CN114223545B - Culture medium for improving germination rate of Pleione bulbocodioides distant hybrid seeds and application and cultivation method thereof - Google Patents
Culture medium for improving germination rate of Pleione bulbocodioides distant hybrid seeds and application and cultivation method thereof Download PDFInfo
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- CN114223545B CN114223545B CN202210029522.9A CN202210029522A CN114223545B CN 114223545 B CN114223545 B CN 114223545B CN 202210029522 A CN202210029522 A CN 202210029522A CN 114223545 B CN114223545 B CN 114223545B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/002—Culture media for tissue culture
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- 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
Abstract
The invention relates to the technical field of Pleione culture, in particular to a culture medium for improving Pleione distant hybridization seed germination rate and an application and a culture method thereof. The culture medium takes 1/2MS culture medium as basic culture medium, and also comprises the following components: 0.5-1 mg/L of NAA, 30g/L of cane sugar, 100mL/L of coconut juice, 7g/L of agar and 1g/L of activated carbon, wherein the pH value of the culture medium is 5.6-5.8. The culture medium provided by the invention obviously improves the germination rate of the Pleione hybrida seeds by compounding coconut juice and activated carbon with proper concentration in the conventional Pleione hybrida culture medium, thereby reducing the cultivation cost of distant hybrid Pleione hybrida.
Description
Technical Field
The invention relates to the technical field of Pleione culture, in particular to a culture medium for improving Pleione distant hybridization seed germination rate and an application and a culture method thereof.
Background
Pleione D.Don belonging to the family Orchidaceae is a native ornamental orchid of China, has a bright color and beautiful flower shape, and has an important position in the international orchid trade. The florescence of the former is generally 3-5 months, and the florescence of the latter is generally 9-10 months.
Although the existing research has been to perform crossbreeding between spring-flower type Pleione and autumn-flower type Pleione, so as to cultivate Pleione capable of blooming in winter, because these two plants belong to different groups (Pleione group and spring-flower Pleione group) of Pleione in taxonomic, the genetic relationship is far, the success rate of hybridization is low, and the embryo development is incomplete, the germination rate of hybrid seed cultivated by the existing method is low, only 2% -5%, and the cultivation cost is high.
Disclosure of Invention
In order to solve the problems, the invention provides a culture medium for improving the germination rate of Pleione bulbocodioides distant hybrid seeds, and an application and a cultivation method thereof. The culture medium provided by the invention can improve the germination rate of the hybrid seeds, thereby reducing the cultivation cost of distant hybrid Pleione bulbocodioides.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a culture medium for improving germination rate of Pleione bulbocodioides distant hybrid seeds, which takes 1/2MS culture medium as basic culture medium and also comprises the following components: 0.5-1 mg/L of NAA, 30g/L of cane sugar, 80-100 mL/L of coconut juice, 6-7 g/L of agar and 1g/L of activated carbon, wherein the pH value of the culture medium is 5.6-5.8.
The invention also provides application of the culture medium in improving the germination rate of the Pleione bulbocodioides hybrid seeds.
The invention also provides application of the culture medium in cultivating the Pleione species flowering in winter.
The invention also provides a cultivation method of the Pleione species flowering in winter, which comprises the following steps:
1) hybridizing the Pleione bulbocodioides variety blooming in spring and the Pleione bulbocodioides variety blooming in autumn to obtain hybrid seeds;
2) adding the hybrid seeds into the culture medium, and sequentially carrying out dark culture and light-dark alternate culture to obtain germinated seedlings;
3) transferring the germinated seedlings into a rooting culture medium, and performing light-dark alternate culture to obtain Pleione bulbocodioides seedlings; the rooting culture medium takes 1/2MS culture medium as a basic culture medium, and also comprises 0.5-1 mg/L, NAA 0.5.5-1 mg/L of 6-BA, 30-50 g/L of banana puree, 30g/L of sucrose and 6-7 g/L of agar; the pH value of the rooting culture medium is 5.6-5.8;
4) transplanting the Pleione seedling to obtain Pleione blooming in winter.
Preferably, the method for hybridizing the spring flowering pleione species and the autumn flowering pleione species in the step 1) comprises the following steps: hybridizing the Pleione bulbocodioides variety blooming in spring and the Pleione bulbocodioides variety blooming in autumn by pollen preservation; the saving method comprises the following steps: standing the pollen for 6-12 h in an environment with the temperature of 20-25 ℃ and the humidity of below 50%, and then sealing and storing at the temperature of 2-8 ℃.
Preferably, the spring flowering Pleione species includes Pleione albolana (P.albicans), Pleione canescens (P.arjuna), Pleione gordonii (P.aurantia), Pleione bulbocodioides (P.bulbocodioides), Pleione chensinensis (P.chuniii), Pleione coronaria (P.coronaria), Pleione formosana (P.formosana), Pleione wrenchea (P.forticii), Pleione major (P.grandiflora), Pleione rougheriana (P.hoorieana), Pleione nana (P.humulis), Pleione kawakianus (P.kakationea), Pleione tetrandra (P.limeri), Pleione americana (P.pleiones), Pleione bulbifera (P.scolyricum), or Pleione bulbifera (P.yunna); the Pleione species in autumn comprises Pleione longata (P. maculota), Pleione bulbocodioides (P.praecox) or Pleione longata (P.saxicola).
Preferably, the conditions of the dark culture in the step 2) include: the temperature is 24-26 ℃, and the time is 30 d;
the conditions of the light-dark alternate culture in the step 2) and the step 3) respectively comprise: the temperature is 24-26 ℃, the illumination time is 12h/d, and the illumination intensity is 2000-3000 lx.
Preferably, the matrix transplanted in the step 4) comprises the following components in percentage by volume: 50 to 70 percent of bark particles, 20 to 30 percent of coconut chaff and 10 to 20 percent of leaf mold.
Preferably, the length of the bark particles is 1-3 cm.
Preferably, before transplanting, hardening off the seedlings; the seedling exercising treatment comprises the following steps: standing the Pleione under the conditions of shading of 45-55%, temperature of 15-25 ℃ and humidity of 60-80% for 5-9 days.
Has the advantages that:
the invention provides a culture medium for improving germination rate of Pleione bulbocodioides distant hybrid seeds, which takes 1/2MS culture medium as basic culture medium and also comprises the following components: 0.5-1 mg/L of NAA, 30g/L of cane sugar, 100mL/L of coconut juice, 7g/L of agar and 1g/L of activated carbon, wherein the pH value of the culture medium is 5.6-5.8. The culture medium provided by the invention obviously improves the germination rate of the Pleione hybrida seeds by compounding coconut juice and activated carbon with proper concentration in the conventional Pleione hybrida culture medium, thereby reducing the cultivation cost of distant hybrid Pleione hybrida.
Drawings
FIG. 1 is a schematic view of the process for cultivating Pleione bulbocodioides which blossoms in winter according to the present invention.
Detailed Description
The invention provides a culture medium for improving germination rate of Pleione bulbocodioides distant hybrid seeds, which takes 1/2MS culture medium as basic culture medium and also comprises the following components: 0.5-1 mg/L of NAA, 30g/L of cane sugar, 80-100 mL/L of coconut juice, 6-7 g/L of agar and 1g/L of active carbon, wherein the pH value of the culture medium is 5.6-5.8. The invention does not require any particular source for the individual components of the culture medium, and commercial products known to those skilled in the art may be used. The culture medium provided by the invention obviously improves the germination rate of the Pleione hybrida seeds by compounding coconut juice and activated carbon with proper concentration in the conventional Pleione hybrida culture medium, thereby reducing the cultivation cost of distant hybrid Pleione hybrida.
The invention also provides application of the culture medium in improving the germination rate of the Pleione bulbocodioides hybrid seeds. According to the invention, the hybrid seeds are inoculated to the culture medium for culture, so that the germination rate of the Pleione bulbocodioides hybrid seeds can be obviously improved. The mechanism of action is the same as above and is not described herein.
The invention also provides application of the culture medium in cultivating Pleione species flowering in winter. According to the invention, the germination rate of the Pleione hybride seeds can be obviously improved by inoculating the hybrid seeds onto the culture medium for culture, so that the cultivation cost of Pleione which blooms in winter is reduced. The mechanism of action is the same as above and is not described herein.
The invention also provides a cultivation method of the Pleione species flowering in winter, which comprises the following steps:
1) hybridizing the Pleione bulbocodioides variety blooming in spring and the Pleione bulbocodioides variety blooming in autumn to obtain hybrid seeds;
2) adding the hybrid seeds into the culture medium, and performing dark culture and light-dark alternate culture in sequence to obtain germinated seedlings;
3) transferring the germinated seedlings into a rooting culture medium, and performing light-dark alternate culture to obtain Pleione bulbocodioides seedlings; the rooting culture medium takes 1/2MS culture medium as basic culture medium, and also comprises 0.5-1 mg/L, NAA 0.5.5-1 mg/L of 6-BA, 30-50 g/L of banana puree, 30g/L of sucrose and 6-7 g/L of agar; the pH value of the rooting culture medium is 5.6-5.8;
4) transplanting the Pleione seedling to obtain Pleione blooming in winter.
The method disclosed by the invention is used for hybridizing the Pleione bulbocodioides variety blooming in spring with the Pleione bulbocodioides variety blooming in autumn to obtain the hybrid seed. The examples of the present invention are given as illustration, but they should not be construed as limiting the scope of the present invention. In the present invention, the spring flowering Pleione species preferably includes Pleione alborona (P.albicans), Pleione canescens (P.arjuna), Pleione gordonii (P.aurantia), Pleione bulbocodioides (P.bulbocodioides), Pleione chensinensis (P.chunigii), Pleione coronaria (P.coronaria), Pleione formosana (P.formosana), Pleione falciparum (P.forrestii), Pleione grandiflorum (P.grandiflorum), Pleione rougheriana (P.hoorieana), Pleione nanensis (P.humulis), Pleione kawae (P.kakayae), Pleione tetrandra (P.limperioperative), Pleione americana (P.pleiones), Pleione uniflora (P.scleocharitis), or Pleione yunnanensis (P.yunnanensis); the variety of the autumn flowering Pleione preferably comprises Pachyione longata (P.maculota), Pleione bulbocodioides (P.praecox) or Pleione bulbocodioides (P.saxicola). The hybrid combination preferably comprises a matching group of Yunan Pleione and autumn Pleione, a matching group of Pleione major and maternal Yangsheng Pleione; more preferably, the Pleione yunnanensis is used as a female parent, the Pleione longans of autumn flower is used as a male parent for hybridization, the Pleione longans of flowers is used as a male parent, and the Pleione longans of rock is used as a female parent for hybridization.
The hybridization method of the present invention preferably comprises: hybridizing the Pleione bulbocodioides variety blooming in spring and the Pleione bulbocodioides variety blooming in autumn by pollen preservation; the stored pollen is preferably pollen taken from 3-7 days after opening. The invention has no special requirement on the pollination mode, and the pollination mode known to the technical personnel can be adopted, and the invention preferably adopts artificial pollination.
The preservation method of the present invention preferably comprises: standing the pollen for 6-12 h in an environment with the temperature of 20-25 ℃ and the humidity of below 50%, and then sealing and storing at the temperature of 2-8 ℃; the temperature of the standing is preferably 20-25 ℃, and more preferably 21-24 ℃; the standing time is preferably 6-12 h, and more preferably 12 h; the temperature of the sealed preservation is preferably 2-8 ℃, and more preferably 2-6 ℃. According to the invention, the pollen is kept stand for a proper time in a shade place (the temperature is 20-25 ℃ and the humidity is below 50 wt.%) for dehydration treatment, and then is stored in a sealed manner at a proper temperature, so that the pollen activity can be ensured to the maximum extent, and the pollination success rate is improved.
In the present invention, the method for obtaining hybrid seeds preferably comprises: hybridizing the Pleione bulbocodioides variety blooming in spring and the Pleione bulbocodioides variety blooming in autumn to obtain hybrid fruit, and sterilizing the hybrid fruit to obtain hybrid seed; the method for disinfecting the hybrid fruit preferably comprises the following steps: soaking in 75 wt.% alcohol for 30s, sterilizing in 0.5 wt.% mercuric chloride solution for 10min, and washing with sterile water for 3 times.
After the hybrid seeds are obtained, the hybrid seeds are added into the culture medium to be subjected to dark culture and light-dark alternate culture in sequence, so that the germinated seedlings are obtained. The conditions for the dark culture in the present invention preferably include: the temperature is 24-26 ℃, and the time is 15-30 d; the light-dark alternate cultivation temperature of the hybrid seeds is preferably 24-26 ℃, the light time of the light-dark alternate cultivation is preferably 12h/d, the light intensity of the light-dark alternate cultivation is preferably 2000-3000 lx, and the light-dark alternate cultivation time is preferably 45-50 d.
After the germinated seedlings are obtained, the germinated seedlings are transferred into a rooting culture medium and are subjected to light-dark alternate culture to obtain Pleione bulbocodioides seedlings; the rooting culture medium takes 1/2MS culture medium as a basic culture medium, and also comprises 0.5-1 mg/L, NAA 0.5.5-1 mg/L of 6-BA, 30-50 g/L of banana puree, 30g/L of sucrose and 6-7 g/L of agar; the pH value of the rooting culture medium is 5.6-5.8. In the present invention, the conditions for the light-dark alternate cultivation of the germinated seedlings preferably include: the temperature is 24-26 ℃, the illumination time is 12h/d, the illumination intensity is 2000-3000 lx, and the culture time is 150-180 d. The invention does not require any particular source for the individual components of the rooting medium, and commercial products well known to those skilled in the art may be used.
After the pleione seedling is obtained, the invention transplants the pleione seedling to obtain the pleione flowering in winter.
In the invention, before transplanting the Pleione seedling, the seedling hardening treatment is preferably further included; the hardening-seedling treatment preferably comprises: standing the Pleione under the conditions of shading light of 45-55%, temperature of 15-25 ℃ and humidity of 50-80% for 5-9 days; more preferably: the Pleione seedling is kept standing for 7 days under the conditions of 50% shading, 20 deg.C and 60% humidity.
In the invention and the application examples, the transplanted substrate preferably comprises the following components in percentage by volume: 50-70% of bark particles, 20-30% of coconut chaff and 10-20% of leaf mold; more preferably 70% bark particles, 20% coconut coir and 10% leaf mold; the length of the bark particles is preferably 1-3 cm. In the present invention, the bark particles preferably comprise pine bark particles.
In the present invention, the culture time of the Pleione seedling in the matrix is preferably 3 years; the culture of the Pleione seedling in the matrix comprises: pouring water for the first time for 3-7 days; spraying 0.5 wt.% of foliar fertilizer once in 14-21 days; the mass ratio of nitrogen, phosphorus and potassium elements of the foliar fertilizer is 1:1: 1.
the invention utilizes pollen block preservation technology to successfully carry out distant hybridization on the unigarlic orchid flowering in spring and autumn which are not in flowering season, and seeds with different hybridization combinations can obtain a plurality of winter flowering varieties through aseptic germination according to the method, thereby breaking the characteristic that the unigarlic orchid can only bloom in spring and autumn, enriching the flowering season of the unigarlic orchid, and simultaneously, the culture medium provided by the invention improves the germination rate of distant hybridization seeds, thereby reducing the cultivation cost of the distant hybridization unigarlic orchid and laying a foundation for the industrialization and commodity popularization of the unigarlic orchid.
To further illustrate the present invention, the culture medium for improving germination rate of Pleione bulbocodioides distant hybrid seeds provided by the present invention, its application and cultivation method are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
A culture medium for improving germination rate of Pleione bulbocodioides distant hybrid seeds takes 1/2MS culture medium as basic culture medium, and only contains the following components: NAA0.5 mg/L, sucrose 30g/L, coconut juice 100mL/L, agar 7g/L and active carbon 1g/L, wherein the pH value of the culture medium is 5.6.
Example 2
A culture medium for improving germination rate of Pleione bulbocodioides distant hybrid seeds takes 1/2MS culture medium as basic culture medium, and only contains the following components: 1mg/L of NAA, 30g/L of cane sugar, 100mL/L of coconut juice, 7g/L of agar and 1g/L of activated carbon, wherein the pH value of the culture medium is 5.8.
Application example 1
Test site: kunming plant institute of Chinese academy of sciences.
A method for cultivating Pleione blossoming in winter comprises the following steps:
(1) and (3) obtaining and storing pollen blocks: selecting healthy autumn Pleione mamulata (Pleione mamulata) flowers in 25 days after 10 months, taking off pollen blocks on the 5 th day of flower opening, standing in a cool room (with the temperature of 20-25 ℃ and the humidity below 50%) for 12h for dehydration, then filling the pollen blocks into a plastic tube, sealing and storing in a refrigerator at the temperature of 2-6 ℃;
(2) artificial hybridization and pollination: in the next 5 days 4 months, the female parent of the Yunnan Mandarin garlic reaches the flowering phase, after flowers bloom, lip flaps of the flowers are picked off (so as to be beneficial to pollination operation), pollen blocks of the male parent stored in a refrigerator are taken, and the pollen blocks are placed on the heads of the adhesive columns of the female parent; pollination is carried out for 5 days, flowers wither, and ovaries begin to expand, which indicates successful pollination;
(3) sterile germination of seeds: taking uncracked fruits pollinated for 6 months, cleaning the surfaces of the uncracked fruits with clear water, soaking the fruits in 75 wt.% of alcohol for 30s, then placing the fruits into 0.5 wt.% of mercuric chloride solution for sterilization for 10min, then washing the fruits with sterile water for 3 times, and finally sowing the fruits on the surfaces of the culture mediums prepared in the example 1 on an ultra-clean workbench; after sowing, putting the seeds into a culture room with the temperature of 25 +/-1 ℃ for culture, firstly performing dark culture (the temperature is 24-26 ℃) for 15 days, and then performing light culture, wherein the illumination time is 12h/d, the illumination intensity is 2500lx, and the temperature of the light culture is 24-26 ℃; seeds begin to germinate to form small protocorms after being sown for two weeks, and small plants with leaves and with the height of about 2cm are formed after two months;
(4) rooting culture of sterile seedlings: transferring the plantlets obtained in the step (3) to a rooting culture medium (wherein 1/2MS culture medium is used as a basic culture medium, and only contains 6-BA 0.5mg/L, NAA 1mg/L, banana puree 50g/L, sucrose 30g/L and agar 7g/L, the pH value of the rooting culture medium is 5.8), transferring 15-20 plantlets in each bottle, placing the bottle in a culture chamber for continuous culture for 4 months to obtain plants with 1-2 leaves, 3-4 roots and 0.5-0.8 cm of pseudobulb diameter;
(5) hardening and transplanting seedlings: the plant with the culture bottle is moved out of a culture room, placed in a greenhouse with 50% of shading for hardening seedlings for about one week, and planted on a substrate of 70% (volume percentage) pine bark particles (with the length of 1-3 cm), 20% (volume percentage) coconut husk and 10% (volume percentage) leaf mold; watering every 5 days in the cultivation process, spraying leaf fertilizer with the concentration of 0.5 wt.% of nitrogen, phosphorus and potassium being 1:1:1 every two weeks, and after planting for 3 years, hybridizing seedlings bloom, wherein the natural flowering phase of the hybridization combination is from 12 months to 1 month in the next year, the single flowering phase is about 20 days, and the specific flow is shown in figure 1.
Application example 2
Test site: kunming plant institute of Chinese academy of sciences.
A method for cultivating Pleione which blossoms in winter comprises the following steps:
(1) and (3) obtaining and storing pollen blocks: selecting healthy Pleione bulbocodioides (P. grandiflora) flowers in 4 months and 10 days, taking off pollen blocks on the 5 th day of blooming, placing in a shade room (with the temperature of 20-25 ℃ and the humidity below 50%) for 12h, then filling the pollen blocks into a plastic pipe, and hermetically storing in a refrigerator at 4-8 ℃;
(2) artificial hybridization and pollination: when the female parent's lithogenous Pleione bulbocola (Pleione saxcola) reaches the flowering stage in 12 days 10 months in the year, picking off the flower lip after the flowers are opened (to facilitate the pollination operation), taking the pollen block of the male parent stored in the refrigerator, and placing the pollen block on the viscous column head of the female parent; when pollination is carried out for 5 days, flowers wither, and ovaries begin to expand, which indicates that the pollination is successful;
the following sterile germination of seeds, rooting culture of sterile seedlings, seedling hardening and transplanting conditions of the seedlings are the same as those of the application example 1, after 3 years of planting, hybrid seedlings bloom, the natural flowering phase of the hybrid combination is from 12 months to 2 months in the next year, and the single flowering phase is about 20 days.
Comparative example 1
A breeding method similar to that of application example 1, except that the dehydration time and storage temperature of pollen were different, and the specific grouping and experimental results are shown in Table 1.
TABLE 1 pollination success rates under different treatment conditions
Note: each treatment pollinates 20 flowers.
As can be seen from Table 1, the treatment mode of the pollen block directly affects the success rate of hybrid pollination, too short or too long dehydration time and too high or too low storage temperature are not beneficial to successful fructification of hybrid combination, and the treatment mode of the pollen block provided by the invention has the highest pollination success rate.
Comparative example 2
A germination culture medium of Pleione bulbocodioides is prepared from 1/2MS culture medium as basic culture medium, and further comprises the following components: NAA0.5 mg/L, sucrose 30g/L and agar 7g/L, and the pH value of the culture medium is 5.6.
Comparative example 3
A method similar to application example 1, except that the medium prepared in example 1 in step (3) was replaced with the germination medium prepared in comparative example 2.
Comparative example 4
A method similar to application example 1 except that steps (1) and (2) are omitted and the seeds in step (3) are replaced with autumn Pleione seeds.
Comparative example 5
A method similar to comparative example 4, except that the medium prepared in example 1 in step (3) was replaced with the germination medium prepared in comparative example 2.
Comparative example 6
The seed germination rates of the application example 1 and the comparative examples 3-5 are respectively counted, and the statistical results are shown in table 2.
TABLE 2 comparison of germination rates of hybrid and parental inbred seeds
| Grouping | Germination rate |
| Application example 1 | 18％～26％ |
| Comparative example 3 | 2％～5％ |
| Comparative example 4 | 86％～94％ |
| Comparative example 5 | 79％～88％ |
As can be seen from Table 2, the seeds involved in the invention belong to distant hybridization, and the embryo development of the seeds is incomplete, so that the sterile germination rate is lower than that of normal seeds.
In conclusion, the culture medium provided by the invention can obviously improve the germination rate of the Pleione hybrid seeds, thereby reducing the cultivation cost of Pleione which blooms in winter; the cultivation method provided by the invention can obviously improve the success rate of hybrid pollination by using a proper pollen preservation method, and the germination rate of the hybrid seed of the Pleione can be obviously improved by inoculating the obtained hybrid seed on the culture medium provided by the invention, so that distant hybridization of the Pleione is possible, the characteristic that the Pleione can only bloom in spring and autumn is broken, the flowering period of the Pleione is enriched, and a foundation is laid for industrialization and commodity popularization of the Pleione.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (9)
1. A culture medium for improving germination rate of Pleione bulbocodioides distant hybrid seeds is characterized in that Pleione bulbocodioides variety blooming in spring and Pleione bulbocodioides variety blooming in autumn are hybridized to obtain hybrid seeds; the culture medium takes 1/2MS culture medium as basic culture medium, and only contains the following components: 0.5-1 mg/L of NAA, 30g/L of cane sugar, 80-100 mL/L of coconut juice, 6-7 g/L of agar and 1g/L of active carbon, wherein the pH value of the culture medium is 5.6-5.8.
2. The use of the medium of claim 1 for increasing the germination rate of Pleione bulbocodioides hybrid seeds.
3. Use of the medium according to claim 1 for breeding a Pleione species flowering in winter.
4. A method for cultivating Pleione blossoming in winter is characterized by comprising the following steps:
1) hybridizing the Pleione bulbocodioides variety which blossoms in spring and the Pleione bulbocodioides variety which blossoms in autumn by using a pollen preservation mode to obtain hybrid seeds; the saving method comprises the following steps: standing the pollen for 6-12 h in an environment with the temperature of 20-25 ℃ and the humidity of below 50%, and then sealing and storing at the temperature of 2-8 ℃;
2) adding the hybrid seeds into the culture medium of claim 1, and performing dark culture and light-dark alternate culture in sequence to obtain germinated seedlings;
3) transferring the germinated seedlings into a rooting culture medium, and performing light-dark alternate culture to obtain Pleione seedling; the rooting culture medium takes 1/2MS culture medium as a basic culture medium, and also comprises 0.5-1 mg/L, NAA 0.5.5-1 mg/L of 6-BA, 30-50 g/L of banana puree, 30g/L of sucrose and 6-7 g/L of agar; the pH value of the rooting culture medium is 5.6-5.8;
4) transplanting the Pleione seedling to obtain Pleione blooming in winter.
5. The method of claim 4, wherein the spring flowering Pleione variety comprises Pleione bulbocodioides (Pleione bulbocodioides) (white flower Pleione)Pleione. albiflora) And (C) Tibet NavyP. arunachalensis) Pleione bulbocodioides (A) of Pleione bulbocodioides (A)P. aurita) Pleione rhizome, herba Pleione rhizomeP. bulbocodioides) Pleione cruda (herba Pleioneae)P. chunii) Pleione rhizome, herba Typhonii gigantei, and radix Et rhizoma RheiP. coronaria) Pleione of Taiwan (1)P. formosana) Pleione odorata (I) and (II)P. forrestii) Pleione rhizome, herba Pleione rhizomeP. grandiflora) Pleione bulbocodioides (L.) Merr (all-grass of Epimedium PursonP. hookeriana) Pleione bulbocodioides (A) and (B)P. humilis) Pleione kadsura (I) C.A.P. kaatiae) Pleione rhizome, herba Typhonii giganteiP. limprichtii) Pleione (R) D.E. ((R))P. pleionoides) Pleione bulbocodioides (B)P. scopulorum) Or Yunan Durio lily (A. Mount Yun)P. yunnanensis) (ii) a The Pleione species in autumn comprises Pleione longbracteata (Laminaria religiosa) in autumnP. maculata) Pleione bulbocodioides (A) and (B)P. praecox) Or rock-grown Pleione (P. saxicola）。
6. The method according to claim 4, wherein the conditions of the dark culture in step 2) include: the temperature is 24-26 ℃, and the time is 30 d;
the conditions of the light-dark alternate culture in the step 2) and the step 3) respectively comprise: the temperature is 24-26 ℃, the illumination time is 12h/d, and the illumination intensity is 2000-3000 lx.
7. The method as claimed in claim 4, wherein the matrix transplanted in the step 4) comprises the following components in percentage by volume: 50% -70% of bark particles, 20% -30% of coconut coir and 10% -20% of leaf mold.
8. The method as claimed in claim 7, wherein the bark particles have a length of 1-3 cm.
9. The method of claim 4, further comprising a hardening-seedling treatment before said transplanting; the seedling exercising treatment comprises the following steps: standing the Pleione bulbocodioides seedling for 5-9 days under the conditions of shading of 45-55%, temperature of 15-25 ℃ and humidity of 60-80%.
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| CN1631102A (en) * | 2004-12-29 | 2005-06-29 | 中国科学院华南植物园 | Pleione test tube breeding ball production technique |
| CN101124892B (en) * | 2007-09-25 | 2010-11-10 | 云南省农业科学院高山经济植物研究所 | Cymbidium edaphic orchids seed aseptic seeding growing seedlings method |
| CN106134986A (en) * | 2016-06-24 | 2016-11-23 | 浙江中医药大学 | A kind of breeding method of pleione bulbocodioides polyploid plant |
| CN106332782B (en) * | 2016-10-19 | 2018-10-19 | 贵州省农作物品种资源研究所 | Pleione bulbocodioides tissue cultures one-step culture method |
| CN108012929A (en) * | 2017-12-27 | 2018-05-11 | 康美药业（文山）药材种植管理有限公司 | A kind of tissue cultures expanding propagation method of pleione bulbocodioides |
| CN108353787A (en) * | 2017-12-27 | 2018-08-03 | 康美药业（文山）药材种植管理有限公司 | A kind of pleione bulbocodioides Initial culture base and preparation method thereof |
| CN112352681B (en) * | 2020-11-23 | 2023-02-03 | 云南省林业和草原科学院 | Method for promoting germination of Pleione macroflora seeds |
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