CN102860260A

CN102860260A - Cryptomeria fortunei tissue culture rapid-propagation method

Info

Publication number: CN102860260A
Application number: CN2012103833836A
Authority: CN
Inventors: 诸葛强; 沙玉清; 徐立安; 王立科; 王章荣; 王福生
Original assignee: Nanjing Forestry University
Current assignee: Nanjing Forestry University
Priority date: 2012-10-11
Filing date: 2012-10-11
Publication date: 2013-01-09
Anticipated expiration: 2032-10-11
Also published as: CN102860260B

Abstract

The invention discloses a cryptomeria fortunei tissue culture rapid-propagation method. The method comprises taking a cryptomeria fortunei explant stem segment, inoculating the cryptomeria fortunei explant stem segment on an inducing adventitious bud culture medium which comprises DCR+ 0.1-2.0mg/L of 6-BA+ 0.1-2.0mg/L of IBA+ 0.5-5.0g/L of AC+ 0.001-0.01mg/L of TDZ+ 30g/L of cane sugar, and performing inducing culture to generate adventitious buds; adopting two stages to take root for rooting culture, namely first placing a rooting culture medium 1 which comprises 1/2 of DCR+ 0.1-2.0mg/L of NAA+ 0.1-2.0mg/L of IBA+0.1-1.0mg/L of 6-BA+ 1.0-10.0g/L of AC+ 10.0-30.0g/L of cane sugar, first performing dark culture for 2-15d, performing culture for 5-30d under illumination, and transferring to a rooting culture medium 2 which comprises DCR+ 0.1-1.0mg/L of NAA+ 0.1-1.0mg/L of 6-BA+ 0.5-5.0g/L of AC+ 30.0g/L of cane sugar. By the method, the tender bud propagation coefficient is over 5.7, and the rooting rate is over 80%. The culture cycle is short, the propagation quantity is large, and the cryptomeria fortunei tissue culture rapid-propagation method is favorable for large-scale production.

Description

A kind of cryptomeria quick breeding method for tissue culture

Technical field

The present invention relates to Plant Tissue Breeding propagation technique field, be specifically related to a kind of cryptomeria quick breeding method for tissue culture, particularly a kind of take cryptomeria stem section as explant material, induce first cryptomeria to produce indefinite bud with the self-control medium, and then root induction obtain regeneration plant.

Background technology

Cryptomeria ( Cryptomeria fortunei) be the Taxodiaceae Cryptomeria ( Cryptomeria D. Don), another name peacock China fir, arbor, up to 4m, it is many that the diameter of a cross-section of a tree trunk 1.3 meters above the ground can reach 2m; The bark rufous, fibrous, be cleaved into strip sheet and come off; Bough is closely verticillate, open and flat or oblique exhibition; Sprig is elongated, and is often sagging, green, and the leaf at branch middle part is longer, often shortens gradually to two ends.Leaf bores shape and slightly curves inwardly, the tip introversion, and there is spiracle line on four limits, long 1 ~ 1.5cm, the leaf of fruit branch is usually shorter, and the leaf of treelet and rudiment branch reaches 2.4cm.

Cryptomeria is one of important commerical tree species of China.Mainly be distributed in the provinces such as Fujian, Zhejiang, Jiangxi in China cryptomeria.Since be subjected to ordering about of economic interests, in recent years cryptomeria resource subject to severe risks of damage, harm and the sick worm of adding acid rain, atmospheric pollution encroach on, and it is large at present that directly cryptomeria is very few.At present cryptomeria is take seed growing as main, and cuttage also can be adopted, but germination rate, qualified seedling rate are low usually.Also main research to the seedling raising technology of research to its breeding.Therefore, utilize biological technique method to carry out artificial Fast-propagation, significant to the protection of resources of cryptomeria.

Zhang Cuiping (2008) Primary Study its tissue culture rapid propagation technique, but this research is take the cryptomeria embryo as explant, the breeding that is not suitable for tool excellent genes type cryptomeria individuality is cultivated, and only having adopted MS in experiment is minimal medium, single factor design has only been adopted in interpolation to exogenous hormone, other minimal mediums and combination hormone is not selected culture experiment.The applicant chooses excellent genes type cryptomeria stem section as explant material, select by experiment DCR to be the suitableeest minimal medium, multifactor design has been carried out in interpolation to exogenous hormone, and adds active carbon etc., for the tissue-culturing quick-propagation of cryptomeria provides effective way.

Summary of the invention

Goal of the invention: for the deficiencies in the prior art, the purpose of this invention is to provide the method for a kind of tissue-culturing quick-propagation cryptomeria.

Technical scheme: for achieving the above object, the technical solution used in the present invention is:

A kind of cryptomeria quick breeding method for tissue culture may further comprise the steps:

(1) gets cryptomeria explant stem section, be inoculated in the evoking adventive bud medium, induce to cultivate to produce indefinite bud; Wherein, the evoking adventive bud medium is: DCR+0.1 ~ 2.0mg/L 6-BA(6-benzyl purine)+and 0.1 ~ 2.0mg/L IBA(indolebutyric acid)+0.5 ~ 5.0g/L AC(active carbon)+0.001 ~ 0.01mg/L TDZ(Thidiazuron)+30.0g/L sucrose;

(2) culture of rootage adopts two stage root of hairs: the healthy and strong tender shoots that will extend and grow is first put into root media 1, after carrying out that 2 ~ 15d is dark and cultivating, puts under the illumination and cultivates 5 ~ 30d; Then transfer in root media 2, continue to be cultured to and take root; Wherein, root media 1 is: 1/2DCR+0.1 ~ 2.0mg/L NAA+0.1 ~ 2.0mg/L IBA+0.1 ~ 1.0mg/L 6-BA+1.0 ~ 10.0g/L AC+10.0 ~ 30.0g/L sucrose, root media 2 is: DCR+0.1 ~ 1.0mg/L NAA+0.1 ~ 1.0mg/L 6-BA+0.5 ~ 5.0g/L AC+30.0g/L sucrose.

In the step (1), described evoking adventive bud medium is: DCR+1.0mg/L 6-BA+0.5mg/L IBA+0.01mg/L TDZ+3.0g/L AC+30g/L sucrose.

In the step (2), described root media 1 is: 1/2DCR+2.0mg/L NAA+1.0mg/L IBA+0.5mg/L 6-BA+0.5mg/L IBA+5.0g/L AC+25g/L sucrose.

In the step (2), described root media 2 is: DCR+0.5mg/L NAA+0.5mg/L 6-BA+3.0g/L AC+30.0g/L sucrose.

In the step (2), in described 1/2DCR culture medium prescription, macroelement reduces by 1/2 amount than conventional DCR.

In the step (2), the light application time under the described illumination is 14 ~ 16h, and intensity of illumination is 1200 ~ 1300Lux, and temperature is 25 ~ 28 ℃.

The method is chosen excellent genes type cryptomeria stem section as explant material, by with DCR for the suitableeest minimal medium, multifactor design has been carried out in the interpolation of exogenous hormone, and has been added active carbon etc., for the tissue-culturing quick-propagation of cryptomeria provides effective way.

Beneficial effect: compared with prior art, remarkable advantage of the present invention is: take the individual stem section of tool excellent genes type cryptomeria as explant, take DCR as minimal medium, the plant growth regulator of additional variety classes, variable concentrations and active carbon induced bud propagation and root induction, pass through this method, the tender shoots growth coefficient reaches more than 5.7, and rooting rate is more than 80%.Cultivation cycle is short, and breeding amount is large, is conducive to large-scale production.

Description of drawings

Fig. 1 induces the cryptomeria indefinite bud of acquisition;

Form the cryptomeria whole plant after Fig. 2 root induction.

Embodiment

The present invention is described further below in conjunction with specific embodiment.

Following examples can make those skilled in the art more fully understand the present invention, but do not limit the present invention in any way.

Embodiment 1

Choose the cryptomeria stem section of robust growth, cut off the needle on the stem section, the stem section is cut into about 4cm, and flowing water flushing 1 ~ 2h places the 75% ethanol 30s that sterilizes, use again 50% 84 thimerosals (to include a small amount of Tween-20, every 50mL adds one) sterilization 10min, aseptic water washing 8 times is cut into the stem section about 2cm again, be inoculated in the evoking adventive bud medium, light induction begins to produce indefinite bud after cultivating 8d; After indefinite bud produces, choosing the healthy and strong tender shoots of growth religion and transferring in root media 1, first dark cultivate 2 ~ 15d after, put into cultivate 2 ~ 30d under the illumination after, transfer again in root media 2, about 10d begins to take root.Wherein, the evoking adventive bud medium is: DCR+0.1 ~ 2.0mg/L 6-BA+0.1 ~ 2.0mg/L IBA+0.001 ~ 0.01mg/L TDZ+0.5 ~ 5.0g/L AC; Root media 1 is: 1/2DCR+0.1 ~ 2.0mg/L NAA+0.1 ~ 2.0mg/L IBA+0.1 ~ 0.5mg/L 6-BA+1.0 ~ 10.0g/L AC+10.0 ~ 30.0g/L sucrose, root media 2 is: DCR+0.1 ~ 1.0mg/L NAA+0.1 ~ 1.0mg/L 6-BA+0.5 ~ 5.0g/LAC+30.0g/L sucrose.

Embodiment 2

Repeat experiment by embodiment 1 described same steps as, induce cultivation stage at each, add respectively various dose active carbon (0.5 ~ 10.0g/L), wherein, add active carbon 0.5 ~ 5.0g/L in the evoking adventive bud medium, add active carbon 1.0 ~ 10.0g/L in the root media 1, add 0.5 ~ 5.0g/L in the root media 2, can obviously promote the cryptomeria robust growth, leaf is green, effectively prevent the generation of brownization of seedling yellow, and in root induction stage hestening rooting.

Embodiment 3

Repeat experiment by embodiment 1 described same steps as, in the root induction stage, the macroelement in the 1/2DCR medium that adopts reduces by 1/2 amount than conventional DCR.The results are shown in Table 1, can obviously promote root induction, make rooting rate bring up to 85%.

The impact that macroelement is taken root on cryptomeria in table 1 medium

Figure 2012103833836100002DEST_PATH_IMAGE001

Embodiment 4

Repeat experiment by embodiment 1 described same steps as, in the root induction stage, sucrose concentration in the medium is reduced to 2% from 3%, to reduce the osmotic pressure of medium, the results are shown in Table 2, effectively promote plant to take root.

The impact that sucrose concentration is taken root on cryptomeria in table 2 medium

Claims

1. a cryptomeria quick breeding method for tissue culture is characterized in that, may further comprise the steps:

(1) gets cryptomeria explant stem section, be inoculated in the evoking adventive bud medium, induce to cultivate to produce indefinite bud; Wherein, the evoking adventive bud medium is: DCR+0.1 ~ 2.0mg/L 6-BA+0.1 ~ 2.0mg/L IBA+0.001 ~ 0.01mg/L TDZ+0.5 ~ 5.0g/L AC+30.0g/L sucrose;

2. the method for cryptomeria tissue-culturing quick-propagation according to claim 1, it is characterized in that: in the step (1), described evoking adventive bud medium is: DCR+1.0mg/L 6-BA+0.5mg/L IBA+0.01mg/L TDZ+3.0g/L AC+30.0g/L sucrose.

3. the method for cryptomeria tissue-culturing quick-propagation according to claim 1, it is characterized in that: in the step (2), described root media 1 is: 1/2DCR+1.0mg/L 6-BA+0.1 ~ 2.0mg/L IBA+2.0mg/L NAA+5.0g/L AC+10.0 ~ 30.0g/L sucrose.

4. the method for cryptomeria tissue-culturing quick-propagation according to claim 1, it is characterized in that: in the step (2), described root media 2 is: DCR+0.1 ~ 1.0mg/L NAA+0.1 ~ 1.0mg/L 6-BA+3.0g/L AC+30.0g/L sucrose.

5. according to claim 1 or the method for 4 described cryptomeria tissue-culturing quick-propagations, it is characterized in that: in the step (2), in described 1/2DCR culture medium prescription, macroelement reduces by 1/2 amount than conventional DCR.

6. the method for cryptomeria tissue-culturing quick-propagation according to claim 1, it is characterized in that: in the step (2), the light application time under the described illumination is 14 ~ 16h, and intensity of illumination is 1200 ~ 1300Lux, and temperature is 25 ~ 28 ℃.