CN110754367B - Method for imitating wild cultivation of seedlings by using anoectochilus formosanus protocorm - Google Patents
Method for imitating wild cultivation of seedlings by using anoectochilus formosanus protocorm Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
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Abstract
The invention discloses a method for imitating wild seedling cultivation of an anoectochilus formosanus protocorm, which comprises the steps of taking mature anoectochilus formosanus capsule as a material, sterilizing, cutting the capsule, sowing seeds on a culture medium, performing propagation culture after the seeds germinate to form protocorm, directly sowing the protocorm in a forest land rich in humus in the field, making small arched sheds with films, allowing the protocorm to grow roots and buds in the small arched sheds with certain humidity, absorbing nutrients in the nature, removing the films when the seedlings grow to 2-3 leaves, and finishing the imitation wild seedling cultivation of the anoectochilus formosanus protocorm. By adopting the method, the seedling time of the anoectochilus formosanus is short, the survival rate is high, the uniformity is good, no variation exists, no plant growth regulator residue is generated during picking, the stress resistance of the plant is good, the growth is fast, the yield is high, the actual problem in the existing production of the anoectochilus formosanus is solved, the supply-demand contradiction of the anoectochilus formosanus can be effectively relieved, and the product has high yield, high quality, good benefit and low cost.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine cultivation, and particularly relates to a method for wild-simulated cultivation of seedlings by using anoectochilus formosanus protocorms.
Background
Anoectochilus formosanus (Anoectochilus formosanus)Anoectochilusroxburghii(Wall.) Lindl is a plant of Orchidaceae, belongs to the genus Orchidaceae, is a traditional precious medicinal material in China, is completely medicinal, contains components such as polysaccharide, flavonoid, alkaloid, trace elements and the like, has the effects of dispelling wind and removing dampness, calming the liver, reducing blood sugar and the like, and has good curative effects on diabetes, hematuria, tumors, various inflammations and the like. Anoectochilus roxburghii is known as Yaowang in folk life and is known as Hakka,Taiwan and Guangdong are often used as high-grade food materials for cooking soup and nourishing.
Along with the continuous improvement of living standard of people, more attention is paid to health and health preservation, the demand of anoectochilus formosanus for both medicine and food is increased day by day, wherein the annual demand of Japan and Korea is more than 1000 tons, and about 700 tons is imported from China basically. The wild anoectochilus formosanus is difficult to germinate in the natural environment due to small seeds, the propagation and growth speed is slow, and the wild anoectochilus formosanus is dug by farmers in a plunder type manner, so that the wild anoectochilus formosanus becomes deficient day by day and is in an endangered state. The contradiction between supply and demand of wild anoectochilus formosanus is increasingly sharp, the market price is high, the fresh product is 800-1000 yuan/kg, the yield per mu is 80kg, and the yield per mu is 6.4-8 ten thousand yuan.
Accelerating the artificial cultivation of the anoectochilus roxburghii is imperative, and the seedlings become the key factor of the artificial cultivation of the anoectochilus roxburghii. Although the current plant tissue culture technology of anoectochilus formosanus breaks through the bottleneck and is better developed, the problems of slower rooting process, long propagation period, high variability, low uniformity, low survival rate, high production cost, residual plant growth regulator and the like still exist, and how to solve the problems of the current tissue culture seedlings of the anoectochilus formosanus is a key for solving the actual production problem of the anoectochilus formosanus.
Disclosure of Invention
During the planting process of anoectochilus formosanus, the inventor finds that a small amount of plant base can generate protocorm and develop into small seedlings, but the amount is small, and the larger the protocorm is, the stronger the seedling is, and the better the growth vigor is. The invention provides a method for cultivating seedlings of anoectochilus formosanus protocorm in a wild imitation way, aiming at solving the problems existing in the current production process of the anoectochilus formosanus tissue culture seedlings and accelerating the wild imitation cultivation of the anoectochilus formosanus.
The invention relates to a method for cultivating seedlings of anoectochilus formosanus protocorm by imitating wild cultivation, which comprises the following steps:
(1) aseptic seeding of mature capsules of anoectochilus formosanus: picking up mature capsule in sunny day, sterilizing with 75% ethanol for 30 s on a superclean workbench, washing with sterile water for 1 time, and sucking out water on the surface of the capsule with sterile paper; 0.1% mercuric chloride (HgCl) was added again2) Sterilizing for 10 min, washing with sterile water for 5 times, and sucking capsule with sterile paperSurface moisture; transversely cutting one end of a capsule, sowing seeds on a primary culture medium, culturing in a culture chamber, performing dark culture for 5 days, performing light culture, and after 15-20 days, enabling the seeds to absorb water and turn round and green; germinating for 30-45 days to form protocorms, wherein the germination rate of seeds is 53.7% -88.5%;
the primary culture medium is MS +6-BA 0.5-2.0 mg/L + GA 0.1-1.0 mg/L + NAA 0.1-0.5 mg/L +10% coconut milk + white sugar 25 g/L + agar 4 g/L;
(2) and (3) performing proliferation culture on protocorms: cutting the protocorm cluster obtained in the step (1) into small clusters, inoculating the small clusters into a proliferation culture medium for culturing, wherein the protocorm begins to proliferate after 10 days, and the proliferation coefficient is counted after 30 days and is 1.8-4.1;
the protocorm proliferation culture medium comprises MS, CPPU 0.05-0.5 mg/L, TDZ 0.1-1.0 mg/L, NAA 0.1-0.5 mg/L, 10% coconut milk, white sugar 25 g/L and agar 4 g/L;
the culture conditions are as follows: the temperature is 25 +/-3 ℃, the illumination time is 6 h/d, and the illumination intensity is 500 lx;
(3) the anoectochilus formosanus protocorm wild-imitating cultivation seedling:
(3.1) selecting forest land and preparing a seedling culture substrate: 90% of canopy density of the forest land, paving a layer of matrix with the thickness of 1 cm on the ground, crushing and uniformly mixing the matrix according to the proportion of 10-30% of sheep manure, 10-40% of cow manure and 30-80% of loess, and spraying water for two days before sowing the original corms for later use;
(3.2) during the period of 4-6 months, taking out the protocorm obtained in the step (2), cleaning the culture medium, sowing the protocorm on a substrate of a forest land, covering a layer of leaves with the thickness of 0.5 cm, making an arch shed by using a film, wherein the humidity in the arch shed is 80%, after 15-25 days, buds begin to grow on the protocorm, after 25-30 days, the bases of the buds protrude, after 30-40 days, the roots begin to grow, after 50-65 days, seedlings with 2-3 roots and 2-3 leaves can grow, and the survival rate of the seedlings is 61.3% -92.6%.
In the method, the primary culture medium in the step (1) is MS +6-BA 0.5-2.0 mg/L + GA 0.1-1.0 mg/L + NAA 0.1-0.5 mg/L +10% coconut juice + white sugar 25 g/L + agar 4 g/; 6-benzylaminopurine (6-BA) in the culture medium mainly has the effects of promoting cell division and increasing cell volume; gibberellin (GA) has the functions of breaking dormancy, promoting cell growth and accelerating seed germination; the naphthylacetic acid (NAA) can promote cell division and enlargement, and promote cell growth; the three plant growth regulators act together to promote the germination of anoectochilus formosanus seeds and the growth of protocorm.
The protocorm proliferation culture medium in the step (2) is MS + CPPU 0.05-0.5 mg/L + TDZ 0.1-1.0 mg/L + NAA 0.1-0.5 mg/L +10% coconut milk + white sugar 25 g/L + agar 4 g/L, and the thiadiazophenyl urea (TDZ) in the culture medium has the special function of dual functions of auxin and cytokinin, can adjust the cell membrane structure, the energy level, the nutrient absorption and the assimilation, and promotes the somatic embryogenesis; forchlorfenuron (CPPU) is a phenylurea plant growth regulator with cytokinin activity, and promotes cell division and growth expansion by accelerating mitosis, increasing cell number, enlarging cells, and the like; the naphthylacetic acid (NAA) can promote cell division and enlargement, and promote cell growth; the three plant growth regulators act together to promote the proliferation of the protocorm of anoectochilus formosanus. The coconut milk is rich in amino acids, hormones, enzymes and other substances, and can promote sterile germination and protocorm proliferation of the anoectochilus formosanus seeds. These substances work together to promote the germination of the anoectochilus formosanus seeds and the growth of protocorm.
In the prior art, a seed seedling is cultivated by using an anoectochilus formosanus protocorm, but the protocorm is proliferated and then is differentiated into a seedling on a culture medium in a culture room, a strong seedling rooting culture and other processes are carried out, and the rooting process of the anoectochilus formosanus tissue culture seedling is slow, the propagation period is long, the variability is high, the survival rate is low, and the production cost is high. The method of the invention has improved various bad indexes.
The following is a comparison table of technical indexes of breeding the method and the common tissue culture seedlings of anoectochilus formosanus:
as can be seen from the above table, the anoectochilus formosanus seedling-forming time is short, the survival rate is high, the uniformity is good, no variation exists, no plant growth regulator residue exists during picking, the stress resistance of the plant is good, the growth is fast, the yield is high, the actual problem in the existing anoectochilus formosanus production is solved, and the supply and demand contradiction of the anoectochilus formosanus can be effectively relieved; the method has the advantages of simple operation, easy control, high safety, good stability, high yield, good quality, good benefit, low cost, and saving of a large amount of resources such as manpower and material resources.
Detailed Description
Example 1:
a method for cultivating seedlings of anoectochilus formosanus protocorm imitating wild comprises the following steps:
(1) aseptic seeding of mature capsules of anoectochilus formosanus: picking up mature capsules (75-90 days after pollination) in a sunny day, sterilizing the capsules for 30 s on a superclean workbench by using 75% ethanol, washing the capsules for 1 time by using sterile water, and sucking the water on the surfaces of the capsules by using sterile paper; 0.1% mercuric chloride (HgCl) was added again2) Sterilizing for 10 min, washing with sterile water for 5 times, and drying the surface water of capsule with sterile paper; transversely cutting one end of a capsule, sowing seeds on a primary culture medium, culturing in a culture chamber, performing dark culture for 5 days, performing light culture, and allowing the seeds to absorb water to turn round and green after about 20 days; the seeds begin to germinate to form protocorms in about 45 days, the protocorms are small, and the germination rate of the seeds is 53.7%;
the primary culture medium is MS +6-BA0.5 mg/L + GA0.1 mg/L + NAA0.1mg/L +10% coconut milk + white sugar 25 g/L + agar 4 g/L;
(2) and (3) performing proliferation culture on protocorms: cutting the protocorm cluster obtained in the step (1) into small clusters, inoculating the small clusters into a proliferation culture medium for culturing, wherein the protocorm begins to proliferate after 10 days, the statistical proliferation coefficient is 1.8 after 30 days, and the head of the protocorm is smaller;
the protocorm proliferation culture medium is MS + CPPU0.05 mg/L + TDZ0.1 mg/L + NAA0.1mg/L +10% coconut milk + white sugar 25 g/L + agar 4 g/L;
the culture conditions of the culture chamber were: the temperature is 25 +/-3 ℃, the illumination time is 6 h/d, and the illumination intensity is 500 lx;
(3) the anoectochilus formosanus protocorm wild-imitating cultivation seedling:
(3.1) selecting forest land and preparing a seedling culture substrate: the canopy density of the forest land is 90%, a layer of matrix with the thickness of 1 cm is paved on the ground, the matrix is crushed and uniformly mixed according to the proportion of 10% of sheep manure, 10% of cow manure and 80% of loess, and water is sprayed for standby two days before the original bulbs are sown;
(3.2) during the period of 4-6 months, taking out the protocorm obtained in the step (2), cleaning a culture medium, sowing the protocorm on a substrate of a forest land, covering a layer of leaves with the thickness of 0.5 cm, making an arch shed by using a film, wherein the humidity in the arch shed is 80%, after 25 days, buds begin to grow on the protocorm, after 30 days, the bases of the buds protrude, after 40 days, the roots begin to grow, after 65 days, seedlings with 2-3 roots and 2-3 leaves can grow, and the survival rate of the seedlings is 61.3%.
Example 2:
a method for cultivating seedlings of anoectochilus formosanus protocorm imitating wild comprises the following steps:
(1) aseptic seeding of mature capsules of anoectochilus formosanus: picking up mature capsules (75-90 days after pollination) in a sunny day, sterilizing the capsules for 30 s on a superclean workbench by using 75% ethanol, washing the capsules for 1 time by using sterile water, and sucking the water on the surfaces of the capsules by using sterile paper; 0.1% mercuric chloride (HgCl) was added again2) Sterilizing for 10 min, washing with sterile water for 5 times, and drying the surface water of capsule with sterile paper; transversely cutting one end of a capsule, sowing seeds on a primary culture medium, culturing in a culture chamber, performing dark culture for 5 days, performing light culture, and after 15 days, enabling the seeds to absorb water and turn round and green; the seeds begin to germinate to form protocorms in 30 days, the protocorms have larger heads, and the germination rate of the seeds is 76.1 percent;
the primary culture medium is MS +6-BA2.0 mg/L + GA1.0 mg/L + NAA0.5 mg/L +10% coconut milk + white sugar 25 g/L + agar 4 g/L;
(2) and (3) performing proliferation culture on protocorms: cutting the protocorm cluster obtained in the step (1) into small clusters, inoculating the small clusters into a proliferation culture medium for culturing, wherein the protocorm begins to proliferate after 10 days, and the proliferation coefficient is counted after 30 days and is 3.6, and the protocorm has larger size but is not uniform enough;
the protocorm proliferation culture medium is MS + CPPU0.5 mg/L + TDZ1.0 mg/L + NAA0.5 mg/L +10% coconut milk + white sugar 25 g/L + agar 4 g/L;
the culture conditions of the culture chamber are as follows: the temperature is 25 +/-3 ℃, the illumination time is 6 h/d, and the illumination intensity is 500 lx;
(3) the anoectochilus formosanus protocorm wild-imitating cultivation seedling:
(3.1) selecting forest land and preparing a seedling culture substrate: the canopy density of the forest land is 90%, a layer of matrix with the thickness of 1 cm is paved on the ground, the matrix is crushed and mixed uniformly according to the proportion of 30% of sheep manure, 40% of cow manure and 30% of loess, and water is sprayed for standby two days before the original bulbs are sown;
(3.2) during the period of 4-6 months, taking out the protocorm obtained in the step (2), cleaning a culture medium, sowing the protocorm on a substrate of a forest land, covering a layer of leaves with the thickness of 0.5 cm, making an arch shed by using a film, wherein the humidity in the arch shed is 80%, after 15 days, buds begin to grow on the protocorm, after 25 days, the bases of the buds protrude, after 30 days, the roots begin to grow, after 50 days, seedlings with 2-3 roots and 2-3 leaves can grow, and the survival rate of the seedlings is 83.9%.
Example 3:
a method for cultivating seedlings of anoectochilus formosanus protocorm imitating wild comprises the following steps:
(1) aseptic seeding of mature capsules of anoectochilus formosanus: picking up mature capsules (75-90 days after pollination) in a sunny day, sterilizing the capsules for 30 s on a superclean workbench by using 75% ethanol, washing the capsules for 1 time by using sterile water, and sucking the water on the surfaces of the capsules by using sterile paper; 0.1% mercuric chloride (HgCl) was added again2) Sterilizing for 10 min, washing with sterile water for 5 times, and drying the surface water of capsule with sterile paper; transversely cutting one end of a capsule, sowing seeds on a primary culture medium, culturing in a culture chamber, performing dark culture for 5 days, performing light culture, and after 17 days, allowing the seeds to absorb water and turn round and green; seeds germinate to form protocorms in about 35 days, the protocorms are large in size, and the germination rate of the seeds is 88.5%;
the primary culture medium is MS +6-BA1.0 mg/L + GA0.5 mg/L + NAA0.3mg/L +10% coconut milk + white sugar 25 g/L + agar 4 g/L;
(2) and (3) performing proliferation culture on protocorms: cutting the protocorm ball obtained in the step (1) into small balls, inoculating the small balls into a proliferation culture medium for culturing, beginning proliferation of the protocorm after about 10 days, counting the proliferation coefficient after 30 days, wherein the proliferation coefficient is 4.1, the head of the protocorm is generally large, and the size of the protocorm is uniform;
the protocorm proliferation culture medium is MS + CPPU0.2 mg/L + TDZ0.4 mg/L + NAA0.3mg/L +10% coconut milk + white sugar 25 g/L + agar 4 g/L;
the culture conditions of the culture chamber were: the temperature is 25 +/-3 ℃, the illumination time is 6 h/d, and the illumination intensity is 500 lx;
(3) the anoectochilus formosanus protocorm wild-imitating cultivation seedling:
(3.1) selecting forest land and preparing a seedling culture substrate: the canopy density of the forest land is 90%, a layer of matrix with the thickness of 1 cm is paved on the ground, the matrix is crushed and uniformly mixed according to the proportion of 20% of sheep manure, 30% of cow manure and 50% of loess, and water is sprayed for standby two days before the original bulbs are sowed;
(3.2) during the period of 4-6 months, taking out the protocorm obtained in the step (2), cleaning a culture medium, sowing the protocorm on a substrate of a forest land, covering a layer of leaves with the thickness of 0.5 cm, making an arch shed by using a film, wherein the humidity in the arch shed is 80%, after 18 days, buds begin to grow on the protocorm, after 26 days, the bases of the buds protrude, after 33 days, the roots begin to grow, after 50 days, seedlings with 2-3 roots and 2-3 leaves can grow, and the survival rate of the seedlings is 92.6%.
From examples 1-3, it can be seen that the concentration of the plant growth regulator in the medium affects the germination rate and protocorm size of the anoectochilus formosanus seeds, while the size of the protocorm of the anoectochilus formosanus affects the germination rate of the seedlings, the larger the protocorm, the higher the germination rate of the seedlings and the higher the survival rate of the seedlings; by combining the above factors, example 3 showed the highest proliferation rate, uniform protocorm size, the highest seedling germination rate and the best survival rate.
Claims (2)
1. A method for cultivating seedlings of anoectochilus formosanus protocorm imitating wild comprises the following steps:
(1) aseptic seeding of mature capsules of anoectochilus formosanus: picking up mature capsule in sunny day, sterilizing with 75% ethanol for 30 s on a superclean workbench, washing with sterile water for 1 time, and sucking out water on the surface of the capsule with sterile paper; sterilizing with 0.1% mercuric chloride for 10 min, washing with sterile water for 5 times, and drying the surface water of capsule with sterile paper; transversely cutting one end of a capsule, sowing seeds on a primary culture medium, performing dark culture for 5 days, performing light culture, and after 15-20 days, enabling the seeds to absorb water and turn round and green; germinating for 30-45 days to form protocorms, wherein the germination rate of seeds is 53.7% -88.5%;
the primary culture medium is MS +6-BA 0.5-2.0 mg/L + GA 0.1-1.0 mg/L + NAA 0.1-0.5 mg/L +10% coconut milk + white sugar 25 g/L + agar 4 g/L;
(2) and (3) performing proliferation culture on protocorms: cutting the protocorm cluster obtained in the step (1) into small clusters, inoculating the small clusters into a proliferation culture medium for culturing, wherein the protocorm begins to proliferate after 10 days, and the proliferation coefficient is counted after 30 days and is 1.8-4.1;
the protocorm proliferation culture medium comprises MS, CPPU 0.05-0.5 mg/L, TDZ 0.1-1.0 mg/L, NAA 0.1-0.5 mg/L, 10% coconut milk, white sugar 25 g/L and agar 4 g/L;
the proliferation culture conditions are as follows: the temperature is 25 +/-3 ℃, the illumination time is 6 h/day, and the illumination intensity is 500 lx;
(3) the anoectochilus formosanus protocorm wild-imitating cultivation seedling:
(3.1) selecting forest land and preparing a seedling culture substrate: 90% of canopy density of the forest land, paving a layer of matrix with the thickness of 1 cm on the ground, crushing and uniformly mixing the matrix according to the proportion of 10-30% of sheep manure, 10-40% of cow manure and 30-80% of loess, and spraying water for two days before sowing the original corms for later use;
(3.2) during the period of 4-6 months, taking out the protocorm obtained in the step (2), cleaning the culture medium, sowing the protocorm on a substrate of a forest land, covering a layer of leaves with the thickness of 0.5 cm, making an arch shed by using a film, wherein the humidity in the arch shed is 80%, after 15-25 days, buds begin to grow on the protocorm, after 25-30 days, the bases of the buds protrude, after 30-40 days, the roots begin to grow, after 50-65 days, seedlings with 2-3 roots and 2-3 leaves grow, and the survival rate of the seedlings is 61.3% -92.6%.
2. The method for cultivating seedlings of anoectochilus formosanus protocorm by imitating wild conditions according to claim 1, wherein the method comprises the following steps:
the primary culture medium in the step (1) is MS +6-BA1.0 mg/L + GA0.5 mg/L + NAA0.3mg/L +10% coconut milk + white sugar 25 g/L + agar 4 g/L;
the protocorm proliferation culture medium in the step (2) is MS + CPPU0.2 mg/L + TDZ0.4 mg/L + NAA0.3mg/L +10% coconut milk + white sugar 25 g/L + agar 4 g/L.
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