CN116349601A - Non-symbiotic sterile germination method for tiny population spots She Biaolan - Google Patents
Non-symbiotic sterile germination method for tiny population spots She Biaolan Download PDFInfo
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Images
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
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
- A01C1/02—Germinating apparatus; Determining germination capacity of seeds or the like
- A01C1/025—Testing seeds for determining their viability or germination capacity
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
- A01G22/60—Flowers; Ornamental plants
- A01G22/63—Orchids
-
- 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
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
-
- 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
-
- 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
- A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
- A01H6/62—Orchidaceae [Orchid family]
Abstract
The invention relates to the field of biotechnology, in particular to a method for non-symbiotic sterile germination of a small population spot She Biaolan; the invention aims to provide an effective path for breeding cypripedium maculosa; the technical scheme adopted specifically comprises the following steps: 1) artificial pollination, 2) capsule picking, 3) low-temperature treatment, 4) seed disinfection, alkaline etching of seed coats, sowing and 5) constant-temperature culture; according to the invention, through field artificial pollination, timely picking seeds, alkaline etching to soften seed coats, removal of inhibiting chemical substances, and influence tests of different sowing culture medium formulas and additives on the germination of the macula She Biaolan, the problem of non-symbiotic sterile germination of the macula She Biaolan is solved, a foundation is laid for artificial domestication, propagation and cultivation of the cypripedium macranthum, and the plant resources of the macula She Biaolan are better protected and reasonably utilized.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a non-symbiotic sterile germination method for a small population spot She Biaolan.
Background
Plaque She Biaolan (Cypripedium margaritaceum) is of the orchid family, cypripedium, class II of the country, important protection plants, the IUCN species red directory endangered species, the very small population, the endangered animal and plant species international trade Convention (CITES) annex class II. The domestic distribution is only on grass slopes or under forests with the altitudes of 2500-3600 meters in the southwest of Sichuan and the northwest of Yunnan. The flower has peculiar appearance, rich and gorgeous color, elegant leaf shape, is the group with the highest ornamental value in the mountain orchid, and has higher gardening value and economic value. The requirements on the growth environment are severe; human activities and grazing cause the damage of habitat environment; the ornamental value is high and the plants are frequently picked and dug, so that the number of field clusters is obviously reduced, the cypripedium plants are mainly bred by dividing plants under the natural condition, the annual breeding rate is extremely low, and the population expansion speed is low; plaque She Biaolan has a low field setting rate, usually less than 10%, due to its harsh living environment, pollination, etc.; plaque She Biaolan seeds are micro-e.g. dust, have no endosperm or cotyledon, have only an immature or incompletely developed spherical embryo in the seed coat, and lack nutrients required for germination; plaque She Biaolan seed is wrapped with embryo, and the seed coat is waterproof, airtight and contains chemicals for preventing germination, so that dormancy is caused, and germination is extremely difficult under natural conditions. The research of the cultivation of the very small population spots She Biaolan is urgent.
No literature report exists in the current research of germination of the plaque She Biaolan seed test tube.
Disclosure of Invention
The invention aims to provide an effective path for breeding cypripedium maculosa.
In order to achieve the above purpose, the invention adopts the following technical scheme: a method for non-symbiotic sterile germination of a very small population of plaques She Biaolan, comprising the steps of:
1) Artificial pollination;
2) Picking capsules;
3) And (3) low-temperature treatment:
4) Seed disinfection, alkali etching of seed coats and sowing;
5) Culturing at constant temperature.
Further, the pollination mode in the step 1) is cross pollination, the pollination time is selected from 7 ten days in month to 7 months, plots with more communities and more flowering plants are selected for pollination, plants which grow healthy and flowers are full of flowers and are not naturally pollinated are selected, and the pollen blocks are provided with a milky yellow color and are sticky.
Further, the specific pollination method in the pollination step is to take off the labial lamella with a blade, take out pollen with forceps and apply the pollen on the stigma, lightly press the pollen, and finish pollination.
Further, the capsules 90-110 days after pollination are collected in the step 2) when the capsules are picked, and the capsules are picked when the seed coats become purple red, white or light brown.
Further, a seed vigor determination is also required prior to step 3).
Further, the specific method in the step 3) is to wipe off fruit surface dirt with 75% alcohol, dry in the shade, wrap with sterile filter paper, put into a tissue culture bottle sterilized at high temperature and added with a drying agent, and put into a constant temperature oven at 4 ℃ for 30 days.
Further, the specific method in the step 4 is to use 75% wine on a sterile operating table of the capsule which is not cracked after the treatment in the step 3)Wiping the surface of the capsule, volatilizing alcohol on the surface, cutting the capsule with a sterilizing blade, taking out the seed, and placing the seed in 0.1% Hgcl 2 Soaking and sterilizing for 10 minutes, washing for 6-8 times by using sterilized distilled water after sterilization, soaking in 0.1mol/L KOH solution for 10 minutes, corroding seed coats, enabling the seed coats to permeate water and breathe so as to promote seed germination, washing for 6-8 times by using sterilized distilled water, placing on a tray filled with sterile filter paper, placing on a sterile operation table for airing, and shaking off the seeds into a non-symbiotic germination culture medium by using a sterile medicine spoon or an inoculating shovel for sowing.
Further, the specific formula of the non-symbiotic germination medium is as follows: the specific formula of the non-symbiotic germination medium is as follows: the modified Knudson C is taken as a basic culture medium, 20-30 g/L sucrose, 20-50 g/L potato, 2g/L tryptone, 0.2-0.6 mg/L6-benzylaminoadenine and 1g/L active carbon are added, and the pH value of the culture medium is regulated to 5.5-5.8 by sodium hydroxide solution and hydrochloric acid.
Further, the modified Knudson C formulation is: KH (KH) 2 PO 4 62.5-125 mg/L of potassium dihydrogen phosphate; ca (NO) 3 ) 2 .4H 2 250-500 mg/L calcium nitrate tetrahydrate; (NH) 4 ) 2 SO 4 125-250 mg/L of ammonium sulfate; mgSO (MgSO) 4 •7H 2 62.5 to 125mg/L of magnesium sulfate heptahydrate; feSO 4 ·7H 2 6.25-12.5 mg/L of O ferrous sulfate heptahydrate; mnSO 4 ·4H 2 1.875-3.75 mg/L of O tetrahydrate manganese sulfate.
Further, placing the culture flask sown in the step 4) in an incubator for culture at 20 ℃ for about 90d in a dark culture way, germinating to form protocorms, transferring the protocorms to a newly configured culture medium, wherein the newly configured culture medium adopts an improved Knudson C formula, the white protocorms grow to form white or light brown root-like stems after extending for about 60 days, carrying out illumination culture for 12h/d, the illumination intensity is 2000Lx, the top end of the root-like stems is differentiated, the bud height is 1.5-3 cm, and the bottle is transplanted when the root length is 2-5 cm.
The beneficial technical effects of the invention are as follows:
according to the invention, through field artificial pollination, timely picking seeds, softening seed coats, removing inhibition chemicals, and influence tests of different sowing culture medium formulas and additives on the germination of the macula She Biaolan, the problem of non-symbiotic sterile germination of the macula She Biaolan is solved, a foundation is laid for artificial domestication, propagation and cultivation of the cypripedium maculosa, and the plant resource of the macula She Biaolan is better protected and reasonably utilized.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only some embodiments of the invention, and that other drawings can be obtained according to these drawings without inventive faculty for a person skilled in the art.
FIG. 1 is a pod and seed from a 110 day artificial pollination;
FIG. 2 is a TTC staining method for checking the viability of the plaque She Biaolan seeds, observing the staining condition of the seeds by an OLYMPUS stereomicroscope, and staining the spherical embryo into red and viable seeds;
FIG. 3 shows a protocorm (1) germinated about 90d by the technique of the invention, after the protocorm germinates, the white protocorm expands to have a root hair (2) about 60 days, and a root stem (3) is formed about 120 days; sprouting around 110 days (4).
Description of the embodiments
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Examples
A method for non-symbiotic sterile germination of a very small population of plaques She Biaolan, comprising the steps of:
1) Artificial pollination
Cross pollination;
pollinating land selection: selecting plots with more communities and more flowering plants for pollination, so that observation and comparison are facilitated;
pollinating season: the spot She Biaolan enters the full bloom period from the middle ten days of 7 months to the bottom ten days of 7 months, so that the optimal time for artificial supplementary pollination is achieved;
pollinated plants and pollen mass selection: selecting plants which grow healthily, have flowers fully bloom and are not naturally pollinated, wherein sepals in the plants which are subjected to natural pollination cover petals and stigmas; selecting a milky yellow sticky pollen mass, and disabling the use of a mildewed or aged pollen mass;
the pollination method comprises the following steps: selecting a sunny day and carrying out the process in the period from 8 to 12 am; the labial lobe is taken off by a blade, pollen is taken out by forceps and is coated on the stigma, and the pollen is lightly pressed to complete pollination. The label is written by pencil and hung on the pollinated mother plant.
2) Picking of capsules
Collecting capsules 90-110 days after pollination, and collecting capsules when the capsules turn to purple red, and the seed coats are white and light brown.
3) Low temperature treatment
Seed vigor determination: before aseptic sowing, the seed vitality is measured to know the development condition and the physiological activity of the embryo, so that the waste of manpower, material resources and time caused by using non-viable seeds is avoided. Most of the seeds were taken out of the same capsule for sowing, and the remaining seeds were used for TTC staining.
And (3) low-temperature treatment: wiping fruit surface dirt with 75% alcohol, and drying in the shade; wrapping with sterile filter paper, and placing into a tissue culture bottle which is sterilized at high temperature and added with a drying agent; placing in a constant temperature box at 4 ℃ for 30 days.
4) Seed disinfection, alkali etching of seed coat and sowing
The capsule which is processed at low temperature and is not cracked is wiped on a sterile operation table by 75% alcohol, after the alcohol on the surface of the capsule is volatilized, the capsule is split by a disinfection blade, the seeds are taken out and placed in 0.1% Hgcl2 for soaking and disinfecting for 10 minutes, after disinfecting, the seeds are washed by sterilized distilled water for 6-8 times, then placed in 0.1mol/L KOH solution for soaking for 10 minutes, seed coats are corroded, so that the seed coats are permeable and breathable, seed germination is promoted, the seeds are washed by sterilized distilled water for 6-8 times, placed on a tray filled with sterile filter paper, placed on the sterile operation table for airing, and the seeds are shaken off into a non-symbiotic germination culture medium by a sterile medicine spoon or a inoculation shovel for sowing.
The specific formula of the non-symbiotic germination medium is as follows: modified Knudson C was used as a minimal medium, and 20g/L sucrose, 50g/L potato, 2g/L tryptone, 0.2 mg/L6-benzylaminoadenine, and 1g/L activated carbon were added, and the pH of the medium was adjusted to 5.5 with sodium hydroxide solution and hydrochloric acid.
The modified Knudson C formulation was: KH (KH) 2 PO 4 125mg/L of potassium dihydrogen phosphate; ca (NO) 3 ) 2 .4H 2 O, 500mg/L calcium nitrate tetrahydrate; (NH) 4 ) 2 SO 4 250mg/L ammonium sulfate; mgSO (MgSO) 4 •7H 2 125mg/L of magnesium sulfate heptahydrate; feSO 4 ·7H 2 12.5mg/L of O ferrous sulfate heptahydrate; mnSO 4 ·4H 2 3.75mg/L of O manganese sulfate tetrahydrate.
5) Constant temperature culture
Placing the sown culture bottle in an incubator for culture, wherein the culture conditions are as follows: culturing in dark at 20 deg.c for 90d to form protocorm, transferring to new culture medium with improved Knudson C recipe, growing white protocorm to form white or light brown root-like stem, culturing in light for 12 hr/d, differentiating bud at the top of root-like stem for 110 days at 1.5-3 cm in height and transplanting in bottle at 2-5 cm in root length.
Examples
The difference between this example and example 1 is that the ratio of the culture medium in this example is different.
The specific formula of the non-symbiotic germination medium is as follows: modified Knudson C is taken as basic culture medium, 25g/L sucrose, 30g/L potato, 2g/L tryptone, 0.4 mg/L6-benzylaminoadenine and 1g/L activated carbon are added, and the pH of the culture medium is regulated to 5.5-5.8 by sodium hydroxide solution and hydrochloric acid.
The modified Knudson C formulation was: KH (KH) 2 PO 4 62.5mg/L of potassium dihydrogen phosphate; ca (NO) 3 ) 2 .4H 2 250mg/L calcium nitrate tetrahydrate; (NH) 4 ) 2 SO 4 125mg/L ammonium sulfate; mgSO (MgSO) 4 •7H 2 62.5mg/L of magnesium sulfate heptahydrate; feSO 4 ·7H 2 Ferrous sulfate heptahydrate of O6.25 mg/L; mnSO 4 ·4H 2 O manganese sulfate tetrahydrate 1.875mg/L.
Examples
The difference between this example and examples 1 and 2 is that the ratio of the culture medium in this example is different.
The specific formula of the non-symbiotic germination medium is as follows: modified Knudson C is taken as basic culture medium, 30g/L sucrose, 50g/L potato, 2g/L tryptone, 0.6 mg/L6-benzylaminoadenine and 1g/L activated carbon are added, and the pH of the culture medium is regulated to 5.5-5.8 by sodium hydroxide solution and hydrochloric acid.
The modified Knudson C formulation was: KH (KH) 2 PO 4 100mg/L of potassium dihydrogen phosphate; ca (NO) 3 ) 2 .4H 2 350mg/L of calcium nitrate tetrahydrate; (NH) 4 ) 2 SO 4 200mg/L ammonium sulfate; mgSO (MgSO) 4 •7H 2 100mg/L of magnesium sulfate heptahydrate; feSO 4 ·7H 2 9.5mg/L of O ferrous sulfate heptahydrate; mnSO 4 ·4H 2 2.5mg/L of O manganese sulfate tetrahydrate.
The technical scheme of the invention is based on years of experience and experiments of the applicant, and the selection of each step depends on experimental data and long-term observation and research practices.
1. Determination of pollination time and time for picking capsules
Plaque She Biaolan has a low field setting rate, typically less than 10%, due to its harsh habitat, pollination, etc. The seed setting rate is improved through artificial pollination, and the seeds are picked timely, so that excellent breeding materials are obtained for non-symbiotic germination of the seeds, and therefore, the time for flowering and flowering is an important link of a non-symbiotic germination path of the spot She Biaolan. Through annual monitoring and recording, the cypripedium hamburger sprouts at the root of the original plant in the next 4 th month of the year, flowers gradually in the middle of 6 th month, and the cypripedium hamburger She Biaolan enters the full bloom period from the middle of 7 th month to the bottom of 7 th month, wherein the period is the optimal period for artificial supplementary pollination. Gradually forming capsules in late 8 months until the capsules are fully mature in the beginning of 12 months, and gradually yellowing plant leaves in the middle of 10 months until the whole plant withers in the end of 12 months.
2. Selection of pollination mode
In the middle ten days of 7 months, the flowering condition is compared by fixedly monitoring 4 spots of cypripedium macranthum, the growth conditions of SL05 spots beside a white jade field and SL03 spots are optimal, the flowering condition is better, artificial supplementary pollination and natural pollination tests are carried out on cypripedium macranthum plants, 28 plants are artificially pollinated, 285 plants are naturally pollinated, and the pollinated plants are marked and recorded in detail. Through continuous observation, about 30 days after pollination, the results are obtained, then statistics is carried out in the collected capsules, 28 plants are cross-pollinated, and 25 plants are obtained; of the natural pollination 285 plants, only 29 plants were found. The artificial pollination result rate is 25/28=89%, and the natural pollination result rate is 29/285=10%. The capsule for artificial pollination is large and full, the size is about 4.0-5.3 cm, the diameter is 1.5-2.1 cm, and the seeds are more; the natural pollination has small capsules, the size of the capsules is about 2.0-3.0 cm, the diameter is 0.8-1.3 cm, and the seeds are few.
TABLE 1 Effect of different pollination modes on the yield of plaque She Biaolan
3. Influence of pollination mode on seed vigor
Before aseptic seeding of orchid, the seed vitality measurement is needed to know the development condition and physiological activity of the embryo, avoiding the waste of manpower, material resources and time caused by improper seeding materials. Determining the physiological activity of seeds by staining reactions is a very important link in orchid care and in vitro culture. Preparing 1% TTC solution according to conventional standard method, dissolving completely, and refrigerating in refrigerator. Putting seeds into glass vials with covers, adding 10mL of TTC solution into each vial, dyeing at 20 ℃ in dark, taking out the seeds after dyeing, and thoroughly washing the seeds with water; and (3) randomly selecting 3 fields of view under the condition of observing seed staining under a stereoscopic microscope, and respectively calculating the total number of seeds, the number of abortive seeds and the number of uncolored seeds of the embryo in each field of view. After TTC staining, the uncolored seed was observed under a microscope, the entire seed coat was stained pale red, and no embryo was observed in the seed. Whereas the colored seeds, which passed through the half of the light brown seed coat, clearly observed the red-colored spherical embryos. Most seeds which develop normally can be dyed in dark red, and the color shades can be slightly different. The seed dyeing rate of artificial pollination is 60-70%.
4. Effect of the inventive Medium on germination browning of plaque She Biaolan
During germination of the seeds of the spot She Biaolan, the brown effect is found to be serious, so that the effect test of the brown effect of the germination of the spots She Biaolan with different addition amounts of active carbon (0 g/L, 0.2g/L, 0.5g/L and 1.0 g/L) is carried out, 20g/L sucrose, 50g/L potato pulp, 2g/L tryptone and 0.2 mg/L6-benzylaminoadenine are added by taking modified Knudson C as a basic culture medium, and the culture temperature is 20 ℃ and the dark culture is carried out.
TABLE 2 Effect of different amounts of activated carbon addition on the germination browning of plaque She Biaolan
The test results show that only 5% of seeds germinate and all the rest of seeds are brown and dead on the culture medium without adding the activated carbon. The browning rate can be obviously reduced by adding 1g/L active carbon into the culture medium, and the browning rate is only 20%.
5. Effect of different media on seed germination of plaque She Biaolan
Seed germination experiments were performed using 5 media: (1) Harvais+6-BA 0.2mg/L, (2) 1/4MS+6-BA 6-BA 0.2mg/L, (3) 1/2MS+6-BA 6-BA 0.2mg/L, (4) KC improvement +6-BA 0.2mg/L, (5) Hyponex (7-6-19) +6-BA 0.2 mg/L. The culture medium contains 1g/L active carbon, 20g/L sucrose and 50g/L potato. The pH value is 5.5, the culture temperature is 20 ℃, and the culture is dark. Seed germination rates were investigated weekly after sowing.
Effect of Medium species on seed germination of plaque She Biaolan
The seeds sown by the improved KC culture medium of day 1 and 14 of 2022 germinate fastest, white granular protocorms can be seen after day 4, day 19 and day 90; the protocorms expand to have root hairs after 6 months, 20 days and 60 days; growing into root-like stems about 10 months, 27 days and 120 days; sprouting was carried out for about 110 days. The culture medium with highest germination rate is modified KC culture medium, and other culture mediums are Harvais, 1/4MS, 1/2MS, and Hyponex (7-6-19) culture medium in sequence from top to bottom.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (10)
1. A method for non-symbiotic sterile germination of a very small population of plaques She Biaolan, comprising the steps of:
1) Artificial pollination;
2) Picking capsules;
3) And (3) low-temperature treatment:
4) Seed disinfection, alkali etching of seed coats and sowing;
5) Culturing at constant temperature.
2. The method for non-symbiotic sterile germination of the minimal population spots She Biaolan according to claim 1, wherein the pollination mode in the step 1) is cross pollination, the pollination time is selected from 7 to 7 months at the end of the middle ten days, plots with a large number of communities and flowering plants are selected for pollination, plants with healthy growth and full bloom and non-natural pollination are selected, and the pollen mass is provided with a yellowish viscosity.
3. A method of non-symbiotic sterile germination of microscopic population spots She Biaolan according to claim 2, wherein the specific pollination method in the pollination step is to remove the labial lamella with a blade, remove pollen with forceps, spread on the stigma, and lightly press to complete pollination.
4. The method for non-symbiotic sterile germination of extremely small population spots She Biaolan according to claim 1, wherein the capsules are collected 90-110 days after pollination in the step 2) and are collected when the capsules turn to mauve, and the seed coats are white or pale brown.
5. A method of non-symbiotic sterile germination of very small population spot She Biaolan according to claim 1 wherein seed vigor determination is also required prior to step 3).
6. The method for non-symbiotic sterile germination of microscopic population spots She Biaolan according to claim 1, wherein the specific method in step 3) is to wipe off fruit surface dirt with 75% alcohol, dry in the shade, wrap with sterile filter paper, put into a tissue culture flask sterilized at high temperature and added with a desiccant, and put into a incubator at 4 ℃ for 30 days.
7. The method for non-symbiotic sterile germination of the minimal population spot She Biaolan according to claim 1, wherein the specific method in step 4 is that the capsule which is not ruptured after the treatment in step 3) is wiped with 75% alcohol on a sterile operating table, after the alcohol on the surface is volatilized, the capsule is split by a sterilizing blade, and the seeds are taken out and placed in 0.1% Hgcl 2 Soaking and sterilizing for 10 minutes, washing for 6-8 times by using sterilized distilled water after sterilization, soaking in 0.1mol/L KOH solution for 10 minutes, corroding seed coats, enabling the seed coats to permeate water and breathe so as to promote seed germination, washing for 6-8 times by using sterilized distilled water, placing on a tray filled with sterile filter paper, placing on a sterile operation table for airing, and shaking off the seeds into a non-symbiotic germination culture medium by using a sterile medicine spoon or an inoculating shovel for sowing.
8. A method for non-symbiotic sterile germination of a very small population spot She Biaolan according to claim 7, wherein said non-symbiotic germination medium is specifically formulated as follows: the modified Knudson C is taken as a basic culture medium, 20-30 g/L sucrose, 20-50 g/L potato, 2g/L tryptone, 0.2-0.6 mg/L6-benzylaminoadenine and 1g/L active carbon are added, and the pH value of the culture medium is regulated to 5.5-5.8 by sodium hydroxide solution and hydrochloric acid.
9. A method of non-symbiotic sterile germination of the microscopic population spot She Biaolan of claim 8 wherein the modified Knudson C formulation is: KH (KH) 2 PO 4 62.5-125 mg/L of potassium dihydrogen phosphate; ca (NO) 3 ) 2 .4H 2 250-500 mg/L calcium nitrate tetrahydrate; (NH) 4 ) 2 SO 4 125-250 mg/L of ammonium sulfate; mgSO (MgSO) 4 •7H 2 62.5 to 125mg/L of magnesium sulfate heptahydrate; feSO 4 ·7H 2 6.25-12.5 mg/L of O ferrous sulfate heptahydrate; mnSO 4 ·4H 2 1.875-3.75 mg/L of O tetrahydrate manganese sulfate.
10. The method for non-symbiotic sterile germination of the minimal population spots She Biaolan according to claim 1, wherein the culture flask sown in the step 4) is placed in an incubator for culture at 20 ℃, the culture temperature is about 90d, the culture flask is germinated to form protocorms, the protocorms are transferred to a newly prepared culture medium, the newly prepared culture medium is an improved Knudson C formula, the white protocorms are elongated and grow to form white or light brown rhizomes with white transparent villi about 60 days, the illumination culture is carried out, the illumination time is 12h/d, the illumination intensity is 2000lx, the top end of the rhizomes differentiate into buds about 110 days, the heights of the buds are 1.5-3 cm, and the rhizomes are transplanted out of the flask when the root length is 2-5 cm.
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| CN110786235A (en) * | 2018-08-02 | 2020-02-14 | 北京林业大学 | Method for aseptically germinating cypripedium guttatum seeds |
| CN113924841A (en) * | 2021-10-20 | 2022-01-14 | 云南省林业和草原科学院 | Non-symbiotic germination method for cypripedium rubrum seeds |
| CN113951144A (en) * | 2021-12-03 | 2022-01-21 | 中国科学院昆明植物研究所 | Method for promoting sterile germination and seedling formation of paphiopedilum sansevieri seeds |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110786235A (en) * | 2018-08-02 | 2020-02-14 | 北京林业大学 | Method for aseptically germinating cypripedium guttatum seeds |
| CN113924841A (en) * | 2021-10-20 | 2022-01-14 | 云南省林业和草原科学院 | Non-symbiotic germination method for cypripedium rubrum seeds |
| CN113951144A (en) * | 2021-12-03 | 2022-01-21 | 中国科学院昆明植物研究所 | Method for promoting sterile germination and seedling formation of paphiopedilum sansevieri seeds |
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