CN101971775A

CN101971775A - Propagation method for directly inducing bulblet from lilium longiflorum aseptic seedling leaf

Info

Publication number: CN101971775A
Application number: CN 201010511389
Authority: CN
Inventors: 张启翔; 石晋芳; 孙明; 程堂仁; 杨炜茹; 孔滢
Original assignee: Beijing Forestry University
Current assignee: Beijing Forestry University
Priority date: 2010-10-18
Filing date: 2010-10-18
Publication date: 2011-02-16
Anticipated expiration: 2030-10-18
Also published as: CN101971775B

Abstract

The invention provides a rapid propagation method for directly inducing a bulblet from a lilium longiflorum tissue culture seedling leaf. The method comprises the following steps of: inoculating a leaf disc of a lilium longiflorum aseptic seedling onto a bulblet inducing culture medium; and inoculating the induced bulblet onto a bulblet swelling culture medium, wherein the bulblet inducing culture medium consists of MS, 2.0 mg/L of 6-butyl acrylate (BA), 0.1 to 0.5 mg/L of naphthyl acetic acid (NAA), 30 g/L of cane sugar and 6.25 g/L of agar; and the bulblet swelling culture medium consists of MS, 2.0 to 2.5 g/L of active carbon, 90 g/L of cane sugar and 6.25 g/L of agar. In propagation method for directly inducing the bulblet from the lilium longiflorum aseptic seedling leaf, one leaf disc can produce 1 to 3 bulblets. The inductivity of a root directly produced by the bulblet is up to 100 percent. Compared with a process in which a cluster bud is directly induced from a scale and then the bulblet is produced, the method shortens time, simplifies steps and is a newly found direct organ generation measure.

Description

The propagation method that lily aseptic seedling blade is directly induced clove

Technical field

The present invention relates to Plant Tissue Breeding, specifically, relate to tissue culture of lily (Liliumlongiflorum) and the method that aseptic seedling blade organ directly takes place.

Background technology

In recent years, lily is large-scale with its flower, rich color, fragrant odour and liked by the people of other countries, occupies cut-flower market, the world rapidly.Wherein, Asia lily hybrid system, oriental hybrid lily hybrid system, Lilium longiflorum hybrid are main cut-flower kind.Cut-flower is produced used kind of ball dependence on import, and this is a undisputable fact.And the import lily bulb tended in 1 year degenerate, so all need import every year.

Though the scale cuttage of lily and the research of tissue rapid propagation have a lot of reports, the scale cuttage causes the problem of kind of sexual involution easily, and that tissue rapid propagation is used in producing again is less, because easy mortality during test-tube seedling transplanting.Because these methods can't satisfy lily ball production demand, therefore, need to explore new lily bulb quick-breeding method, and form relevant kind ball production technology system.

Summary of the invention

The purpose of this invention is to provide propagation method and application thereof that a kind of lily aseptic seedling blade is directly induced clove.

The propagation method that lily aseptic seedling blade is directly induced clove, comprise that the leaf dish with aseptic seedling is seeded to the clove inducing culture, again the clove of inducing is seeded to clove and expands medium, wherein said clove inducing culture is: MS+6-BA2.0mg/L+NAA0.1～0.5mg/L+ sucrose 25～35g/L+ agar 6.25g/L, described clove is expanded medium and is: MS+ active carbon (AC) 2.0～2.5g/L+ sucrose 90g/L+ agar 6.25g/L.

Wherein, secretly cultivate after the described Bulbus Lilii leaf dish inoculation, described clove is seeded to clove and expands and carry out illumination cultivation behind the medium, and described illumination condition is that natural scattering light 2500～3000Lux adds artificial fill-in light 1500～2000Lux, and light application time is 12h/d.

The propagation method that lily aseptic seedling blade provided by the invention is directly induced clove, also comprise preparation lily aseptic seedling, be specially: will be seeded to the inducing clumping bud medium behind the lily bud scale surface sterilizing, induce and produce the bud of growing thickly, the simple bud of the bud of growing thickly of inducing is seeded to the adventitious buds proliferation medium, obtains a large amount of aseptic seedling.Wherein said inducing clumping bud medium is MS+6-BA2.0mg/L+NAA 0.1～0.5mg/L+ sucrose 25～35g/L+ agar 6.25g/L, or MS+6-BA2.0mg/L+2,4-D 0.1～0.5mg/L+ sucrose 25～35g/L+ agar 6.25g/L, or MS+6-BA2.0mg/L+IAA 0.1mg/L+ sucrose 25～35g/L+ agar 6.25g/L, be preferably MS+6-BA2.0mg/L+NAA 0.1mg/L+ sucrose 30g/L+ agar 6.25g/L, wherein said adventitious buds proliferation medium is: MS+6-BA1.0～2.5mg/L+NAA 0.1～0.5mg/L+ sucrose 25～35g/L+ agar 6.25g/L is preferably MS+6-BA2.0mg/L+NAA 0.5mg/L+ sucrose 30g/L+ agar 6.25g/L.

Lily aseptic seedling blade provided by the invention directly induces the propagation method of clove can use quick breeding with lily bulb.

Compare with traditional lily bulb generation approach, blade directly induces the method that produces clove that following advantage is arranged:

(1) on the basis of inducing clumping bud, enlarged inductivity once more, (simple bud can produce 3～8 blades, and each blade can produce 1～3 clove again).Therefore, 3～24 times have been improved than traditional inducing clumping bud clove inductivity.

(2) shortened the incubation time that bulb expands, traditional simple bud is cultivated into clove needs the long time, and the clove that blade directly produces, and size is even, neat and consistent, expand grew one month on the medium after, promptly produce scaly leaf.

(3) the direct clove that produces of blade has long root, does not need the special root induction stage.

Description of drawings

Fig. 1 is that scale is inoculated the situation of inducing after 45 days.

Fig. 2 is 45 days growing states afterwards of indefinite bud subculture that scale is induced.

Fig. 3 is that simple bud is induced 90 days growing states afterwards.

Fig. 4 is the induce situation of leaf dish at MS+6-BA2.0mg/L+NAA 0.5mg/L+ sucrose 30g/L+ agar 6.25g/L.

Fig. 5 is that the clove that induces inoculates one month growing state at MS+AC2.0g/L+ sucrose 90g/L+ agar 6.25g/L.

Embodiment

Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.

Embodiment 1

Lily bud scale is induced: the purchase of choosing no damage by disease and insect is from ' Siberia ' of Beijing Lv Er Co., Ltd lily bud scale, after the liquid detergent cleaning, under running water, wash 30min, with 75% alcohol-pickled 35s, twice of aseptic water washing, again with 0.1% mercuric chloride sterilization different time, be outer scale sterilization 15min, middle level scale sterilization 12min, internal layer scale sterilization 10min, aseptic water washing 4～5 times, stripping and slicing is standby in the culture dish of aseptic filter paper then.

The lily bud scale of above-mentioned surface sterilizing is seeded to MS+6-BA2.0mg/L+NAA0.5mg/L; MS+6-BA2.0mg/L+NAA 0.1mg/L; MS+6-BA2.0mg/L+2,4-D 0.5mg/L; MS+6-BA2.0mg/L+2,4-D 0.1mg/L; MS+6-BA2.0mg/L+IAA0.1mg/L.Wherein, in the above-mentioned medium, cane sugar content is 30g/L, and agar content is 6.25g/L.

The result shows, after ' Siberia ' outer scale adopted mercuric chloride sterilization 15min, adventitious bud induction frequency was up to 48.56%, is higher than 28.18% adventitious bud induction frequency of 39.26% and internal layer scale of middle level scale.

The comparison of the different number of plies coefficient of differentiations of scale: on the inducing culture of MS+6-BA2.0mg/L+NAA0.5mg/L+ sucrose 30g/L+ agar 6.25g/L, the average coefficient of differentiation of the middle level scale in ' Siberia ' is 5.2, outer scale is 3.0, and the internal layer scale is 2.73.Wherein, average coefficient of differentiation is meant the mean of the indefinite bud that scale broke up that each has broken up.

' Siberia ' scale is seeded in above-mentioned 5 kinds of inducing cultures, wherein the medium of MS+6-BA2.0mg/L+NAA 0.1mg/L+ sucrose 30g/L+ agar 6.25g/L induce differentiation rate the highest (Fig. 1), be 42.87%.The indefinite bud that induces is carried out subculture in above-mentioned medium, indefinite bud continues propagation (Fig. 2).

Embodiment 2

The bud of growing thickly that differentiates on the scale is cut into simple bud to be seeded in following eight kinds of medium:

A：MS+6-BA1.0mg/L+NAA0.1mg/L；

B：MS+6-BA1.0mg/L+NAA0.5mg/L；

C：MS+6-BA1.5mg/L+NAA0.1mg/L；

D：MS+6-BA1.5mg/L+NAA0.5mg/L；

E：MS+6-BA2.0mg/L+NAA0.1mg/L；

F：MS+6-BA2.0mg/L+NAA0.5mg/L；

G：MS+6-BA2.5mg/L+NAA0.1mg/L；

H：MS+6-BA2.5mg/L+NAA0.5mg/L。

Wherein, in the above-mentioned medium, cane sugar content is 30g/L, and agar content is 6.25g/L.

The bud growth factor of growing thickly on the A medium is 2.76, and seedling grows fine; The bud growth factor of growing thickly on the B medium is 3.17, and the seedling growing way is better; The bud growth factor of growing thickly on the C medium is 2.97, and the seedling growing way is poor; The bud growth factor of growing thickly on the D medium is 3.96, and seedling grows fine; The bud growth factor of growing thickly on the E medium is 3.20, and the seedling growing way is better; The bud growth factor of growing thickly on the F medium is 4.23, and seedling grows fine; The bud growth factor of growing thickly on the G medium is 4.13, the seedling vitrifying; The bud growth factor of growing thickly on the H medium is 3.12, the seedling vitrifying.

Therefore, from simple bud coefficient of differentiation and the quality of bud of growing thickly, ' Siberia ' coefficient of differentiation on medium MS+6-BA2.0mg/L+NAA 0.5mg/L+ sucrose 30g/L+ agar 6.25g/L reaches 4.23, seedling grow fine (Fig. 3).

Embodiment 3

The aseptic seedling blade is directly induced the screening of clove medium: choose ' Siberia ' aseptic seedling of robust growth, blade is cut into leaf dish about 3cm, be seeded in following 4 kinds of medium, wherein culture vessel is a culture dish:

MS+Pic (the poison rust is fixed) 2.0mg/L+ sucrose 30g/L+ agar 6.25g/L;

MS+Pic (the poison rust is fixed) 2.0mg/L+KT0.5mg/L+ sucrose 30g/L+ agar 6.25g/L;

MS+6-BA2.0mg/L+NAA 0.1mg/L+ sucrose 30g/L+ agar 6.25g/L;

MS+6-BA2.0mg/L+NAA 0.5mg/L+ sucrose 30g/L+ agar 6.25g/L.

The culture dish of inoculating the leaf dish is placed in the paper box, paper box is placed in the climatic cabinate of complete dark again.The cultivation temperature of tissue cultivating seedling is 20～23 ℃ in the whole process.

Wherein, on medium MS+Pic (the poison rust is fixed) 2.0mg/L+ sucrose 30g/L+ agar 6.25g/L, the inductivity of simple bud is 2.77%, and on medium MS+Pic (the poison rust is fixed) the 2.0mg/L+KT0.5mg/L+ sucrose 30g/L+ agar 6.25g/L, the simple bud inductivity is 16.16%.On medium MS+6-BA2.0mg/L+NAA 0.1mg/L+ sucrose 30g/L+ agar 6.25g/L, can directly induce clove, but inductivity is 14.32%, the clove inductivity reaches 29.58% on medium MS+6-BA2.0mg/L+NAA 0.5mg/L+ sucrose 30g/L+ agar 6.25g/L, the derivative coefficient of clove is 1～3, and direct growth goes out thin and delicate and long root (Fig. 4) on clove.

Embodiment 5

Induce the clove of acquisition to cut down embodiment 4, be seeded to and expand among medium MS+AC (active carbon) the 2.0g/L+ sucrose 90g/L+ agar 6.25g/L, after one month, it is very fast that bulb expands speed, and differentiate scaly leaf (Fig. 5) rapidly.

If no specified otherwise, illumination condition is that natural scattering light 2500～3000Lux adds artificial fill-in light 1500～2000Lux in the above-mentioned incubation, and light application time is 12h/d, and cultivation temperature is 20～23 ℃.

The above only is a preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the technology of the present invention principle; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.

Claims

1. lily aseptic seedling blade is directly induced the propagation method of clove, comprise that the leaf dish with the lily aseptic seedling is seeded to the clove inducing culture, again the clove of inducing is seeded to clove and expands medium, wherein said clove inducing culture is: MS+6-BA2.0mg/L+NAA 0.1～0.5mg/L+ sucrose 25～35g/L+ agar 6.25g/L, described clove is expanded medium and is: MS+ active carbon 2.0～2.5g/L+ sucrose 85～95g/L+ agar 6.25g/L.

2. method according to claim 1 is characterized in that, described clove inducing culture is: MS+6-BA2.0mg/L+NAA 0.5mg/L+ sucrose 30g/L+ agar 6.25g/L.

3. method according to claim 1 is characterized in that, it is MS+ active carbon 2.0g/L+ sucrose 90g/L+ agar 6.25g/L that described clove is expanded medium.

4. method according to claim 1 is characterized in that, secretly cultivates after described leaf dish is seeded to the clove inducing culture.

5. method according to claim 1 is characterized in that, also comprises the described lily aseptic seedling of preparation, and its step comprises:

The inducing clumping bud medium will be seeded to behind the lily bud scale surface sterilizing, induce and produce the bud of growing thickly, the simple bud of the bud of growing thickly of inducing is seeded to the adventitious buds proliferation medium, obtain aseptic seedling, wherein said inducing clumping bud medium is MS+6-BA2.0mg/L+NAA0.1～0.5mg/L+ sucrose 25～35g/L+ agar 6.25g/L, or MS+6-BA2.0mg/L+2,4-D 0.1～0.5mg/L+ sucrose 25～35g/L+ agar 6.25g/L, or MS+6-BA2.0mg/L+IAA 0.1mg/L+ sucrose 25～35g/L+ agar 6.25g/L, wherein said adventitious buds proliferation medium is: MS+6-BA1.0～2.5mg/L+NAA 0.1～0.5mg/L+ sucrose 25～35g/L+ agar 6.25g/L.

6. method according to claim 5 is characterized in that, described inducing clumping bud medium is MS+6-BA2.0mg/L+NAA 0.1mg/L+ sucrose 30g/L+ agar 6.25g/L.

7. method according to claim 5 is characterized in that, described adventitious buds proliferation medium is MS+6-BA2.0mg/L+NAA 0.5mg/L+ sucrose 30g/L+ agar 6.25g/L.

8. method according to claim 5 is characterized in that, described scale is an outer scale.

9. the application of each described method of claim 1～8 in lily bulb is bred fast.