CN102792890B

CN102792890B - Rapid propagation method of tissue culture for zelkova schneideriana hand.-Mazz

Info

Publication number: CN102792890B
Application number: CN201210273106.XA
Authority: CN
Inventors: 金晓玲; 何平; 胡希军; 张日清; 廖飞勇; 汪灵丹
Original assignee: Central South University of Forestry and Technology
Current assignee: Central South University of Forestry and Technology
Priority date: 2012-08-03
Filing date: 2012-08-03
Publication date: 2014-06-25
Anticipated expiration: 2032-08-03
Also published as: CN102792890A

Abstract

The invention discloses a rapid propagation method of tissue culture for zelkova schneideriana hand.-Mazz. According to the method, selecting stem segments with buds as explants, and appropriate culture mediums for each stage for the tissue culture, a large number of nursery stocks, which maintain the qualified characteristics of a mother stock, can be obtained; and the genotype and all the qualified characteristics of an original plant can be 100% maintained, thereby greatly improving the propagation coefficient.

Description

Beech tree quick breeding method for tissue culture

Technical field

The invention belongs to plant propagation technical field, be specifically related to the tissue culture and rapid propagation method of a kind of beech tree.

Background technology

Beech tree zelkova schneiderianahand.-Mazz. be Ulmaceae Zeldova.Be precious hard broad-leaved commerical tree species, the wood quality of beech tree is hard flexible, and beautiful texture is glossy, and without special odor, has become the preferred material that many families carry out interior decoration and manufacture top-grade furniture, liked by consumers in general.Meanwhile, it is tree-like attractive in appearance that beech are set, and leaf can, with presenting different colors season, have very high landscape architecture and ornamental value.And beech tree damage by disease and insect is few, has very strong resistance against diseases; Tree crown and root system are all very huge, and fallen leaves amount is many, have good water and soil conservation and Effect of improving soil, play a very important role in protection with in improving the ecological environment.But due to too much felling and shortage protection, the wild resource of beech tree is quite deficient, beech tree is listed in Chinese Second Class Key Protected Plant.

The new pursuit to living standard along with expanding economy and people, current quarter in all parts of the country, park, roadside greening are in the ascendant, and China market is to greening seedling, and particularly the demand of rare tree species is increasing, and imbalance between supply and demand is outstanding.Add that China's forest plantation seeds unification and " needle " phenomenon are very serious, caused the series of problems such as the ecosystem is unbalance and low in economic efficiency, develop good hard broad-leaved forest plantation and caused the great attention of forest department of China.In forest plantation Tree Structure of new generation is adjusted, select material good, fast-growing performance and the stronger rare tree species of insect resistance capacity be as beech tree afforestation has become the common recognition of brainstrust, the demand of high-quality fast-growing beech sapling wood is constantly increased.But to the research of beech tree, as the research of the aspects such as introduction and acclimatization, breeding of new variety, breeding and popularization relatively lags behind, the very different phenomenon of seed seedling-raising is very serious, and the science and technology support of production application and market supply can not be provided.Therefore, the research of the quick propagating technology to beech tree is particularly important.

The research report of cultivating about beech tree tissue at present mainly contains the tissue of terminal bud and cultivates and cultivate by the tissue of blade evoked callus.It is poor that the tissue of terminal bud is cultivated the Multiple Buds nursery stock comparison of coherence obtaining, reproduction coefficient low (27 ~ 125); The tissue of blade evoked callus cultivates the regeneration plant of generation and the uniformity of maternal plant is poor.

Summary of the invention

The present invention is intended to overcome the deficiencies in the prior art, and a kind of method of beech tree tissue-culturing quick-propagation is provided.

In order to achieve the above object, technical scheme provided by the invention is:

Beech tree quick breeding method for tissue culture, comprises the steps:

(1) beech are set to young shoot alcohol disinfecting 25 ~ 35s of 75%, with aseptic water washing, with 0.1% mercuric chloride, the 8 ~ 12min that sterilizes, then used aseptic water washing;

(2) will be cut into the stem section that long 0.8 ~ 1.2cm at least contains an axillalry bud through step (1) branch after treatment, and implant in inducing clumping bud medium and cultivate 28 ~ 32 days, obtain Multiple Buds; Wherein, described inducing clumping bud medium is: WPM+1.0 ~ 3.0 mg.L ^-1bA+0 ~ 1.0 mg.L ^-1nAA, agar 6.5 g.L ^-1, sucrose 20 g.L ^-1, pH value is 5.7;

(3) Multiple Buds growing after step (2) induction is cultivated is proceeded in subculture medium and cultivated 60 ~ 65 days, carry out the propagation of bud; Wherein, described subculture medium is: WPM+2.0 ~ 3.0 mg.L ^-1bA+1.0 ~ 2.0 mg.L ^-1nAA, agar 6.5 g.L ^-1, sucrose 20 g.L ^-1, pH value is 5.7;

(4) cutting the height growing after step (3) subculture is cultivated is beech tree healthy seedlings more than 2.0 cm, implants in root media and cultivates 45 ~ 50 days, obtains regeneration plant; Wherein, described root media is: root media is: WPM+0.5 ~ 1.0 mg.L ^-1iBA+2.0 g.L ^-1aC, agar 7.0 mg.L ^-1, sucrose 20 g.L ^-1, pH value is 5.7;

(5) regeneration plant that will obtain through step (4) is cleaned, and is transplanted in mixed-matrix be made up of peat soil and perlite hardening cultivation 7 ~ 10 days, more outdoor cultivation 90 ~ 100 days, can be moved to field production, shades one week when field production.

Preferably, to set young shoot be to give birth to be then with bud children shoot spring to the described beech of step (1).

Preferably, it is that the healthy and strong branch of the beech trees fallen leaves in winter containing axillalry bud is placed in to the beaker that distilled water is housed that the described beech of step (1) are set young shoot, carries out water planting after 10 ~ 15 days, the young shoot being formed by axillary bud sprouting at 25 ℃.

Preferably, in the mixed-matrix described in step (5), peat soil and perlitic volume ratio are 1:1.

Preferably, the condition of culture in step (2) to (4) is: 24 ± 2 ℃ of room temperatures, intensity of illumination PPFD=65.5 μ .moL.m ^-2.s ^-1, 16 hours every days of light application time.

Wherein, in step of the present invention (2), the inductivity of Multiple Buds is 80 ~ 90%; The bud growth coefficient that in step (3), bud propagation is cultivated is 7 ~ 10; In step (4), the rooting rate of culture of rootage is 86 ~ 95%; In step (5), transplant to the survival rate of field production be 95 ~ 100%.

Compared with prior art, beneficial effect of the present invention is:

1, the inventive method select tape leaf stem section is explant, and selects suitable each stage medium, organizes cultivation, and a large amount of nursery stocks of the maternal plant merit that can be maintained can the 100% former plant genotype of maintenance and all good characteristics thereof.

2, the present invention can realize factorial seedling growth, and produces the beech sapling wood of neat and consistent in enormous quantities, increases substantially reproduction coefficient (343 ~ 1000).The calculating of reproduction coefficient is: breeding a time is 4 months, within 1 year 12 months, can breed 3 times, and growth coefficient is once 7 ~ 10, and the reproduction coefficient of a year is 7 × 7 × 7 ~ 10 × 10 × 10(343 ~ 1000 so).

Embodiment

Below in conjunction with embodiment, the present invention is described in further detail.

embodiment 1

(1) the beech tree of choosing with alcohol disinfecting 30 ~ 35s of 75% for bud children shoot, with aseptic water washing 3 times, with the 0.1% mercuric chloride 8min that sterilizes, then is used to aseptic water washing 3 times then raw spring;

(2) will be cut into the stem section that long 1cm at least contains an axillalry bud through step (1) branch after treatment, and implant in bud inducing culture and cultivate 28 ~ 30 days, obtain Multiple Buds; Wherein, described bud inducing culture is: WPM+3.0 mg.L ^-1bA, agar 6.5 g.L ^-1, sucrose 20 g.L ^-1, pH value is 5.7, medium autoclaving 20min; The inductivity of Multiple Buds is 84 ~ 90%;

(3) Multiple Buds growing after step (2) induction is cultivated is proceeded in subculture medium and cultivated 60 days, carry out the propagation of bud, bud growth coefficient is 8 ~ 10; Wherein, described subculture medium is: WPM+3.0 mg.L ^-1bA+2.0 mg.L ^-1nAA, agar 6.5 g.L ^-1, sucrose 20 g.L ^-1, pH value is 5.7, medium autoclaving 20min;

(4) cutting the height growing after step (3) subculture is cultivated is beech tree healthy seedling more than 2.0cm, implants in root media and cultivates 45 days, obtains regeneration plant, and rooting rate is 89 ~ 95%; Wherein, described root media is: root media is: WPM+0.5 mg.L ^-1iBA+2.0 g.L ^-1aC, agar 7.0 mg L ^-1, sucrose 20 g.L ^-1, pH value is 5.7, medium autoclaving 20min;

(5) regeneration plant that will obtain through step (4) is cleaned, be transplanted to by hardening in peat soil and the perlite mixed-matrix that 1:1 forms by volume and cultivate 7 days, more outdoor cultivation 90 days, can be moved to field production, when field production, shade one week, transplanting survival rate is 100%.

Condition of culture in above-mentioned steps (2) to (4) is: 24 ± 2 ℃ of room temperatures, intensity of illumination PPFD=65.5 μ .moL.m ^-2.s ^-1, 16 hours every days of light application time.

Result of the test shows, a large amount of nursery stocks of the maternal plant merit that can be maintained can 100% keep former plant genotype and all good characteristics thereof, have higher reproduction coefficient (512 ~ 1000).

embodiment 2

The healthy and strong branch of the beech trees fallen leaves in winter containing axillalry bud is placed in to the beaker that distilled water is housed, carries out water planting after 15 ~ 30 days at 25 ℃, obtain the young shoot that axillary bud sprouting forms, then carry out following steps;

(1) beech are set to young shoot alcohol disinfecting 25 ~ 30s of 75%, with aseptic water washing 3 times, with the 0.1% mercuric chloride 12min that sterilizes, then used aseptic water washing 3 times;

(2) will be cut into the stem section that long 1.2cm at least contains an axillalry bud through step (1) branch after treatment, and implant in inducing clumping bud medium and cultivate 30 ~ 32 days, obtain Multiple Buds; Wherein, described inducing clumping bud medium is: WPM+1.0 mg.L ^-1bA+1.0 mg.L ^-1nAA, agar 6.5 g.L ^-1, sucrose 20 g.L ^-1, pH value is 5.7, medium autoclaving 20min;

(3) Multiple Buds growing after step (2) induction is cultivated is proceeded in subculture medium and cultivate the propagation cultivation of carrying out bud for 65 days, bud growth coefficient is 7 ~ 9; Wherein, described subculture medium is: WPM+2.0 mg.L ^-1bA+1.0 mg.L ^-1nAA, agar 6.5 g.L ^-1, sucrose 20 g.L ^-1, pH value is 5.7, medium autoclaving 20min;

(4) cutting the height growing after step (3) subculture is cultivated is beech tree healthy seedlings more than 2.0 cm, implants in root media and cultivates 50 days, obtains regeneration plant, and rooting rate is 86%; Wherein, described root media is: root media is: WPM+1.0 mg.L ^-1iBA+2.0 g.L ^-1aC, agar 7.0 mg L ^-1, sucrose 20 g.L ^-1, pH value is 5.7, medium autoclaving 20min;

(5) regeneration plant that will obtain through step (4) is cleaned, be transplanted to by hardening in peat soil and the perlite mixed-matrix that 1:1 forms by volume and cultivate 10 days, more outdoor cultivation 100 days, can be moved to field production, when field production, shade one week, transplanting survival rate is 95%.

Result of the test shows, a large amount of nursery stocks of the maternal plant merit that can be maintained can 100% keep former plant genotype and all good characteristics thereof, have higher reproduction coefficient (343 ~ 729).

Claims

1. a beech tree quick breeding method for tissue culture, comprises the steps:

(1) the beech tree of choosing with alcohol disinfecting 30～35s of 75% for bud children shoot, with aseptic water washing 3 times, with the 0.1% mercuric chloride 8min that sterilizes, then is used to aseptic water washing 3 times then raw spring;

(2) will be cut into the stem section that long 1cm at least contains an axillalry bud through step (1) branch after treatment, and implant in bud inducing culture and cultivate 28～30 days, obtain Multiple Buds; Wherein, described bud inducing culture is: WPM+3.0 mg L ^-1bA, agar 6.5 gL ^-1, sucrose 20 gL ^-1, pH value is 5.7, medium autoclaving 20min; The inductivity of Multiple Buds is 84～90%;

(3) Multiple Buds growing after step (2) induction is cultivated is proceeded in subculture medium and cultivated 60 days, carry out the propagation of bud, bud growth coefficient is 8～10; Wherein, described subculture medium is: WPM+3.0 mgL ^-1bA+2.0 mgL ^-1nAA, agar 6.5 gL ^-1, sucrose 20 gL ^-1, pH value is 5.7, medium autoclaving 20min;

(4) cutting the height growing after step (3) subculture is cultivated is beech tree healthy seedling more than 2.0cm, implants in root media and cultivates 45 days, obtains regeneration plant, and rooting rate is 89～95%; Wherein, described root media is: WPM+0.5 mgL ^-1iBA+2.0 gL ^-1aC, agar 7.0 mgL ^-1, sucrose 20 gL ^-1, pH value is 5.7, medium autoclaving 20min;

(5) regeneration plant that will obtain through step (4) is cleaned, is transplanted to by hardening cultivation in peat soil and the perlite mixed-matrix that 1:1 form by volume 7 days, more outdoor cultivation 90 days, move to field production;

Condition of culture in above-mentioned steps (2) to (4) is: 24 ± 2 ℃ of room temperatures, intensity of illumination PPFD=65.5 μ moLm ^-2s ^-1, 16 hours every days of light application time.