Summary of the invention
Purpose of the present invention be exactly provide in order to overcome the defective that above-mentioned prior art exists a kind of transplanting survival rate can be up to 95%, keep the method for tissue culture of the heavenly bamboo ' flame ' of parent present situation.
Purpose of the present invention can be achieved through the following technical solutions:
The tissue culture method of heavenly bamboo flame, it is characterized in that, this method is by after carrying out aseptic process to the new budlet that sprouts of heavenly bamboo flame of winning, be inoculated in successively again cultivate in budlet inducing culture, adventitious bud proliferation medium, strong seedling culture base and the root media and place the greenhouse hardening after, can transplant outdoorly, obtain heavenly bamboo ' flame ' plant.
This method specifically may further comprise the steps:
(1) acquisition of sterilizable material
Win the new budlet that sprouts of heavenly bamboo flame, with behind the running water flushing 2h on superclean bench, behind the alcohol immersion 20-40s and 1wt ‰ mercuric chloride solution immersion 10-20min with 75wt%, utilize aseptic water washing 5-6 time and blot surface moisture, get the budlet base portion and be cut into 1cm and be inoculated in the pH value on the budlet inducing culture of 5.5-6.0, the control cultivation temperature is 24-26 ℃, and illumination is 70-90 μ mol/ms, carries out the bud inducing culture;
(2) differentiation of bud and propagation
Budlet was inoculated on the budlet inducing culture after 4 weeks, budlet begins to expand, the yellow green projection appears, the visible significantly callus in 2 week backs, continue to cultivate 1 month, downcut band bud callus and be inoculated in the pH value on the adventitious bud proliferation medium of 5.5-6.0, the control cultivation temperature is 24-26 ℃, illumination is 70-90 μ mol/ms, carries out enrichment culture;
(3) indefinite bud strong seedling culture
After in the adventitious bud proliferation medium, inducing the bud of growing thickly, the bud of will growing thickly is divided into Xiao Cong or simple bud and is inoculated in the pH value in the strong seedling culture base of 5.5-6.0, the control cultivation temperature is 24-26 ℃, illumination is 70-90 μ mol/ms, carry out strong seedling culture, indefinite bud extends rapidly, can grow 2-3cm after 30 days;
(4) culture of rootage
Get and highly be the plantlet of 2-3cm, it is inoculated in the pH value in the root media of 5.5-6.0, the control cultivation temperature is 24-26 ℃, illumination is 70-90 μ mol/ms, carry out culture of rootage, the seedling base section dissolves the root original hase of many whites after 10 days, can grow to 4-6cm after 30 days;
(5) hardening and transplanting
Continue culture of rootage 30-50 days, and when root system grows to 1-2cm, selected the aseptic seedling of well developed root system robust growth, indoor uncork hardening 7 days is taken out afterwash root agar, and domestication is after 40 days in the greenhouse, can transplant outdoorly, give rich water quality management, can obtain the product plant.
The basis of described budlet inducing culture comprises sucrose 30g/L, agar 6g/L, and nutrient component is MS+6-BA 1.0~5.0mg/L+NAA0.1~0.5mg/L+IBA1.0~5.0mg/L.
The preferred MS+6-BA5.0mg/L+NAA0.5mg/L+IBA5.0mg/L of the nutrient component of described budlet inducing culture.
The basis of described adventitious bud proliferation medium comprises sucrose 30g/L, agar 6g/L, and nutrient component is 1/2MS+6-BA1.0~3.0mg/L+NAA0.1~0.3mg/L+IBA0.1~0.5mg/L.
The preferred 1/2MS+6-BA2.0mg/L+NAA0.2mg/L+IBA 0.3mg/L of the nutrient component of described adventitious bud proliferation medium.
The basis of described strong seedling culture base comprises sucrose 30g/L, agar 6g/L, and nutrient component is MS+6-BA0.5~2.0mg/L+NAA0.1~0.2mg/L+IBA0.3~0.5mg/L.
The preferred MS+6-BA0.5mg/L+NAA0.1mg/L+IBA0.3mg/L of the nutrient component of described strong seedling culture base.
The basis of described root media comprises sucrose 30g/L, agar 6g/L, and nutrient component is 1/2MS+NAA 0.1~1.0mg/L+IBA3.0~8.0mg/L.
The preferred 1/2MS+NAA0.5mg/L+IBA5.0mg/L of the nutrient component of described root media.
Compared with prior art, transplanting survival rate of the present invention can improve the regularity of reproduction speed and nursery stock greatly up to 95%, keeps original maternal character better.
Embodiment
The present invention is described in detail below in conjunction with specific embodiment.
Embodiment 1
The method for tissue culture of heavenly bamboo ' flame ', this method may further comprise the steps:
(1) acquisition of sterilizable material
Win the new budlet that sprouts of heavenly bamboo flame, with behind the running water flushing 2h on superclean bench, behind the alcohol immersion 20s and 1wt ‰ mercuric chloride solution immersion 10min with 75wt%, utilize aseptic water washing 5-6 time and blot surface moisture, get the budlet base portion and be cut into 1cm and be inoculated in the budlet inducing culture, the composition of this budlet inducing culture is MS+6-BA1.0mg/L+NAA0.1mg/L+IBA1.0mg/L;
(2) differentiation of bud and propagation
Budlet was inoculated on the budlet inducing culture after 4 weeks, budlet begins to expand, the yellow green projection appears, the visible significantly callus in 2 week backs, budlet is many on the base portion callus, but do not influence the propagation of indefinite bud, the indefinite bud growth there is no bad phenomenon such as vitrifying rapidly, it is good to grow in this cultivation, continue to cultivate 1 month, downcut band bud callus and be inoculated on the adventitious bud proliferation medium, the composition of this adventitious bud proliferation medium is 1/2MS+6-BA1.0mg/L+NAA0.1mg/L+IBA0.1mg/L;
(3) indefinite bud strong seedling culture
After in the adventitious bud proliferation medium, inducing the bud of growing thickly, the bud of will growing thickly is divided into Xiao Cong or simple bud and is inoculated in the strong seedling culture base, indefinite bud extends rapidly, can grow 2cm after 30 days, and the composition of this strong seedling culture base is MS+6-BA0.5mg/L+NAA0.1mg/L+IBA 0.3mg/L;
(4) culture of rootage
Get and highly be it to be inoculated in the root media plantlet of 2cm, the composition of this root media is 1/2MS+NAA0.1mg/L+IBA3.0mg/L, and the seedling base section dissolves the root original hase of many whites after 10 days, can grow to 4cm after 30 days;
(5) hardening and transplanting
Continued culture of rootage 30 days, and when root system grows to 1cm, selected the aseptic seedling of well developed root system robust growth, indoor uncork hardening 7 days is taken out afterwash root agar, and domestication after 40 days in the greenhouse can be transplanted outdoorly, gives rich water quality management, can obtain the product plant.
The composition of all above-mentioned medium also comprises sucrose 30g/L, agar 6g/L, and the cultivation temperature of this medium is 24 ℃, and illumination is 70 μ mol/ms, and the pH value is 5.5.
Embodiment 2
The method for tissue culture of heavenly bamboo ' flame ', this method may further comprise the steps:
(1) acquisition of sterilizable material
Win the new budlet that sprouts of heavenly bamboo flame, with behind the running water flushing 2h on superclean bench, behind the alcohol immersion 40s and 1wt ‰ mercuric chloride solution immersion 20min with 75wt%, utilize aseptic water washing 6 times and blot surface moisture, get the budlet base portion and be cut into 1cm and be inoculated in the budlet inducing culture, the composition of this budlet inducing culture is MS+6-BA3.0mg/L+NAA0.3mg/L+IBA3.0mg/L;
(2) differentiation of bud and propagation
Budlet was inoculated on the budlet inducing culture after 4 weeks, budlet begins to expand, the yellow green projection appears, the visible significantly callus in 2 week backs, budlet is many on the base portion callus, but do not influence the propagation of indefinite bud, the indefinite bud growth there is no bad phenomenon such as vitrifying rapidly, it is good to grow in this cultivation, continue to cultivate 1 month, downcut band bud callus and be inoculated on the adventitious bud proliferation medium, the composition of this adventitious bud proliferation medium is 1/2MS+6-BA 3.0mg/L+NAA0.3mg/L+IBA0.5mg/L;
(3) indefinite bud strong seedling culture
After in the adventitious bud proliferation medium, inducing the bud of growing thickly, the bud of will growing thickly is divided into Xiao Cong or simple bud and is inoculated in the strong seedling culture base, indefinite bud extends rapidly, can grow 3cm after 30 days, and the composition of this strong seedling culture base is MS+6-BA2.0mg/L+NAA0.2mg/L+IBA0.5mg/L;
(4) culture of rootage
Get and highly be it to be inoculated in the root media plantlet of 3cm, the composition of this root media is 1/2MS+NAA1.0mg/L+IBA8.0mg/L, and the seedling base section dissolves the root original hase of many whites after 10 days, can grow to 6cm after 30 days;
(5) hardening and transplanting
Continued culture of rootage 50 days, and when root system grows to 2cm, selected the aseptic seedling of well developed root system robust growth, indoor uncork hardening 7 days is taken out afterwash root agar, and domestication after 40 days in the greenhouse can be transplanted outdoorly, gives rich water quality management, can obtain the product plant.
The composition of all above-mentioned medium also comprises sucrose 30g/L, agar 6g/L, and the cultivation temperature of this medium is 26 ℃, and illumination is 90 μ mol/ms, and the pH value is 6.0.
Embodiment 3
The method for tissue culture of heavenly bamboo ' flame ', this method may further comprise the steps:
(1) acquisition of sterilizable material
Win the new budlet that sprouts of heavenly bamboo flame, with behind the running water flushing 2h on superclean bench, behind the alcohol immersion 30s and 1wt ‰ mercuric chloride solution immersion 15min with 75wt%, utilize aseptic water washing 5 times and blot surface moisture, get the budlet base portion and be cut into 1cm and be inoculated in the budlet inducing culture, the composition of this budlet inducing culture is MS+6-BA5.0mg/L+NAA0.5mg/L+IBA5.0mg/L;
(2) differentiation of bud and propagation
Budlet was inoculated on the budlet inducing culture after 4 weeks, budlet begins to expand, the yellow green projection appears, the visible significantly callus in 2 week backs, the budlet of continuing on the base portion callus is many, but do not influence the propagation of indefinite bud, the indefinite bud growth there is no bad phenomenon such as vitrifying rapidly, it is good to grow in this cultivation, continue to cultivate 1 month, downcut band bud callus and be inoculated on the adventitious bud proliferation medium, the composition of this adventitious bud proliferation medium is 1/2MS+6-BA2.0mg/L+NAA0.2mg/L+IBA 0.3mg/L;
(3) indefinite bud strong seedling culture
After in the adventitious bud proliferation medium, inducing the bud of growing thickly, the bud of will growing thickly is divided into Xiao Cong or simple bud and is inoculated in the strong seedling culture base, the composition of this strong seedling culture base is MS+6-BA0.5mg/L+NAA0.1mg/L+IBA0.3mg/L, and indefinite bud extends rapidly, can grow 3cm after 30 days;
(4) culture of rootage
Get and highly be it to be inoculated in the root media plantlet of 3cm, the composition of this root media is 1/2MS+NAA0.5mg/L+IBA5.0mg/L, and the seedling base section dissolves the root original hase of many whites after 10 days, can grow to 6cm after 30 days;
(5) hardening and transplanting
Continued culture of rootage 40 days, and when root system grows to 2cm, selected the aseptic seedling of well developed root system robust growth, indoor uncork hardening 7 days is taken out afterwash root agar, and domestication after 40 days in the greenhouse can be transplanted outdoorly, gives rich water quality management, can obtain the product plant.
The composition of all above-mentioned medium also comprises sucrose 30g/L, agar 6g/L, and the cultivation temperature of this medium is 25 ℃, and illumination is 80 μ mol/ms, and the pH value is 5.8.