CN1224315C - Stem inductive method for microbody reproduction of Japan dahurian larch - Google Patents
Stem inductive method for microbody reproduction of Japan dahurian larch Download PDFInfo
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Abstract
The present invention relates to a technology for forest propagation and plant regeneration in forest science, particularly to a stem inducing method for the micropropagation of Japanese larch. In the method, the branches of clone plants at different ages of the Japanese larch, which grow for one year, are selected as materials, and the axillary buds of the branches are taken out so as to realize the regeneration of stem organs through adventitious stem formation, elongation growth, successive transfer culturing, etc. The present invention provides a propagation method with the advantages of high speed, short period, seasonal influence prevention and high proliferation efficiency for the regeneration plants of the Japanese larch.
Description
Technical field
The present invention relates to forest tree propagation and plant regeneration technique in the forest-science, specifically a kind of stem abductive approach of larch-tree micropropagation.
Background technology
Larch subfamily (Laricodeae) in the Larch Pinaceae (Pinaceae), this genus germ plasm resource is abundant, according to document announcement, total 25 kinds of larch in the whole world and some mutation, hybrid, wherein 17 kinds are distributed in China (document 1: the king fight (chief editor), Zhang Songyun (associate editor), 1992, the China larch forest, Beijing: China Forest publishing house, pp1-3).They distribute wide, spread all over mountain area, subtropics, mountain area, temperate zone and the cool temperate zone flat area in the Northern Hemisphere.Larch has that adaptability is strong, fast growth, grow into forest early, fine quality such as disease and insect resistance and strength of wood height, rotproofness are strong, have been widely used and can be industrial as important paper making raw material.Because larchen above-mentioned characteristic can be used as the large tracts of land reproducting tree species.
Larch-tree (Larix Kaempferi (Lamb) Carr.) originates in middle part, island, Japanese Honshu, in world many countries introducing and planting has been arranged so far, and it is in 1884 that China introduces a fine variety larch-tree the earliest, plants on a small quantity in the Qingdao Laoshan.Go through a century, introduction range has expanded to North gets Linkou County, Heilungkiang, reaches Anhua, Hunan in the south, and east is from the Qingdao Laoshan, and to Hami, but cultural area mainly concentrates on the Liao Dynasty, Ji, Shandong, Hubei Province, river, Shan, province such as sweet.Contrast and larch-tree provenance are united to test and are shown between twice larch kind of carrying out the seventies and the eighties, alpine region in mountain area, temperate zone more than China rainfall 500mm and the subtropics, larch-tree is compared with other sibling species, as larix olgensis (L.olgensis Henry), Larix principis-rupprechtii (L.principis-rupprechtii Mayr.), Dahurian larch (L.gmelini Rupr.) etc., has bigger growth vigor (document 2: Ma Changgeng, Wang Jianhua, 1992, the discussion of China's development larch-tree problem, the selection of larch kind and provenance, Beijing: publishing house of Beijing Agricultural University, pp22-29).But because larch-tree seed year rareness, solid amount is few, and seed should not be gathered and weakness such as germination rate is low, utilizes natural seed can not meet the needs of production far away, and therefore, people have carried out a series of improvement work to it.The breeding work of domestic larch-tree starts from the sixties in 20th century, basically experience kind and provenance and selected experimental stage, seed orchard stage and clone stage, now selected a collection of plant with merit, but because individual amount is few, so be difficult to satisfy the demand of commodity forestry.Last century, the mid-80 began the research of asexual cuttage technique, and the treelet cuttage is obtaining certain achievement, but the cuttage problem of Cheng Shu is still still unresolved.Therefore, impel people to explore new stock breeding approach again.
Micropropagation is as an important component part of modern biotechnology, the characteristics that have that speed is fast, the cycle is short, be not subjected to seasonal effect, offspring's stability are high, now obtained important achievement, and be used widely at aspects such as agricultural, forestry, medicine, genetic breedings.The larch micropropagation mainly concentrates on European larch (L.decidua Mill.) and Ou Ri hybrid larch (on two seeds of L. * eurolepis), as: Bonga (document 3:J.M.Bonga, 1982, Shoot formation in callusfrom the stalks of young female strobili of Larix deciduas, Can.J.Bot., 60 (8): 1357-1359) form branch through callus, from tender tissue, form indefinite bud from European larch female cone part; The most great breakthrough is Nagimani (document 4:R.Nagmani; J.M.Bonga, 1985, Embryo in subcultured callus of Larix deciduas.Can.J.For.Res., 15 (6): 1088-1091) utilize egagametophyte to induce the European larch regeneration plant of formation, Laliberte and Lalonde (document 5:A.Laliberte; M.Lalonde, 1988, Sustained caulogenesis incallus cultures of Larix * eurolepis initiated from short shoot buds of a12-year-old tree, Am.J.Bot., 75 (6): 767-777) the hybrid larch regeneration plant of Pei Yanging, and Klimaszewska (document 6:K.Klimaszewska, 1989, Recovery of somaticembryos and plantlets from protoplast cultures of Larix * eurolepis, Plant CellReports, 8 (8): first somatic embryo regeneration plant in the Larch that 440-444) obtains--the regeneration plant of hybrid larch immature zygotic embryos.The nineties in last century, Ewald (document 7:D.Ewald, 1998, Advances in tissue culture of adult larch, Vitro Cellular andDevelopmental Biology Plant, 34 (4): 325-330) wait on problems such as the micropropagation of Cheng Shu such as root induction and obtained breakthrough, find material continuous subculture on the medium of no hormone, or micrografting, can make the material of growing up recover juvenile character.So far, though in the treelet micropropagation of European larch, set up lasting micropropagation system, but they or reproduction rate are low, utilized terminal bud (the document 8:N.Itahana of larch-tree as Itahana, 1995, Manual for budculture of Japanese larch, Bulletin of the National Forest Tree Breeding Center, 13:145-149), and each branch only has 1 terminal bud; Perhaps only pay attention to problem in science, therefore the test (seeing document 6) as Klimaszewska utilizes the protoplast approach regeneration plant that the hybrid larch carries out still has suitable distance apart from production application.At present in China, the micropropagation of larch-tree also there is not relevant report.
Summary of the invention
The object of the present invention is to provide a kind of stem abductive approach of larch-tree micropropagation, it can improve the proliferate efficiency of elite germplasm material, satisfies the quantity demand of commodity production of forestry to good nursery stock.
To achieve these goals, technical scheme of the present invention is as follows:
1 year living branch of larch-tree clone of choosing the different age of stands is a material, gets its axillalry bud, forms, extends and induce through indefinite stem, reaches processes such as successive transfer culture, realizes the regeneration of stem organ; Specifically can operate as follows:
1) collection of material and preliminary treatment:
Acquisition time is sprouted preceding or when sprouting for going into the early spring in Winter Solstice bud bud, before the sampling bough water planting to bud bud is put brightly, and water temperature is a room temperature;
2) preparation of medium:
(1) with conventional method preparation improvement MS, WPM or SH medium, wherein WPM or SH induce the generation effect obvious to stem;
(2) the packing medium horizontal high voltage steam sterilizing of going forward side by side, the cooling back is standby;
3) inoculation axillalry bud:
Axillalry bud is cut in the branch flowing water washing that bud bud after the water planting is shinny, and aseptic condition is sterilization down, and WPM or SH wherein add plant growth regulating substance as the stem inducing culture, and the inoculation of medium axillalry bud carries out the generation of indefinite stem; Wherein the axillalry bud of being cut is of a size of 0.5～1cm;
4) elongation growth of indefinite stem:
The indefinite stem that generates is transferred in having added plant growth regulating substance and having promoted in the modified MS medium of thing, culturing room's temperature is 22～28 ℃, and light intensity is 2000～3000lux, and light/dark cycle is 14～20h/4～10h, last 25～30 days, finish the regeneration of stem organ; Wherein said light/dark cultivation cycle is best with 16h/8h; For root induction or preservation material, after step 4), can carry out successive transfer culture, promote the childrenization of indefinite stem material.
Growth regulatory substance of the present invention is respectively: the zeatin in the basic element of cell division (Zea), six benzyl purines (BA), methyl in the growth hormone (NAA), heteroauxin (IAA) and/or indolebutyric acid (IBA), rate of branching out during these are induced stem and stem elongation effect are obvious; Described promotion thing is active carbon (AC);
Described additional plant growth regulating substance consumption is: BA 0.1～0.5mg/l, Zea0.01～0.5mg/l, NAA 0.01～0.1mg/l, IBA 0.1～0.5mg/l and/or IAA 0.5～1.0mg/l; Promote the thing consumption to be: AC 0.5～10.0g/l;
The generation phase of indefinite stem, the best of breed of medium supplemented plant growth regulating substance is: WPM+Zea+IAA or SH+Zea+IAA; In the elongation growth stage of indefinite stem, the best of breed of medium supplemented plant growth regulating substance and promotion thing is: MS+NAA+IBA+AC.
The present invention has following advantage:
1. micropropagation speed is fast.Determined by biological property, the larch nursery stock is through just entering the generative growth phase that blossoms and bears fruit the vegetative growth phase in 13～15 years, the time that its offspring's merit also need be no less than 1/3 life cycle (30～40 years) can show, and make one's options (needing 8～12 years approximately), but this moment, the asexual multiplication ability of plant significantly descended, and the solution still not yet in effect of conventional cottage propagation technology recovers the problem of rootability.After the present invention finishes by inducing of the axillalry bud of micropropagation, the formation of indefinite stem and elongation, the method of the stem of inducing elongation being carried out successive transfer culture has promoted one-tenth material in age to recover the juvenile growth characteristic, be to improve the highly effective approach of branching out of organizing with rooting rate, its with nature under take the conventional method of seminal propagation or cottage propagation to compare, utilize method of the present invention, can make to shorten over half the performance period of good nursery stock population.
2. micropropagation reproductive efficiency height.Only utilize terminal bud to breed with prior art and compare, the present invention has utilized a large amount of axillalry buds of 1 year living branch explant of elite stand, can breed out a large amount of filial generation seedling, under the technology maturation condition, with 80m
2To cooperate necessary culture facility be example in the small test chamber, can produce per year more than 200,000 strains, and reproductive process can not be subjected to natural season to influence (needing artificial facility condition guarantee), can satisfy the quantity demand of commodity production of forestry to good nursery stock.
3. the perfect technology path of larch-tree genetic improvement.Through practice for many years, larch-tree has been established the genetic improvement route of " sexual creation, asexual utilization ", but select breeding, the only obtainable a small amount of good seed of crossbreeding, adopt the present invention can make full use of a spot of good seed, by the fast breeding incubation, form large batch of asexual propagation material, thereby a perfect major technique link in the larch-tree improved variety process has satisfied in the commodity production of forestry number needs to good nursery stock and has wanted.
Description of drawings
Figure 1A is the stem inducing culture in the embodiment of the invention 1, shows to rigidly connect the axillalry bud of planting.
Figure 1B is for inducing the indefinite stem of generation among the embodiment of the invention l.
Fig. 1 C is an indefinite stem of finishing the stem neomorph in the embodiment of the invention 1, black has shown medium supplemented active carbon.
Fig. 2 A is the axillalry bud in the stem generative process in the embodiment of the invention 2.
Fig. 2 B is the indefinite stem in the elongation growth process in the embodiment of the invention 2.
Fig. 3 A finishes the sample that indefinite stem is induced generative process in the embodiment of the invention 3.
Fig. 3 B is a sample of finishing indefinite stem elongation growth process in the embodiment of the invention 3.
Embodiment
Below in conjunction with the drawings and specific embodiments in detail the present invention is described in detail.
Embodiment 1
Choose the clone plant of larch-tree, growing body outward with 1 year living branch before the early spring, the bud bud was sprouted is material, get its axillalry bud, the formation of indefinite stem, extend and induce, and successive transfer culture, realize the regeneration of stem organ; Concrete operations are as follows:
1. the collection of material and preliminary treatment
The larch-tree of choosing forest farm, the Five Dragons, Fushun County, Liaoning Province seed orchard is towards No. 85 clones, the age of tree 12 years, gather 1 year living tree crown middle part branch, acquisition time is before the early spring, the bud bud was sprouted, this moment, bud bud bract scale changed glossiness light brown or filbert into by lacklustre burgundy or crineous, preserved down, took out back normal temperature two weeks of water planting for 4 ℃, end when shinny to bud bud, standby.
2. preparation medium
1) the conventional WPM medium of preparation, the dosage of its constituent is: Ca (No
3)
24H
2O556mg/l, NH
4NO
3400mg/l, K
2SO
4990mg/l, KH
2PO
4170mg/l, CaCl
22H
2O96mg/l, MgSO
47H
2O 370mg/l, FeSO
47H
2O 27.8mg/l, Na
2EDTA2H
2O37.3mg/l, H
3BO
36.2mg/l, ZnSO
47H
2O 8.6mg/l, MnSO
44H
2O 22.5mg/l, Na
2MoO
42H
2O 0.25mg/l, CuSO
45H
2O 0.25mg/l, inositol 100mg/l, glycine 2.0mg/l, thiamine hydrochloride 1.0mg/l, nicotinic acid 0.5mg/l, puridoxine hydrochloride 0.5mg/l, agar 6000mg/l;
Prepare modified MS medium again, the dosage of its constituent is: KNO
3950mg/l, NH
4NO
3825mg/l, KH
2PO
4170mg/l, CaCl
22H
2O 220mg/l, MgSO
47H
2O 370mg/l, FeSO
47H
2O 27.8mg/l, Na
2EDTA2H
2O 37.3mg/l, H
3BO
36.2mg/l, ZnSO
4.7H
2O8.6mg/l, MnSO
4.4H
2O 22.3mg/l, Na
2MoO
42H
2O 0.25mg/l, KI 0.83mg/l, CuSO
45H
2O 0.025mg/l, CoCl
26H
2O 0.025mg/l, inositol 100mg/l, glycine 2.0mg/l, thiamine hydrochloride 0.1mg/l, nicotinic acid 0.5mg/l, puridoxine hydrochloride 0.5mg/l, agar 6000mg/l.
2) respectively with two kinds of medium packing, high pressure steam sterilization, the cooling back is standby.
3. inoculation axillalry bud
The shinny branch that grows axillalry bud of bud bud after the water planting is divided into the segment of 1～2cm, after washing in the flowing water, immerse in 75% ethanolic solution and sterilized 1 minute, take out the axillalry bud that is of a size of 0.5～1cm on the branch segment, peel off the scale that is coated on the axillalry bud, axillalry bud drops into 0.1% mercuric chloride or 3% hydrogenperoxide steam generator was sterilized 10～30 minutes, and aseptic water washing for several times then; Described WPM medium supplemented plant growth regulating substance: Zea 0.5mg/l, IAA 1.0mg/l, the axillalry bud of the bacterium of will having gone out transfer in the triangular flask that described WPM medium is housed for preparing, and connect 3～6 axillalry buds in every bottle, induce the generation of indefinite stem.
Figure 1A is the stem inducing culture, and expression rigidly connects the axillalry bud of planting; Figure 1B shows the generation of finishing stem.
4. the elongation growth of indefinite stem
In described modified MS medium, add NAA, IBA and AC, the dosage that adds is: NAA0.1mg/l, IBA 0.3mg/l, AC 1.0g/l, again with induce in the step 3 generation＞the indefinite stem of 2cm takes out from the WPM medium, be placed in the sterile water, cut aging bottom or the callus of examination material, be inoculated in the triangular flask that is loaded with modified MS medium, 3～5 indefinite stems of every bottle graft enter the indefinite stem elongation growth stage.
The condition of culture of inducing indefinite stem to generate and extending is: 23 ℃ of culturing room's temperature, and light intensity 2,000lux, light dark period 16h/8h respectively through 30 days, finishes the regeneration of stem organ.
Fig. 1 C is an indefinite stem of finishing the stem neomorph, and black is represented the active carbon that adds.
5. for root induction or preservation material, can carry out successive transfer culture, promote the childrenization of indefinite stem material.Before the subinoculation, excise the brownization part of indefinite basal part of stem, 1～2 month blanking time of subculture, repeating step 4.Change over to when taking root, stop the successive transfer culture of indefinite stem.
Principle of the present invention is:
The most basic principle of Plant Tissue Breeding is plant cell tool " totipotency ", and promptly a cell contains a whole set of gene that bion grows.The key that the larch-tree stem is induced is growth regulatory substance kind and concentration, minimal medium and the selection of additional promotion thing and the reasonable control of environmental condition.Wherein, the basic element of cell division and growth hormone inducing and growing and all play an important role to bud.It is generally acknowledged that the tendency of organ differentiation depends on the balance of endogenous cell mitogen and growth hormone, for reaching this balance, according to original Endogenous Hormones in the different plant explants, need add a certain amount of growth regulatory substance.Inducing and growing of stem is exactly in fact the division and the elongation of cell, aspect the promotion cell division, the basic element of cell division is the triggering thing of division of cytoplasm, and it is synthetic for mitosis, nuclear division and RNA to be essential, and growth hormone then is to influence doubling and mitosis of DNA; Promoting to have experiment to show aspect the cell elongation, six benzyl purines have the metabolism of the intracellular energy of enhancing, quicken the synthetic and transportation of intracellular organic matter, improve the permeability of cell wall, maintain the effect that cell enlarges the required turgescence of growth.Mostly active carbon is that in medium non-selectivity absorbs, and it can absorb the harmful substance that produces through autoclaving, also can absorb the metabolite of cell in plant growth regulating class material and the group training process, thereby the selection optimum content also is the key that stem is induced.In a word, the process of tissue culture is exactly the expression that promotes specific gene in the specific period after all, regulates the synthetic of specified protein, thereby influences division, the atomization of whole cell.
According to its physiological action, the present invention induces with indefinite stem growth the indefinite stem of larch-tree and has carried out medium, growth regulatory substance, the additional selection test that promotes thing, on the WPM that selects, SH, three kinds of minimal mediums of improvement MS, the basic element of cell division, growth hormone and the active carbon of variety classes, variable concentrations have been added respectively, under the suitable culture condition, finish the regeneration induction of larch-tree stem.
Result: in 270 axillalry buds, have 48.15% axillalry bud to finish the regeneration of stem organ for examination.
Embodiment 2
Difference from Example 1 is:
1) choose No. 38 plant of larch-tree clone of 28 years age of trees, gathering full 1 year living branch of hibernaculum March is material;
2) preparation medium SH, medium component is: KNO
32500mg/l, NH
4H
2PO
3300mg/l, CaCl
22H
2O 200mg/l, MgSO
47H
2O 400mg/l, FeSO
47H
2O 15.0mg/l, Na
2EDTA2H
2O 20.0mg/l, H
3BO
35.0mg/l, ZnSO
47H
2O 6.0mg/l, MnSO
44H
2O10.0mg/l, Na
2MoO
42H
2O 0.1mg/l, KI 1.0mg/l, CoCl
26H
2O 0.1mg/l, inositol 1000mg/l,, thiamine hydrochloride 5.0mg/l, nicotinic acid 5.0mg/l, puridoxine hydrochloride 0.5mg/l, agar 6000mg/l.
3) inoculation axillalry bud
At described SH medium supplemented plant growth regulating substance: Zea 0.2mg/l, IAA 0.5mg/l, the axillalry bud of the bacterium of will having gone out transfer in the triangular flask that described SH medium is housed for preparing, and connect 3～6 axillalry buds in every bottle, induce the generation of indefinite stem.
Fig. 2 A is the axillalry bud that is in the stem generative process.
4) induce the elongation of indefinite stem
Add NAA 0.5mg/l, IBA 0.1mg/l and AC 10.0g/l in the described modified MS medium;
Induce the generation condition of culture of indefinite stem to be: 28 ℃ of room temperatures, light intensity 3000lux, light dark period 18h/6h; Induce the elongation condition of culture of indefinite stem to be: 26 ℃ of culturing room's temperature, light intensity 2500lux, light dark period 20h/4h respectively through 25～30 days, finishes the regeneration of stem organ.
Present embodiment does not have the successive transfer culture step.
Result: in 128 axillalry buds, have 47.06% axillalry bud to finish the regeneration of stem organ for examination.
Fig. 2 B is the indefinite stem that is in the stem elongation growth process.
Embodiment 3
Repeat the test of embodiment 1 and embodiment 2, medium and condition of culture are respectively with above-mentioned, additional growth regulatory substance is BA 0.1mg/l or Zea 0.01mg/l or NAA0.01mg/l at the stem induction period, in the stem elongation stage is IBA 0.3mg/l, NAA 0.1mg/l and AC 10g/l, sampling at the beginning of 4 months.In 181 axillalry buds, there is 80.06% axillalry bud to finish the regeneration of stem organ for examination.Fig. 3 A, 3B, 3C, 3D show the result of above-mentioned two embodiment repeated tests, and wherein Fig. 3 A is the root media of present embodiment 1, and Fig. 3 B and 3C are the checking result of embodiment 1, and Fig. 3 D is the checking result of present embodiment 2.
Fig. 3 is the indefinite stem in the present embodiment, shows that the BA of optimal dose among independent use the present invention or Zea or NAA also can finish induce (Fig. 3 A) of stem, finish the regeneration (Fig. 3 B) of stem organ.
Present embodiment does not have the successive transfer culture step.
Claims (8)
1. the stem abductive approach of a larch-tree micropropagation, it is characterized in that: choose the larch-tree clone, 1 year living branch of all ages and classes plant is a material, get its axillalry bud,, realize the regeneration of stem organ through indefinite stem formation, elongation growth, successive transfer culture etc.; Specifically can operate as follows:
1) collection of material and preliminary treatment:
Acquisition time is sprouted preceding or when sprouting, is put bough water planting to bud bud bright before using for going into the early spring in Winter Solstice bud bud;
2) preparation of medium:
(1) conventional method preparation improvement MS, WPM or SH;
(2) the packing medium horizontal high voltage steam sterilizing of going forward side by side, the cooling back is standby;
3) inoculation axillalry bud:
Axillalry bud is cut in the branch flowing water washing that bud bud after the water planting is shinny, sterilizes under aseptic condition, and additional plant growth regulating substance is inoculated axillalry bud in the stem inducing culture on WPM or SH medium, carries out the generation of indefinite stem;
4) elongation growth of indefinite stem:
The indefinite stem that generates is transferred in having added plant growth regulating substance and having promoted in the modified MS medium of thing, and control culturing room temperature is 22～28 ℃, and light intensity is 2000～3000lux, and light/dark cycle is 14～20h/4～10h, finishes the regeneration of stem organ.
2. according to the stem abductive approach of the described larch-tree micropropagation of claim 1, it is characterized in that: for root induction or preservation material, after step 4), can carry out successive transfer culture, promote the childrenization of indefinite stem material.
3. according to the stem abductive approach of the described larch-tree micropropagation of claim 1, it is characterized in that described growth regulatory substance is: the Zea in the basic element of cell division, BA, the NAA in the growth hormone, IAA and/or IBA; Described promotion thing is AC.
4. according to the stem abductive approach of the described larch-tree micropropagation of claim 3, it is characterized in that: described additional plant growth regulating substance consumption is: BA 0.1～0.5mg/l, Zea0.01～0.5mg/l, NAA 0.01～0.1mg/l, IBA 0.1～0.5mg/l and/or IAA 0.5～1.0mg/l; Promote the thing consumption to be: AC 0.5～10.0g/l.
5. according to the stem abductive approach of the described larch-tree micropropagation of claim 1, it is characterized in that: the axillalry bud of being cut in the described step 3) is of a size of 0.5～1cm.
6. according to the stem abductive approach of claim 2 or 3 described larch-tree micropropagations, it is characterized in that: at the generation phase of indefinite stem, the best of breed of medium supplemented plant growth regulating substance is: WPM+Zea+IAA or SH+Zea+IAA.
7. according to the stem abductive approach of the described larch-tree micropropagation of claim 1, it is characterized in that: in the elongation growth stage of indefinite stem, the best of breed of medium supplemented plant growth regulating substance and promotion thing is: MS+NAA+IBA+AC.
8. according to the stem abductive approach of the described larch-tree micropropagation of claim 1, it is characterized in that: light described in the described step 4)/secretly cultivate optimal period is 16h/8h.
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