentially identical with MS culture mediums (be such as same in standard MS medium for any other compositions The about 80-130% of component content, preferably 90-120%).

In another preference, one or more (or whole) of the sub-step (d1), (d2), (d3) and/or (d4) In, the regeneration is in low nitrogen, low NH₄ ⁺Culture medium in carry out.

In another preference, described " low nitrogen " refers to the nitrogen content N of the culture medium or condition of culture_LWith MS culture mediums The ratio between middle nitrogen content N0 (N_L/ N0) it is 0.3-0.9, preferably 0.35-0.8, more preferably 0.3-0.6.

In another preference, described " low NH₄ ⁺" refer to the NH of the culture medium or condition of culture₄ ⁺Content M_LCultivated with MS NH in base₄ ⁺The ratio between content M0 (M_L/ M0) it is 0.2-0.7, preferably 0.25-0.6, more preferably 0.3-0.5.

In another preference, in the step (d), the culture medium is that based on MS culture mediums, and nitrogen content is (dense Degree) it is the 1/4-1/2 of nitrogen content in MS culture mediums, it is preferred that 1/3-1/2.

In another preference, the culture medium contains NH₄Cl、NH₄NO₃And KNO₃。

In another preference, in the step (d) or one or more sub-step, in the culture medium, NH₄Cl's Concentration is 300-650mg/l, it is preferred that 400-600mg/l.

In another preference, in the step (d) or one or more sub-step, in the culture medium, NH₄NO₃'s Concentration is 400-700mg/l, it is preferred that 500-650mg/l.

In another preference, in the step (d) or one or more sub-step, in the culture medium, KNO₃'s Concentration is 600-1000mg/l, it is preferred that 700-900mg/l.

In another preference, in the step (d) or one or more sub-step, in the culture medium, with Ca (NO₃)₂·2H₂The quality meter of O, Ca (NO₃)₂·2H₂The concentration of O is 400-800mg/l, it is preferred that 500-700mg/l.

In another preference, in the step (d1), the hypocotyl segment is placed in containing 0.08-20mg/l (preferably Ground, 0.1-10mg/l, more preferably, 0.3-8mg/l, most preferably, 0.5-3mg/l) BA and 0.01-5mg/l is (it is preferred that 0.05- 3mg/l, more preferably, 0.08-1mg/l, most preferably, 0.1-0.5mg/l) NAA culture medium on, in 23 ± 1 DEG C of cultures, it is described under The nearly cotyledon end of plumular axis starts to expand, and obtains callus.

In another preference, in the step (d2), by the callus be placed in containing 0.05-10mg/l (it is preferred that 0.08-8mg/l, more preferably, 0.1-5mg/l) BA and 0.01-5mg/l (it is preferred that 0.05-3mg/l, more preferably, 0.08- 1mg/l) on the culture medium of NAA, cultivated 8-12 weeks in 23 ± 1 DEG C, obtain original adventitious bud, i.e. bud former base.

In another preference, in the step (d3), will the callus group with original adventitious bud (i.e. bud former base) Knit be placed in containing 0.05-10mg/l (it is preferred that 0.08-8mg/l, more preferably, 0.1-6mg/l) BA, 0-0.1mg/l (it is preferred that 0.001-0.08mg/l, more preferably, 0.005-0.05mg/l) TDZ and 0.01-5mg/l (it is preferred that 0.05-3mg/l, more preferably Ground, 0.08-1mg/l) NAA culture medium on cultivate 6-8 week, in 23 ± 1 DEG C cultivate, induce form clearly adventitious bud.

In another preference, in the step (d4), the adventitious bud of the elongation is placed in containing 0-6mg/l (preferably Ground, 0.01-3mg/l, more preferably, 0.05-1mg/l, most preferably, 0.1-0.6mg/l) BA, 0-0.6mg/l be (it is preferred that 0.01- 0.5mg/l, more preferably, 0.001-0.4mg/l, most preferably, 0.002-0.2mg/l) NAA and 0-6mg/l is (it is preferred that 0.01- 4mg/l, more preferably, 0.05-3mg/l, most preferably, 0.1-2mg/l) GA₃Culture medium on, carry out light culture 2-8 in 22 ± 1 DEG C My god (preferably 3-7 days).

In another preference, in the step (d4), by the adventitious bud after the light culture (it is preferred that separate list Strain adventitious bud), removal morphology lower end blade, with 5-100mg/l (it is preferred that 10-80mg/l, more preferably, 20-60mg/l) IBA pre-processes 0.2-36h (preferably 0.5-24h).

In another preference, in the step (d4), the pretreated adventitious bud is (it is preferred that separate individual plant Adventitious bud), excision morphology lower end stem section, containing 0.01-5mg/l (it is preferred that 0.05-3mg/l, more preferably, 0.08-1mg/ L, most preferably, 0.1-0.5mg/l) IBA 1/2MS culture mediums (wherein nitrogen content and NH₄Content is kept to about the 1/5 of MS culture mediums Continue to cultivate to 1/4) middle.

In another preference, in the step (d4), the length of the lower end stem section of the excision is 0.3-5mm, preferably Ground, 0.5-4mm, more preferably, 0.6-2mm.

In another preference, in the step (d4), in 1/2MS culture mediums (wherein nitrogen content and NH₄Content is kept to MS About the 1/5 to 1/4 of culture medium) in continue cultivate 4-6 week, in 24 ± 1 DEG C culture inductions obtain adventitious root.

In another preference, in the step (d4), the adventitious root of acquisition is incubated at 25 ± 2 DEG C, after cultivating 1-2 weeks Obtain the larch plant of Regeneration in Vitro.

In another preference, after methods described also includes for the plant that step (d) is obtained carrying out hardening, cultivation is implanted into The step of being cultivated in soil.

In another preference, the temperature cultivated in the cultivation soil is 25 ± 2 DEG C.

In another preference, the matrix of the cultivation soil includes peat, vegetable garden soil, and/or perlite.

In another preference, the peat, vegetable garden soil, the ratio of perlite are 3:6:1.

In another preference, the category that the larch plant is selected from the group：Pinaceae category, Larch or its combination.

In another preference, the larch plant is selected from the group：Larch-tree, Larix principis-rupprechtii, white fallen leaves long Pine, Yunnan larch, Xinjiang Luobupo, Larix kaempferi×L.olgensis hybridization (hybridization larch) or its combination.

In another preference, in the step (a), the Germination Temperature is 25 ± 1 DEG C.

In another preference, in the step (a), the sprout time is 5-30 days, it is preferred that 8-20 days, more preferably Ground, 7-15 days.

Second aspect present invention provides a kind of propagation method of larch plant, including step：

A the seed of the larch plant is carried out sprouting treatment by (), so as to obtain the seedling through sprouting；

B the seedling is carried out culture 4-20 days by (), obtain the seedling through cultivating；

C () cuts to the hypocotyl of the seedling, obtain hypocotyl segment, and the hypocotyl segment is 1-6mm；

D () is cultivated described hypocotyl segment, obtain callus；

E () carries out Fiber differentiation to described callus, obtain the callus with adventitious bud；With

F () is cultivated the adventitious bud that step (e) is obtained, acquisition larch plant of taking root.

In another preference, in the step (b), culture 5-18 days is carried out to the seedling, it is preferred that 6-16 days.

In another preference, in one or more (or all) steps of step (d)-(f), in low nitrogen and/or Low NH₄ ⁺Culture medium in cultivated.

In another preference, in the step (d), the hypocotyl segment is placed in containing 0.08-20mg/l (preferably Ground, 0.1-10mg/l, more preferably, 0.3-8mg/l, most preferably, 0.5-3mg/l) BA and 0.01-5mg/l is (it is preferred that 0.05- 3mg/l, more preferably, 0.08-1mg/l, most preferably, 0.1-0.5mg/l) NAA culture medium on, in 23 ± 1 DEG C of cultures, it is described under The nearly cotyledon end of plumular axis starts to expand, and obtains callus.

In another preference, the step (e) includes step：

(e1) callus that step (d) is obtained is cultivated, is obtained healing with original adventitious bud (i.e. bud former base) Injured tissue；

(e2) Fiber differentiation is carried out to the callus with original adventitious bud (i.e. bud former base) that step (e1) is obtained, is obtained Obtain adventitious bud.

In another preference, in the step (e1), by the callus be placed in containing 0.05-10mg/l (it is preferred that 0.08-8mg/l, more preferably, 0.1-5mg/l) BA and 0.01-5mg/l (it is preferred that 0.05-3mg/l, more preferably, 0.08- 1mg/l) on the culture medium of NAA, cultivated 8-12 weeks in 23 ± 1 DEG C, obtain original adventitious bud, i.e. bud former base.

In another preference, in the step (e2), will the callus group with original adventitious bud (i.e. bud former base) Knit be placed in containing 0.05-10mg/l (it is preferred that 0.08-8mg/l, more preferably, 0.1-6mg/l) BA, 0-0.1mg/l (it is preferred that 0.001-0.08mg/l, more preferably, 0.005-0.05mg/l) TDZ and 0.01-5mg/l (it is preferred that 0.05-3mg/l, more preferably Ground, 0.08-1mg/l) NAA culture medium on cultivate 6-8 week, in 23 ± 1 DEG C cultivate, induce form clearly adventitious bud.

In another preference, the step (f) includes step：

(f1) adventitious bud is cultivated, obtains the adventitious bud of elongation；

(f2) adventitious bud of the elongation that separating step (f1) is obtained, continues to cultivate to acquisition larch plant of taking root.

In another preference, in the step (f1), by the adventitious bud be placed in containing 0-6mg/l (it is preferred that 0.01-3mg/l, more preferably, 0.05-1mg/l, most preferably, 0.1-0.6mg/l) BA, 0-0.6mg/l be (it is preferred that 0.01- 0.5mg/l, more preferably, 0.001-0.4mg/l, most preferably, 0.002-0.2mg/l) NAA and 0-6mg/l is (it is preferred that 0.01- 4mg/l, more preferably, 0.05-3mg/l, most preferably, 0.1-2mg/l) GA₃Culture medium on, in 23 ± 1 DEG C culture, after 4-6 weeks, The adventitious bud for being extended.

In another preference, in the step (f2), by the adventitious bud of elongation be placed in containing 0-6mg/l (it is preferred that 0.01-3mg/l, more preferably, 0.05-1mg/l, most preferably, 0.1-0.6mg/l) BA, 0-0.6mg/l be (it is preferred that 0.01- 0.5mg/l, more preferably, 0.001-0.4mg/l, most preferably, 0.002-0.2mg/l) NAA and 0-6mg/l is (it is preferred that 0.01- 4mg/l, more preferably, 0.05-3mg/l, most preferably, 0.1-2mg/l) GA₃Culture medium on, carry out light culture, 3- in 22 ± 1 DEG C 7 days.

In another preference, in the step (f2), by the adventitious bud after the light culture (it is preferred that separate list Strain adventitious bud), removal morphology lower end blade, with 5-100mg/l (it is preferred that 10-80mg/l, more preferably, 20-60mg/l) IBA pre-processes 0-36h (preferably 0.5-24h).

In another preference, in the step (f2), the pretreated adventitious bud is (it is preferred that separate individual plant Adventitious bud), excision morphology lower end stem section, containing 0.01-5mg/l (it is preferred that 0.05-3mg/l, more preferably, 0.08-1mg/ L, most preferably, 0.1-0.5mg/l) IBA 1/2MS culture mediums (wherein nitrogen content and NH₄Content is kept to about 1/5 to 1/4) relaying Continuous culture.

In another preference, in the step (f2), the length of the lower end stem section of the excision is 0.3-5mm, preferably Ground, 0.5-4mm, more preferably, 0.6-2mm.

In another preference, in the step (f2), in 1/2MS culture mediums (wherein nitrogen content and NH₄Content is kept to about 1/5 to 1/4) continue to cultivate 4-6 weeks in, cultivating induction in 24 ± 1 DEG C obtains adventitious root.

In another preference, in the step (f2), the adventitious root of acquisition is incubated at 25 ± 2 DEG C, after cultivating 1-2 weeks Obtain the complete plantlets (i.e. larch plant) of Regeneration in Vitro.

In another preference, after methods described also includes for the plant that step (f) is obtained carrying out hardening, cultivation is implanted into The step of being cultivated in soil.

It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment) Can be combined with each other between each technical characteristic of body description, so as to constitute new or preferred technical scheme.As space is limited, exist This no longer tires out one by one states.

Brief description of the drawings

Fig. 1 shows larch hypocotyl regenerating system, wherein scheming A：The hypocotyl cut-out expanded, bar:2mm.Figure B：Just Level embryo callus, bar:4mm.Figure C：Callus rich in a large amount of bud former bases, bar:6mm.Figure D：High-frequency synchronousization clump Bud, bar:8mm.Figure E：The sprout of elongation, bar:1cm.Figure F：The plantlet of off-body kidney, bar:4cm.

Specific embodiment

The present inventor is screened, first with falling for being prepared using special process by extensively and in depth studying by a large amount of The hypocotyl segment of leaf pine plant seedling as initial explant, in specific low nitrogen, low NH₄ ⁺Trained under the condition of culture of content Support, can induce out the callus containing many cells,primordials, and can further efficient evoking adventive bud generation, and by not The elongation of normal bud, take root and transplant seedling after, a large amount of fallen leaves that regeneration plant phenotype uniformity is high, genetic stability is good can be obtained Loose plant.The larch plant obtained with breeding method of the invention, can be carried out as one of parent of somatic hybridization Cell hydridization, so as to the larch new germ plasm different from former parent can be developed efficiently and rapidly.On this basis, this hair is completed It is bright.

Culture medium

MS culture mediums used herein be this area at present use most common culture medium, Murashige and Skoog in 1962 is tobacco cell Training Design, is characterized in that inorganic salts and ion concentration are higher, is that more stable ionic equilibrium is molten Liquid, its nitrate content is high, and the quantity and ratio of its nutrient are suitable, can meet the nutrition and physiological requirements of plant cell, because And the scope of application is wider, most plants tissue-culturing quick-propagation uses it as the minimal medium (Murashige of culture medium and Skoog A revised medium for rapid growth and bio assays with tobacco tissue cultures.Physiol Plant,1962.15(3):473.).Unless otherwise indicated, using known in the art Formula prepares MS culture mediums or MS solid mediums.

In the present invention, the culture medium for being used can enter on the basis of the conventional medium of the larch plant of this area Row builds, and the total nitrogen content in conventional medium is adjusted simultaneously so that the total nitrogen content (concentration) in culture medium is low In conventional medium.

Preferably, the preferred culture medium of a class (i.e. larch culture medium) of the invention is low nitrogen and/or low NH₄ ⁺Culture Base.

As used herein, " low nitrogen " refers to the nitrogen content N of the culture medium or condition of culture_LWith nitrogen content N0 in MS culture mediums The ratio between (Nl/N0) be 0.3-0.9, preferably 0.35-0.8, more preferably 0.3-0.6.

As used herein, " low NH₄ ⁺" refer to the NH of the culture medium or condition of culture₄ ⁺Content M_LWith NH in MS culture mediums₄ ⁺Contain The ratio between amount M0 (Ml/M0) is 0.2-0.7, preferably 0.25-0.6, more preferably 0.3-0.5.

In the present invention, described low nitrogen and/or low NH₄ ⁺Culture medium can be based on the conventional culture medium in this area (such as MS culture mediums, ML culture mediums) prepared.

Typically, the particularly preferred larch culture medium of a class is low nitrogen and/or low NH based on MS culture mediums₄ ⁺Culture Base.In another preference, in the low nitrogen and/or low NH₄ ⁺Culture medium in, except nitrogen and/or NH₄ ⁺Outside other into Point and content and MS culture mediums it is essentially identical (be such as same composition in standard MS medium for any other compositions The about 80-130% of content, preferably 90-120%).

In the present invention, one or more or whole sub-step of the sub-step (d1), (d2), (d3) and/or (d4) In, in low nitrogen, low NH₄ ⁺Culture medium in regenerated.

Generally, in culture medium of the invention, total nitrogen content (concentration) is the 1/4- of nitrogen content (concentration) in MS culture mediums 1/2, it is preferred that 1/3-1/2.Additionally, K⁺、Ca²⁺Concentration and MS culture mediums in K⁺、Ca²⁺Concentration it is essentially identical, preferably Ground, with total restatement of culture medium, K⁺、Ca²⁺Concentration be 80-120%.

In a preferred embodiment, in culture medium of the invention, NH₄The concentration of Cl is 300-650mg/l, preferably Ground, 400-600mg/l.

In a preferred embodiment, in culture medium of the invention, NH₄NO₃Concentration be 400-700mg/l, preferably Ground, 500-650mg/l.

In a preferred embodiment, in culture medium of the invention, KNO₃Concentration be 600-1000mg/l, preferably Ground, 700-900mg/l.

In a preferred embodiment, in culture medium of the invention, with Ca (NO₃)₂·2H₂The quality meter of O, Ca (NO₃)₂·2H₂The concentration of O is 400-800mg/l, it is preferred that 500-700mg/l.

6-benzyladenine

As used herein, term " 6-benzyladenine ", " 6- benzyladenines ", " 6- benzyls adenine ", " 6-BA ", " benzyladenine ", " BA " etc. can be with used interchangeablies.

6-benzyladenine is a kind of broad spectrum activity plant growth regulator, can promote plant cell growth, suppresses leaves of plants Green element, nucleic acid, the decomposition of protein, improve the content of amino acid, Delaying Leaf-Senescence, by amino acid, auxin, inorganic salts etc. To various efficiency such as treatment site allocation and transportation, each stage from germinateing to harvesting of agricultural, fruit tree and garden crop is widely used in.

One of ordinary skill in the art can obtain 6-benzyladenine using conventional method, and such as market is bought or with often Rule method is produced.

Gibberellin

As used herein, term " gibberellin " can be with " GA ", " GA₃" used interchangeably.

Gibberellin is the plant hormone that a class is widely present, and chemical constitution is more complicated, belongs to Diterpenes acid, by Fourth Ring bone Frame derives and obtains.Gibberellin all contains gibberellane skeleton.The nearest precursor of gibberellin is commonly considered as shellfish in higher plant Shell China fir alkene.The basic structure of gibberellin is gibberellane, there is 4 rings.On gibberellane, due to double bond, hydroxy number and position Put difference, form various gibberellin, it is known that at least 38 kinds of gibberellin class.Different gibberellin biological activities is different, red Mould acid (GA₃) active highest.Activity compound high must have a red mould loop systems (ring ABCD), there is carboxyl on C-7, There is a lactonic ring on A rings.

Gibberellin is now widely used for agricultural production, such as improves vintage, or flower is adjusted in breeding of hybrid rice Phase is so that Parent flower synchronization etc.；There is significant effect of increasing production to paddy rice, cotton, vegetables, melon and fruit, green manure etc..

One of ordinary skill in the art can obtain various gibberellin using conventional method, and such as market is bought or with routinely Method is produced.A kind of method that routine produces gibberellin is to be extracted with methyl alcohol.Different gibberellin can use various chromatography Technology is separated.

Methyl α-naphthyl acetate

As used herein, term " methyl α-naphthyl acetate " can be with " NAA " used interchangeably.

Methyl α-naphthyl acetate is broad spectrum type plant growth regulator, can promote cell division and expand, and induced synthesis adventitious root increases Fruit setting, prevents shedding, changes female, male flower ratio etc..Can be through blade, the tender epidermis of branch, seed is entered into plant, with nutrition Flow transporting to complete stool.

One of ordinary skill in the art can obtain methyl α-naphthyl acetate using conventional method, and such as market is bought or uses conventional method Produce.

IBA

As used herein, term " IBA ", " indolebutyric acid " are used interchangeably.

Indolebutyric acid (IBA) is obligate root-promoting hormone, and is a kind of indoles plant growth regulator of wide spectrum, is good Good root-growing agent, can promote what draft and woody ornamentais transplanted to take root.Can be additionally used in the fruit setting of melon and fruit, improve percentage of fertile fruit.

In the present invention, the purpose of addition IBA is to accelerate the reproduction conversion of plant for hestening rooting with effect.

One of ordinary skill in the art can obtain IBA using conventional method, and such as market is bought or uses conventional method system Take.

TDZ

As used herein, term " TDZ ", " Thidiazuron (thidiazuron) " are used interchangeably.

TDZ is a kind of new plant growth regulator, with very strong cytokine activity (CTK), its CTK activity Tens times higher than general CTK to hundred times, research shows, it can promote the regeneration and breeding of plant sprout, break bud stop Eye, promotes seed to sprout, and promotes callus growth, delays plant senescence etc..

In the present invention, the purpose of addition TDZ is to promote adventitious buds differentiation.

One of ordinary skill in the art can obtain TDZ using conventional method, and such as market is bought or uses conventional method system Take.

The features described above that the present invention is mentioned, or the feature that embodiment is mentioned can be in any combination.Disclosed in this case specification All features can be used in combination with any combinations thing form, each feature disclosed in specification, can by it is any provide it is identical, The alternative characteristics substitution of impartial or similar purpose.Therefore except there is special instruction, disclosed feature is only impartial or similar spy The general example levied.

Main advantages of the present invention include：

(1) direct adventitious bud of the present invention first to larch plant is studied in vitro, and is obtained higher The regeneration plant of frequency.

(2) present invention is first initial explant with the seedling hypocotyl of larch plant, can induce out and contains many The callus of cells,primordial, and can further efficient evoking adventive bud generation, and by the elongation of adventitious bud, take root and move After planting seedling, the larch that a large amount of regeneration plant phenotype uniformity are high, genetic stability is good, hereditary conservation is high can be obtained and planted Thing.

(3) present invention firstly discovers that larch hypocotyl regenerating system of the invention is also used as the parent of somatic hybridization One of this, carries out cell hydridization, can create the larch new germ plasm different from former parent.

(4) method of the present invention can breed larch plant by rapid, high volume in vitro, reproducible.

(5) method of the present invention can in a short time produce substantial amounts of larch high quality seedling, and this seedling Generation is not subject to seasonal restrictions, and can produce throughout the year.

(6) present invention makes larch hypocotyl culture successful regeneration into plant first.In the present inventive method in triplicate Experiment, average each hypocotyl cut-out can produce about 20-30 plants of regeneration plant, and high with parental phenotypes uniformity and indefinite Bud induction rate is (about 90% or higher) high, and rooting rate is (about 95%) high, transplants survival rate (about 90% or higher) high

(7) larch hypocotyl regenerating system of the invention can be used to save endangered larch germ plasm resource.

With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention Rather than limitation the scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, generally according to conventional strip Part such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) described in condition, or according to the condition proposed by manufacturer.Unless otherwise indicated, otherwise percentage and Number is calculated by weight.

Unless otherwise defined, all specialties used in text and scientific words and meaning familiar to one skilled in the art institute Justice is identical.Additionally, during any method similar to described content or impartial and material all can be applied to the inventive method.Wen Zhong Described preferable implementation only presents a demonstration with material and is used.

Unless otherwise stated, material used in the embodiment of the present invention and reagent are commercially available prod.

Embodiment 1

The preparation of larch culture medium No.1-3

Larch plant culture is prepared, based on the formula of MS culture mediums, to KNO₃、NH₄NO₃、NH₄Cl and Ca (NO₃)₂·2H₂The content of O is adjusted (as shown in table 1), and other compositions are not adjusted.To 5.8,121 DEG C sterilize regulation pH value 15-25 minutes.So as to obtain low nitrogen/low NH₄ ⁺Culture medium No.1, No.2 and No.3.The induction and elongation of adventitious bud are used Described low nitrogen/low NH₄ ⁺Culture medium (may be simply referred to as " larch culture medium ").

Simultaneously using commercially available MS culture mediums as control medium.

Table 1

The preparation of the regeneration plant of embodiment 2

In the present embodiment, for the larch culture medium No.1-3 and the MS culture mediums of control that are prepared in embodiment 1, Hypocotyl segment (or control segment material) to being prepared through special process carries out regeneration treatment.Wherein, weight under each experiment condition It is multiple three times.Specific experiment method is as follows：

Experimental technique：

(1) larix olgensis seed (being provided by Northeast Forestry University) is soaked 1 week with running water, removal necrotic lesion Seed.Seed is placed on the absorbent cotton of moistening, drenched gauze is covered on seed, 28 DEG C of greenhouses are sprouted, and note moisturizing, 10- Emerged after 15 days.

(2) the larch seedling emerged 7-15 days is cleaned with running water, surface water drops is blotted, on superclean bench With ethanol disinfection 30-90 seconds of 70%, 0.1% mercuric chloride soaked 5-8 minutes, and aseptic water washing 5-8 times, aseptic filter paper blots nothing The bacterium material surface globule, the segment that hypocotyl is cut into 2-4 millimeters is with standby.(control material is the hypocotyl segment of 10mm)

(3) hypocotyl segment is inoculated in described in the embodiment 1 of attached BA containing 0.5-3mg/l and 0.1-0.5mg/l NAA After being cultivated 7-15 days in culture medium, the nearly cotyledon end of hypocotyl cut-out starts to expand (Fig. 1 schemes A), and 4- is cultivated on this culture medium After 6 weeks, initial explant is substantially by fine and close callus parcel (Fig. 1 schemes B).Condition of culture：Illumination (60 μm of ol.m^- ².s^-1) 16h/d cultures, cultivation temperature is 23 ± 1 DEG C.

(4) by the fine and close callus obtained by step (3) attached BA's containing 0.5-3mg/l and 0.1-0.5mg/l NAA On culture medium described in embodiment 1 cultivate 8-12 week after, base output existing form obscure original adventitious bud, i.e., bud former base (Fig. 1, Figure C).Illumination (60 μm of ol.m^-2.s^-1) 16h/d cultures, cultivation temperature is 23 ± 1 DEG C, and once, culture medium is constant for 4 weeks subcultures.After For the frequency, 4 weeks/generation.

(5) by the callus of incidentally original adventitious bud in attached BA containing 0.5-3mg/l, 0-0.01mg/l TDZ and 0.1- After being cultivated 6-8 weeks on culture medium described in the embodiment 1 of 0.5mg/l NAA, form clearly adventitious bud (Fig. 1, figure is induced D).Illumination cultivation (60 μm of ol.m^-2.s^-1) 16h/d, cultivation temperature is 23 ± 1 DEG C.

(6) adventitious bud is transferred to attached BA containing 0-0.6mg/l, 0-0.06mg/l NAA and 0-0.6mg/l GA₃Embodiment After 4-6 weeks, adventitious bud obtains effectively elongation (Fig. 1 schemes E), illumination (60 μm of ol.m to medium culture described in 1^-2.s^-1)16h/ D, 22 ± 1 DEG C of cultivation temperature.

(7) sprout that will be extended light culture (22 ± 1 DEG C) 3-7 days in the culture medium of step (5).

(8) sprout after light culture is separated into individual plant, peels off seedling morphology lower end blade, located in advance with 50mg/l IBA Reason 0-36h, after the 1mm stem sections of excision morphology lower end, is transferred to the fallen leaves described in attached 1/2 embodiment 1 containing 0.1-0.5mg/l IBA Adventitious root is induced out (Fig. 1 schemes F) after being cultivated 4-6 weeks in loose culture medium (the larch culture medium for halving).Root media Additional saccharose 2%, and solidified with 0.25% gellan gum (gelrite).Condition of culture：Illumination (80 μm of ol.m^-2.s^-1) 16h/d, 24 ± 1 DEG C of cultivation temperature.

(9) step (7) gained rooted seedling is transferred to 28 ± 2 DEG C, illumination (80 μ together with culture medium, blake bottle etc. mol.m^-2.s^-1) 16h/d condition of culture under carry out tolerance culture 1-2 week after, open culture bottle cover, inject 10-20ml without Bacterium water or deionized water, hardening 5-8 days.

(10) seedling that will take root is transplanted in the matrix of peat, vegetable garden soil and perlite (3: 6: 1), in 28 ± 2 DEG C, nature Well-grown in the greenhouse of illumination, as the larch regeneration plant consistent with growth seedling naturally.

Experimental result：

During using conventional MS culture mediums (control medium), during using the hypocotyl segment of 10mm as vegetable material, The larch plant of regeneration cannot be obtained.During using conventional MS culture mediums (control medium), even if using through special process The hypocotyl segment of preparation is simultaneously counted stage by stage, and resulting larchen adventitious bud induction frequency is only 20%, and rooting rate is only 10%, it is 75% to transplant survival rate.Average each hypocotyl cut-out is only capable of producing regeneration plant≤0.3 plant.

In contrast, using larch hypocotyl segment prepared by special process of the invention specifically and specific low Nitrogen/low NH₄ ⁺Culture medium (culture medium No.1,2 or 3 in embodiment 1), can greatly improve the yield of regeneration plant.

During the larch culture medium No.1,2 or 3 prepared using the hypocotyl segment and use embodiment 1 of 2-4mm, each Result in culture medium is essentially identical.For larch plant, average each hypocotyl cut-out can produce regeneration plant 20- 30 plants (compared with using MS medium controls, improve about 60-90 times), and it is high with parental phenotypes uniformity and indefinite Bud induction rate 90-92%, rooting rate about 94-96%, transplant survival rate 90%.Compared with using MS medium controls, fallen leaves The Plantlet Morphological Formation of loose culture medium group is more excellent.

Embodiment 3

The step of the present embodiment, is substantially the same manner as Example 2, and difference is replaced length and fallen in vain using Larix principis-rupprechtii seed Leaf pine seed.

(average each hypocotyl cut-out is only capable of producing again with the control group using conventional MS culture mediums (control medium) Raw plant≤0.3 plant) compare, using larch hypocotyl segment prepared by special process of the invention specifically and specific low Nitrogen/low NH₄ ⁺Culture medium (culture medium No.1,2 or 3 in embodiment 1), can greatly improve the yield of regeneration plant.

During the larch culture medium No.1,2 or 3 prepared using the hypocotyl segment and use embodiment 1 of 2-4mm, each Result in culture medium is essentially identical.For larch plant, average each hypocotyl cut-out can produce regeneration plant 22- 32 plants (compared with using MS medium controls, improve about 70-96 times), and it is high with parental phenotypes uniformity and indefinite Bud induction rate 89-92%, rooting rate about 90-95%, transplant survival rate about 90%.

Embodiment 3

The step of the present embodiment, is substantially the same manner as Example 2, and difference is replaced length and fallen in vain using Yunnan Larch Seed Leaf pine seed.

(average each hypocotyl cut-out is only capable of producing again with the control group using conventional MS culture mediums (control medium) Raw plant≤0.32 plant) compare, using larch hypocotyl segment prepared by special process of the invention specifically and specific Low nitrogen/low NH₄ ⁺Culture medium (culture medium No.1,2 or 3 in embodiment 1), can greatly improve the yield of regeneration plant.

During the larch culture medium No.1,2 or 3 prepared using the hypocotyl segment and use embodiment 1 of 2-4mm, each Result in culture medium is essentially identical.For larch plant, average each hypocotyl cut-out can produce regeneration plant 22- 28 plants (compared with using MS medium controls, improve at least about 60 times), and it is high with parental phenotypes uniformity and indefinite Bud induction rate about 91%, rooting rate about 94% transplants survival rate ＞ 90%.

The all documents referred in the present invention are all incorporated as reference in this application, independent just as each document It is incorporated as with reference to such.In addition, it is to be understood that after above-mentioned instruction content of the invention has been read, those skilled in the art can Made various changes or modifications with to the present invention, these equivalent form of values equally fall within the model that the application appended claims are limited Enclose.

Claims

1. a kind of propagation method of larch plant, it is characterised in that including step：

C () cuts to the hypocotyl of the seedling, obtain hypocotyl segment, and the length of the hypocotyl segment is 1- 6mm；With

D () regenerates to described hypocotyl segment, so as to obtain larch plant.

2. the method for claim 1, it is characterised in that in step (d), including following one or more independent sons Step：

(d1) described hypocotyl segment is cultivated, obtains callus；

3. the method for claim 1, it is characterised in that in the step (c), the length of the hypocotyl segment is 1- 5mm, it is preferred that 2-4mm.

4. the method for claim 1, it is characterised in that in the step (d), the regeneration is in low nitrogen and/or low NH₄ ⁺ Culture medium in carry out.

5. method as claimed in claim 4, it is characterised in that described low nitrogen and/or low NH₄ ⁺Culture medium be based on MS training Support the low nitrogen and/or low NH of base₄ ⁺Culture medium.

6. method as claimed in claim 4, it is characterised in that described " low nitrogen " refers to the nitrogen of the culture medium or condition of culture Content N_LWith the ratio between nitrogen content N0 (N in MS culture mediums_L/ N0) it is 0.3-0.9, preferably 0.35-0.8, more preferably 0.3-0.6.

7. method as claimed in claim 4, it is characterised in that described " low NH₄ ⁺" refer to the culture medium or condition of culture NH₄ ⁺Content M_LWith NH in MS culture mediums₄ ⁺The ratio between content M0 (M_L/ M0) it is 0.2-0.7, preferably 0.25-0.6, more preferably 0.3- 0.5。

8. the method for claim 1, it is characterised in that methods described also includes carrying out the plant that step (d) is obtained After hardening, it is implanted into cultivation soil the step of cultivate.

9. method as claimed in claim 8, it is characterised in that the matrix of the cultivation soil include peat, vegetable garden soil, and/or Perlite.

10. the method for claim 1, it is characterised in that the larch plant is selected from the group：Larch-tree, China Northern larch, larix olgensis, Yunnan larch, Xinjiang Luobupo, Larix kaempferi×L.olgensis hybridization (hybridization larch), Or its combination.