CN116584380A - Tissue culture method suitable for Liupu tea mountain-sill purple stock seedlings - Google Patents
Tissue culture method suitable for Liupu tea mountain-sill purple stock seedlings Download PDFInfo
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Classifications
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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
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/40—Afforestation or reforestation
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Abstract
The application discloses a tissue culture method suitable for preparing Liupu tea mountain-leaf holly stock seedlings, which 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 insert cotyledon and hypocotyl into a callus induction culture medium for induction culture for 30-35 d; the callus induction culture medium is 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 is 6.8-7.0. The Liupu tea mountain purple tissue culture seedling has the advantages of thick leaves, she Zise, thick stems, low variation rate, multiplication coefficient of 6-7 times and hardening-seedling survival rate of more than 90 percent, and is suitable for providing the seedling for Liupu tea planting. The asexual propagation keeps the excellent characteristics of the mountain purple parent, has low cost and little pollution, can be produced in four seasons, and is suitable for large-scale asexual seedling.
Description
Technical Field
The application belongs to the technical field of plant propagation, and particularly relates to a tissue culture method suitable for preparing Liupu tea mountain-leaf holly stock seedlings.
Background
Liupu tea (Camellia nitidissima cv. Liubaocha) is a evergreen shrub or small arbor of Camellia genus of Theaceae family, she Chengchang elliptic needle shape, she Sehe black light moist, yellow flower points in the middle, and reddish brown leaf bottom. Liupu tea essence is known as red, dense, old and mellow, and has long and tight strip rope, red dense soup color, chen Hou aroma and sweet and delicious taste; liupu town and its surrounding area of the state of Guangxi in China, it is one of Chinese black tea representatives, it is the national geographic marking products, its traditional preparation method has already been listed in the national non-material cultural heritage directory, liupu tea ship ancient way is the new sea silk road culture, it is the special dominant industry of Guangxi. "Kangxi Sanqing" (a Chinese character of Qing Kangxi) sixteen years, 1697 edition): the Liupu tea is characterized by being suitable for people who are in the world and being famous in Liupu town of the Guangxi city, and the quality characteristics of red, thick, old and mellow are taken as the quality characteristics of the red, thick, old and mellow. The production history can be traced back to 1500 years, and the product mainly contains 74 and 80 volatile aroma components and the like, wherein the Zn, cu, sr, ni, se content is higher, and Liupu tea medicine and food homologous products are utilized in traditional diet therapy in China.
At present, more than 90% of Liupu tea raw materials in the domestic market come from Guangxi province, and Liupu town is taken as a core production area. Therefore, the method of artificial propagation and seedling culture is necessary for protecting the wild Liupu tea resources and meeting the requirement of large-scale production. Liupu tea is a treasured in whole, and besides the medicinal value of the rhizome and the leaf of Liupu tea, liupu tea is also a tree species with extremely high economic value for controlling mountain areas and barren lands and forestry production. However, the Liupu tea seedling raising is a bottleneck for inhibiting the Liupu tea industry, and the application and popularization of the Liupu tea seedling raising are limited due to the problems that the Liupu tea is one of tree species which are difficult to root in cutting, the common cutting is difficult to root, the seed germination rate is low and the like.
At present, the planting and cultivation of Liupu tea mainly adopts seed seedlings for seedling raising, and as the tea trees are frequently picked up to be bud, the seed setting rate of the conventional field seeds 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 application aims at: the mountain-sill purple belongs to a high-quality original rare variety of Liupu tea, aims to solve the problems of optimization of fine variety, stable fine variety character maintenance, high breeding efficiency, large-scale production and the like in original old tea trees, and provides a tissue culture method suitable for preparing Liupu tea mountain-sill purple original seed seedlings.
In order to achieve the above object, the present application adopts the following technical scheme:
a tissue culture method suitable for Liupu tea mountain purple stock seedling 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 insert cotyledon and hypocotyl into a callus induction culture medium for induction culture for 30-35 d; the callus induction culture medium is 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 is 6.8-7.0.
Preferably, the aseptic seeding comprises the steps of:
(1) Harvesting seeds: harvesting mature fruits of good single plants in 10-12 months, and selecting large, full, uncracked, mildew-free and pest-free seeds, wherein the seeds are suitable for sowing as the seeds are picked;
(2) Surface sterilization: soaking seeds in 0.1% neutral detergent water solution for 20-30 min, and then flushing the seeds in running water under running water for 10-30 min; on an ultra-clean workbench, soaking seeds in 75% alcohol for 40-60 s, 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: embedding seeds into sterile river sand, wetting the sterile river sand with sterile water, covering a film for moisturizing, and placing the film at the temperature of 25+/-2 ℃ with the illumination intensity of: accelerating germination in a culture room with illumination time of 14-16h/d and with illumination time of 1500-2000 LUX;
(4) Obtaining of explants: after the seeds germinate, selecting aseptic seedlings with 2 cotyledons and true leaves which are not spread, cutting cotyledons and hypocotyls or splicing new buds which germinate after the first time, the second time and the third time, or picking Liupu tea mountain threshold purple tender buds outdoors in the afternoon on a sunny day, as explants.
Preferably, the bud differentiation culture is to culture the callus on a differentiation culture medium for 30-35 d; the differentiation culture 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 is 6.8-7.0.
Preferably, the cluster bud induction culture is to culture adventitious buds differentiated by the callus on a cluster bud culture medium for 30-35 d; 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 is 6.8-7.0.
Preferably, the proliferation culture is to cut buds with the length of about 1-2 cm after the induction of clustered buds or segments into stem segments with one bud, and then to culture the stem segments on a proliferation culture medium for 30-35 d; the proliferation culture 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 is 6.8-7.0.
Preferably, the rooting culture is to cut tissue culture buds which grow robustly and have the height of 3-4 cm from adventitious buds or cluster buds obtained by multiplication culture, and then to put the tissue culture buds on a rooting culture medium for 20-30 d; the rooting culture 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 is 6.8-7.0.
Preferably, the seedling hardening is to select tissue culture seedlings with root, stem and leaf, plant height of 3.0-4.0 cm, leaves of 2-6 pieces, root number of more than 3-5 pieces and root length of 1-2 cm, firstly placing the tissue culture seedlings with bottles in an environment with room temperature of 15-28 ℃ and illumination intensity of 1500-3000 LUX and air relative humidity of more than or equal to 85% for 2-7 d, and then removing bottle caps and continuously placing the tissue culture seedlings for 1-2 d to harden the seedlings.
Preferably, the culture temperature of the callus induction culture, callus differentiation culture, cluster bud induction culture, proliferation culture and rooting culture is 23-30 ℃, the illumination intensity is 1800-2 000LUX, and the photoperiod is 14-16 h.
In summary, due to the adoption of the technical scheme, the beneficial effects of the application are as follows:
(1) The application takes primary high-quality Liupu tea mountain purple seeds or buds of Liupu tea core producing areas as explants, and prepares special culture media for induction, differentiation, cluster bud culture, rooting culture and the like through standard steps of aseptic seeding, callus induction culture, callus differentiation culture, cluster bud induction, proliferation culture, rooting culture and seedling hardening by screening culture media, thereby successfully establishing a complete set of system for Liupu tea mountain purple tissue culture seedling raising and providing a reliable technology and method for rapid propagation of Liupu tea mountain purple.
(2) The proliferation coefficient of the Liupu tea reaches 3-4 times by adopting a tissue culture method, so that 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 Liupu tea adventitious buds are rich in leaves and thick in stems, and after a plurality of rounds of subculture, the tissue culture seedlings grow normally.
(3) Compared with the similar method, the method has great advantages, such as difficult rooting of branch cutting propagation and low survival rate although the cutting seedling is easy to operate; in the seed seedling raising method, because the seeds of Liupu tea are fewer, the seed coats are hard, the germination rate of the conventional field is less than 4%, so that a great amount of seeds are wasted and the Liupu tea seedlings are lack in forestation; the tissue culture method is adopted to breed Liupu tea mountain-sill purple seedlings, the sterile seedling multiplication coefficient is more than 3-4 times, the seedling hardening survival rate is more than 80%, the efficient cultivation management technology is realized, one-year tree planting can be realized, the effect is seen in the second year, a quick and effective way is provided for large-scale Liupu tea breeding, and the method has strong practical significance in factory scale seedling cultivation production.
(4) The Liupu tea mountain purple tissue culture seedling has the advantages of thick leaves, she Zise, thick stems, low mutation rate and high multiplication coefficient, and is suitable for providing seedlings for Liupu tea planting; the application maintains the excellent characteristics of the parents through asexual propagation, has low cost and little pollution, can be produced in four seasons, and is suitable for large-scale asexual seedling.
Drawings
FIG. 1 is a Liupu tea mountain Violet tissue culture seedling of the present application, comprising (a) and (b);
FIG. 2 shows rooting of Liupu tea mountain purple tissue culture seedlings of the application;
FIG. 3 is a cluster of tissue culture seedlings of Liupu tea mountain Violet according to the present application;
FIG. 4 is a Liupu tea mountain side sill spring tip of the present application;
FIG. 5 is a purple bud of the Liupu tea mountain of the present application sprouting purple;
FIG. 6 is a Liupu tea mountain purple flower of the present application;
FIG. 7 is a Liupu tea mountain range purple fruit of the present application;
FIG. 8 is a Liupu tea mountain purple tree of the present application.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be described in further detail by referring to preferred embodiments. It should be noted, however, that many of the details set forth in the description are merely provided to provide a thorough understanding of one or more aspects of the application, and that these aspects of the application may be practiced without these specific details.
In the application, the following components are added: 6-BA is 6-benzylaminoadenine, NAA is alpha-naphthylacetic acid, IBAK is indole potassium butyrate salt, GA is gibberellin, and TDZ is N-phenyl-N-1, 2, 3-thiadiazole-5-urea.
Example 1
A tissue culture method suitable for preparing Liupu tea mountain purple stock seedlings comprises the following steps:
(1) And (3) aseptic sowing:
1) Harvesting seeds: mature fruits of excellent single plants are harvested in 10-12 months, and seeds which are large, full, not cracked, free of mildew and insect damage are selected, and the seeds are preferably harvested and sown at the same time;
2) Surface sterilization: soaking seeds in 0.1% neutral detergent water solution for 25min, and then flushing the seeds in running water for 20min; on an ultra-clean workbench, soaking seeds in 75% alcohol for 45s, washing with sterile water for 4 times, soaking in 0.1% mercuric chloride solution for 18min, and washing with sterile water for 4 times;
3) Seed treatment and sowing: embedding seeds into sterile river sand, wetting the sterile river sand with sterile water, covering a film for moisturizing, and placing the film 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 of explants: after the seeds germinate, selecting aseptic seedlings with 2 cotyledons and true leaves which are not spread, cutting cotyledons and hypocotyls or splicing new buds which germinate after the first time, the second time and the third time, or picking tender buds of Liupu tea outdoors in the afternoon on a sunny day as explants;
(2) Callus induction culture
Inoculating cotyledon and hypocotyl to callus induction culture medium for induction culture for 32d; the callus induction culture medium is MS+6-BA0.5 mg/L+2,4-D0.5 mg/L+sucrose 30 g/L+agar 5.5g/L, and the pH is 6.8;
(3) Callus bud differentiation culture
Inoculating the callus to an adventitious bud differentiation culture medium for culture for 32 days; the differentiation culture medium is MS+6-BA 2mg/L+NAA 0.10mg/L+TDZ 0.015 mg/L+sucrose 30 g/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 32d; the cluster bud culture medium is MS+6-BA 2mg/L+NAA 0.10mg/L+TDZ 0.02 mg/L+gibberellin 0.15 mg/L+sucrose 30 g/L+agar 5.0g/L, and the pH is 6.8;
(5) Proliferation culture
Cutting adventitious buds or segments with the length of about 1-2 cm after cluster bud induction into stem segments with one bud, and inoculating the stem segments to a proliferation culture medium for culturing for 30d; the proliferation culture medium is MS+6-BA0.6mg/L+NAA0.06 mg/L+IBA0.06 mg/L+sucrose 30 g/L+agar 4.8g/L, and the pH is 6.8;
(6) Rooting culture
Cutting tissue culture buds which grow robustly and have the height of 3-4 cm from adventitious buds or cluster buds obtained by multiplication culture, inoculating the tissue culture buds to a rooting culture medium for culture for 25 days; the rooting culture medium is 1/2MS+0.80mg/L IBA+30 g/L sucrose+4.8 g/L agar, and the pH is 6.8;
(7) Hardening off seedlings
Selecting tissue culture seedlings with root length of 1-2 cm, wherein the plant height is 3.0-4.0 cm, the number of leaves is 2-6, the number of the leaves is more than 3-5, the tissue culture seedlings are firstly placed in an environment with room temperature of 20 ℃ and illumination intensity of 2500LUX and air relative humidity of more than or equal to 85% for 5 days with a bottle, and then the tissue culture seedlings are continuously placed for more than 1 day after the bottle cover is opened 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 15h.
Example 2
Other process parameters of this example were consistent with those of example 1 except that: step (1), aseptic seeding includes the steps of:
1) Harvesting seeds: harvesting mature fruits of excellent single plants in 10-12 months, and selecting seeds which are large, full, not cracked, free of mildew and insect damage, and sowing along with harvesting;
2) Surface sterilization: soaking seeds in 0.1% neutral detergent water solution for 20min, and then flushing the seeds in running water for 10min; on an ultra-clean workbench, soaking seeds in 75% alcohol for 40s, washing 3 times with sterile water, soaking in 0.1% mercuric chloride solution for 16min, and washing 4 times with sterile water;
3) Seed treatment and sowing: embedding seeds into sterile river sand, wetting the sterile river sand with sterile water, covering a film for moisturizing, and placing the film at the temperature of 25+/-2 ℃ with the illumination intensity of: accelerating germination in a culture room with illumination time of 8-10h/d and 1500 LUX;
4) Obtaining of explants: after the seeds germinate, selecting aseptic seedlings with 2 cotyledons and true leaves which are not spread, cutting cotyledons and hypocotyls or splicing new buds which are germinated after the first time, the second time and the third time, or picking tender buds of Liupu tea outdoors in the afternoon on sunny days as explants.
Example 3
Other process parameters of this example were consistent with those of example 1 except that: step (1), aseptic seeding includes the steps of:
1) Harvesting seeds: harvesting mature fruits of excellent single plants in 10-12 months, and selecting seeds which are large, full, not cracked, free of mildew and insect damage, and sowing along with harvesting;
2) Surface sterilization: soaking seeds in 0.1% neutral detergent water solution for 30min, and then flushing the seeds in running water for 30min; on an ultra-clean workbench, soaking seeds in 75% alcohol for 60s, washing 3 times with sterile water, soaking in 0.1% mercuric chloride solution for 20min, and washing 4 times with sterile water;
3) Seed treatment and sowing: embedding seeds into sterile river sand, wetting the sterile river sand with sterile water, covering a film for moisturizing, and placing the film at the temperature of 25+/-2 ℃ with the illumination intensity of: accelerating germination in a culture room with the illumination time of 2000LUX and 10-12 h/d;
4) Obtaining of explants: after the seeds germinate, selecting aseptic seedlings with 2 cotyledons and true leaves which are not spread, cutting cotyledons and hypocotyls or splicing new buds which are germinated after the first time, the second time and the third time, or picking tender buds of Liupu tea outdoors in the afternoon on sunny days as explants.
Example 4
Other process parameters of this example were consistent with those of example 1 except that: step (2), callus induction culture: inoculating cotyledon and hypocotyl onto callus induction culture medium for induction culture for 30d; the callus induction culture medium is MS+6-BA0.1 mg/L+2,4-D0.1 mg/L+sucrose 25 g/L+agar 5.0g/L, and the pH is 7.0.
Example 5
Other process parameters of this example were consistent with those of example 1 except that: step (2), callus induction culture: inoculating cotyledon and hypocotyl to callus induction culture medium for induction culture for 35d; the callus induction culture medium is MS+6-BA0.8 mg/L+2,4-D0.8 mg/L+sucrose 35 g/L+agar 5.8g/L, and the pH is 7.0.
Example 6
Other process parameters of this example were consistent with those of example 1 except that: step (3), callus bud differentiation culture: inoculating the callus onto a differentiation culture medium for culture for 30 days; the differentiation medium is MS+6-BA1mg/L+NAA0.05 mg/L+TDZ0.01 mg/L+sucrose 20 g/L+agar 4.5g/L, and the pH is 6.8.
Example 7
Other process parameters of this example were consistent with those of example 1 except that: step (3), callus bud differentiation culture: inoculating the callus onto a differentiation culture medium for culture for 35d; the differentiation medium is MS+6-BA 3mg/L+NAA0.15mg/L+TDZ 0.02 mg/L+sucrose 40 g/L+agar 5.6g/L, and the pH is 7.0.
Example 8
Other process parameters of this example were consistent with those of example 1 except that: step (4), cluster bud induction culture: inoculating the callus subjected to callus differentiation culture to a cluster bud culture medium for culture for 30 days; the cluster bud culture medium is MS+6-BA1mg/L+NAA0.05 mg/L+TDZ0.01 mg/L+gibberellin 0.10 mg/L+sucrose 20 g/L+agar 4.5g/L, and the pH is 7.0.
Example 9
Other process parameters of this example were consistent with those of example 1 except that: step (4), cluster bud induction culture: inoculating adventitious buds subjected to callus differentiation culture to a cluster bud culture medium for culture for 30d; the cluster bud culture medium is MS+6-BA3mg/L+NAA0.15 mg/L+TDZ0.03 mg/L+gibberellin 0.20 mg/L+sucrose 40 g/L+agar 6.0g/L, and the pH is 7.0.
Example 10
Other process parameters of this example were consistent with those of example 1 except that: step (5), proliferation culture: cutting the bud or segment with the length of about 1-2 cm after cluster bud induction into stem segments with one bud, and inoculating the stem segments to a proliferation culture medium for culturing for 30d; the proliferation culture medium is MS+6-BA0.2mg/L+NAA0.02 mg/L+IBA0.02 mg/L+sucrose 20 g/L+agar 4.5g/L, and the pH is 6.8.
Example 11
Other process parameters of this example were consistent with those of example 1 except that: step (5), proliferation culture: cutting adventitious buds or segments with the length of about 1-2 cm after cluster bud induction into stem segments with one bud, and inoculating the stem segments to a proliferation culture medium for culturing for 35d; the proliferation culture medium is MS+6-BA0.8mg/L+NAA0.08mg/L+IBA0.08mg/L+sucrose 40 g/L+agar 6.0g/L, and the pH is 7.0.
Example 12
Other process parameters of this example were consistent with those of example 1 except that: step (6), rooting culture: cutting tissue culture buds which grow robustly and have the height of 3-4 cm from adventitious buds or cluster buds obtained by multiplication culture, inoculating the tissue culture buds to a rooting culture medium for culture for 20d; the rooting culture medium is 1/2MS+0.50mg/L IBA+20 g/L sucrose+4.5 g/L agar, and the pH is 6.8.
Example 13
Other process parameters of this example were consistent with those of example 1 except that: step (6), rooting culture: cutting tissue culture buds which grow robustly and have the height of 3-4 cm from adventitious buds or cluster buds obtained by multiplication culture, inoculating the tissue culture buds to a rooting culture medium for culture for 30d; the rooting culture medium is 1/2MS+1.00mg/L IBA+40 g/L sucrose+6.0 g/L agar, and the pH is 7.0.
Example 14
Other process parameters of this example were consistent with those of example 1 except that: and (7) hardening off, namely selecting tissue culture seedlings with the root length of 1-2 cm, the root height of 3.0-4.0 cm, the leaf length of 2-6 leaves and the root number of more than 3-5, placing the tissue culture seedlings with the root length in an environment with the room temperature of 15 ℃ and the illumination intensity of 1500LUX for 2d and the air relative humidity of more than or equal to 85%, and then opening a bottle cover to continue placing the tissue culture seedlings for more than 1d for hardening off.
Example 15
Other process parameters of this example were consistent with those of example 1 except that: and (7) hardening seedlings, namely selecting tissue culture seedlings with the root length of 1-2 cm, the root height of 3.0-4.0 cm, the leaf length of 2-6 leaves and the root number of more than 3-5, placing the tissue culture seedlings with the root length in an environment with the room temperature of 28 ℃ and the illumination intensity of 3000LUX for 7d and the air relative humidity of more than or equal to 85%, and then opening a bottle cover to continue placing the tissue culture seedlings for more than 2d for hardening seedlings.
The Liupu tea mountain threshold purple stock seedling is an existing tea seed. At the end of nineties of the last century, the old tea farmers of the mountain range group of the Liupu mountain range, guangxi, county found a number of wild old tea plants distributed in a dispersed manner in the mountain range group (place name) elder Lin which was two hours away from their home. Wherein, a old tree with a stake diameter of 23 cm is broken by a mountain pig or wild beast, and a plurality of new branches grow out from the side surface of the trunk. Because of the long-term use in the wild, the middle part of the stump is also damaged by worms. In order to protect the old tea tree, the old tea farmer digs the old tea tree back from the deep mountain and cares for the tea mountain planted at home. Several years ago, the old tree was evaluated by an expert, with a tree age of around 160 years.
The old tea tree named as mountain purple for distinguishing and commemorating the plant is one of the rare original species of Liupu town in Hashimeji county, and is the middle leaf species of shrubs.
The mountain purple old tea tree is developed in tree pose, and branches are dense. The original height of the tea tree is estimated to be more than 3.5 meters, the height of newly germinated lateral branches is 98 cm, the tree width is about 85 cm, and the internodes are 3 cm. The blade is inclined upwards; the length of the formed leaf reaches 7.8 cm, the width of the formed leaf reaches 3.2 cm, the leaf shape is elliptic and elongated, the leaf bud is purple, three and four leaves turn green, the leaf surface is micro-bulge, part of the bulge is obvious, the luster is medium, the leaf margin is micro-wave, the saw teeth are thin, shallow and dense, the leaf body is flat and slightly inwards coiled, the leaf quality is medium, and the leaf tip is pointed; side pulses 8-11 pairs. The young bud tip is light purple, one bud has two three leaves and purple, the fuzz is little, the germination is dense, and the tenderness is strong; the germination density is 68/square ruler. Sprouting in the middle of 3 months, mining in the early april, belonging to early sprouting seeds, and the sprouting termination period is in the middle of 10 months. The quality tracking research of the tea in 5 years proves that the ancient tree variety of mountain purple has excellent indexes, mellow taste, pleasant fragrance, rich layering and long aftertaste.
After hardening seedlings, the Liupu tea of examples 1 to 15 is subjected to tissue culture seedling hardening, seedling management and the like. Wherein the domestication of the tissue culture seedling 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%; (2) a matrix: the substrate adopts imported peat sterilized by 50% carbendazim wettable powder and bacillus subtilis, the pH value is 5.5-6.5, and the substrate is filled into seedling raising plug after composting fermentation for 5-10 d; the transplanting container adopts a 50 or 72-hole seedling tray, and the specification of the tray is 54cm multiplied by 28cm; (3) cleaning: taking out the tissue culture seedlings from the culture bottle, cleaning the root culture medium, soaking the tissue culture seedlings in 0.1-1% potassium permanganate aqueous solution for 10-20min, draining, and airing the tissue culture seedlings in a shade place for 10-20 min; (4) Transplanting 1 plant in each hole, planting the seedlings in a matrix, and spraying and moisturizing; (5) post-transplanting management: the transplanted tissue culture seedling is placed in a seedbed, covered with a film, and shading is carried out by adopting a shading net with shading rate of 60% -80%. Spraying and moisturizing in daytime according to the dry and wet conditions, and removing the film after transplanting for 30-40 d. The seedling management comprises water management, fertilization management and disease and pest control, wherein the fertilization management is that 0.1% monopotassium phosphate aqueous solution is sprayed on leaf surfaces for 1 time every 10 d-15 d, or 0.1% -0.3% urea and compound fertilizer N-P are used every 10 d-15 d 2 O 5 -K 2 O (15-15-15) water solution is alternatively sprayed for 1 time.
FIGS. 1-8 are, respectively, flowers, fruits and trees of Liupu tea mountain Violet tissue culture seedlings, rooting, cluster buds, spring tips, violet buds and mountain Violet original plants obtained by the tissue culture method of example 1 of the present application. The application adopts a tissue culture method to breed seedlings, the multiplication coefficient of the mountain ultra violet sterile seedlings is more than 3-4 times, the survival rate of the hardening seedlings reaches more than 80%, the efficient cultivation management technology is realized, one-year tree planting can be realized, the effect is seen in the second year, a quick and effective way is provided for large-scale Liupu tea breeding, and the application has strong practical significance in factory scale seedling production.
The foregoing is merely a preferred embodiment of the present application and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present application, which are intended to be comprehended within the scope of the present application.
Claims (8)
1. A tissue culture method suitable for preparing Liupu tea mountain purple stock seedlings is characterized by comprising the following steps: including 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 insert cotyledon and hypocotyl into a callus induction culture medium for induction culture for 30-35 d; the callus induction culture medium is 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 is 6.8-7.0.
2. The tissue culture method for the suitable Liupu tea mountain range purple stock seedlings according to claim 1, wherein the method comprises the following steps: the aseptic seeding comprises the following steps:
(1) Harvesting seeds: harvesting mature fruits of good single plants in 10-12 months, and selecting large, full, uncracked, mildew-free and pest-free seeds, wherein the seeds are suitable for sowing as the seeds are picked;
(2) Surface sterilization: soaking seeds in 0.1% neutral detergent water solution for 20-30 min, and then flushing the seeds in running water under running water for 10-30 min; on an ultra-clean workbench, soaking seeds in 75% alcohol for 40-60 s, 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: embedding seeds into sterile river sand, wetting the sterile river sand with sterile water, covering a film for moisturizing, and placing the film at the temperature of 25+/-2 ℃ with the illumination intensity of: accelerating germination in a culture room with illumination time of 14-16h/d and with illumination time of 1500-2000 LUX;
(4) Obtaining of explants: after the seeds germinate, selecting aseptic seedlings with 2 cotyledons and true leaves which are not spread, cutting cotyledons and hypocotyls or splicing new buds which germinate after the first time, the second time and the third time, or picking Liupu tea mountain threshold purple tender buds outdoors in the afternoon on a sunny day, as explants.
3. The tissue culture method for the suitable Liupu tea mountain range purple stock seedlings according to claim 1, wherein the method comprises the following steps: the bud differentiation culture is to culture the callus on an adventitious bud differentiation culture medium for 30-35 d; the bud differentiation culture 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 is 6.8-7.0.
4. The tissue culture method for the suitable Liupu tea mountain range purple stock seedlings according to claim 1, wherein the method comprises the following steps: the cluster bud induction culture is to culture adventitious buds subjected to callus differentiation culture on a cluster bud culture medium for 30-35 d; 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 is 6.8-7.0.
5. The tissue culture method for the suitable Liupu tea mountain range purple stock seedlings according to claim 1, wherein the method comprises the following steps: the proliferation culture is to cut adventitious buds or segments with the length of about 1-2 cm after cluster bud induction into stem segments with one bud, and then to culture the stem segments on a proliferation culture medium for 30-35 d; the proliferation culture 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 is 6.8-7.0.
6. The tissue culture method for the suitable Liupu tea mountain range purple stock seedlings according to claim 1, wherein the method comprises the following steps: the rooting culture is to cut tissue culture buds which grow robustly and have the height of 3-4 cm from adventitious buds or cluster buds obtained by multiplication culture, and then inoculating the tissue culture buds to a rooting culture medium for culture for 20-30 d; the rooting culture 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 is 6.8-7.0.
7. The tissue culture method for the suitable Liupu tea mountain range purple stock seedlings according to claim 1, wherein the method comprises the following steps: the seedling hardening is to select tissue culture seedlings with root, stem and leaf height of 3.0-4.0 cm, leaves of 2-6 pieces, root number of more than 3-5 and root length of 1-2 cm, firstly placing the tissue culture seedlings with bottles in an environment with room temperature of 15-28 ℃ and illumination intensity of 1500-3000 LUX and air relative humidity of more than or equal to 85% for 2-7 d, and then removing bottle caps and continuously placing the tissue culture seedlings for 1-2 d to hardening the seedlings.
8. The tissue culture method for the adapted Liupu tea mountain purple stock seedlings according to any one of claims 1 to 7, wherein: the culture temperature of the callus induction culture, callus differentiation culture, cluster bud induction culture, proliferation culture and rooting culture is 23-30 ℃, the illumination intensity is 1800-2 000LUX, and the photoperiod is 14-16 h.
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