CN107896987B - Breeding method of Guangxi wild adinandra nitida tissue culture seedlings - Google Patents
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Abstract
The invention discloses a tissue culture breeding method of Guangxi wild adinandra nitida, which comprises the following steps: the method for breeding the wild cliff tea tissue culture seedlings is simple to operate, high in survival rate, capable of keeping the excellent properties of the wild cliff tea, suitable for breeding industrial tissue culture seedlings, and has important significance and immeasurable commercial value for popularization of the wild cliff tea which is a precious species.
Description
Technical Field
The invention belongs to the field of crop breeding, and particularly relates to a breeding method of Guangxi wild adinandra nitida tissue culture seedlings.
Background
Adinandra nitida, entitled "Liangyehuangrui", a tree of the genus Yangtze. Mainly grows in the mountain area in the south of the autonomous county of Jinxiyao nationality in Guangxi Zhuang district, likes the cliff or mountain edge of a mountain with short sunshine, large temperature difference and shady and damp, and the tea is longer than the cliff and the wall, so that the tea can absorb spirits of heaven and earth and essence of daybreak; the method has unique ecological environment, breeds pure natural beverage with unique style, picks the tamed monkey in ancient times, and is also called as picking the tea by the folk as "picking the monkey". According to the detection of relevant departments, the adinandra nitida tea does not contain caffeine, does not affect sleep after being drunk, is rich in flavonoids and has a good health-care effect, so the adinandra nitida tea is popular with consumers. Identification of plant experts of China university in Guangdong of cliff tea: the Pingle wild adinandra nitida tea is a daphne giraldii tea family in the genus of adinandra nitida, and comprises the following main components: 20 percent of flavonoids, 23.4 percent of tea polyphenol, 9.73 percent of amino acids, wild alkaloids and other elements beneficial to human bodies are the plants which are found to have the highest content of flavonoids in natural plants at present and are rare in tea of original ecological pure natural green plants which are rare in China. In addition, the wild adinandra nitida tea is dark green like jade, fragrant and tasty, and low in theophylline content. The product has endless aftertaste, does not influence sleep, is drunk in the morning and evening for a long time, has the effects of clearing heat and detoxicating, protecting liver and improving eyesight, diminishing inflammation and moistening lung, beautifying, lowering blood pressure, reducing fat, invigorating stomach and promoting digestion, has obvious effects on pharyngolaryngitis, obesity, diabetes, hypertension and hyperlipidemia, and can be called a treasure in the mountain of Dayao mountain.
The breeding survival rate of wild Adinandra nitida grown in Jinxiu county is extremely low, the breeding of wild Adinandra nitida also becomes a research hotspot of Adinandra nitida researchers, and the following breeding method data of Adinandra nitida are searched:
1. chinese patent: patent publication No. CN102487706A discloses a method for growing seedlings of Adinandra nitida, which is to pick mature Adinandra nitida fruits before and after every year of Frost, store and treat seeds by adopting a dry storage method and a sand storage method, and then breed the Adinandra nitida fruits during 3-4 months of the next year. The breeding method is complex and sexual, the offspring is easy to mutate, the original characteristics of the cliff tea are not kept, the requirement on the environment is high, and the market demand cannot be met.
2. Chinese patent: patent grant No. CN103202165B, name: a method for cutting and seedling raising of cliff tea comprises the following steps: the invention discloses a method for raising seedlings of adinandra nitida by cutting, which comprises the steps of selecting single plants with excellent quality, taking the middle upper parts of annual twigs, shearing the middle upper parts into special cuttings, soaking in a medicament to promote root growth, soaking in a nutrient solution, establishing a seedbed, preparing special covering soil, cutting, covering and the like. The method breaks through the technical difficulty that the cuttage breeding of the cliff tea tree is extremely low in survival rate or can not be performed at all, starts from five aspects of branch selection, branch pruning, branch treatment, cutting bed matrix preparation, management and protection after cuttage and the like, and enables the cliff tea to be high in survival rate, fast in outplanting and high in outplanting rate. The cuttage technology has low propagation efficiency, can improve the propagation survival rate only by manually operating the technology quantity and maturity, is suitable for the operation of farmers and is not suitable for factory or large-area propagation.
Disclosure of Invention
Compared with the traditional breeding method, the breeding method of the Guangxi wild Adinandra nitida tissue culture seedlings provided by the invention has the advantages that the excellent property genes of the wild Adinandra nitida can be maintained to the maximum extent, the operation is convenient, the survival rate is high, the breeding method is suitable for large-batch or industrial seedling breeding, and the breeding method has social value and commercial value for breeding the rare species of the wild Adinandra nitida.
The technical scheme of the invention is realized as follows:
a tissue culture breeding method of Guangxi wild Adinandra nitida comprises the following steps:
(1) picking explants: in 5-6 months of each year, selecting wild Adinandra nitida which grows on the cliff in all years and has no disease and pest infection and has an altitude of more than 800 meters, and picking tender shoots of the wild Adinandra nitida.
(2) And (3) disinfection: repeatedly washing the tender shoots with purified water for 3-4 times, disinfecting with mercuric chloride solution for 10-20 min, and blotting with disinfected filter paper for later use.
(3) Callus culture: the following operations are carried out on the clean bench: and transversely cutting a bud section with the length of 1.2-1.5 cm by using a sterilized scalpel, putting the bud section into a callus culture medium for culturing, and carrying out dark culture for 15-18 days at the temperature of 28-30 ℃ and the humidity of 60-85% to obtain the callus.
(4) Culturing aseptic cliff tea sprouts: selecting non-polluted callus, transferring the non-polluted callus to a subculture multiplication medium for bud induction culture, and culturing for 30-35 days; the culture conditions are as follows: the temperature is 26 +/-2 ℃, and the illumination intensity is 2000-2500 lux; and finishing when axillary buds are continuously generated.
(5) And (3) rooting culture of aseptic Adinandra nitida seedlings: selecting subculture seedlings with the length of 1.5-2.5 cm, and transferring the subculture seedlings to a rooting culture medium for culture, wherein the culture conditions are as follows: the temperature is 28-30 ℃, and the illumination intensity is 1200-1500 lux; and after culturing for 25-30 days, finishing culturing when main side roots are obvious.
The callus culture medium in the step (3) has the formula of MS + N6 benzyladenine 0.3-0.5 mg/L + naphthylacetic acid 0.8-1.5 mg/L + vitamin C2.0 mg/L + sucrose 30g/L + agar 4.0g/L, and has the pH value of 6.8-7.2.
The formula of the subculture medium in the step (4) is MS + sucrose 4.0g/L + N6-benzylpurine 0.3-0.5 mg/L + naphthylacetic acid 0.3-0.8 mg/L + agar 5g/L, and the pH value is 6.0-6.5.
The rooting medium in the step (5) is prepared from MS, 3-6 g/L of sucrose, 0.5-1.4 mg/L of indoleacetic acid, 0.5-1.3 mg/L of naphthylacetic acid, 0.1mg/L of IBA, 0.5-1.5 g/L of manganese sulfate, 6-8 g/L of agar and 6.0-6.4 of pH.
The invention has the beneficial effects that:
wild Adinandra nitida in Jinxiu county in Guangxi grows on cliff all the year round, is a rare species which changes from the natural environment for thousands of years and is superior to the rare species which survives, and due to the lack of research on the biological characteristics, growth rules and conditions of the wild Adinandra nitida, people cannot succeed in the breeding method of grafting and cuttage. According to the method, through the investigation of biological characteristics and gene research of the wild Adinandra nitida, tender shoots which grow robustly and keep excellent properties of the wild Adinandra nitida are used as explant tissues for culture, and wild Adinandra nitida seedlings which are high in survival rate and can keep excellent properties in later growth are cultured by inducing axillary buds to grow and carrying out rooting culture. The method for breeding the wild adinandra nitida tissue culture seedlings is simple to operate, is suitable for breeding industrialized tissue culture seedlings, and has important significance and immeasurable commercial value for popularizing the wild adinandra nitida as a precious species.
Detailed Description
Example 1
A tissue culture breeding method of Guangxi wild Adinandra nitida comprises the following steps:
(1) picking explants: in 5-6 months of each year, selecting wild Adinandra nitida which grows on the cliff in all years and has no disease and pest infection and has an altitude of more than 800 meters, and picking tender shoots of the wild Adinandra nitida.
(2) And (3) disinfection: repeatedly washing the tender shoots with purified water for 3-4 times, disinfecting with mercuric chloride solution for 10-20 min, and blotting with disinfected filter paper for later use.
(3) Callus culture: the following operations are carried out on the clean bench: transversely cutting a bud section with the length of 1.2-1.5 cm by using a sterilized scalpel, putting the bud section into a callus culture medium for culturing, and carrying out dark culture for 15 days at the temperature of 28-30 ℃ and the humidity of 60-85% to obtain callus; the formula of the callus culture medium is MS + N6 benzyladenine 0.3mg/L + naphthylacetic acid 0.8mg/L + vitamin C2.0 mg/L + sucrose 30g/L + agar 4.0g/L, and the pH value is 6.8.
(4) Culturing aseptic cliff tea sprouts: selecting non-polluted callus, transferring the non-polluted callus to a subculture multiplication medium for bud induction culture for 30 days; the culture conditions are as follows: the temperature is 26 +/-2 ℃, and the illumination intensity is 2000-2500 lux; ending when axillary buds are continuously generated; the formulation of the subculture medium comprises MS, sucrose 4.0g/L, N6-benzylpurine 0.3mg/L, naphthylacetic acid 0.3mg/L, agar 5g/L and pH 6.0.
(5) And (3) rooting culture of aseptic Adinandra nitida seedlings: selecting subculture seedlings with the length of 1.5-2.5 cm, and transferring the subculture seedlings to a rooting culture medium for culture, wherein the culture conditions are as follows: the temperature is 28-30 ℃, and the illumination intensity is 1200-1500 lux; after 25 days of culture, finishing the culture when main lateral roots are obvious; the formula of the rooting medium is MS, 3g/L of sucrose, 0.5mg/L of indoleacetic acid, 0.5mg/L of naphthylacetic acid, 0.1mg/L of IBA0.1mg/L, 0.5g/L of manganese sulfate and 6g/L of agar, and the pH value is 6.0.
Through the breeding method of the tissue culture seedlings, the survival rate of the wild Adinandra nitida tissue culture seedlings is 92.5%, the transplanting and afforestation are carried out after the tissue culture seedlings are cultured, the transplanting survival rate is 86.78%, and 157 mu of forestation is successfully transplanted at present.
Example 2
A tissue culture breeding method of Guangxi wild Adinandra nitida comprises the following steps:
(1) picking explants: in 5-6 months of each year, selecting wild Adinandra nitida which grows on the cliff in all years and has no disease and pest infection and has an altitude of more than 800 meters, and picking tender shoots of the wild Adinandra nitida.
(2) And (3) disinfection: repeatedly washing the tender shoots with purified water for 3-4 times, disinfecting with mercuric chloride solution for 10-20 min, and blotting with disinfected filter paper for later use.
(3) Callus culture: the following operations are carried out on the clean bench: transversely cutting a bud section with the length of 1.2-1.5 cm by using a sterilized scalpel, putting the bud section into a callus culture medium for culturing, and carrying out dark culture for 18 days at the temperature of 28-30 ℃ and the humidity of 60-85% to obtain callus; the formula of the callus culture medium is MS + N6 benzyladenine 0.5mg/L + naphthylacetic acid 1.5mg/L + vitamin C2.0 mg/L + sucrose 30g/L + agar 4.0g/L, and the pH value is 7.2.
(4) Culturing aseptic cliff tea sprouts: selecting non-polluted callus, transferring the non-polluted callus to a subculture multiplication medium for bud induction culture for 35 days; the culture conditions are as follows: the temperature is 26 +/-2 ℃, and the illumination intensity is 2000-2500 lux; ending when axillary buds are continuously generated; the formulation of the subculture medium comprises MS, sucrose 4.0g/L, N6-benzylpurine 0.5mg/L, naphthylacetic acid 0.8mg/L, agar 5g/L and pH 6.5.
(5) And (3) rooting culture of aseptic Adinandra nitida seedlings: selecting subculture seedlings with the length of 1.5-2.5 cm, and transferring the subculture seedlings to a rooting culture medium for culture, wherein the culture conditions are as follows: the temperature is 28-30 ℃, and the illumination intensity is 1200-1500 lux; after 30 days of culture, finishing the culture when main lateral roots are obvious; the formula of the rooting medium is MS + 6g/L sucrose + 1.4mg/L indoleacetic acid + 1.3mg/L naphthylacetic acid + 0.1mg/L IBA0 + 1.5g/L manganese sulfate + 6-8 g/L agar, and the pH value is 6.4.
By adopting the method, the survival rate of the wild adinandra nitida tissue culture seedlings is 91.2 percent, the transplanting and afforestation are carried out after the tissue culture seedlings are cultured, the transplanting survival rate is 83.4 percent, and the wild adinandra nitida tissue culture seedlings are successfully transplanted and afforested for 225 mu at present.
Example 3
A tissue culture breeding method of Guangxi wild Adinandra nitida comprises the following steps:
(1) picking explants: in 5-6 months of each year, selecting wild Adinandra nitida which grows on the cliff in all years and has no disease and pest infection and has an altitude of more than 800 meters, and picking tender shoots of the wild Adinandra nitida.
(2) And (3) disinfection: repeatedly washing the tender shoots with purified water for 3-4 times, disinfecting with mercuric chloride solution for 10-20 min, and blotting with disinfected filter paper for later use.
(3) Callus culture: the following operations are carried out on the clean bench: transversely cutting a bud section with the length of 1.2-1.5 cm by using a sterilized scalpel, putting the bud section into a callus culture medium for culture, and carrying out dark culture for 168 days at the temperature of 28-30 ℃ and the humidity of 60-85% to obtain callus; the formula of the callus culture medium is MS + N6 benzyladenine 0.4mg/L + naphthylacetic acid 0.8mg/L + vitamin C2.0 mg/L + sucrose 30g/L + agar 4.0g/L, and the pH value is 6.9.
(4) Culturing aseptic cliff tea sprouts: selecting non-polluted callus, transferring the non-polluted callus to a subculture multiplication medium for bud induction culture for 32 days; the culture conditions are as follows: the temperature is 26 +/-2 ℃, and the illumination intensity is 2000-2500 lux; ending when axillary buds are continuously generated; the formulation of the subculture medium comprises MS, sucrose 4.0g/L, N6-benzylpurine 0.3mg/L, naphthylacetic acid 0.5mg/L, agar 5g/L and pH 6.3.
(5) And (3) rooting culture of aseptic Adinandra nitida seedlings: selecting subculture seedlings with the length of 1.5-2.5 cm, and transferring the subculture seedlings to a rooting culture medium for culture, wherein the culture conditions are as follows: the temperature is 28-30 ℃, and the illumination intensity is 1200-1500 lux; after culturing for 26 days, finishing culturing when main lateral roots are obvious; the rooting medium comprises MS, sucrose 5g/L, indoleacetic acid 1.0mg/L, naphthylacetic acid 0.8mg/L, IBA0.1mg/L, manganese sulfate 0.8g/L and agar 6-8 g/L, and the pH value is 6.2.
By using the method, the survival rate of the wild adinandra nitida tissue culture seedlings reaches 95.5%, and the tissue culture seedlings of the batch are not outplanted for forestation.
Example 4
A tissue culture breeding method of Guangxi wild Adinandra nitida comprises the following steps:
(1) picking explants: in 5-6 months of each year, selecting wild Adinandra nitida which grows on the cliff in all years and has no disease and pest infection and has an altitude of more than 800 meters, and picking tender shoots of the wild Adinandra nitida.
(2) And (3) disinfection: repeatedly washing the tender shoots with purified water for 3-4 times, disinfecting with mercuric chloride solution for 10-20 min, and blotting with disinfected filter paper for later use.
(3) Callus culture: the following operations are carried out on the clean bench: transversely cutting a bud section with the length of 1.2-1.5 cm by using a sterilized scalpel, putting the bud section into a callus culture medium for culturing, and carrying out dark culture for 15 days at the temperature of 28-30 ℃ and the humidity of 60-85% to obtain callus; the formula of the callus culture medium is MS + N6 benzyladenine 0.5mg/L + naphthylacetic acid 1.5mg/L + vitamin C2.0 mg/L + sucrose 30g/L + agar 4.0g/L, and the pH value is 7.0.
(4) Culturing aseptic cliff tea sprouts: selecting non-polluted callus, transferring the non-polluted callus to a subculture multiplication medium for bud induction culture for 35 days; the culture conditions are as follows: the temperature is 26 +/-2 ℃, and the illumination intensity is 2000-2500 lux; ending when axillary buds are continuously generated; the formulation of the subculture medium comprises MS, sucrose 4.0g/L, N6-benzylpurine 0.3mg/L, naphthylacetic acid 0.3mg/L, agar 5g/L and pH 6.2.
(5) And (3) rooting culture of aseptic Adinandra nitida seedlings: selecting subculture seedlings with the length of 1.5-2.5 cm, and transferring the subculture seedlings to a rooting culture medium for culture, wherein the culture conditions are as follows: the temperature is 28-30 ℃, and the illumination intensity is 1200-1500 lux; after 28 days of culture, finishing the culture when main lateral roots are obvious; the rooting medium comprises MS, sucrose 6g/L, indoleacetic acid 1.0mg/L, naphthylacetic acid 1.0mg/L, IBA0.1mg/L, manganese sulfate 0.8g/L and agar 6-8 g/L, and the pH value is 6.2.
By using the method, the survival rate of the wild adinandra nitida tissue culture seedlings is 96.1 percent, the transplanting and afforestation are carried out after the tissue culture seedlings are cultured, the transplanting survival rate is 80.2 percent, and 55 mu of forestation is successfully transplanted at present.
Example 5
A tissue culture breeding method of Guangxi wild Adinandra nitida comprises the following steps:
(1) picking explants: in 5-6 months of each year, selecting wild Adinandra nitida which grows on the cliff in all years and has no disease and pest infection and has an altitude of more than 800 meters, and picking tender shoots of the wild Adinandra nitida.
(2) And (3) disinfection: repeatedly washing the tender shoots with purified water for 3-4 times, disinfecting with mercuric chloride solution for 10-20 min, and blotting with disinfected filter paper for later use.
(3) Callus culture: the following operations are carried out on the clean bench: transversely cutting a bud section with the length of 1.2-1.5 cm by using a sterilized scalpel, putting the bud section into a callus culture medium for culturing, and carrying out dark culture for 18 days at the temperature of 28-30 ℃ and the humidity of 60-85% to obtain callus; the formula of the callus culture medium is MS + N6 benzyladenine 0.3mg/L + naphthylacetic acid 0.8mg/L + vitamin C2.0 mg/L + sucrose 30g/L + agar 4.0g/L, and the pH value is 6.8.
(4) Culturing aseptic cliff tea sprouts: selecting non-polluted callus, transferring the non-polluted callus to a subculture multiplication medium for bud induction culture for 35 days; the culture conditions are as follows: the temperature is 26 +/-2 ℃, and the illumination intensity is 2000-2500 lux; ending when axillary buds are continuously generated; the formulation of the subculture medium is MS + sucrose 4.0g/L + N6-benzylpurine 0.5mg/L + naphthylacetic acid 0.4mg/L + agar 5g/L, and the pH value is 6.5.
(5) And (3) rooting culture of aseptic Adinandra nitida seedlings: selecting subculture seedlings with the length of 1.5-2.5 cm, and transferring the subculture seedlings to a rooting culture medium for culture, wherein the culture conditions are as follows: the temperature is 28-30 ℃, and the illumination intensity is 1200-1500 lux; after culturing for 26 days, finishing culturing when main lateral roots are obvious; the rooting medium comprises MS, sucrose 6g/L, indoleacetic acid 0.5mg/L, naphthylacetic acid 0.5mg/L, IBA0.1mg/L, manganese sulfate 0.5g/L and agar 6-8 g/L, and the pH value is 6.3.
By using the method, the survival rate of the wild adinandra nitida tissue culture seedlings is 94.0 percent, the transplanting and afforestation are carried out after the tissue culture seedlings are cultured, the transplanting survival rate is 86.25 percent, and the transplanting and afforestation are carried out for 332 mu successfully at present.
Claims (2)
1. A tissue culture breeding method of Guangxi wild Adinandra nitida is characterized in that: the method comprises the following steps:
(1) picking explants: selecting wild Adinandra nitida at an altitude of more than 800 meters and growing on cliff all year round without being infected by diseases and insect pests in 5-6 months every year, and picking tender shoots of the wild Adinandra nitida;
(2) and (3) disinfection: repeatedly washing tender shoots for 3-4 times by using purified water, disinfecting for 10-20 min by using a mercuric chloride solution, and sucking dry by using a disinfected filter paper sheet for later use;
(3) callus culture: the following operations are carried out on the clean bench: transversely cutting a bud section with the length of 1.2-1.5 cm by using a sterilized scalpel, putting the bud section into a callus culture medium for culturing, and carrying out dark culture for 15-18 days at the temperature of 28-30 ℃ and the humidity of 60-85% to obtain callus;
(4) culturing aseptic cliff tea sprouts: selecting non-polluted callus, transferring the non-polluted callus to a subculture multiplication medium for bud induction culture, and culturing for 30-35 days; the culture conditions are as follows: the temperature is 26 +/-2 ℃, and the illumination intensity is 2000-2500 lux; ending when axillary buds are continuously generated;
(5) and (3) rooting culture of aseptic Adinandra nitida seedlings: selecting subculture sprouts with the sprout length of 1.5-2.5 cm, transferring the subculture sprouts to a rooting culture medium for culture, wherein the culture conditions are as follows: the temperature is 28-30 ℃, and the illumination intensity is 1200-1500 lux; after culturing for 25-30 days, finishing culturing when main lateral roots are obvious;
the callus culture medium in the step (3) has a formula of MS + N6 benzyladenine 0.3-0.5 mg/L + naphthylacetic acid 0.8-1.5 mg/L + vitamin C2.0 mg/L + sucrose 30g/L + agar 4.0g/L, and has a pH of 6.8-7.2;
the formula of the subculture medium in the step (4) is MS + sucrose 4.0g/L + N6-benzylpurine 0.3-0.5 mg/L + naphthylacetic acid 0.3-0.8 mg/L + agar 5g/L, and the pH value is 6.0-6.5.
2. The tissue culture breeding method of Guangxi wild Adinandra nitida as claimed in claim 1, which is characterized in that: the rooting medium in the step (5) is prepared from MS, 3-6 g/L of sucrose, 0.5-1.4 mg/L of indoleacetic acid, 0.5-1.3 mg/L of naphthylacetic acid, 0.1mg/L of IBA, 0.5-1.5 g/L of manganese sulfate, 6-8 g/L of agar and 6.0-6.4 of pH.
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| CN102007867A (en) * | 2009-09-07 | 2011-04-13 | 湖南省林业科学院 | Efficient rooting method for oil tea clone tissue culture seedlings |
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| CN102422810A (en) * | 2011-09-02 | 2012-04-25 | 安徽农业大学 | In-vitro regeneration culture method for tea clones |
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