CN108834898A - Pad the method for tissue culture of willow - Google Patents

Abstract

The present invention provides a kind of method for tissue culture for padding willow.This method mainly carries out tissue cultures using two kinds of band axillary bud branch, the leaflet tablet explants of pad willow, and experimental result determines 2000lux intensity of illumination, and 26 DEG C of temperature, callus inducing medium：MS+0.1mg/L 6-BA+0.1mg/L NAA, tufted seedling induced medium：MS+3mg/L 6-BA+0.5mg/L NAA+0.05mg/L GA, root media：1/2 MS+0.1mg/L NAA is the condition of culture for being well suited to pad willow tissue cultures.

Description

Pad the method for tissue culture of willow

Fields：

The invention belongs to field of plant tissue culture technique, and in particular to a kind of group of pad willow (Salix lindlayana) Knit cultural method.

Background technique：

It pads willow (Salix lindlayana) and belongs to Salicaceae sallow, cushion shrub.Entire plant height does not often surpass 30 centimetres are crossed, trunk crawls and takes root, it is distributed in height above sea level 4300-4700 Mead lattice, Ganzi, Kowloon, wood, the ground such as Maerkang, It is common in the extremely severe area of the ecological environments such as Alpine screes or alpine scrub, is a kind of very special willow in Salix. Researcher speculates, with the continuous grand liter of Qinghai-Tibet Platean, in order to adapt to continually changing various habitats, the willow to live herein Violent species differentiation also occurs for platymiscium, thus a large amount of new species out that develop, pad willow is one of them, more other willows Platymiscium, their plant are in shape of crawling, and branch is intensive, and blade is minimum, are the ideal material for carrying out Salix genetics research, base Because the development of functional study needs to establish an efficient genetic conversion system.Agrobacterium-mediated genetic transformation technology is extensive For various plants, the research fields such as it is plant growth and development, Resistance Physiology and organ occur provide many good test materials And effective way, but mature Dian Liu tissue culturing system not yet establishes, and establishing good acceptor material regenerating system is to plant The basis of object genetic transformation.It is had not been reported at present for padding the tissue cultures of willow.

Summary of the invention：

The present invention is intended to provide a kind of method for tissue culture for padding willow.

In order to realize above-mentioned purpose of the invention, the present invention provides the following technical solutions：

It is a kind of pad willow method for tissue culture, this method using pad willow band axillary bud branch, two kinds of explants of leaflet tablet into Row tissue cultures are cultivated at 2000lux intensity of illumination, 26 DEG C of temperature, and callus inducing medium is：MS+ 0.1mg/L 6-BA+0.1mg/L NAA, tufted seedling induced medium are：MS+3mg/L 6-BA+0.5mg/L NAA+0.05mg/ L GA, root media are：1/2MS+0.1mg/L NAA.

A kind of method for tissue culture padding willow, this method include the following steps：

(1) it cultivates：It takes the pad willow plant of robust growth, no disease and pests harm to plant in the greenhouse, white sprout just has been spat to lateral bud Until；

(2) explant selects：It uses blade to take 1-2cm of branch, blade 0.5cm × 0.5cm with axillary bud as explant, uses 70% alcohol disinfecting 30s, with sterile water wash 3 times, until being divided under super bacterium workbench aseptic condition with 20% hypochlorite disinfectant 15 Clock is blotted on explant surface with the filter paper Jing Guo sterilization treatment with aseptic water washing 5 times；

(3) callus is cultivated：By the branch with axillary bud and blade inoculation on the culture medium of evoked callus, explant It shows consideration for into culture medium, culture medium prescription is：MS minimal medium+6-BA0.1-0.3mg/L+NAA 0.1-0.3mg/L, 30g/L Sucrose, pH:5.8,8g/L agar are placed under common fluorescent light source, intensity of illumination 2000Lux, and daily light application time is 12 hours, temperature was 25 DEG C, after culture 15 days, induced green calli；

(4) tufted seedling induces：The callus of green is subjected to squamous subculture, subculture medium is：MS minimal medium+ The sucrose of 6-BA 1-3mg/L+NAA 0.3-0.5mg/L+0.05mg/L GA, 30g/L, pH:5.8,8g/L agar, are put into illumination Intensity is to cultivate under 2000lux illumination, and temperature is 25 DEG C, and daily light application time is 12h, every two week subculture 1 time；

(5) seedling cultivation and rooting：Single plant is cut into Multiple Buds separation, base portion is sheared, is linked into root media, it is raw Root culture medium is：1/2MS+0.1g/L NAA+1g/L active carbon, 10g/L sucrose, pH:5.8, it is placed in the ring of common daylight lamp source Under border, intensity of illumination 2000lux, daily light application time is 20 hours, 25 DEG C of temperature, is cultivated 30 days；

(6) hardening：After test tube seedling corkage is saved 2 days, takes out test tube seedling and clean, be transplanted in soil.

Beneficial effects of the present invention are：The present invention passes through band axillary bud branch, two kinds of explants of leaflet tablet carry out tissue cultures, It successfully induces callus and forms regrowth, so as to illustrate that the tissue cultures for padding willow have been carried out.The present invention discloses One kind by tissue culture and inducement regenerate seedling-growing method it is easy to operate, inductivity is high, low in cost, for further utilize biotechnology side The molecular mechanism on the method research pad plateau Liu Shiying has established technical foundation.

Specific embodiment：

Essentiality content of the invention is further illustrated with the embodiment of the present invention below, but this is not limited with this Invention.

Embodiment 1：

Pad the tissue cultures of willow：

In the tissue culture procedures of pad willow, the factor for influencing tissue cultures success or not includes explant source, culture The Multiple factors such as based component, culture illumination, cultivation temperature.The present invention is explant using band axillary bud branch, the blade of pad willow, is ground Different culture medium member condition induces differentiation into the influence of seedling to it in Jiu Liao culturing room.

1, material and method

1.1 research material

It takes the pad willow of field acquisition to cultivate in ordinary greenhouse, is put into after taking light green band axillary bud branch and leaflet tablet sterilizing Induced medium:MS minimal medium+6-BA (0.1mg/L, 0.2mg/L, 0.3mg/L)+NAA (0.1mg/L, 0.2mg/L, 0.3mg/L), 30g/L sucrose, 8.0g/L agar adjust pH value to evoked callus in 5.8, and condition of culture is temperature 25 DEG C, intensity of illumination 2000lux, light application time 12h, relative humidity 60%, after culture 15 days, it is semi-transparent that induction generates green Bright callus, to be transplanted to research induction differentiation seedling under different culture medium ingredient.

1.2 method

1.2.1 squamous subculture in differential medium

The callus induced is accessed in differential medium, the composition of differential medium is：MS minimal medium+6- BA (1mg/L, 2mg/L, 3mg/L)+NAA (0.3mg/L, 0.4mg/L, 0.5mg/L)+GA (0.05mg/L), adds 30g/L's Sucrose, 8.0g/L agar adjust pH value to 5.8, and every ware is inoculated with the similar callus of growing way, is put into culturing room's culture.

1.2.2 culture of rootage

The seedling that will be differentiated, which moves on to, carries out rooting induction in root media, root media is that 1/2MS is trained substantially Support base+0.1mg/L NAA+1g/L active carbon, 10g/L sucrose, 8g/L agar, pH：5.8.

1, best-of-breed technology scheme

The pad willow that field acquisition is returned is planted in greenhouse, chooses the branch disinfection with axillary bud and is put into callus tissue culture In base, the culture medium of several gradients can induce callus, and effect is best to be：MS minimal medium+6-BA (0.1mg/ L)+NAA (0.1mg/L), 30g/L sucrose, 8g/L agar, pH：5.8 evoked callus, condition of culture are 25 DEG C of temperature, light According to 2000lux, light application time 12h, relative humidity 60% induces green calli, by this callus group after culture 15 days It knits after substituting into the vial for filling differential medium, the best culture medium of effect is：MS minimal medium+6-BA (3mg/L)+ NAA (0.5mg/L)+GA (0.05mg/L), 8g/L agar, pH：5.8,25 DEG C of temperature, illumination 2000lux, light application time 12h, Relative humidity 60%, every fortnight subculture are primary.The Cong Miao that will be differentiated after 30 days is separated into single plant shearing base portion and is inserted into life It takes root on root culture medium, root media is：1/2MS minimal medium+NAA (0.1mg/L)+1g/L active carbon, 10g/L sugarcane Sugar, agar 8g/L, pH：5.8.

Claims (2)

1. a kind of method for tissue culture for padding willow, this method is carried out using two kinds of band axillary bud branch, leaflet tablet explants of pad willow Tissue cultures are cultivated at 2000lux intensity of illumination, 26 DEG C of temperature, and callus inducing medium is：MS+0.1mg/L 6-BA+0.1mg/L NAA, tufted seedling induced medium are：MS+3mg/L 6-BA+0.5mg/L NAA+0.05mg/L GA is raw Root culture medium is：1/2MS+0.1mg/L NAA.

2. a kind of method for tissue culture for padding willow, it is characterised in that this method includes the following steps：

(1) it cultivates：, take robust growth, no disease and pests harm pad willow plant plantation in the greenhouse, just spat to lateral bud it is white sprout for Only；

(2) explant selects：Blade is used to take 1-2cm of branch, blade 0.5cm × 0.5cm with axillary bud as explant, with 70% Alcohol disinfecting 30s, with sterile water wash 3 times, until under super bacterium workbench aseptic condition, with 20% hypochlorite disinfectant 15 minutes, With aseptic water washing 5 times, explant surface is blotted with the filter paper Jing Guo sterilization treatment；

(3) callus is cultivated：By the branch with axillary bud and blade inoculation on the culture medium of evoked callus, explant is shown consideration for Into culture medium, culture medium prescription is：The sugarcane of MS minimal medium+6-BA0.1-0.3mg/L+NAA 0.1-0.3mg/L, 30g/L Sugar, pH:5.8,8g/L agar are placed under common fluorescent light source, intensity of illumination 2000Lux, and daily light application time is 12 small When, temperature is 25 DEG C, after culture 15 days, induces green calli；

(4) tufted seedling induces：The callus of green is subjected to squamous subculture, subculture medium is：MS minimal medium+6-BA The sucrose of 1-3mg/L+NAA 0.3-0.5mg/L+0.05mg/L GA, 30g/L, pH:5.8,8g/L agar, are put into intensity of illumination To cultivate under 2000lux illumination, temperature is 25 DEG C, and daily light application time is 12h, every two week subculture 1 time；

(5) seedling cultivation and rooting：Single plant is cut into Multiple Buds separation, base portion is sheared, is linked into root media, training of taking root Feeding base is：1/2MS+0.1g/L NAA+1g/L active carbon, 10g/L sucrose, pH:5.8, it is placed in the environment of common daylight lamp source, Intensity of illumination is 2000lux, and daily light application time is 20 hours, 25 DEG C of temperature, is cultivated 30 days；

(6) hardening：After test tube seedling corkage is saved 2 days, takes out test tube seedling and clean, be transplanted in soil.