CN114766366A - Tissue culture method suitable for Liupao tea Shannu purple original seed seedlings - Google Patents
Tissue culture method suitable for Liupao tea Shannu purple original seed seedlings 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
- 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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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
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Abstract
The invention discloses a tissue culture method for preparing Liupu tea Shanjia purple original seed seedlings, which comprises aseptic seeding, callus induction culture, bud differentiation culture, cluster bud induction culture, multiplication culture, rooting culture and seedling hardening; the callus induction culture is to inoculate cotyledons and hypocotyls to a callus induction culture medium for induction culture for 30-35 days; the callus induction culture medium comprises MS, 0.1-0.8 mg/L of 6-BA and 2, 0.1-0.8 mg/L of 4-D, 25-35 g/L of sucrose and 5.0-5.8 g/L of agar, and the pH value is 6.8-7.0. The Liupao tea Shanshen purple tissue culture seedling has thick leaves, thick stems, low mutation rate, 6-7 times of multiplication coefficient and more than 90% of hardening seedling survival rate, is suitable for providing seedlings for Liupao tea planting, performs asexual propagation, maintains the excellent characteristics of the Shanshen purple parent, has low cost and little pollution, can be produced in four seasons, and is suitable for large-scale asexual seedling culture.
Description
Technical Field
The invention belongs to the technical field of plant propagation, and particularly relates to a tissue culture method suitable for Liupao tea Shannu purple original seed seedlings.
Background
Liupao tea (Camellia nitidissima cv. Liubaocha) is evergreen shrub or small arbor in Camellia of Theaceae, with oval scalded leaves, brownish black and smooth leaves, yellow spots in the middle, and reddish brown leaves. The Liupao tea extract is known to be red, thick, old and mellow, and has long and compact strip, red and thick soup color, old and thick aroma and sweet and delicious taste; the Shansill purple belongs to a variety of Liupu tea, is originally produced in Liupu town of Wuzhou in Guangxi province of China and peripheral areas thereof, is one of Chinese black tea representatives, is a national geographical sign product, is listed in a national non-material culture heritage list in the traditional manufacturing process, is a new marine silk road culture, and is a Guangxi characteristic dominant industry. Canon prefecture county journal (thirty-six years of Qingkang xi, 1697 edition) records: the Liupu tea is mainly produced in sages and does not change in taste, and has good tea color, fragrance and taste, the Liupu tea is not reputed to be inside and outside by the special areca fragrance and certain health care effect in Qingjiaqing years, the fermentation process in the Liupu tea can be classified into post-fermentation tea which is famous because the Liupu tea is originally produced in the town of Liupu county in Guomian city in Guangxi, and the Liupu tea is famous for the old people by the quality characteristics of red, thick, old and mellow. The production history can be traced back to 1500 years, the Liupao tea mainly contains 74 and 80 volatile aroma components and other components, wherein the contents of Zn, Cu, Sr, Ni and Se are high, and Liupao tea medicinal and edible products are utilized in traditional food therapy in China.
At present, more than 90% of Liupu tea raw materials in domestic markets come from Guangxi, and Liupu tea takes Liupu town as a core production area. 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 Liupao tea mainly adopts seed seedlings to grow seedlings, because the tea trees are usually subjected to bud picking, 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 quick large-scale industrial planting and cultivation are 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 character of the improved variety, efficient breeding, large-scale production and the like, the tissue culture method suitable for preparing the Liupu tea Shannu purple primordial seed seedlings is provided, the multiplication coefficient of aseptic seedlings is 3-4 times, the hardening survival rate is more than 80%, and the problem that the existing Liupu tea seed seedlings cannot be efficiently and quickly planted and cultured in a large-scale industrialized mode can be solved.
In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:
a tissue culture method for preparing Liupu tea Shanjia purple original seed germchit comprises aseptic seeding, callus induction culture, bud differentiation culture, cluster bud induction culture, proliferation culture, rooting culture and seedling hardening; the callus induction culture is to inoculate cotyledons and hypocotyls to a callus induction culture medium for induction culture for 30-35 days; the callus induction culture medium comprises MS +6-BA 0.1-0.8 mg/L +2, 4-D0.1-0.8 mg/L + sucrose 25-35 g/L + agar 5.0-5.8 g/L, and the pH value is 6.8-7.0.
Preferably, the aseptic seeding comprises the following steps:
(1) seed harvesting: harvesting mature fruits of excellent single plants in 10-12 months, selecting seeds which are large in size, full, not cracked, free of mildew and insect damage, and sowing the seeds at any time;
(2) surface sterilization: soaking the seeds in 0.1 percent neutral detergent aqueous solution for 20-30 min, and then flushing the seeds for 10-30 min under running water; soaking the seeds in 75% alcohol for 40-60 s on a superclean workbench, washing with sterile water for 3-5 times, soaking in 0.1% mercuric chloride solution for 16-20 min, and washing with sterile water for 3-5 times;
(3) seed treatment and sowing: burying the seeds in sterile river sand, moistening the sterile river sand by sterile water, covering a film to preserve moisture, and standing at the temperature of 25 +/-2 ℃ with the illumination intensity of: 1500-2000LUX, the illumination time is 14-16 h/d;
(4) obtaining an explant: after the seeds germinate, selecting sterile seedlings with 2 cotyledons and true leaves which are not developed, and cutting cotyledons and hypocotyls or cutting new buds which sprout after first, second and third times of cutting or picking tender buds of the Liupao tea outdoors in the afternoon of sunny days as explants.
Preferably, the bud differentiation culture is to inoculate the callus onto a differentiation culture medium for culture, and the culture time is 30-35 d; the differentiation medium is MS +6-BA 1-3 mg/L + NAA 0.05-0.15 mg/L + TDZ 0.01-0.02 mg/L + sucrose 20-40 g/L + agar 4.5-5.6 g/L, and the pH value is 6.8-7.0.
Preferably, the cluster bud induction culture is to inoculate the callus subjected to callus differentiation culture onto a cluster bud culture medium for culture for 30-35 days; the cluster bud culture medium is MS +6-BA 1-3 mg/L + NAA 0.05-0.15 mg/L + TDZ 0.01-0.03 mg/L + gibberellin 0.1-0.2 mg/L + sucrose 20-40 g/L + agar 4.5-6.0 g/L, and the pH value is 6.8-7.0.
Preferably, the enrichment culture is to inoculate adventitious buds with the length of about 1-2 cm after the induction of the cluster buds or cut stem sections with one bud into an enrichment culture medium for culture for 30-35 d; the enrichment medium is MS +6-BA 0.2-0.8 mg/L + NAA 0.02-0.08 mg/L + IBA 0.02-0.08 mg/L + sucrose 20-40 g/L + agar 4.5-6.0 g/L, and the pH value is 6.8-7.0.
Preferably, the rooting culture is to cut out a tissue culture bud which is strong and 3-4 cm high from the adventitious bud or the cluster bud obtained by the propagation culture, and to culture the tissue culture bud on a rooting culture medium for 20-30 days; the rooting medium is 1/2MS + 0.50-1.00 mg/L IBA + 20-40 g/L sucrose + 4.5-6.0 g/L agar, and the pH value is 6.8-7.0.
Preferably, the seedling exercising is to select tissue culture seedlings with complete roots, stems and leaves, the plant height of 3.0-4.0 cm, 2-6 leaves, more than 3-5 roots and 1-2 cm root length, the tissue culture seedlings are firstly placed in an environment with the room temperature of 15-28 ℃, the illumination intensity of 1500-3000 LUX and the relative air humidity of more than or equal to 85% for 2-7 days with a bottle, and then the bottle cover is removed and the tissue culture seedlings are continuously placed for 1-2 days for exercising.
Preferably, the culture temperature of the callus induction culture, the callus differentiation culture, the cluster bud induction culture, the proliferation culture and the rooting culture is 23-30 ℃, the illumination intensity is 1800-2000 LUX, and the photoperiod is 14-16 h.
In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:
(1) the invention takes high-quality Liupu tea Shanshen purple seeds in the Liupu tea core production area as explants, prepares special culture mediums for induction, differentiation, cluster bud culture, rooting culture and the like through the standard steps of sterile sowing, callus induction culture, callus differentiation culture, cluster bud induction, proliferation culture, rooting culture and seedling hardening and screening of the culture mediums, successfully establishes a complete system for culturing and breeding Liupu tea Shanshen purple tissues and provides a reliable technology and method for the rapid propagation of Liupu tea Shanshen purple mass tissue culture.
(2) According to the invention, by adopting a tissue culture method, the multiplication coefficient reaches 3-4 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 accelerated; after induced proliferation, most of Liupu tea adventitious bud leaves are 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 easy operation of cutting seedling, difficult rooting of branch cutting propagation and low survival rate; 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 tissue culture method is adopted to breed the Liupao tea Shannu purple seedlings, the multiplication coefficient of aseptic seedlings is more than 3-4 times, the hardening survival rate of seedlings reaches more than 80%, the efficient cultivation management technology is realized, the trees can be planted in one year, the effect can be realized in the second year, a quick and effective way is provided for large-scale Liupao tea breeding, and the tissue culture method has strong practical significance in large-scale seedling culture production in factories.
(4) The Liupao tea hill purple tissue culture seedling has the advantages of thick leaves and thick stems, low variation rate and high multiplication coefficient, and is suitable for providing seedlings for Liupao tea planting; the invention keeps the excellent characteristics of the parent through asexual propagation, 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. However, it should be noted that the numerous details set forth in the description are merely intended to provide a thorough understanding of one or more aspects of the present invention, even though such aspects of the invention 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 tissue culture method suitable for Liupu tea Shannu purple basic seed seedlings comprises the following steps:
(1) and (3) sterile sowing:
1) seed harvesting: harvesting mature fruits of excellent single plants in 10-12 months, selecting seeds which are large in size, full, not cracked, free of mildew and insect damage, and sowing the seeds at any time;
2) surface sterilization: soaking the seeds in 0.1% neutral detergent aqueous solution for 25min, and then washing the seeds for 20min under tap water; soaking the seeds in 75% alcohol for 45s on a clean bench, washing with sterile water for 4 times, soaking with 0.1% mercuric chloride solution for 18min, and washing with sterile water for 4 times;
3) seed treatment and sowing: burying the seeds in sterile river sand, moistening the sterile river sand by sterile water, covering a film to preserve moisture, and standing at the temperature of 25 +/-2 ℃ with the illumination intensity of: 1800LUX, and accelerating germination in a culture room with the illumination time of 15 h/d;
4) obtaining an explant: after the seeds germinate, selecting sterile seedlings with 2 cotyledons and undeveloped true leaves, and cutting out cotyledons and hypocotyls or cutting new buds which sprout after first, second and third times of cutting or picking tender buds of Liupao tea outdoors in the afternoon of sunny days as explants;
(2) callus induction culture
Inoculating the cotyledon and the hypocotyl to a callus induction culture medium for induction culture for 32 d; the callus induction culture medium is MS +6-BA0.5 mg/L +2,4-D0.5 mg/L + sucrose 30g/L + agar 5.5g/L, and the pH value is 6.8;
(3) differentiation culture of callus bud
Inoculating the callus onto adventitious bud differentiation culture medium for culturing for 32 d; the differentiation medium is MS +6-BA 2mg/L + NAA0.10 mg/L + TDZ 0.015mg/L + sucrose 30g/L + agar 5.0g/L, and the pH is 6.8;
(4) cluster bud induction
Inoculating adventitious buds subjected to callus differentiation culture to a cluster bud culture medium for culture for 32 d; the cluster bud culture medium is MS +6-BA 2mg/L + NAA0.10 mg/L + TDZ0.02 mg/L + gibberellin 0.15mg/L + sucrose 30g/L + agar 5.0g/L, and the pH value is 6.8;
(5) multiplication culture
Cutting adventitious buds of 1-2 cm after induction of the multiple buds or stem segments with one bud into a proliferation culture medium for culture for 30 d; the proliferation culture medium is MS +6-BA0.6mg/L + NAA0.06 mg/L + IBA0.06mg/L + sucrose 30g/L + agar 4.8g/L, and the pH is 6.8;
(6) rooting culture
Cutting strong tissue culture buds with the height of 3-4 cm from the adventitious buds or cluster buds obtained by propagation culture, inoculating the tissue culture buds to a rooting culture medium for culture for 25 d; the rooting medium is 1/2MS, 0.80mg/L IBA, 30g/L sucrose and 4.8g/L agar, and the pH value is 6.8;
(7) hardening off the seedling
Selecting tissue culture seedlings with complete roots, stems and leaves, 3.0-4.0 cm of plant height, 2-6 leaves, more than 3-5 roots and 1-2 cm of root length, placing the tissue culture seedlings with bottles in an environment with the room temperature of 20 ℃, the illumination intensity of 2500LUX and the air relative humidity of more than or equal to 85% for 5 days, and then removing the bottle caps and continuing to place for 1 day for hardening the seedlings.
The culture temperature of callus induction culture, bud differentiation culture, cluster bud induction culture, proliferation culture and rooting culture is 28 ℃, the illumination intensity is 1900LUX, and the photoperiod is 15 h.
Example 2
Other process parameters of the embodiment are consistent with those of embodiment 1, except that: step (1), sterile seeding comprises the following steps:
1) seed harvesting: harvesting mature fruits of excellent single plants in 10-12 months, selecting seeds which are large in size, full, not cracked, free of mildew and insect damage, and sowing the seeds at any time;
2) surface sterilization: soaking the seeds in 0.1% neutral detergent water solution for 20min, and then flushing the seeds for 10min under tap water; soaking the seeds in 75% alcohol for 40s on a clean bench, washing with sterile water for 3 times, soaking with 0.1% mercuric chloride solution for 16min, and washing with sterile water for 4 times;
3) seed treatment and sowing: burying the seeds in sterile river sand, moistening the sterile river sand with sterile water, covering a film, keeping moisture, and standing at the temperature of 25 +/-2 ℃ under the illumination intensity of: accelerating germination in a culture room with 1500LUX and illumination time of 8-10 h/d;
4) obtaining an explant: after the seeds germinate, selecting sterile seedlings with 2 cotyledons and undeveloped true leaves, and cutting out cotyledons and hypocotyls or cutting new buds which sprout after first, second and third times or picking tender buds of the Liupao tea outdoors in the afternoon of sunny days as explants.
Example 3
Other process parameters of the embodiment are consistent with those of the embodiment 1, and the difference is that: step (1), sterile seeding comprises the following steps:
1) seed harvesting: harvesting mature fruits of excellent single plants in 10-12 months, selecting seeds which are large in size, full, not cracked, free of mildew and insect damage, and sowing the seeds at any time;
2) surface sterilization: soaking the seeds in 0.1% neutral detergent water solution for 30min, and then flushing the seeds with running water for 30 min; soaking the seeds in 75% alcohol for 60s on a clean bench, washing with sterile water for 3 times, soaking with 0.1% mercuric chloride solution for 20min, and washing with sterile water for 4 times;
3) seed treatment and sowing: burying the seeds in sterile river sand, moistening the sterile river sand by sterile water, covering a film to preserve moisture, and standing at the temperature of 25 +/-2 ℃ with the illumination intensity of: accelerating germination in a culture room with illumination time of 10-12h/d at 2000 LUX;
4) obtaining an explant: after the seeds germinate, selecting sterile seedlings with 2 cotyledons and undeveloped true leaves, and cutting out cotyledons and hypocotyls or cutting new buds which sprout after first, second and third times or picking tender buds of the Liupao tea outdoors in the afternoon of sunny days as explants.
Example 4
Other process parameters of the embodiment are consistent with those of embodiment 1, except that: and (2) callus induction culture: inoculating the cotyledon and the hypocotyl to a callus induction culture medium for induction culture for 30 d; the callus induction culture medium comprises MS +6-BA0.1 mg/L +2,4-D0.1 mg/L + sucrose 25g/L + agar 5.0g/L, and the pH value is 7.0.
Example 5
Other process parameters of the embodiment are consistent with those of the embodiment 1, and the difference is that: step (2), callus induction culture: inoculating the cotyledon and the hypocotyl to a callus induction culture medium for induction culture for 35 d; the callus induction culture medium is MS +6-BA0.8mg/L +2,4-D0.8mg/L + sucrose 35g/L + agar 5.8g/L, and the pH value is 7.0.
Example 6
Other process parameters of the embodiment are consistent with those of the embodiment 1, and the difference is that: and (3) callus bud differentiation culture: inoculating the callus onto a differential culture medium for culture for 30 d; the differentiation medium is MS +6-BA1mg/L + NAA0.05 mg/L + TDZ0.01 mg/L + sucrose 20g/L + agar 4.5g/L, and the pH value is 6.8.
Example 7
Other process parameters of the embodiment are consistent with those of embodiment 1, except that: and (3) callus bud differentiation culture: inoculating the callus onto a differential culture medium for culturing for 35 d; the differentiation medium is MS +6-BA3mg/L + NAA0.15 mg/L + TDZ0.02 mg/L + sucrose 40g/L + agar 5.6g/L, and the pH is 7.0.
Example 8
Other process parameters of the embodiment are consistent with those of the embodiment 1, and the difference is that: step (4), cluster bud induction culture: inoculating the callus after callus differential culture into a cluster bud culture medium for culture for 30 d; the culture medium of the cluster buds is MS +6-BA1mg/L + NAA0.05 mg/L + TDZ0.01 mg/L + gibberellin 0.10mg/L + sucrose 20g/L + agar 4.5g/L, and the pH value is 7.0.
Example 9
Other process parameters of the embodiment are consistent with those of embodiment 1, except that: step (4), carrying out induced culture on the cluster buds: inoculating the callus after callus differential culture into a cluster bud culture medium for culture for 30 d; the culture medium of the cluster buds is MS +6-BA3mg/L + NAA0.15 mg/L + TDZ0.03 mg/L + gibberellin 0.20mg/L + sucrose 40g/L + agar 6.0g/L, and the pH value is 7.0.
Example 10
Other process parameters of the embodiment are consistent with those of the embodiment 1, and the difference is that: and (5) proliferation culture: cutting adventitious buds of 1-2 cm after induction of the multiple buds or stem segments with one bud into a proliferation culture medium for culture for 30 d; the multiplication culture medium is MS +6-BA0.2mg/L + NAA0.02 mg/L + IBA0.02mg/L + sucrose 20g/L + agar 4.5g/L, and the pH value is 6.8.
Example 11
Other process parameters of the embodiment are consistent with those of embodiment 1, except that: and (5) proliferation culture: cutting adventitious buds of 1-2 cm in length after induction of the multiple buds or stem segments with one bud into a proliferation culture medium for culture for 35 d; the proliferation culture medium is MS +6-BA0.8mg/L + NAA0.08mg/L + IBA0.08mg/L + sucrose 40g/L + agar 6.0g/L, and the pH value is 7.0.
Example 12
Other process parameters of the embodiment are consistent with those of the embodiment 1, and the difference is that: step (6), rooting culture: cutting strong tissue culture buds with the height of 3-4 cm from the adventitious buds or cluster buds obtained by propagation culture, inoculating the tissue culture buds to a rooting culture medium for culture for 20 d; the rooting medium is 1/2MS +0.50mg/L IBA + 20g/L sucrose + 4.5g/L agar, and the pH value is 6.8.
Example 13
Other process parameters of the embodiment are consistent with those of the embodiment 1, and the difference is that: step (6), rooting culture: cutting strong tissue culture buds with the height of 3-4 cm from the adventitious buds or cluster buds obtained by propagation culture, inoculating the tissue culture buds to a rooting culture medium for culture for 30 d; the rooting medium is 1/2MS +1.00mg/L IBA + 40g/L sucrose + 6.0g/L agar, and the pH value is 7.0.
Example 14
Other process parameters of the embodiment are consistent with those of embodiment 1, except that: and (7) hardening the seedlings, namely selecting tissue culture seedlings with complete roots, stems and leaves, plant height of 3.0-4.0 cm, 2-6 leaves, more than 3-5 roots and root length of 1-2 cm, putting the tissue culture seedlings with bottles in an environment with room temperature of 15 ℃, illumination intensity of 1500LUX and air relative humidity of more than or equal to 85% for 2 days, removing the bottle caps, and continuously putting the tissue culture seedlings for 1 day to harden the seedlings.
Example 15
Other process parameters of the embodiment are consistent with those of the embodiment 1, and the difference is that: and (7) hardening seedlings, namely selecting tissue culture seedlings with complete roots, stems and leaves, plant height of 3.0-4.0 cm, 2-6 leaves, more than 3-5 roots and root length of 1-2 cm, putting the tissue culture seedlings with bottles in an environment with room temperature of 28 ℃, illumination intensity of 3000LUX and air relative humidity of more than or equal to 85% for 7 days, removing the bottle caps, and continuously putting the tissue culture seedlings for hardening the seedlings for 2 days.
In this example 1-15 Liupu tea, after refining seedlings, domestication of tissue culture seedlings and seedling management and the like are also performed. The domestication of the tissue culture seedlings is specifically as follows: (1) environmental conditions: the ambient temperature is 15-28 ℃, and the relative humidity of air is more than or equal to 85 percent; (2) matrix: the substrate is imported peat disinfected by 50% carbendazim wettable powder and bacillus subtilis, the pH value is 5.5-6.5, and the substrate is filled into a seedling culture plug tray after retting, stacking and fermenting for 5-10 days; transplanting containers adopt seedling raising hole trays with 50 or 72 holes, and the specification of the hole trays is 54cm multiplied by 28 cm; (3) cleaning of: taking out the tissue culture seedlings from the culture bottle, cleaning the culture medium at the root, soaking the tissue culture seedlings in 0.1-1% potassium permanganate aqueous solution for 10-20min, draining water, and airing the tissue culture seedlings in a shade for 10-20 min; (4) transplanting 1 plant in each hole, planting the plantlet in the matrix, and spraying for moisturizing; (5) managing after transplanting: and placing the transplanted tissue culture seedlings on a seedbed, covering a film, and shading by adopting a shading net with shading rate of 60-80%. Spraying water in daytime according to dry and wet conditions, transplanting for 30-40 days, and removing the film. The seedling management comprises water management, fertilization management and prevention and control of diseases, pests and weeds, wherein the fertilization management comprises that 0.1 percent monopotassium phosphate aqueous solution is sprayed on the leaf surfaces of the seedlings for 1 time every 10 days to 15 days, or 0.1 percent to 0.3 percent urea and compound fertilizer N-P are sprayed on the leaves of the seedlings for every 10 days to 15 days2O5-K2The water solution of O (15-15-15) is applied by spraying for 1 time alternately.
The tissue culture method is adopted to breed the seedlings, the propagation coefficient of aseptic seedlings of the Shannu purple is more than 3-4 times, the hardening seedling survival rate is more than 80%, the efficient cultivation management technology is realized, the tree planting in one year can be realized, the effect can be realized in the second year, a quick and effective way is provided for the large-scale Liupao tea breeding, and the tissue culture method has strong practical significance in the large-scale seedling culture production of factories.
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 (8)
1. A tissue culture method suitable for Liupao tea Shannu purple stock seedlings is characterized by comprising the following steps: comprises aseptic seeding, callus induction culture, bud differentiation culture, cluster bud induction culture, proliferation culture, rooting culture and seedling hardening; the callus induction culture is to inoculate cotyledons and hypocotyls to a callus induction culture medium for induction culture for 30-35 days; the callus induction culture medium comprises MS +6-BA 0.1-0.8 mg/L +2, 4-D0.1-0.8 mg/L + sucrose 25-35 g/L + agar 5.0-5.8 g/L, and the pH value is 6.8-7.0.
2. The tissue culture method for preparing the Liupu tea Shannu purple elite seedlings according to the claim 1, which is characterized in that: the aseptic seeding comprises the following steps:
(1) seed harvesting: harvesting mature fruits of excellent single plants in 10-12 months, selecting seeds which are large in size, full, not cracked, free of mildew and insect damage, and sowing the seeds at any time;
(2) surface sterilization: soaking the seeds in 0.1 percent neutral detergent aqueous solution for 20-30 min, and then flushing the seeds for 10-30 min under running water; soaking the seeds in 75% alcohol for 40-60 s on a clean bench, washing with sterile water for 3-5 times, soaking in 0.1% mercuric chloride solution for 16-20 min, and washing with sterile water for 3-5 times;
(3) seed treatment and sowing: burying the seeds in sterile river sand, moistening the sterile river sand by sterile water, covering a film to preserve moisture, and standing at the temperature of 25 +/-2 ℃ with the illumination intensity of: 1500-2000LUX, the illumination time is 14-16 h/d;
(4) obtaining an explant: after the seeds germinate, selecting sterile seedlings with 2 cotyledons and true leaves which are not developed, and cutting cotyledons and hypocotyls or cutting new buds which sprout after first, second and third times of cutting or picking tender buds of the Liupao tea outdoors in the afternoon of sunny days as explants.
3. The tissue culture method for preparing the Liupu tea Shannu purple elite seedlings according to the claim 1, which is characterized in that: the bud differentiation culture is to inoculate the callus onto an adventitious bud differentiation culture medium for culture, wherein the culture time is 30-35 d; the bud differentiation medium is MS +6-BA 1-3 mg/L + NAA 0.05-0.15 mg/L + TDZ 0.01-0.02 mg/L + sucrose 20-40 g/L + agar 4.5-5.6 g/L, and the pH value is 6.8-7.0.
4. The tissue culture method for preparing the Liupu tea Shannu purple elite seedlings according to the claim 1, which is characterized in that: the cluster bud induction culture is to inoculate adventitious buds subjected to callus differentiation culture onto a cluster bud culture medium for culture for 30-35 days; the cluster bud culture medium is MS +6-BA 1-3 mg/L + NAA 0.05-0.15 mg/L + TDZ 0.01-0.03 mg/L + gibberellin 0.1-0.2 mg/L + sucrose 20-40 g/L + agar 4.5-6.0 g/L, and the pH value is 6.8-7.0.
5. The tissue culture method of adapted Liupao tea Shannu purple stock seedlings according to claim 1, characterized in that: the enrichment culture is to cut adventitious buds of about 1-2 cm after induction of the cluster buds or stem segments with one bud in a segmented mode into an enrichment culture medium for culture for 30-35 d; the enrichment medium comprises MS + 0.2-0.8 mg/L of 6-BA, 0.02-0.08 mg/L of NAA, 0.02-0.08 mg/L of IBA, 20-40 g/L of sucrose and 4.5-6.0 g/L of agar, and the pH value is 6.8-7.0.
6. The tissue culture method of adapted Liupao tea Shannu purple stock seedlings according to claim 1, characterized in that: the rooting culture is to cut out a tissue culture bud which is strong in growth and 3-4 cm high from an adventitious bud or a cluster bud obtained by the proliferation culture, and to culture the tissue culture bud on a rooting culture medium for 20-30 days; the rooting medium is 1/2MS, 0.50-1.00 mg/L IBA, 20-40 g/L sucrose and 4.5-6.0 g/L agar, and the pH value is 6.8-7.0.
7. The tissue culture method of adapted Liupao tea Shannu purple stock seedlings according to claim 1, characterized in that: the seedling exercising method comprises the steps of selecting tissue culture seedlings with complete roots, stems and leaves, the plant height of 3.0-4.0 cm, 2-6 blades, more than 3-5 blades and 1-2 cm root length, placing the tissue culture seedlings with bottles in an environment with the room temperature of 15-28 ℃, the illumination intensity of 1500-3000 LUX and the relative air humidity of more than or equal to 85% for 2-7 days, removing the bottle caps, and continuing to place for 1-2 days to exercise the seedlings.
8. The tissue culture method for preparing the Liupu tea mountain sill purple stock seed seedling as claimed in any one of claims 1 to 7, wherein the tissue culture method comprises the following steps: the culture temperature of the callus induction culture, the callus differentiation culture, the cluster bud induction culture, the proliferation culture and the rooting culture is 23-30 ℃, the illumination intensity is 1800-2000 LUX, and the photoperiod is 14-16 h.
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| CN110338063A (en) * | 2019-08-19 | 2019-10-18 | 昆明理工大学 | A kind of method, production method and the twisting device of tea tissue cultures of looking for one's roots |
| WO2021258738A1 (en) * | 2021-01-14 | 2021-12-30 | 中国科学院华南植物园 | Tissue culture and rapid propagation method for high-quality hippeastrum vittatum seedlings |
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| CN110338063A (en) * | 2019-08-19 | 2019-10-18 | 昆明理工大学 | A kind of method, production method and the twisting device of tea tissue cultures of looking for one's roots |
| WO2021258738A1 (en) * | 2021-01-14 | 2021-12-30 | 中国科学院华南植物园 | Tissue culture and rapid propagation method for high-quality hippeastrum vittatum seedlings |
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