CN108094213A - A kind of green bamboo tissue culture culture medium and its method for tissue culture - Google Patents
A kind of green bamboo tissue culture culture medium and its method for tissue culture Download PDFInfo
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- CN108094213A CN108094213A CN201810019346.4A CN201810019346A CN108094213A CN 108094213 A CN108094213 A CN 108094213A CN 201810019346 A CN201810019346 A CN 201810019346A CN 108094213 A CN108094213 A CN 108094213A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
Abstract
The present invention relates to a kind of green bamboo tissue culture culture medium and its method for tissue culture, including following culture medium：Bud inducement cultivation base：3/4MS+6‑BA 3‑5mg·L‑1+KT 0.5‑1.5mg·L‑1+TDZ 0.5g·L‑1+ sucrose 30gL‑1+ agar 5gL‑1+ coconut palm breast 100mlL‑1；Bud subculture multiplication medium：1/2MS+6‑BA 1.5‑2.5mg·L‑1+KT 0.5‑1mg·L‑1+NAA 0.1‑0.4mg·L‑1+ sucrose 30gL‑1+ agar 5gL‑1+ coconut palm breast 100ml.L‑1+CPPU0‑0.5g·L‑1；Root media：1/2MS+IBA 0.2‑0.6mg·L‑1The 15 35ppm+AC 0.1mgL of+root sun‑1+ sucrose 20gL‑1+ agar 6gL‑1；Select, sterilize through explant, bud induction, the culture of bud shoot proliferation, culture of rootage, bottle seedling hardening, transplanting, obtain tissue-cultured seedling.The present invention is in bud induction period through that can form bud clump after subculture 3 times, in bud shoot proliferation cultivation stage, multiplication is fast, growth is fast, the less generation of browning, in the culture of rootage stage, cane is chosen to lead to directly, what blade was unfolded, an old one new bud point plant of plant band carries out culture of rootage, and rooting rate is 60%~80%.
Description
Technical field
The present invention relates to a kind of green bamboo tissue culture culture medium and its method for tissue culture.
Background technology
Green bamboo (Bambusa oldhami) is grass family perennial evergreen bamboo plant.Green bamboo bamboo pole is generally taller and bigger, closely
Uprightly, construction timber can be done or split thin bamboo strip knitted article；Green bamboo bamboo fibre is longer, can be used as paper making raw material；Dendrocalamopsis oldhami matter is tender delicious,
For eating raw, dried bamboo shoots or can be also processed into；Green bamboo caulis bambusae in taenian can do Chinese medicinal material, widely be cultivated for south China excellent
One of Cluster Bamboo.
Fujian warmer climate, abundant rainfall grow suitable for bamboo.Fujian bamboo resource is extremely abundant.Development of Bamboo produces,
Shake off poverty and set out on the road to prosperity for mountain area, increase farmers' income and be of great significance.But since good seed is limited, bamboo production is constrained
Development.It is optimal approach by tissue-culturing quick-propagation, breediness is kept not make a variation degeneration, neat and consistent is provided
Nursery stock is widely applied plantation in a short time.Plant tissue culture technique can provide high-quality kind a large amount of for production unit in a short time
Seedling.The Preliminary Results of green bamboo tissue cultures are reported herein.
The content of the invention
It is an object of the invention to provide a kind of green bamboo tissue culture culture medium and its method for tissue culture, expand numerous and work for green bamboo
Industryization provides technical support.
What the present invention was realized in：
Present invention firstly provides a kind of green bamboo tissue culture culture medium, including following culture medium：
Bud inducement cultivation base：3/4MS (a great number of elements reduces to 3/4 in MS culture mediums, remaining is constant)+6-BA (3-5) mg
L-1+KT(0.5-1.5)mg·L-1+TDZ0.5g·L-1+ sucrose 30gL-1+ agar 5gL-1+ coconut palm breast 100mlL-1, favorably
In the induction of bud；
Bud subculture multiplication medium：1/2MS (a great number of elements reduces to 1/2 in MS culture mediums, remaining is constant)+6-BA (1.5-
2.5)mg·L-1+KT(0.5-1)mg·L-1+NAA(0.1-0.4)mg·L-1+ sucrose 30gL-1+ agar 5gL-1+ coconut palm breast
100ml.L-1+CPPU 0-0.5g·L-1.Wherein CPPU intervals use, and every 5 generation, interval was using once using once
CPPU0.5g·L-1, promote cell division, enhance the activity of bud.Because CPPU is activity is most strong in the ureas basic element of cell division one
Kind, so cannot be used continuously.After 5 generation of shoot proliferation, fluid nutrient medium is converted into cultivate, and is more advantageous to the training of bud shoot proliferation
It supports；
Root media：1/2MS (a great number of elements reduces to 1/2 in MS culture mediums, remaining is constant)+IBA (0.2-0.6) mg
L-1+ root the sun (15-35) ppm+AC 0.1mgL-1+ sucrose 20gL-1+ agar 6gL-1, be conducive to take root.
The present invention also provides a kind of green bamboo method for tissue culture, include the following steps：
(1) explant selects
Maternal by tissue cultures of adult tree carpentery workshop, during March~September, it is explant to choose and give birth to rudiment bar then；
(2) sterilize
Explant is cut into the stem section of 1 axillary bud of band, is then subsequently disinfected with after 75% alcohol wipe；
(3) bud induces
The inducing culture of bud is 3/4MS (a great number of elements reduces to 3/4 in MS culture mediums, remaining is constant)+6-BA (3-5)
mg·L-1+KT(0.5-1.5)mg·L-1+TDZ0.5g·L-1+ sucrose 30gL-1+ agar 5gL-1+ coconut palm breast 100mlL-1's
Culture medium, pH value 5.8；
Condition of culture：Light culture, 26 DEG C~28 DEG C of cultivation temperature；
(4) bud shoot proliferation culture
The formula of bud subculture multiplication medium：1/2MS (wherein a great number of elements reduces to 1/2, remaining is constant)+6-BA (1.5-
2.5)mg·L-1+KT(0.5-1)mg·L-1+NAA(0.1-0.2)mg·L-1+ sucrose 30gL-1+ agar 5gL-1+ coconut palm breast
100ml·L-1+CPPU 0-0.5g·L-1Culture medium is cultivated, pH value 5.8；Wherein CPPU intervals use, and every 5 generation uses one
It is secondary；
Condition of culture：Daily continuous illumination 10h~12h, 24 DEG C~28 DEG C of cultivation temperature, intensity of illumination 2000lux~
3000lux；After 5 generation of shoot proliferation, fluid nutrient medium is converted into cultivate；
(5) culture of rootage
The formula of root media：1/2MS+IBA(0.2-0.6)mg·L-1+ root the sun (15-35) ppm+AC0.1mg
L-1+ sucrose 20gL-1+ agar 6gL-1, pH value 5.8；
Condition of culture：Daily continuous illumination 8h~10h, 28 DEG C~30 DEG C of cultivation temperature, intensity of illumination 1000lux~
2000lux；
(6) bottle seedling hardening
The tissue-cultured seedling taken root is shifted into hardening 15~20 days in greenhouse, tissue-cultured seedling is taken out, base portion is rinsed well with tap water
Culture medium, be put into 0.1~0.5gL-1Liquor potassic permanganate in impregnate 1~3min, then with tap water rinse one time or use
500 times of carbendazim impregnated base portion after 10-15 seconds, covered wet towel moisturizing in case transplanting；
(7) transplant
Tissue-cultured seedling is transplanted in the nutrient bag equipped with matrix, root water of drenching in time after transplanting, and covers plastic film,
Plastic film is opened after a week, is managed after being transplanted；Transplanting time is in annual March-June or September-November.
Wherein step (2) described disinfection the step of include：With the sterilizing of 5g/L is net or 84 medicining liquid dipping 5min, tap water
Half an hour is rinsed, then aseptically carries out following operation：
A. after sterile water wash 2 times, with 75% alcohol disinfecting 10s, with aseptic water washing 3 times；
B. 0.2%Hg Cl are used230min is impregnated in solution vibration, with aseptic water washing 3 times；
C. 0.2%HgCl is used again2Solution left standstill impregnates 5~7min, and processing time is different due to material tender degree always, and use is sterile
Water rinses 5~7 times.
Step (6) described greenhouse is the glasshouse that degree of shading is 50%.
Step (7) described matrix is the mixture that is made of thin river sand, peat soil, quality proportioning 5:5.
Step (7) described transplanting, before tissue culture transplantation of seedlings to nutrient bag, first with 3~5gL-1Liquor potassic permanganate disappear
Poison is equipped with the nutrient bag of matrix.
It manages, comprises the following steps after step (7) described transplanting：
In preceding 30d controlled at 26 ± 2 DEG C, humidity 85%；Transplanting after two weeks, periodically sprays weekly NITROGEN IN LOW CONCENTRATION phosphorus
K composite fertilizer (400~500ppm) and foliar fertilizer.Depending on disease, insect pest occurrence degree, fungicide and insecticide are periodically sprinkled.Two months
Afterwards outdoor is moved to together with nutrient bag.Seedling stage needs to shade, and avoids burning sun exposure, pays attention to ventilation and water and fertilizer management work.
Seedling length after step (7) is transplanted in nutrient bag is to 15cm, you can goes out garden and go up a hill afforestation or to cultivate to adopt fringe mother
Strain.
The invention has the advantages that：
(1) in bud induction period, sterilizable material is inoculated on bud inducement cultivation base, through bud can be formed after subculture 3 times
Clump.
(2) in bud shoot proliferation cultivation stage, agar solidified culture medium is commonly used to cultivate the method for vegetable material, although side
Method equipment is simple, easy, but Nutrient distribution is uneven, and the speed of growth is unbalanced, and often has browning intoxicating phenomenon.Along with more
Brown stain occurs for Dai Houyi, in order to improve this case, after 5 generation of shoot proliferation, a small amount of fluid nutrient medium can be used to cultivate, green bamboo
Multiplication is fast, and growth is fast, the less generation of browning.
(3) in the culture of rootage stage, selection cane leads to directly, what blade was unfolded, and an old one new bud point plant of plant band carries out
Culture of rootage, rooting rate are 60%~80%.
Specific embodiment
Embodiment 1
The induction childrenization culture of 1.1 green bamboo clone explants.
1.1.1 select the adult tree carpentery workshop of excellent Genetic Performance type maternal for tissue cultures.During March~September, explant
Disinfecting for body is afternoon in fine day, and from elite plant base portion, it is explant to choose and give birth to rudiment bar then.
1.1.2 explant is cut into the stem section of 1 axillary bud of band with after twice of 75% alcohol wipe.With the sterilizing of 5g/L it is net or
84 medicining liquid dipping 5min, put and half an hour are rinsed in flowing water.
1.1.3 following operation is aseptically carried out：(1) after sterile water wash 2 times, with 75% alcohol disinfecting 10s,
With aseptic water washing 3 times；(2) 30min is impregnated with the vibration of 0.2%Hgcl2 solution, with aseptic water washing 3 times；(3) again with 0.2%
Hgcl2 solution left standstills impregnate 5~7min, and processing time is different due to material tender degree always, with aseptic water washing 5~7 times.
1.1.4 the Fiber differentiation based formulas of bud is to use 3/4MS+6-BA3mgL-1+KT0.5mg·L-1+ sucrose 30gL-1
+ agar 5gL-1+ coconut palm breast 100ml.L-1.Its pH value is 5.8.Hormone-content is relatively low at this time, inductivity 70%.
Condition of culture：Light culture, 26 DEG C~28 DEG C of cultivation temperature；Temperature is high, no light, be conducive to green bamboo sprouting sprout and
Normal growth.
1.2 clone bud shoot proliferation cultures.
1.2.1 minimal medium is MS, adjusts its a great number of elements, hormone combination etc..The subculture multiplication medium of green bamboo is matched somebody with somebody
Side is improvement MS (wherein a great number of elements reduces to 1/2)+6-BA1.5mgL-1+KT0.5mg·L-1+NAA0.1mg·L-1+ sucrose
30g·L-1+ agar 5gL-1+ coconut palm breast 100ml.L-1, pH value 5.8.Hormone-content is relatively low at this time, and proliferation rate is 1.5 times.
More be not in vitreous shoot with MS is improved than common MS.Agar solidified culture medium is commonly used to cultivate the method for vegetable material, although
Method equipment is simple, easy, but Nutrient distribution is uneven, and the speed of growth is unbalanced, and often has browning intoxicating phenomenon.It adds
Brown stain mostly occurs for rear Ben Shengyi, in order to improve this case, after 5 generation of shoot proliferation, fluid nutrient medium can be used to cultivate, it is green
Bamboo multiplication is fast, and growth is fast, the less generation of browning.
1.2.2 condition of culture：Optical culture cycle 10h~12h, 24 DEG C~28 DEG C of temperature, intensity of illumination 2000lux~
3000lux.Illumination is strong, is conducive to the normal leaf growth of green bamboo.
1.2.3 often for cultivation cycle be 25d.
1.3 clone culture of rootage.
1.3.1 the prescription of rooting medium of green bamboo is to use 1/2MS+IBA0.2mgL-1+ root sun 15ppm+AC0.1mg
L-1+ sucrose 20gL-1+ agar 6gL-1, pH value 5.8.1/2MS is conducive to Furcation defects and seedling growth, and root is thin, rooting rate
For 60%.
1.3.2 condition of culture：Under the conditions of 28 DEG C~30 DEG C, carry out 8h~10h optical cultures, take root the cycle for 15d~
25d。
1.3.3 choose cane to lead to directly, blade is unfolded, and an old one new bud point plant of plant band carries out culture of rootage.
1.4 bottle seedling hardenings and transplanting
1.4.1 the tissue-cultured seedling taken root is moved on into hardening 15~20 days in the glasshouse that degree of shading is 50%, takes out tissue culture
Seedling rinses the culture medium of base portion well with tap water, is put into 0.1~0.5gL-1Liquor potassic permanganate in impregnate 1~3min,
After being rinsed one time with tap water again or impregnated base portion 10-15 seconds with 500 times of carbendazim, wet towel moisturizing is covered in case transplanting.
Before tissue culture transplantation of seedlings to nutrient bag, first with 3~5gL-1Disinfecting solution of potassium permanganate be equipped with matrix nutrient bag, then will cleaning
Tissue-cultured seedling afterwards is directly transplanted in nutrient bag.It drenches in time after transplanting root water, and covers plastic film.It gradually beats after a week
Plastic film is opened, while pays attention to the control of humiture.
1.4.2 transplanting time annual March to June and September are to November.The mixing that matrix is made of thin river sand, peat soil
Object, quality proportioning 5:5.
1.4.3 managed after transplanting, be most to pay attention to water and fertilizer management in preceding 30d, keep ventilated, moisturizing work especially weighs
It will.Changed according to environment temperature, humidity, adjust temperature at any time at 26 ± 2 DEG C, humidity is in 85% scope.It transplants after two weeks, weekly
Periodically spray NITROGEN IN LOW CONCENTRATION phosphorus potassium complex fertilizer (400~500ppm) and foliar fertilizer.Depending on disease, insect pest occurrence degree, periodically sprinkle and kill
Microbial inoculum and insecticide.Seedling stage needs to shade, and avoids burning sun exposure, pays attention to ventilation and water and fertilizer management work.
1.4.4 quality requirement：Survival rate can be 85% or so.It goes up a hill when the seedling length in nutrient bag to 15cm can go out garden
Afforestation is cultivated as mother plant for cutting.
Embodiment 2
The induction childrenization culture of 2.1 green bamboo clone explants.
2.1.1 select the adult tree carpentery workshop of excellent Genetic Performance type maternal for tissue cultures.During March~September, explant
Disinfecting for body is afternoon in fine day, and from elite plant base portion, it is explant to choose and give birth to rudiment bar then.
2.1.2 explant is cut into the stem section of 1 axillary bud of band with after twice of 75% alcohol wipe.With the sterilizing of 5g/L it is net or
84 medicining liquid dipping 5min, put and half an hour are rinsed in flowing water.
2.1.3 following operation is aseptically carried out：(1) after sterile water wash 2 times, with 75% alcohol disinfecting 30s,
With aseptic water washing 3 times；(2) 3min is impregnated with the vibration of 0.2%Hgcl2 solution, with aseptic water washing 3 times；(3) again with 0.2%
Hgcl2 solution left standstills impregnate 5~7min, and processing time is different due to material tender degree always, with aseptic water washing 5~7 times.
2.1.4 the Fiber differentiation based formulas of bud is 3/4MS+6-BA 4mgL-1+KT1mg·L-1+ sucrose 30gL-1+ fine jade
Fat 5gL-1.Its pH value is 5.8.Hormone is moderate, and inductivity 85% is also beneficial to Multiple Buds and is formed.Sterilizable material is inoculated with
Onto bud inducement cultivation base, through bud clump can be formed after subculture 3 times.
Condition of culture：Light culture, 26 DEG C~28 DEG C of cultivation temperature；Temperature is high, is conducive to green bamboo sprouting and sprouts and normal life
It is long.
2.2 clone bud shoot proliferation cultures.
2.2.1 minimal medium is MS, adjusts its a great number of elements, hormone combination etc..The subculture multiplication medium of green bamboo is matched somebody with somebody
Side is improvement MS (wherein a great number of elements reduces to 1/2)+6-BA2mgL-1+KT 0.8mg·L-1+NAA0.2mg·L-1+ sucrose
30g·L-1+ agar 5gL-1+ coconut palm breast 100ml.L-1, pH value 5.8.Hormone is moderate, and proliferation rate is 2 times, and robust seedling of growing thickly carries
A small amount of callus.More be not in vitreous shoot with MS is improved than common MS.Agar solidified culture medium is commonly used to cultivate plant material
The method of material, easy although method equipment is simple, Nutrient distribution is uneven, and the speed of growth is unbalanced, and often has browning poisoning now
As occurring.Along with brown stain mostly occurs for rear Ben Shengyi, in order to improve this case, after 5 generation of shoot proliferation, liquid training can be used
Foster base is cultivated, and green bamboo multiplication is fast, and growth is fast, browning less generation.
2.2.2 condition of culture：Optical culture cycle 10h~12h, 24 DEG C~28 DEG C of temperature, intensity of illumination 2000lux~
3000lux.Illumination is strong, is conducive to the normal leaf growth of green bamboo.
2.2.3 often for cultivation cycle be 25d.
2.3 clone culture of rootage.
2.3.1 the prescription of rooting medium of green bamboo is to use 1/2MS+IBA0.4mgL-1+ root sun 25ppm+AC0.1mg
L-1+ sucrose 20gL-1+ agar 6gL-1, pH value 5.8.1/2MS is conducive to Furcation defects and seedling growth, and root is thicker, root system
Uniformly, rooting rate 72%.
2.3.2 condition of culture：Under the conditions of 28 DEG C~30 DEG C, 8h~10h optical cultures, cycle 15d~25d of taking root are carried out.
2.3.3 cane is chosen to lead to directly, what blade was unfolded, an old one new bud point plant of plant band carries out culture of rootage.
2.4 bottle seedling hardenings and transplanting
2.4.1 the tissue-cultured seedling taken root is moved on into hardening 15~20 days in the glasshouse that degree of shading is 50%, takes out tissue culture
Seedling rinses the culture medium of base portion well with tap water, is put into 0.1~0.5gL-1Liquor potassic permanganate in impregnate 1~3min,
After being rinsed one time with tap water again or impregnated base portion 10-15 seconds with 500 times of carbendazim, wet towel moisturizing is covered in case transplanting.
Before tissue culture transplantation of seedlings to nutrient bag, first with 3~5gL-1Disinfecting solution of potassium permanganate be equipped with matrix nutrient bag, then will cleaning
Tissue-cultured seedling afterwards is directly transplanted in nutrient bag.It drenches in time after transplanting root water, and covers plastic film.It gradually beats after a week
Plastic film is opened, while pays attention to the control of humiture.
2.4.2 transplanting time annual March to June and September are to November.The mixing that matrix is made of thin river sand, peat soil
Object, quality proportioning 5:5.
2.4.3 managed after transplanting, be most to pay attention to water and fertilizer management in preceding 30d, keep ventilated, moisturizing work especially weighs
It will.Changed according to environment temperature, humidity, adjust temperature at any time at 26 ± 2 DEG C, humidity is in 85% scope.It transplants after two weeks, weekly
Periodically spray NITROGEN IN LOW CONCENTRATION phosphorus potassium complex fertilizer (400~500ppm) and foliar fertilizer.Depending on disease, insect pest occurrence degree, periodically sprinkle and kill
Microbial inoculum and insecticide.Seedling stage needs to shade, and avoids burning sun exposure, pays attention to ventilation and water and fertilizer management work.
2.4.4 quality requirement：Survival rate can be 85% or so.It goes up a hill when the seedling length in nutrient bag to 15cm can go out garden
Afforestation is cultivated as mother plant for cutting.
Embodiment 3
The induction childrenization culture of 3.1 green bamboo clone explants.
3.1.1 select the adult tree carpentery workshop of excellent Genetic Performance type maternal for tissue cultures.During March~September, explant
Disinfecting for body is afternoon in fine day, and from elite plant base portion, it is explant to choose and give birth to rudiment bar then.
3.1.2 explant is cut into the stem section of 1 axillary bud of band with after twice of 75% alcohol wipe.With the sterilizing of 5g/L it is net or
84 medicining liquid dipping 10min, put and half an hour are rinsed in flowing water.
3.1.3 following operation is aseptically carried out：(1) after sterile water wash 2 times, with 75% alcohol disinfecting 30s,
With aseptic water washing 3 times；(2) 30min is impregnated with the vibration of 0.2%Hgcl2 solution, with aseptic water washing 3 times；(3) again with 0.2%
Hgcl2 solution left standstills impregnate 5~7min, and processing time is different due to material tender degree always, with aseptic water washing 5~7 times.
3.1.4 the Fiber differentiation based formulas of bud is 3/4MS+6-BA 5mgL-1+KT1.5mg·L-1+ sucrose 30gL-1+
Agar 5gL-1+TDZ0.5g·L-1.Its pH value is 5.8.Hormone-content is relatively high, and inductivity, which increases to 90%, TDZ, to be had very
Strong cell division activity can promote the regeneration and breeding of bud, be also beneficial to Multiple Buds and be formed.Sterilizable material is inoculated into bud
On inducing culture, through bud clump can be formed after subculture 3 times.
Condition of culture：Light culture, 26 DEG C~28 DEG C of cultivation temperature；Temperature is high, is conducive to green bamboo sprouting and sprouts and normal life
It is long.
3.2 clone bud shoot proliferation cultures.
3.2.1 minimal medium is MS, adjusts its a great number of elements, hormone combination etc..The subculture multiplication medium of green bamboo is matched somebody with somebody
Fang Weiwei improves MS (wherein a great number of elements reduces to 1/2)+6-BA2.5mgL-1+KT1mg·L-1+NAA0.4mg·L-1+ sucrose
30g·L-1+ agar 5gL-1+ coconut palm breast 100ml.L-1+CPPU0.5g·L-1, pH value 5.8.Hormone is high, and callus hyperplasia is bright
Aobvious, invalid bud can also increase, and can be spaced using a CPPU0.5gL-1, promote cell division, enhance the activity of bud.Because
CPPU is one kind that activity is most strong in the ureas basic element of cell division, so cannot be used continuously.More will not with improvement MS than common MS
There is vitreous shoot.Agar solidified culture medium is commonly used to cultivate the method for vegetable material, it is easy although method equipment is simple, it supports
It is unevenly distributed, the speed of growth is unbalanced, and often has browning intoxicating phenomenon.Along with mostly for rear Ben Shengyi occur brown stain,
In order to improve this case, after 5 generation of shoot proliferation, fluid nutrient medium can be used to cultivate, green bamboo multiplication is fast, and growth is fast, browning
Less generation.
3.2.2 condition of culture：Optical culture cycle 10h~12h, 24 DEG C~28 DEG C of temperature, intensity of illumination 2000lux~
3000lux.Illumination is strong, is conducive to the normal leaf growth of green bamboo.
3.2.3 often for cultivation cycle be 25d.
3.3 clone culture of rootage.
3.3.1 the prescription of rooting medium of green bamboo is to use 1/2MS+IBA0.6mgL-1+ root sun 35ppm+AC0.1mg
L-1+ sucrose 20gL-1+ agar 6gL-1, pH value 5.8.1/2MS be conducive to Furcation defects and seedling growth, hormone coca slightly compared with
It is crisp, rooting rate 80%.
3.3.2 condition of culture：Under the conditions of 28 DEG C~30 DEG C, 8h~10h optical cultures, cycle 15d~25d of taking root are carried out.
3.3.3 cane is chosen to lead to directly, what blade was unfolded, an old one new bud point plant of plant band carries out culture of rootage.
3.4 bottle seedling hardenings and transplanting
3.4.1 the tissue-cultured seedling taken root is moved on into hardening 15~20 days in the glasshouse that degree of shading is 50%, takes out tissue culture
Seedling rinses the culture medium of base portion well with tap water, is put into 0.1~0.5gL-1Liquor potassic permanganate in impregnate 1~3min,
After being rinsed one time with tap water again or impregnated base portion 10-15 seconds with 500 times of carbendazim, wet towel moisturizing is covered in case transplanting.
Before tissue culture transplantation of seedlings to nutrient bag, first with 3~5gL-1Disinfecting solution of potassium permanganate be equipped with matrix nutrient bag, then will cleaning
Tissue-cultured seedling afterwards is directly transplanted in nutrient bag.It drenches in time after transplanting root water, and covers plastic film.It gradually beats after a week
Plastic film is opened, while pays attention to the control of humiture.
3.4.2 transplanting time annual March to June and September are to November.The mixing that matrix is made of thin river sand, peat soil
Object, quality proportioning 5:5.
3.4.3 managed after transplanting, be most to pay attention to water and fertilizer management in preceding 30d, keep ventilated, moisturizing work especially weighs
It will.Changed according to environment temperature, humidity, adjust temperature at any time at 26 ± 2 DEG C, humidity is in 85% scope.It transplants after two weeks, weekly
Periodically spray NITROGEN IN LOW CONCENTRATION phosphorus potassium complex fertilizer (400~500ppm) and foliar fertilizer.Depending on disease, insect pest occurrence degree, periodically sprinkle and kill
Microbial inoculum and insecticide.Seedling stage needs to shade, and avoids burning sun exposure, pays attention to ventilation and water and fertilizer management work.
3.4.4 quality requirement：Survival rate can be 85% or so.It goes up a hill when the seedling length in nutrient bag to 15cm can go out garden
Afforestation is cultivated as mother plant for cutting.
Although specific embodiments of the present invention have been described above, those familiar with the art should manage
Solution, our described specific embodiments are merely exemplary rather than for the restriction to the scope of the present invention, are familiar with this
The equivalent modification and variation that the technical staff in field is made in the spirit according to the present invention, should all cover the present invention's
In scope of the claimed protection.
Claims (7)
1. a kind of green bamboo tissue culture culture medium, it is characterised in that：Including following media：
(1) bud inducement cultivation base：3/4MS+6-BA 3-5mg·L-1+KT 0.5-1.5mg·L-1+TDZ 0.5g·L-1+ sucrose
30g·L-1+ agar 5gL-1+ coconut palm breast 100mlL-1；
(2) bud subculture multiplication medium：1/2MS+6-BA 1.5-2.5mg·L-1+KT 0.5-1mg·L-1+NAA 0.1-
0.4mg·L-1+ sucrose 30gL-1+ agar 5gL-1+ coconut palm breast 100ml.L-1+CPPU0-0.5g·L-1；Wherein CPPU intervals make
With every 5 generation is using once；
(3) root media：1/2MS+IBA 0.2-0.6mg·L-1+ root sun 15-35ppm+AC 0.1mgL-1+ sucrose
20g·L-1+ agar 6gL-1。
2. a kind of method for tissue culture of green bamboo, it is characterised in that：Include the following steps：
(1) explant selects
Maternal by tissue cultures of adult tree carpentery workshop, during March~September, it is explant to choose and give birth to rudiment bar then；
(2) sterilize
Explant is cut into the stem section of 1 axillary bud of band, is then subsequently disinfected with after 75% alcohol wipe；
(3) bud induces
The inducing culture of bud is 3/4MS+6-BA 3-5mgL-1+KT 0.5-1.5mg·L-1+TDZ 0.5g·L-1+ sucrose
30g·L-1+ agar 5gL-1+ coconut palm breast 100mlL-1Culture medium, pH value 5.8；
Condition of culture：Light culture, 26 DEG C~28 DEG C of cultivation temperature；
(4) bud shoot proliferation culture
The formula 1/2MS+6-BA 1.5-2.5mgL of bud subculture multiplication medium-1+KT 0.5-1mg·L-1+NAA 0.1-
0.4mg·L-1+ sucrose 30gL-1+ agar 5gL-1+ coconut palm breast 100ml.L-1+CPPU0-0.5g·L-1, pH value 5.8；Wherein
CPPU intervals use, and every 5 generation is using once；
Condition of culture：Daily continuous illumination 10h~12h, 24 DEG C~28 DEG C of cultivation temperature, intensity of illumination 2000lux~
3000lux；After 5 generation of shoot proliferation, fluid nutrient medium is converted into cultivate；
(5) culture of rootage
The formula of root media：1/2MS+IBA 0.2-0.6mg·L-1+ root sun 15-35ppm+AC 0.1mgL-1+ sucrose
20g·L-1+ agar 6gL-1, pH value 5.8；
Condition of culture：Daily continuous illumination 8h~10h, 28 DEG C~30 DEG C of cultivation temperature, intensity of illumination 1000lux~2000lux；
(6) bottle seedling hardening
The tissue-cultured seedling taken root is shifted into hardening 15~20 days in greenhouse, tissue-cultured seedling is taken out, rinses the training of base portion well with tap water
Base is supported, is put into 0.1~0.5gL-1Liquor potassic permanganate in impregnate 1~3min, then rinsed with tap water one time or with 500 times
Carbendazim impregnated base portion after 10-15 seconds, covered wet towel moisturizing in case transplanting；
(7) transplant
Tissue-cultured seedling is transplanted in the nutrient bag equipped with matrix, root water of drenching in time after transplanting, and covers plastic film, one week
After open plastic film, managed after being transplanted；Transplanting time is in annual March-June or September-November.
3. the method for tissue culture of green bamboo according to claim 2, it is characterised in that：The step of step (2) described disinfection, wraps
It includes：It is net or 84 medicining liquid dipping 5min, tap water rinse half an hour with the sterilizing of 5g/L, it then aseptically carries out following
Operation：
A. after sterile water wash 2 times, with 75% alcohol disinfecting 10s, with aseptic water washing 3 times；
B. 0.2%Hg Cl are used230min is impregnated in solution vibration, with aseptic water washing 3 times；
C. 0.2%HgCl is used again2Solution left standstill impregnates 5~7min, and processing time is different due to material tender degree always, is rushed with sterile water
It washes 5~7 times.
4. the method for tissue culture of green bamboo according to claim 2, it is characterised in that：Step (6) described greenhouse is degree of shading
For 50% glasshouse.
5. the method for tissue culture of green bamboo according to claim 2, it is characterised in that：Step (7) described matrix is by thin river
Husky, peat soil composition mixture, quality proportioning 5:5.
6. the method for tissue culture of green bamboo according to claim 2, it is characterised in that：Step (7) described transplanting, in tissue-cultured seedling
Before transplanting to nutrient bag, first with 3~5gL-1Disinfecting solution of potassium permanganate be equipped with matrix nutrient bag.
7. the method for tissue culture of green bamboo according to claim 2, it is characterised in that：It manages, wraps after step (7) described transplanting
Include following steps：
In preceding 30d controlled at 26 ± 2 DEG C, humidity 85%；Transplanting after two weeks, periodically sprays weekly 400~500ppm nitrogen
Phosphorus potassium complex fertilizer and foliar fertilizer.Depending on disease, insect pest occurrence degree, fungicide and insecticide are periodically sprinkled；After two months together with nutrition
Bag moves to outdoor；Seedling stage needs to shade, and avoids burning sun exposure, pays attention to ventilation and water and fertilizer management work.
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