CN114946655A - Liupao tea seedling tissue culture method - Google Patents
Liupao tea seedling tissue culture method Download PDFInfo
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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
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G17/00—Cultivation of hops, vines, fruit trees, or like trees
- A01G17/005—Cultivation methods
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G31/00—Soilless cultivation, e.g. hydroponics
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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
- A01H4/002—Culture media for tissue culture
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Abstract
The invention discloses a tissue culture method for Liupu tea seedlings, which mainly comprises the steps of culturing Liupu tea seeds or terminal buds or axillary buds on a germination culture medium to grow germinated plantlets, inoculating the germinated plantlets on a cluster bud induction culture medium for propagation, carrying out propagation subculture, growing cluster buds and adventitious buds to obtain rootless seedlings, carrying out adventitious root induction culture on the rootless seedlings, carrying out hardening acclimation and transplanting for planting. The culture medium of the invention is a germination culture medium, and 6-BA and NAA or GA are added on MS; adding 6-BA, NAA and TDZ into MS in the cluster bud induction culture medium, and adding 6-BA, NAA and IBAK into MS in the adventitious root induction culture medium. The Chinese Liupao tea tissue culture seedling has the advantages of thick leaves, thick stems, low mutation rate and high multiplication coefficient, the survival rate of the acclimatized seedling reaches more than 90 percent, and the seedling culture method is suitable for providing seedlings planted in the Chinese Liupao tea.
Description
Technical Field
The invention belongs to the technical field of plant propagation, and particularly relates to a Liupu tea seedling tissue culture method.
Background
Liupao tea (Camellia nitidissima cv. Liubaocha) Theaceae Camellia belongs to evergreen shrub or small arbor, is originally produced in Liupao town of Sterculia urensis in Guangxi of China and peripheral areas thereof, is one of Chinese dark tea representatives, can be classified into post-fermented tea in the Liupao tea fermentation process, is named as the Liupao tea originally produced in Liupao town of Xanthium county in Guangxi Sterculia city, and is named as the old people by the quality characteristics of 'red, thick, old and mellow'. The production history can be traced back to 1500 years. Mainly contains 74 and 80 volatile aroma components and other components, wherein the contents of Zn, Cu, Sr, Ni and Se are higher, and Liupao tea medicinal and edible products are utilized in the traditional food therapy of China.
At present, more than 90% of Liupu tea raw materials in domestic markets come from Guangxi, and Liupu tea is produced in a core production area in Liupu town. Therefore, the method for artificially propagating and raising seedlings is very necessary for protecting the wild Liupu tea resources and meeting the requirement of large-scale production. The Liupao tea trees are good in whole body, and the Liupao tea is also a tree species with extremely high economic value for treating rocky mountain areas and wastelands and producing forestry besides the root, stem and leaf medicinal value of the Liupao tea. However, the seedling raising of the Liupu tea is a bottleneck for inhibiting the Liupu tea industry, and the application and popularization of the Liupu tea are limited due to the problems that the Liupu tea belongs to one of the tree species which are difficult to root by cutting, the common cutting is difficult to root, the seed germination rate is low and the like.
At present, the planting and cultivation of the Chinese Liupao tea mainly adopts seed seedlings for seedling culture, because the tea trees frequently pick off buds, the fructification is extremely little, the conventional field seed germination rate is less than 4%, the growth period is long, the occupied area is large, and the efficient and rapid large-scale industrialized planting and cultivation is difficult to realize.
Disclosure of Invention
The invention aims to: aiming at the problems, in order to solve the problems of optimized improved variety, stable improved variety character, high breeding efficiency, large-scale production and the like, the tissue culture method for the Liupu tea seedlings is provided, the culture method comprises cluster bud induction culture and adventitious root induction culture, after several rounds of subculture, the buds grow normally, the hardening-off survival rate reaches more than 90%, and the survival rate of the Liupu tea seedlings can be remarkably improved; and can solve the problem that the existing seedling of the Chinese Liupao tea seed cannot be efficiently and quickly planted and cultivated in a large-scale industrialized way.
In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:
a Liupu tea seedling tissue culture method comprises pretreatment, cluster bud induction culture, adventitious root induction culture, hardening seedling domestication and transplanting planting; the tissue culture method comprises the following steps:
(1) pretreatment: inoculating the seeds, terminal buds or axillary buds of the Liupu tea into test tubes or culture bottles containing a germination culture medium after aseptic treatment for germination culture, inoculating one seed, terminal bud or axillary bud in each test tube or culture bottle, and then placing the test tubes or culture bottles in a culture room;
(2) and (3) inducing and culturing cluster buds: transferring the germinated plantlets obtained by culturing in the germination culture medium into a cluster bud induction culture medium for culturing, and performing propagation subculture after 5-6 weeks to obtain rootless plantlets;
(3) adventitious root induction culture: transferring the rootless seedlings into an adventitious root induction culture medium, and culturing for 5-7 weeks;
(4) hardening and domesticating seedlings: removing the seedlings with the root length of 0.4-1.5cm obtained by adventitious root induction culture from a culture room, half opening a bottle cover, placing in a sun-shading greenhouse for 5-10d, transplanting into a peat and perlite mixed matrix, culturing for 1-5 weeks, after the plants develop 3-5 functional leaves, planting in a nutrition cup, and placing in an outdoor seedling field for continuous culture;
(5) transplanting and planting: after the cluster roots and the second-level root systems are attached with mud and are solid, planting the cluster roots and the second-level root systems in a tea production area in spring and summer or winter and spring, digging ridges, removing nutrition cups during planting, covering soil and backfilling, slightly pressing soil after root lifting, watering root fixing water during dry days, and determining whether to water or open drip irrigation water retention according to the drought conditions of the tea planting area.
Preferably, in step (1), the aseptic treatment is: using a soft brush to dip water solution added with a small amount of detergent to lightly wipe seeds or branches and axillary buds thereof, washing with sterile water for 3-4 times, sterilizing with 65-75% alcohol by volume fraction concentration for 20-40s in a super-clean workbench, washing with sterile water for 3-4 times, sterilizing with 0.1-0.2% mercury bichloride for 10-18min, and washing with sterile water for 3-5 times.
Preferably, in step (1), the germination medium is: MS basal medium +6-BA 2.0 mg.L -1 +NAA 0.2mg·L -1 + sucrose 30 g.L -1 + agar powder 5.3 g.L -1 pH 5.8-7.0; or the germination culture medium is MS basal culture medium +6-BA 2.0 mg.L -1 +GA 5-30mg·L -1 + sucrose 20 g.L -1 + agar powder 5.3 g.L -1 ，pH 5.8-7.0。
Preferably, in step (2), the clumpy bud induction medium is: MS basic culture medium +6-BA 1.0-2.5 mg.L -1 +NAA 0.1-0.5mg·L -1 +TDZ 0.1-4mg·L -1 + sucrose 30 g.L -1 + agar 5.3 g.L -1 ，pH 5.8-7.0。
Preferably, in step (3), the adventitious root induction medium is: 1/2MS basic culture medium +6-BA 1.0-2.5 mg.L -1 +NAA 0.1-0.5mg·L -1 +IBAK 0.1-0.2mg·L -1 + sucrose 30 g.L -1 + agar 5.3 g. L -1 ，pH 5.8-7.0。
Preferably, the conditions of germination culture are that the temperature is 23-27 ℃, the illumination time is 12-14h/d, and the illumination intensity is 1500-; the conditions of the cluster bud induction culture are that the temperature is 23-27 ℃, the illumination time is 14-16h/d, and the illumination intensity is 1500-; the adventitious root induction culture condition is that the temperature is 23-27 ℃, the illumination time is 10-12h/d, and the illumination intensity is 2000-25001 ux.
Preferably, in step (2), the height of the germinated plantlet is 2.0-6.0cm, and after being cut into 10-20mm long stem sections with leaves, the germinated plantlets are transferred into a cluster bud induction culture medium for induction culture.
In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:
(1) the invention takes the high-quality Liupu tea seeds or terminal buds or axillary buds in the core production area of the Chinese Liupu tea as explants, prepares the special culture medium for induction through screening of the culture medium, successfully establishes a complete system for tissue culture and seedling culture of the Chinese Liupu tea, and provides a reliable technology and method for rapid propagation of mass tissue culture of the Chinese Liupu tea.
(2) By adopting a tissue culture method, the propagation coefficient reaches 6-7 times, a large number of high-quality seedlings can be provided in a short period, and the popularization and application speed of the Liupu tea is increased; after induced proliferation, most of the Liupu tea adventitious bud leaves are thick and thick, the stems are thick, and after several rounds of subculture, the buds grow normally.
(3) Compared with the similar method, the method has great advantages, such as the operation is easy for cutting seedling raising, but the rooting of the branch cutting propagation is difficult, and the survival rate is low; in the seedling raising method, because the Liupao tea seeds are small, the seed coats are hard, and the germination rate in the conventional field is less than 4 percent, a large amount of seeds are wasted, and the Liupao tea seedlings in forestation are short; the invention adopts a tissue culture method to breed the seedlings, realizes an efficient cultivation management technology, can realize tree planting in one year and effect in the second year, provides a quick and effective way for large-scale Liupao tea breeding, and has strong practical significance in factory large-scale seedling production.
(4) The Liupao tea tissue culture seedling has the advantages of thick leaves and thick stems, low mutation rate and high multiplication coefficient, the survival rate of the hardening seedlings reaches over 90 percent, and the Liupao tea tissue culture seedling is suitable for providing seedlings planted in the Liupao tea; the invention keeps the excellent characteristics of the parents by asexual breeding, has low cost and little pollution, can be produced in four seasons and is suitable for large-scale asexual seedling culture.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail below with reference to preferred embodiments. It should be noted, however, that the numerous details set forth in the description are merely for the purpose of providing the reader with a thorough understanding of one or more aspects of the present invention, which may be practiced without these specific details.
In the invention: 6-BA is 6-benzylamino adenine, NAA is alpha-naphthylacetic acid, IBAK is potassium indolebutyrate, GA is gibberellin, and TDZ is N-phenyl-N-1, 2, 3-thiadiazole-5-urea.
Example 1
A Liupao tea seedling tissue culture method comprises the following steps:
(1) pretreatment: picking up top buds or axillary buds of healthy and plump Chinese Liupu tea, dipping the materials with a soft brush and adding a small amount of water solution of detergent to lightly wipe seeds or branches and axillary buds of the seeds or branches, washing the seeds or branches with tap water for 1h, sterilizing the seeds or branches and the axillary buds with 70% alcohol by volume fraction concentration for 20s in a super clean workbench, washing the seeds or branches with sterile water for 4 times, sterilizing the seeds or branches with 0.1% mercuric chloride by mass fraction concentration for 18min, and washing the seeds or branches and the axillary buds with the sterile water for 5 times; inoculating the material subjected to surface sterilization treatment into test tubes containing a germination culture medium for germination culture, inoculating one terminal bud or axillary bud in each test tube, and then placing the test tubes into a culture chamber, wherein the germination culture medium is as follows: MS basal medium +6-BA 2.0 mg.L -1 +NAA0.2mg·L -1 + sucrose 30 g.L -1 + agar powder 5.3 g.L -1 pH 5.8; culturing at 25 deg.C, illumination time 12h/d, illumination intensity 15001ux for 5 weeks, and removing contaminated material;
(2) and (3) inducing and culturing cluster buds: taking a germinated plantlet with the height of 5.0cm, cutting the germinated plantlet into stem sections with axillary buds with the length of 15mm, and transferring the stem sections to a cluster bud induction culture medium for induction culture, wherein the cluster bud induction culture medium comprises: MS basic culture medium +6-BA 1.0 mg.L -1 +NAA 0.3mg·L -1 +TDZ 0.1mg·L -1 + sucrose 30 g.L -1 + agar 5.3 g.L -1 pH 5.8; the culture conditions are that the temperature is 25 ℃, the illumination time is 14h/d, and the illumination intensity is 20001 ux; after 5 weeks, carrying out propagation subculture to obtain a rootless seedling;
(3) adventitious root induction culture: transferring the rootless seedling into adventitious root induction culture medium, and performing adventitious root induction cultureThe nutrient base is: 1/2MS basic culture medium +6-BA 1.0 mg.L -1 +NAA 0.3mg·L -1 +IBAK 0.2mg·L -1 + sucrose 30 g.L -1 + agar 5.3 g.L -1 pH 5.8; culturing at 25 deg.C for 10h/d with illumination intensity of 20001ux for 6 weeks;
(4) hardening and domesticating seedlings: removing the seedlings with the root length of 1.0cm obtained by adventitious root induction culture from a culture chamber, half opening a bottle cover, placing the seedlings in a sun-shading greenhouse for 8 days, transplanting the seedlings into a peat and perlite mixed matrix, culturing for 3 weeks under the conditions of 20 ℃ temperature, 65% humidity and natural illumination, after 4 functional leaves develop, planting the plants into a 9 x 12cm nutrition cup, placing the plants into an outdoor seedling field, and continuously culturing for 4 weeks, wherein the survival rate of the plants can reach 95%, and after 4 functional leaves develop, planting the plants into the nutrition cup, placing the plants into the outdoor seedling field, and continuously culturing;
(5) transplanting and planting: after the cluster roots and the secondary roots are attached with mud and are fruited, planting the cluster roots and the secondary roots in a tea production area in spring and summer or winter and spring, digging ridges according to 20-15 cm, removing nutrition cups during planting, covering soil and backfilling, lightly pressing soil after root extraction, watering root fixing water during dry weather, and then determining whether to water or open drip irrigation water retention according to the drought condition of the tea planting area.
Example 2
A Liupu tea seedling tissue culture method comprises the following steps:
(1) pretreatment: collecting healthy and plump Liupao tea seed materials, removing episperm, sterilizing with 65% alcohol for 40s on a superclean bench, washing with sterile water for 3 times, sterilizing with 0.2% mercury bichloride for 10min, and washing with sterile water for 3 times; inoculating the material subjected to surface sterilization treatment into a culture bottle containing a germination culture medium for germination culture, inoculating one seed into each culture bottle, and then placing the culture bottle into a culture chamber, wherein the germination culture medium is as follows: MS basal medium +6-BA 2.0 mg.L -1 +NAA0.1mg·L -1 + sucrose 20 g.L -1 + agar powder 5.3 g.L -1 Placing the mixture on a germination culture medium with the pH of 7.0 in a culture room for dark culture for 5 weeks, and counting the germination rate by taking the grown young leaves as the germination standard;
(2) and (3) inducing and culturing cluster buds: the seedlings are taken to be germinated when the height of the seedlings is 2.0cmCutting the seedling into stem sections with axillary buds and leaves with the length of 10mm, transferring the stem sections into a cluster bud induction culture medium for induction culture, wherein the cluster bud induction culture medium comprises: MS basal medium +6-BA 2.5 mg.L -1 +NAA 0.1mg·L -1 +TDZ 4mg·L -1 + sucrose 30 g.L -1 + agar 5.3 g. L -1 pH 7.0; the culture conditions are that the temperature is 27 ℃, the illumination time is 16h/d, the illumination intensity is 15001ux, and after 6 weeks, the propagation subculture is carried out to obtain the rootless seedlings;
(3) adventitious root induction culture: transferring the rootless seedlings into an adventitious root induction culture medium, wherein the adventitious root induction culture medium comprises: 1/2MS basic culture medium +6-BA 2.5 mg.L -1 +NAA0.1mg·L -1 +IBAK0.2mg·L -1 + 0.5g/L of active carbon + 30 g.L of sucrose -1 + agar 5.3 g.L -1 pH 7.0; culturing at 27 deg.C, illumination time 12h/d, illumination intensity 25001ux for 7 weeks;
(4) hardening and domesticating seedlings: the seedlings with the root length of 0.4cm obtained by adventitious root induction culture are moved out of a culture room, after a bottle cover is half opened, the seedlings are placed in a sunshade greenhouse for 10 days and then are transplanted into a peat and perlite mixed matrix, the seedlings are cultured for 5 weeks under the conditions of 16 ℃ of temperature, 60% of humidity and natural illumination, and the survival rate of the plants can reach 92%; after the plants develop 3 functional leaves, the plants are planted into a nutrition cup and placed in an outdoor seedling field for continuous culture;
(5) transplanting and planting: after the cluster roots and the secondary roots are attached with mud and are fruited, planting the cluster roots and the secondary roots in a tea production area in spring and summer or winter and spring, digging ridges according to 20-15 cm, removing nutrition cups during planting, covering soil and backfilling, lightly pressing soil after root extraction, watering root fixing water during dry weather, and then determining whether to water or open drip irrigation water retention according to the drought condition of the tea planting area.
Example 3
A Liupu tea seedling tissue culture method comprises the following steps:
(1) pretreatment: picking the top bud or axillary bud material of healthy and plump Chinese Liupu tea, lightly wiping branches and axillary buds thereof with water solution added with a small amount of detergent by using a soft brush, washing with tap water for 1.5h, sterilizing in an ultraclean workbench for 30s with 75% alcohol by volume fraction concentration, washing with sterile water for 4 times, sterilizing with 0.2% mercury bichloride for 8min,washing with sterile water for 5 times; inoculating the surface sterilized material into test tubes containing germination culture medium for germination culture, and inoculating one terminal bud L in each test tube -1 Or axillary buds, and then placing in a culture chamber, wherein the germination culture medium is as follows: MS basal medium +6-BA 2.0 mg.L -1 +NAA0.2mg·L -1 + sucrose 30 g.L -1 + agar powder 5.3 g.L -1 pH 5.8; culturing for 5 weeks at 27 deg.C, 16h/d illumination time and 20001ux illumination intensity, and removing the contaminated material;
(2) and (3) inducing and culturing cluster buds: taking a germinated plantlet with the height of 6.0cm, cutting the germinated plantlet into stem sections with axillary buds with the length of 20mm, and transferring the stem sections to a cluster bud induction culture medium for induction culture, wherein the cluster bud induction culture medium comprises: MS basic culture medium +6-BA 1.0 mg.L -1 +NAA 0.2mg·L -1 +TDZ 0.1mg·L -1 + sucrose 30 g.L -1 + agar 5.3 g.L -1 pH 7.0; the culture conditions are that the temperature is 27 ℃, the illumination time is 16h/d, and the illumination intensity is 15001 ux; after 5 weeks, carrying out propagation subculture to obtain a rootless seedling;
(3) adventitious root induction culture: transferring the rootless seedlings into an adventitious root induction culture medium, wherein the adventitious root induction culture medium comprises: 1/2MS basic culture medium +6-BA 1.0 mg.L -1 +NAA 0.2mg·L -1 +IBAK 0.2mg·L -1 + sucrose 30 g.L -1 + agar 5.3g/L, pH 5.8; culturing at 25 deg.C, illumination time 10h/d, illumination intensity 20001ux for 6 weeks;
(4) hardening and domesticating seedlings: the seedlings with the root length of 1.5cm obtained by adventitious root induction culture are moved out of a culture room, after a bottle cover is half opened, the seedlings are placed in a sunshade greenhouse for 10 days and then are transplanted into a peat and perlite mixed matrix, the seedlings are cultured for 5 weeks under the conditions of 25 ℃ of temperature, 70% of humidity and natural illumination, and the survival rate of the plants can reach 93%; after 5 functional leaves develop, the plants are planted into a 10 x 12cm nutrition cup and placed outside a seedling field for continuous culture.
(5) Transplanting and planting: after the cluster roots and the secondary roots are attached with mud and are fruited, planting the cluster roots and the secondary roots in a tea production area in spring and summer or winter and spring, digging ridges according to the specification of 20-15 cm, removing nutrition cups during planting, covering soil and backfilling, lightly pressing soil after root extraction, watering root fixing water during dry weather, and then determining whether to water or open drip irrigation water retention according to the drought condition of the tea planting area.
Example 4
Other process parameters of the embodiment are consistent with those of embodiment 1, except that: the germination culture medium comprises: MS basal medium +6-BA 2.0 mg.L -1 +GA 5mg·L -1 + sucrose 30 g.L -1 + agar powder 5.3 g.L -1 ，pH 5.8。
After the seedling hardening and domesticating, the survival rate of the plants can reach 92 percent.
Example 5
Other process parameters of the embodiment are consistent with those of embodiment 1, except that: the germination culture medium comprises: MS basal medium +6-BA 2.0 mg.L -1 +GA 30mg·L -1 + sucrose 30 g.L -1 + agar powder 5.3 g.L -1 ，pH 7.0。
After the seedling hardening and domesticating, the survival rate of the plants can reach 90 percent.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should also be considered as the protection scope of the present invention.
Claims (9)
1. A Liupao tea seedling tissue culture method is characterized by comprising the following steps: comprises pretreatment, cluster bud induction culture, adventitious root induction culture, acclimatization and transplantation; the tissue culture method comprises the following steps: :
(1) pretreatment: inoculating the seeds, terminal buds or axillary buds of the Liupu tea into test tubes or culture bottles containing a germination culture medium after aseptic treatment for germination culture, inoculating one seed, terminal bud or axillary bud in each test tube or culture bottle, and then placing the test tubes or culture bottles in a culture room;
(2) and (3) inducing and culturing cluster buds: transferring the germinated plantlets cultured in the germination culture medium into a cluster bud induction culture medium for induction culture, and performing propagation subculture after 5-6 weeks to obtain rootless plantlets;
(3) adventitious root induction culture: transferring the rootless seedlings into an adventitious root induction culture medium, and culturing for 5-7 weeks;
(4) hardening and domesticating seedlings: removing the seedlings with the root length of 0.4-1.5cm obtained by adventitious root induction culture from a culture room, half opening a bottle cover, placing in a sun-shading greenhouse for 5-10d, transplanting into a peat and perlite mixed matrix, culturing for 1-5 weeks, after the plants develop 3-5 functional leaves, planting in a nutrition cup, and placing in an outdoor seedling field for continuous culture;
(5) transplanting and planting: after the clustered roots and the second-level root systems are attached with mud and fructified, planting the tea production areas in spring and summer or winter and spring, digging ridges, removing the nutrition cups when planting, covering soil and backfilling, lightly pressing the soil after root lifting, watering root fixing water when drought occurs, and determining whether to water or open drip irrigation water retention or spray water retention according to drought conditions and weather forecast of the tea planting areas.
2. The Liupu tea seedling tissue culture method according to claim 1, is characterized in that: in step (1), the aseptic treatment is: lightly wiping seeds or branches and axillary buds thereof with a solution prepared by dipping a soft brush with a small amount of detergent, washing with sterile water for 3-4 times, sterilizing with 65-75% alcohol by volume fraction concentration for 20-40s in a super clean bench, washing with sterile water for 3-4 times, sterilizing with 0.1-0.2% mercury bichloride for 10-18min, and washing with sterile water for 3-5 times.
3. The tissue culture method of Liupu tea seedlings according to claim 1, characterized in that: in step (1), the germination medium is: MS basal medium +6-BA 2.0 mg.L -1 +NAA 0.2mg·L -1 + sucrose 30 g.L -1 + agar powder 5.3 g.L -1 pH 5.8-7.0; or the germination culture medium is MS basal culture medium +6-BA 2.0 mg.L -1 +GA 5-30mg·L -1 + sucrose 20 g.L -1 + agar powder 5.3 g.L -1 ，pH 5.8-7.0。
4. The Liupu tea seedling tissue culture method according to claim 1, is characterized in that:
in step (2), the cluster bud induction medium is: MS basal medium +6-BA 1.0-2.5mg·L -1 +NAA 0.1-0.5mg·L -1 +TDZ 0.1-4mg·L -1 + sucrose 30 g.L -1 + agar 5.3 g.L -1 ，pH 5.8-7.0。
5. The Liupu tea seedling tissue culture method according to claim 1, is characterized in that:
in step (3), the adventitious root induction medium is: 1/2MS basic culture medium +6-BA 1.0-2.5 mg.L -1 +NAA 0.1-0.5mg·L -1 +IBAK 0.1-0.2mg·L -1 + sucrose 30 g.L -1 + agar 5.3 g.L -1 ，pH 5.8-7.0。
6. The Liupu tea seedling tissue culture method according to any one of claims 1 to 5, wherein the Liupu tea seedling tissue culture method comprises the following steps: the conditions of the germination culture are that the temperature is 23-27 ℃, the illumination time is 12-14h/d, and the illumination intensity is 1500-; the conditions for inducing and culturing the cluster buds are that the temperature is 23-27 ℃, the illumination time is 14-16h/d, and the illumination intensity is 1500-.
7. The Liupu tea seedling tissue culture method according to claim 6, is characterized in that: in the step (2), the height of the germinated plantlets is 2.0-6.0cm, the seedlings are cut into 10-20mm long single-bud stem sections with leaves, and then the stem sections are transferred into a cluster bud induction culture medium for induction culture.
8. The Liupao tea seedling tissue culture method according to claim 6, is characterized in that: in the step (3), the height of the germinated plantlets is 4.0-8.0cm, the buds are cut into bud sections with leaves and the length of the bud sections is 2-4cm, and then the bud sections are transferred into a rooting induction culture medium for induction culture. The adventitious root induction culture conditions are 23-27 ℃, the illumination time is 10-12h/d, and the illumination intensity is 2000-25001 ux.
9. The Liupu tea seedling tissue culture method according to claim 6, is characterized in that: in the step (3), the seedlings are transplanted into the peat and perlite mixed matrix and cultured for 1-5 weeks under the conditions of 16-25 ℃ of temperature, 60-70% of humidity and natural illumination.
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| CN106718890A (en) * | 2016-12-02 | 2017-05-31 | 河池学院 | A kind of method for tissue culture suitable for Chinese herbal medicine murraya paniculataJack seedling |
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