CN116195516B - Non-symbiotic germination medium and breeding method for anoectochilus formosanus homozygotic plant seeds - Google Patents
Non-symbiotic germination medium and breeding method for anoectochilus formosanus homozygotic plant seeds Download PDFInfo
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Abstract
The invention belongs to the technical field of plant seedling culture, in particular to a method for cultivating plant seedlingsDiscloses a non-symbiotic germination culture medium and a breeding method of anoectochilus formosanus homonymous plant seeds, wherein the culture medium is an improved B5 culture medium and comprises the following components: 113.24 mg.L ‑1 Anhydrous calcium chloride, 150 mg.L ‑1 Sodium dihydrogen phosphate, 2500 mg.L ‑1 Potassium nitrate 134 mg.L ‑1 Ammonium sulfate 122.09 mg L ‑1 Anoectochilus roxburghii belongs to plants including Gao Jinxian orchid, herba Anoectochilus roxburghii, herba Castaneae, and the like, the culture medium formula and the breeding method can enable various kinds of Anoectochilus roxburghii seeds to germinate into seedlings, the germination time is 10 days earlier, the germination rate is far higher than that of the existing culture medium, stem node proliferation induction culture is added in research, and therefore, anoectochilus roxburghii seedlings can be obtained in a large amount, the requirement on wild resources of Anoectochilus roxburghii is relieved, and the purpose of promoting the protection of the species is really achieved.
Description
Technical Field
The invention belongs to the technical field of plant seedling culture, and particularly relates to a non-symbiotic germination culture medium and a breeding method for anoectochilus formosanus homozygotic plant seeds.
Background
Anoectochilus spp (also known as Anoectochilus roxburghii) is a perennial herb of Orchidaceae, alchezia, mainly distributed in China, japan, spearmint, india, nepal and other countries, and mainly distributed in Yunnan, fujian, zhejiang, guangdong, guangxi, jiangxi and other subtropical and tropical regions in China, 40 species are available worldwide, and 20 species and 2 varieties are available in China. The anoectochilus formosanus is a rare Chinese herbal medicine in China, and has the names of medicine king and golden grass, all the herbs can be used as medicines, have sweet taste and slight cold property, have the effects of clearing heat and detoxicating, cooling blood and calming gall, and the like, and have very long traditional medicine history in China. The anoectochilus formosanus contains various active ingredients such as polysaccharide, trace elements, cardiac glycosides, amino acids, organic acids, steroid compounds, flavonoid compounds, alkaloids and the like, and is widely used for treating hepatitis, nephritis, pneumonia, infantile convulsion and hyperpyrexia, diabetes, tumor resistance, rheumatism and the like. A plurality of anoectochilus formosanus under the genus of Anoectochilus have been used as basic plants for producing the anoectochilus formosanus, and traditional Chinese patent medicines such as compound anoectochilus formosanus capsules, anoectochilus formosanus spray and the like, which take the anoectochilus formosanus as main raw materials, have been clinically used for treating related diseases.
The anoectochilus formosanus belongs to the same plant, has small plant type, beautiful leaf shape, golden or silvery veins, is arranged in a net shape, and is an indoor leaf-viewing precious product with extremely high ornamental value. The anoectochilus formosanus has higher medicinal and ornamental values, the wild resources of the anoectochilus formosanus are exhausted due to artificial long-term unordered excessive excavation, and the anoectochilus formosanus has low natural reproduction rate, strict requirements on ecological environment and poor adaptability, so that the species is almost extinct. The wild resources need to be protected, the market demand for the anoectochilus roxburghii is high, and only a large number of anoectochilus roxburghii seedlings can be artificially bred to meet the utilization, so that the demand for the wild resources is reduced.
The method adopted by the artificial seedling breeding of orchid is divided propagation, tissue culture and seed non-symbiotic propagation, the divided propagation technology is not applicable to the orchid which does not generate lateral buds, the tissue culture is the most common propagation method of the orchid and is reported most but limited by the collection quantity of plant tissues, the culture mediums used by different tissues of different varieties are different, the large-scale multi-variety culture of the factory is not facilitated in the large-scale culture process of the factory, and the purchase cost, the complex procedure prepared by a plurality of culture mediums and the labor cost are increased under the condition of simultaneous culture of a plurality of varieties. At present, the existing literature and patents report that the breeding methods of anoectochilus formosanus (Anoectochilus roxburghii), anoectochilus formosanus (Anoectochilus formosanus), anoectochilus formosanus (Anoectochilus chapaensis), anoectochilus formosanus (Anoectochilus caLcareus Aver), anoectochilus formosanus (Anoectochilus emeiensis), anoectochilus formosanus (Anoectochilus xingrenensis) and the like are respectively tried to carry out breeding by different culture mediums (MS, 1/2MS, 1/4MS, H1, H2, H3, N6, KC and the like), the germination effect and the germination rate are uneven, and the anoectochilus formosanus or different tissue parts are replaced by different culture mediums and breeding methods. By exploring the non-symbiotic germination technology of seeds, a general culture medium formula and a breeding method suitable for non-symbiotic germination of the seeds of different anoectochilus formosanus are screened for mainly cultivated anoectochilus formosanus homonymous plants, so that the cost is reduced, the time is saved, a large number of various anoectochilus formosanus seedlings meeting market demands can be obtained in a short period, and the aims of utilizing promotion and protection are really achieved, so that an efficient mode is provided for development, utilization and conservation of anoectochilus formosanus.
Disclosure of Invention
The invention mainly aims to provide a germination medium and a breeding method for large-scale multi-variety anoectochilus roxburghii seeds, which adopt a seed non-symbiotic germination technology to screen a general culture medium formula and a breeding method suitable for non-symbiotic germination of different anoectochilus roxburghii seeds, reduce cost and save time, and are hopeful to obtain a large number of various anoectochilus roxburghii seedlings meeting market demands in a short period, so that the purpose of utilizing promotion and protection is truly achieved, and an efficient mode is provided for development, utilization and conservation of anoectochilus roxburghii.
In order to achieve the above object, the present invention provides the following technical solutions:
a non-symbiotic germination medium for a seed of a syngeneic plant of anoectochilus formosanus, the medium being a modified B5 medium comprising: 113.24 mg.L -1 Anhydrous calcium chloride, 150 mg.L -1 Sodium dihydrogen phosphate, 2500 mg.L -1 Potassium nitrate 134 mg.L -1 Ammonium sulfate 122.09 mg L -1 Anhydrous magnesium sulfate, 27. mg.L -1 Ferrous sulfate, 37.3 mg.L -1 Disodium ethylenediamine tetraacetate, 10.0 mg L -1 Manganese sulfate, 2.0 mg.L -1 Zinc sulfate, 0.25 mg.L -1 Sodium molybdate, 0.025 mg L -1 Cobalt chloride, 3.0 mg.L -1 Boric acid, 0.75 mg.L -1 Potassium iodide, 0.025 mg.L -1 Copper sulfate, 10.0 mg.L -1 Vitamin B 1 、1.0 mg·L -1 Vitamin B 6 、1.0 mg·L -1 Nicotinic acid, 100.0 mg.L -1 Inositol, 10000 mg.L -1 Coconut powder 20000 mg L -1 Sucrose, 7000 mg.L -1 Agar;
the anoectochilus formosanus homogenus plant comprises anoectochilus formosanus (AnoectochiLus elatus), anoectochilus formosanus (AnoectochiLus calcareus Aver), anoectochilus formosanus (Anoectochilus malipoensis), anoectochilus formosanus (AnoectochiLus roxburghii), anoectochilus formosanus (Anoectochilus chapaensis), anoectochilus formosanus (Anoectochilus brevilabris), anoectochilus formosanus (Anoectochilus xingrenensis) and anoectochilus yunnanensis (Anoectochilus burmannicus).
Further, the invention provides a non-symbiotic germination culture method for the anoectochilus roxburghii homonymous plant seeds, which comprises the following steps:
(1) Pod harvesting: picking the mature undeployed pods of 90-110 d after artificial cross pollination;
(2) Performing aseptic treatment and transverse cutting on the pods to obtain seeds;
(3) Protocorm acquisition: adopting an improved B5 culture medium, and sowing the seed of the anoectochilus roxburghii homogenus plant in the improved B5 culture medium to obtain the protocorm after seed germination;
(4) Subculture of protocorms: the secondary culture medium is modified B5 culture medium added with 0.2-0. g.L -1 Activated carbon, 0.2-0.5 mg.L -1 Naphthalene acetic acid, pH 5.4-5.6;
(5) Proliferation culture: taking out the seedling body with 2-3 knots from the subculture to 6-7cm, keeping the knots under aseptic condition, cutting into 2-3 knots, and placing the knots in a proliferation culture medium for proliferation culture; the proliferation medium is modified B5 medium added with 0.2-0. g.L -1 Activated carbon, 2-3 mg.L -1 6-benzylaminoadenine, 0.5-1. mg.L -1 Naphthalene acetic acid, pH 5.4-5.6;
(6) Strong seedling and rooting culture: transferring the anoectochilus formosanus seedling growing to 7-8 cm high after proliferation and subculture into strong seedling and rooting culture medium for rooting and strong seedling culture, wherein the strong seedling culture medium is modified B5 culture medium, and 1.0-2.0 g.L is added -1 Activated carbon, 0.5-1. mg.L -1 Naphthalene acetic acid, 50-150 g.L -1 Homogenizing potato, and pH is 5.4-5.6;
(7) Hardening and transplanting: after the strong seedlings and the rooting culture are carried out for 30-60 days, when 2-3 roots are arranged, the root length is 7-8 cm, and the seedling height is 10-12 cm, the seedling can be acclimatized and transplanted.
Preferably, the culture conditions of step (3) are: 25. the culture is carried out under illumination at the temperature of +/-2 ℃ for 8h/d, and the illumination is 800-1000 Lx or dark culture.
Preferably, the culture conditions of step (4) are: the illumination is 12 h/d, the illumination is 1200-1500 Lx, and the culture temperature is 25+/-2 ℃.
Preferably, the culture conditions in step (5) are: the illumination is 8h/d, the illumination is 1000-1200Lx, and the culture temperature is 25+/-2 ℃.
Preferably, the culture conditions in step (6) are: the illumination is 14 h/d, the illumination is 1500-2000Lx, and the culture temperature is 25+/-2 ℃.
The invention achieves the technical effects that:
compared with the existing literature control culture medium, the culture medium formula and the breeding method in the research can enable various anoectochilus formosanus seeds to germinate into seedlings, the germination time is 10 days earlier, the germination rate is far higher than that of the existing culture medium, the stem node proliferation induction culture is also added in the research, the anoectochilus formosanus seedlings can be obtained in a large quantity, the requirement on wild resources of the anoectochilus formosanus is relieved, and the protection of the species is really realized by utilizing and promoting.
Drawings
FIG. 1 shows the fruit pod of the same genus of anoectochilus formosanus (A: gaoenleaf; B: makeup anoectochilus formosanus; C: armillaria matsutake; D: anoectochilus formosanus);
FIG. 2 shows a diagram of asepsis treatment of the pod of Massa Medicata Fermentata;
FIG. 3 is a graph showing the non-symbiotic germination effect of Massa Medicata Fermentata seed (germination of Massa Medicata Fermentata seed in modified B5 medium, 1/4MS and H1 medium, respectively, from left to right);
FIG. 4 shows the differentiation of leaves of the original bulb of Massa Medicata Fermentata in modified B5 medium;
FIG. 5 shows the effect of subculture of Anoectochilus roxburghii;
FIG. 6 shows the proliferation culture effect of anoectochilus formosanus;
FIG. 7 shows the effect of strong seedling and rooting culture of Anoectochilus gracilis;
FIG. 8 shows a bottle-out planting view of Anoectochilus gracilis.
Detailed Description
For a better explanation of the present invention, the main content of the present invention is further elucidated below in conjunction with the specific examples, but the content of the present invention is not limited to the following examples only. The technical scheme of the invention is conventional in the field, and the reagents or materials are all derived from commercial sources and all used chemical reagents are analytically pure, if not specified.
Example one Castanea mollissima seed non-symbiotic germination culture
The Anoectochilus roxburghii Anoectochilus malipoensis is land herb, is about 15 cm high, is grown under evergreen broad-leaved forests in limestone mountain areas, has an altitude of 1600-1700 m, and is produced in China in Ma chestnut slope county of Yunnan province and has a flowering period of 7-8 months.
(1) Obtaining of Anoectochilus roxburghii pods
And (3) pollinating the Machestnut slope anoectochilus formosanus in the full-bloom stage of 7 months by adopting an artificial cross pollination technology, recording pollination time, observing pod change, picking (97, d) when the pod is ripe and becomes red and not cracked, and sowing in a refrigerator at 4 ℃ as soon as possible after being bagged by kraft paper.
(2) Asepsis treatment of Anoectochilus roxburghii pod
Cleaning the pod of the Machestnut hill anoectochilus formosanus, sterilizing with 75% alcohol for 30 s, soaking in 10% sodium hypochlorite solution for 8 minutes, washing with sterile water, sucking off surface water, and transversely cutting the pod into two halves in a culture dish for later use.
(3) Non-symbiotic germination medium formula
Non-symbiotic germination medium (modified B5 medium): 113.24 mg.L -1 Anhydrous calcium chloride, 150 mg.L -1 Sodium dihydrogen phosphate, 2500 mg.L -1 Potassium nitrate 134 mg.L -1 Ammonium sulfate 122.09 mg L -1 Anhydrous magnesium sulfate, 27. mg.L -1 Ferrous sulfate, 37.3 mg.L -1 Disodium ethylenediamine tetraacetate, 10.0 mg L -1 Manganese sulfate, 2.0 mg.L -1 Zinc sulfate, 0.25 mg.L -1 Sodium molybdate, 0.025 mg L -1 Cobalt chloride, 3.0 mg.L -1 Boric acid, 0.75 mg.L -1 Potassium iodide, 0.025 mg.L -1 Copper sulfate, 10.0 mg.L -1 Vitamin B 1 、1.0 mg·L -1 Vitamin B 6 、1.0 mg·L -1 Nicotinic acid, 100.0 mg.L -1 Inositol, 10000 mg.L -1 Coconut powder 20000 mg L -1 Sucrose, 7000 mg.L -1 Agar.
Control germination medium: 1/4MS (containing 10000 mg.L) -1 Coconut powder 20000 mg L -1 Sucrose, 7000 mg.L -1 Agar, 0.2 g.L -1 Activated carbon), H1 (containing 1000 mg.L -1 Huabao first number 10000 mg·L -1 Coconut powder, 0.2 g.L -1 Activated carbon 20000 mg L -1 Sucrose, 7000 mg.L -1 Agar).
(4) Non-symbiotic germination culture method for seed of Machestnut slope anoectochilus formosanus
Sowing the sterilized seed of the Machestnut slope anoectochilus formosanus in a non-symbiotic germination medium (modified B5 medium) and a control germination medium, sowing about 50 seeds per bottle, sowing 5 bottles of each medium, culturing under illumination (culturing 8h/d, and illuminance of 800-1000 Lx) at 25+/-2 ℃, and recording the germination time and germination rate of the seeds. The breaking of the seed coat by the expanded embryo of the seed was confirmed as the germination of the seed, and the germination rate was divided by the total number of seeds to be germinated and multiplied by 100. The Massa Medicata Fermentata began to germinate after 30 days of culture in the modified B5 medium, the germination rate was 95%, and none of the Massa Medicata Fermentata had germinated in the control 1/4MS and H1 medium (Table 1, table 2).
(5) Subculture of Machestnut slope anoectochilus formosanus
Adding 0. g.L to modified B5 culture medium -1 Activated carbon, 0. mg.L -1 Naphthalene acetic acid (pH 5.4-5.6) is used as a secondary culture medium of the Anoectochilus roxburghii, the Anoectochilus roxburghii germinates in the improved B5 culture medium, white protocorms are formed after germination, leaves are differentiated from the protocorms in the process of growing and lengthening, and the protocorms start to turn green. When leaves of anoectochilus formosanus turn green, the seedlings are transferred into a subculture medium for subculture, wherein the culture condition is that the illumination is 12 h/d, the illumination is 1200-1500 Lx, the culture temperature is 25+/-2 ℃, and the subculture is carried out once every 60 d for 2 times.
(6) Proliferation culture of Machestnut slope anoectochilus formosanus
Taking out the Machestnut hill anoectochilus formosanus with 2-3 knots, keeping the knots under aseptic condition, cutting into 2-3 sections, and placing in proliferation culture medium for proliferation culture. The proliferation medium is modified B5 medium added with 0. g.L -1 Activated carbon, 3 mg L -1 6-Benzylaminoadenine, 0.5 mg.L -1 Naphthalene acetic acid (pH 5.4-5.6) is cultured under the conditions of illumination of 8h/d, illumination of 1000-1200Lx and culture temperature of 25+ -2deg.C. Culturing stem node of herba Anoectochili Roxburghii in proliferation medium 32 d, and inducing from nodeMany cluster buds with multiplication coefficient of 5.8 are transferred to a secondary culture medium for continuous culture.
(7) Strong seedling and rooting culture of Machestnut slope anoectochilus formosanus
Transferring the seedling of the Anoectochilus roxburghii growing to 7-8 cm high after proliferation and subculture into strong seedling and rooting culture medium for rooting and strong seedling culture, wherein the strong seedling culture medium is modified B5 culture medium, and 2.0 g.L is added -1 Activated carbon, 0.5 mg.L -1 Naphthalene acetic acid, 120 g.L -1 The potato homogenate (pH 5.4-5.6) is cultured under the conditions of illumination of 14 h/d, illumination of 1500-2000Lx and culture temperature of 25+ -2deg.C. After the Machestnut slope anoectochilus formosanus seedlings are cultured in a strong seedling and rooting culture medium for 56 d, root systems grow long and thick, and rooting rate is 98%. When 2-3 roots are arranged, the root length is 7-8 cm, and the seedling height is 10-12 cm, the seedling can be acclimatized and transplanted.
Example two Amaryloxide film seed non-symbiotic germination culture
Anoectochilus gracilis AnoectochiLus caLcareus Aver is produced in the China under evergreen broad-leaved forest in Ma chestnut hill county, guangxi county, napo county and 7-8 months in flowering phase.
(1) Obtaining of Anoectochilus gracilis pods
Artificial cross pollination technology is adopted to pollinate the anoectochilus formosanus in the full bloom stage of 7 months, pollination time is recorded, pod change is observed, picking is carried out when the pod is ripe and becomes purplish red and not cracked (105 d), kraft paper bags are used for being put into a refrigerator of 4 ℃ for sowing as soon as possible.
(2) Aseptic treatment of Anoectochilus gracilis pods
Cleaning herba Anoectochili Roxburghii pods, sterilizing with 75% alcohol for 30 s, soaking in 10% sodium hypochlorite solution for 8 min, washing with sterile water, sucking off surface water, and cutting pods into two halves in culture dish.
(3) Non-symbiotic germination medium formula
Non-symbiotic germination medium (modified B5 medium): 113.24 mg.L -1 Anhydrous calcium chloride, 150 mg.L -1 Sodium dihydrogen phosphate, 2500 mg.L -1 Potassium nitrate 134 mg.L -1 Ammonium sulfate,122.09 mg·L -1 Anhydrous magnesium sulfate, 27. mg.L -1 Ferrous sulfate, 37.3 mg.L -1 Disodium ethylenediamine tetraacetate, 10.0 mg L -1 Manganese sulfate, 2.0 mg.L -1 Zinc sulfate, 0.25 mg.L -1 Sodium molybdate, 0.025 mg L -1 Cobalt chloride, 3.0 mg.L -1 Boric acid, 0.75 mg.L -1 Potassium iodide, 0.025 mg.L -1 Copper sulfate, 10.0 mg.L -1 Vitamin B 1 、1.0 mg·L -1 Vitamin B 6 、1.0 mg·L -1 Nicotinic acid, 100.0 mg.L -1 Inositol, 10000 mg.L -1 Coconut powder 20000 mg L -1 Sucrose, 7000 mg.L -1 Agar.
Control germination medium: 1/4MS (containing 10000 mg.L) -1 Coconut powder 20000 mg L -1 Sucrose, 7000 mg.L -1 Agar, 0.2 g.L -1 Activated carbon), H1 (containing 1000 mg.L -1 Huabao first number 10000 mg L -1 Coconut powder, 0.2 g.L -1 Activated carbon 20000 mg L -1 Sucrose, 7000 mg.L -1 Agar).
(4) Non-symbiotic germination culture method for anoectochilus gracilis seeds
The sterilized anoectochilus formosanus seeds are sown in a non-symbiotic germination medium (modified B5 medium) and a control germination medium, about 50 seeds are sown in each bottle, 5 bottles of each medium are sown, the medium is placed in total darkness for culture, and the seed germination time and germination rate are recorded. The breaking of the seed coat by the expanded embryo of the seed was confirmed as the germination of the seed, and the germination rate was divided by the total number of seeds to be germinated and multiplied by 100. The anoectochilus formosanus can germinate in the modified B5 culture medium, the 1/4MS culture medium and the H1 culture medium, the germination time is 31 d, 47 d and 56 d, the germination rate is 93%, 69% and 59%, and the germination effect of the anoectochilus formosanus in the modified B5 culture medium is better than that of the control germination culture medium from the aspects of the germination time and the germination rate (table 1 and table 2).
(5) Subculture of Anoectochilus gracilis
Adding 0. g.L to modified B5 culture medium -1 Activated carbon, 0. mg.L -1 Naphthalene acetic acid (pH 5.4-5.6) as secondary culture medium, anoectochilus gracilis inGermination is carried out in the modified B5 culture medium, white protocorms are formed after germination, the protocorms are cultured under light, leaves are differentiated from the protocorms in the process of growing and lengthening, and the protocorms start to turn green. When leaves of the anoectochilus formosanus turn green, the seedlings are transferred into a subculture medium for subculture, wherein the culture condition is that the illumination is 12 h/d, the illumination is 1200-1500 Lx, the culture temperature is 25+/-2 ℃, and the subculture is carried out once every 60 d for 2 times.
(6) Proliferation culture of anoectochilus formosanus
And (3) taking out the anoectochilus formosanus with 2-3 knots from the secondary culture to 6-7cm, keeping the knots under aseptic conditions, cutting into 2-3 sections, and placing the sections into a proliferation culture medium for proliferation culture. The proliferation medium is modified B5 medium added with 0. g.L -1 Activated carbon, 2.5 mg.L -1 6-Benzylaminoadenine, 0. mg.L -1 Naphthalene acetic acid (pH 5.4-5.6) is cultured under the conditions of illumination of 8h/d, illumination of 1000-1200Lx and culture temperature of 25+ -2deg.C. After the stem node of the anoectochilus formosanus is cultured in a proliferation medium for 40 d, a plurality of cluster buds are induced from the stem node, the proliferation coefficient is 4.6, and the cluster buds are transferred to a secondary culture medium for continuous culture.
(7) Strong seedling and rooting culture of anoectochilus formosanus
Transferring the seedling of Anoectochilus gracilis growing to 7-8 cm high after proliferation and subculture into strong seedling and rooting culture medium for rooting and strong seedling culture, wherein the strong seedling culture medium is modified B5 culture medium, and 1.0 g.L is added -1 Activated carbon, 0. mg.L -1 Naphthalene acetic acid, 50 g.L -1 The potato homogenate (pH 5.4-5.6) is cultured under the conditions of illumination of 14 h/d, illumination of 1500-2000Lx and culture temperature of 25+ -2deg.C. After the anoectochilus formosanus seedlings are cultured in a strong seedling and rooting culture medium for 60 d, the root system is long, thick and long, and the rooting rate is 96%. When 2-3 roots are arranged, the root length is 7-8 cm, and the seedling height is 10-12 cm, the seedling can be acclimatized and transplanted.
Example three high Anoectochilus roxburghii seed non-symbiotic germination culture
The anoectochilus formosanus AnoectochiLus eLatus is produced in evergreen broad-leaved forest, and is produced in Dai nationality of Jingfu city in double-plate of Yunnan province in the country of Dai mountain in the dry dam village of Dai mountain, and the flowering period is 11 months to 1 month in the next year.
(1) Obtaining of pod of Anoectochilus formosanus
Artificial cross pollination technology is adopted to pollinate the anoectochilus formosanus in the full bloom stage of 11 months, the pollination time is recorded, the pod change is observed, the fruit pod is picked (100 d) when the fruit pod is ripe and becomes red and not cracked, and the fruit pod is put into a refrigerator of 4 ℃ by kraft paper bags for sowing as soon as possible.
(2) Aseptic treatment of Anoectochilus roxburghii pods
Cleaning the pod of the anoectochilus formosanus, sterilizing with 75% alcohol for 30 s, soaking in 10% sodium hypochlorite solution for 8 minutes, washing with sterile water, sucking off surface water, and transversely cutting the pod into two halves in a culture dish for later use.
(3) Non-symbiotic germination medium formula
Non-symbiotic germination medium (modified B5 medium): 113.24 mg.L -1 Anhydrous calcium chloride, 150 mg.L -1 Sodium dihydrogen phosphate, 2500 mg.L -1 Potassium nitrate 134 mg.L -1 Ammonium sulfate 122.09 mg L -1 Anhydrous magnesium sulfate, 27. mg.L -1 Ferrous sulfate, 37.3 mg.L -1 Disodium ethylenediamine tetraacetate, 10.0 mg L -1 Manganese sulfate, 2.0 mg.L -1 Zinc sulfate, 0.25 mg.L -1 Sodium molybdate, 0.025 mg L -1 Cobalt chloride, 3.0 mg.L -1 Boric acid, 0.75 mg.L -1 Potassium iodide, 0.025 mg.L -1 Copper sulfate, 10.0 mg.L -1 Vitamin B 1 、1.0 mg·L -1 Vitamin B 6 、1.0 mg·L -1 Nicotinic acid, 100.0 mg.L -1 Inositol, 10000 mg.L -1 Coconut powder 20000 mg L -1 Sucrose, 7000 mg.L -1 Agar.
Control germination medium: 1/4MS (containing 10000 mg.L) -1 Coconut powder 20000 mg L -1 Sucrose, 7000 mg.L -1 Agar, 0.2 g.L -1 Activated carbon), H1 (containing 1000 mg.L -1 Huabao first number 10000 mg L -1 Coconut powder, 0.2 g.L -1 Activated carbon 20000 mg L -1 Sucrose, 7000 mg.L -1 Agar).
(4) Non-symbiotic germination culture method for anoectochilus formosanus seeds
The sterilized Gaojinlan orchid seeds are sown in a non-symbiotic germination culture medium (a modified B5 culture medium) and a control germination culture medium, about 50 seeds are sown in each bottle, 5 bottles of each culture medium are sown, the culture medium is placed in total darkness for culture, and the germination time and the germination rate of the seeds are recorded. The breaking of the seed coat by the expanded embryo of the seed was confirmed as the germination of the seed, and the germination rate was divided by the total number of seeds to be germinated and multiplied by 100. Gaoenlan orchid began to germinate after 33 days of culture in modified B5 medium, at 86% germination rate, and did not germinate in both control 1/4MS and H1 medium (Table 1, table 2).
(5) Subculture of Gaoenlan orchid
Adding 0. g.L to modified B5 culture medium -1 Activated carbon, 0.5 mg.L -1 Naphthalene acetic acid (pH 5.4-5.6) is used as a secondary culture medium, the anoectochilus formosanus germinates in the modified B5 culture medium, white protocorms are formed after germination, the white protocorms are cultured under light, leaves are differentiated from the protocorms in the process of growing and lengthening, and the protocorms start to turn green. When Gao Jinxian blue leaves turn green, the seedlings are transferred into a subculture medium for subculture under the culture conditions of 12 h/d illumination, 1200-1500 Lx illumination, 25+/-2 ℃ culture temperature, and 3 times of subculture every 60 d.
(6) Proliferation culture of anoectochilus formosanus
Taking out the anoectochilus formosanus with 2-3 knots from the subculture to 6-7cm, keeping the knots under aseptic condition, cutting into 2-3 sections, and placing in proliferation culture medium for proliferation culture. The proliferation medium is modified B5 medium added with 0. g.L -1 Activated carbon, 3 mg L -1 6-Benzylaminoadenine, 1.0 mg.L -1 Naphthalene acetic acid (pH 5.4-5.6) is cultured under the conditions of illumination of 8h/d, illumination of 1000-1200Lx and culture temperature of 25+ -2deg.C. After the stem node of the anoectochilus formosanus is cultured in a proliferation medium for 42 d, a plurality of cluster buds are induced from the stem node, the proliferation coefficient is 5.2, and the cluster buds are transferred to a secondary culture medium for continuous culture.
(7) Strong seedling and rooting culture of anoectochilus formosanus
Gao Jinxian blue seedlings which are grown to 7-8 cm high through proliferation and subcultureTransferring into strong seedling and rooting culture medium for rooting and strong seedling culture, wherein the strong seedling culture medium is modified B5 culture medium, and 2. g.L is added -1 Activated carbon, 1. mg.L -1 Naphthalene acetic acid, 150 g.L -1 The potato homogenate (pH 5.4-5.6) is cultured under the conditions of illumination of 14 h/d, illumination of 1500-2000Lx and culture temperature of 25+ -2deg.C. After the Machestnut slope anoectochilus formosanus seedlings are cultured in a strong seedling and rooting culture medium for 60 d, root systems grow long and thick, and rooting rate is 95%. When 2-3 roots are arranged, the root length is 7-8 cm, and the seedling height is 10-12 cm, the seedling can be acclimatized and transplanted.
Example four Roxburgh anoectochilus seed non-symbiotic germination culture
Anoectochilus roxburghii AnoectochiLus roxburghii is produced in evergreen broad-leaved forest or valley wet places, and is produced in Zhejiang, jiangxi, fujian, hunan, guangdong, hainan, guangxi, sichuan, yunnan and southeast part of Tibet (ink drop), the altitude is 50-1600 m, and the flowering period is 8-11 months.
(1) Obtaining of Anoectochilus roxburghii fruit pod
Artificial cross pollination technology is adopted to pollinate anoectochilus roxburghii in the full bloom stage of 8 months, the pollination time is recorded, the pod change is observed, picking is carried out when the pod is ripe and yellow or red is not cracked (92 d), and the anoectochilus roxburghii is put into a refrigerator at 4 ℃ for sowing as soon as possible in kraft paper bags.
(2) Anoectochilus roxburghii pod aseptic treatment
Cleaning herba Anoectochili Roxburghii pods, sterilizing with 75% alcohol for 30 s, soaking in 10% sodium hypochlorite solution for 8 min, washing with sterile water, absorbing surface water, and cutting pods into two halves in culture dish.
(3) Non-symbiotic germination medium formula
Non-symbiotic germination medium (modified B5 medium): 113.24 mg.L -1 Anhydrous calcium chloride, 150 mg.L -1 Sodium dihydrogen phosphate, 2500 mg.L -1 Potassium nitrate 134 mg.L -1 Ammonium sulfate 122.09 mg L -1 Anhydrous magnesium sulfate, 27. mg.L -1 Ferrous sulfate, 37.3 mg.L -1 Disodium ethylenediamine tetraacetate, 10.0 mg L -1 Manganese sulfate, 2.0 mg.L -1 Zinc sulfate, 0.25 mg.L -1 Sodium molybdate, 0.025 mg L -1 Cobalt chloride, 3.0 mg.L -1 Boric acid, 0.75 mg.L -1 Potassium iodide, 0.025 mg.L -1 Copper sulfate, 10.0 mg.L -1 Vitamin B 1 、1.0 mg·L -1 Vitamin B 6 、1.0 mg·L -1 Nicotinic acid, 100.0 mg.L -1 Inositol, 10000 mg.L -1 Coconut powder 20000 mg L -1 Sucrose, 7000 mg.L -1 Agar.
Control germination medium: 1/4MS (containing 10000 mg.L) -1 Coconut powder 20000 mg L -1 Sucrose, 7000 mg.L -1 Agar, 0.2 g.L -1 Activated carbon), H1 (containing 1000 mg.L -1 Huabao first number 10000 mg L -1 Coconut powder, 0.2 g.L -1 Activated carbon 20000 mg L -1 Sucrose, 7000 mg.L -1 Agar).
(4) Non-symbiotic germination culture method for anoectochilus roxburghii seeds
The sterilized anoectochilus formosanus seeds are sown in a non-symbiotic germination culture medium (modified B5 culture medium) and a control germination culture medium, about 50 seeds are sown in each bottle, 5 bottles of the culture medium are sown, the culture medium is placed under illumination (culture 8h/d, the illumination is 800-1000 Lx) at 25+/-2 ℃ for culture, and the germination time and the germination rate of the seeds are recorded. The breaking of the seed coat by the expanded embryo of the seed was confirmed as the germination of the seed, and the germination rate was divided by the total number of seeds to be germinated and multiplied by 100. The anoectochilus formosanus can germinate in the improved B5 culture medium, 1/4MS and H1, the germination time is 29d, 43d and 53d, the germination rate is 96%, 76% and 68%, and the germination effect of the anoectochilus formosanus in the improved B5 culture medium is superior to that of a control germination culture medium (table 1 and table 2) from the viewpoints of the germination time and the germination rate.
(5) Subculture of anoectochilus formosanus
The secondary culture medium is modified B5 culture medium added with 0. g.L -1 Activated carbon, 0.3 mg.L -1 Naphthalene acetic acid, pH 5.4-5.6. The anoectochilus formosanus germinates in the improved B5 culture medium, white protocorms are formed after germination, and leaves are differentiated from the protocorms in the process of growing and lengthening, and the protocorms start to turn green. When leaves of anoectochilus formosanus turn green, seedlings are transferred into a secondary culture medium for secondary culture, and culture strips are cultivatedThe parts are 12-h/d light, 1200-1500 Lx light intensity, and the culture temperature is 25+/-2 ℃, and the times of the culture are carried out every 60 d times, and the total times of the culture are 2 times.
(6) Proliferation culture of anoectochilus formosanus
Taking out the anoectochilus formosanus with 2-3 knots, keeping the knots under aseptic condition, cutting into 2-3 sections, and placing in proliferation culture medium for proliferation culture. The proliferation medium is modified B5 medium added with 0. g.L -1 Activated carbon, 2 mg L -1 6-Benzylaminoadenine, 0. mg.L -1 Naphthylacetic acid, pH5.4-5.6, and culturing at 25+ -2deg.C under the conditions of illumination of 8 hr/d and illumination of 1000-1200 Lx. After the stem node of the anoectochilus formosanus is cultured in a proliferation medium for 28 d, a plurality of cluster buds are induced from the stem node, the proliferation coefficient is 6.5, and the cluster buds are transferred to a secondary culture medium for continuous culture.
(7) Roxburgh anoectochilus strong seedling and rooting culture
Transferring the anoectochilus formosanus seedling growing to 7-8 cm high after proliferation and subculture into strong seedling and rooting culture medium for rooting and strong seedling culture, wherein the strong seedling culture medium is modified B5 culture medium, and 1.5 g.L is added -1 Activated carbon, 0. mg.L -1 Naphthalene acetic acid, 80 g.L -1 The potato is homogenized, the pH is 5.4-5.6, the culture condition is that the illumination is 14-h/d, the illumination is 1500-2000Lx, and the culture temperature is 25+/-2 ℃. After the anoectochilus formosanus seedlings are cultured in a strong seedling and rooting culture medium for 48 d, root systems grow long and thick, and the rooting rate is 99%. When 2-3 roots are arranged, the root length is 7-8 cm, and the seedling height is 10-12 cm, the seedling can be acclimatized and transplanted.
Under the germination culture medium and the breeding method, the anoectochilus formosanus homogenus plant seeds can be germinated into seedlings and planted in a bottle, and the culture medium can be used as a universal culture medium for the anoectochilus formosanus seedling breeding research and is suitable for industrial large-scale rapid propagation of the anoectochilus formosanus homogenus plants.
TABLE 1 germination time of Anoectochilus Roxburghii homozygous plant seeds in different media
TABLE 2 germination Rate of Anoectochilus Roxburghii seed in different Medium
The foregoing is merely illustrative of the present invention, and the present invention is not limited thereto, and any person skilled in the art will readily recognize that variations or substitutions are within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (6)
1. The non-symbiotic germination culture medium for the anoectochilus formosanus homonymous plant seeds is characterized in that the culture medium is an improved B5 culture medium, and is as follows: 113.24 mg.L -1 Anhydrous calcium chloride, 150 mg.L -1 Sodium dihydrogen phosphate, 2500 mg.L -1 Potassium nitrate 134 mg.L -1 Ammonium sulfate 122.09 mg L -1 Anhydrous magnesium sulfate, 27. mg.L -1 Ferrous sulfate, 37.3 mg.L -1 Disodium ethylenediamine tetraacetate, 10.0 mg L -1 Manganese sulfate, 2.0 mg.L -1 Zinc sulfate, 0.25 mg.L -1 Sodium molybdate, 0.025 mg L -1 Cobalt chloride, 3.0 mg.L -1 Boric acid, 0.75 mg.L -1 Potassium iodide, 0.025 mg.L -1 Copper sulfate, 10.0 mg.L -1 Vitamin B 1 、1.0 mg·L -1 Vitamin B 6 、1.0 mg·L -1 Nicotinic acid, 100.0 mg.L -1 Inositol, 10000 mg.L -1 Coconut powder 20000 mg L -1 Sucrose, 7000 mg.L -1 Agar;
the anoectochilus formosanus homogenus plant comprises anoectochilus formosanus, anoectochilus formosanus or anoectochilus formosanus.
2. The non-symbiotic germination method for the anoectochilus formosanus homonymous plant seeds is characterized by comprising the following steps of:
(1) Pod harvesting: picking the mature undeployed pods of 90-110 d after artificial cross pollination;
(2) Performing aseptic treatment and transverse cutting on the pods to obtain seeds;
(3) Protocorm acquisition: preparing the improved B5 culture medium by adopting the formula in claim 1, and sowing the seed of the anoectochilus formosanus homonymous plant in the improved B5 culture medium to obtain a protocorm after seed germination;
(4) Subculture of protocorms: the secondary culture medium is modified B5 culture medium added with 0.2-0. g.L -1 Activated carbon, 0.2-0.5 mg.L -1 Naphthalene acetic acid, pH 5.4-5.6;
(5) Proliferation culture: taking out the seedling body with 2-3 knots from the subculture to 6-7cm, keeping the knots under aseptic condition, cutting into 2-3 knots, and placing the knots in a proliferation culture medium for proliferation culture; the proliferation medium is modified B5 medium added with 0.2-0. g.L -1 Activated carbon, 2-3 mg.L -1 6-benzylaminoadenine, 0.5-1. mg.L -1 Naphthalene acetic acid, pH 5.4-5.6;
(6) Strong seedling and rooting culture: transferring the seedlings which grow to 7-8 cm high through proliferation and subculture into strong seedlings and rooting culture medium for rooting and strong seedlings culture, wherein the culture medium is modified B5 culture medium, and 1.0-2.0 g.L is added -1 Activated carbon, 0.5-1. mg.L -1 Naphthalene acetic acid, 50-150 g.L -1 Homogenizing potato, and pH is 5.4-5.6;
(7) Hardening and transplanting: after the strong seedlings and the rooting culture are carried out for 30-60 days, when 2-3 roots are arranged, the root length is 7-8 cm, and the seedling height is 10-12 cm, the seedling can be acclimatized and transplanted.
3. The method of claim 2, wherein the culturing conditions of step (3) are: 25. the culture is carried out under illumination at the temperature of +/-2 ℃ for 8h/d, and the illumination is 800-1000 Lx or dark culture.
4. The method of claim 2, wherein the culturing conditions of step (4) are: the illumination is 12 h/d, the illumination is 1200-1500 Lx, and the culture temperature is 25+/-2 ℃.
5. The method of claim 2, wherein the culturing conditions of step (5) are: the illumination is 8h/d, the illumination is 1000-1200Lx, and the culture temperature is 25+/-2 ℃.
6. The method of claim 2, wherein the culturing conditions of step (6) are: the illumination is 14 h/d, the illumination is 1500-2000Lx, and the culture temperature is 25+/-2 ℃.
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| CN103461118A (en) * | 2013-07-31 | 2013-12-25 | 湖州市农业科学研究院 | Industrialized production method for anoectochilus roxburghii seedlings |
| CN104823847A (en) * | 2015-04-29 | 2015-08-12 | 福建省农业科学院农业生物资源研究所 | Zhejiang anoectochilus roxburghii seed tissue culture and rapid seedling raising method |
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| CN101715732A (en) * | 2010-01-19 | 2010-06-02 | 烟台汇鹏生物科技有限公司 | Method for producing anoectochilus formosanus by tissue culture |
| CN103461118A (en) * | 2013-07-31 | 2013-12-25 | 湖州市农业科学研究院 | Industrialized production method for anoectochilus roxburghii seedlings |
| CN104823847A (en) * | 2015-04-29 | 2015-08-12 | 福建省农业科学院农业生物资源研究所 | Zhejiang anoectochilus roxburghii seed tissue culture and rapid seedling raising method |
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