CN109964817B - Rapid propagation method of Emei glauca - Google Patents
Rapid propagation method of Emei glauca Download PDFInfo
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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
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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 discloses a method for obtaining capsules of zephyr omeiensis and breeding seeds of the capsules to obtain seedlings, which belongs to the technical field of plant breeding, wherein capsules are obtained by artificial pollination among plants with a certain genetic distance, and the maturing rate is 100%; a rapid propagation system of the zephyranthes omeiensis is established, the propagation coefficient of the system reaches more than 7 times, the rooting and seedling rate reaches more than 90%, and a scientific and technical basis is laid for protecting wild resources of the species; the obtained excellent coerulea sylvestris seedling provides resource foundation and technical support for sustainable utilization of the coerulea sylvestris seedling, actively promotes the industrial development of plant resources, and has extremely important practical significance and economic benefit.
Description
Technical Field
The invention belongs to the technical field of plant propagation, and particularly relates to a method for obtaining zephyranthes omeiensis capsules and a method for rapidly propagating seeds of zephyranthes omeiensis capsules.
Background
Zealand omeei (herb of Emei)Holcoglossum omeiense) The type of the plants in the genus of Vanilla is low mountain, the type of the plants is a special species in the Emei mountain area of China, and the plants are only distributed in the middle and low mountain areas of the area. The coerulea omeiensis belongs to rare or endangered species due to extremely narrow distribution region and extremely small population and individual number, and is listed in 'minimal population (narrow distribution) protection species', 'Chinese biodiversity red directory (higher plant volume)', 'Chinese species red directory (plant part)' and 'Chinese rare or endangered plant pictorial identification'. At present, the zephyranthes omeiensis is a wild plant with a national minimum population, and is listed in 120 species with minimum population protection, namely national forestry bureau and national development and development of national minimum population wild plant rescue and protection engineering plan, which is printed by national development and development committee, and belongs to one of 11 wild plants listed in the rescue plan and distributed in Sichuan province.
The Zealand omelania omeiensis is perennial herb of genus Zealand of family Orchidaceae, and is attached to the trunk of forest. At present, the number of wild plants of the zephyranthes omeiensis is very small, and the wild plants are rare and endangered wild plants with extremely small population and have extremely high scientific research value; in addition, the form of the Emei tongue is elegant and elegant, the flower color is pure white and elegant, and the garden ornament has extremely high gardening and ornamental values. At present, with the development of economy and Emei tourism, scientific research and the collection of orchid enthusiasts, the ecological environment of the plant is damaged and the plant is excessively excavated, so that the wild population is greatly threatened.
The research on the plants of the gladiolus sylvestris in China is extremely lacking, only rough information such as narrow distribution areas, roughly distributed places, extremely rare quantity and the like of the gladiolus sylvestris is known, the research on the propagation aspect of the gladiolus sylvestris is blank, and currently, researchers in China cannot successfully propagate the gladiolus sylvestris plants.
Disclosure of Invention
Aiming at the situation that the plant is a tiny population plant, the quantity of field resources is very small, zephyranthes omeiensis capsules cannot be collected in the field, and the systematic and rapid propagation method of zephyranthes omeiensis in the prior art is lacked, the invention aims to provide a method for obtaining zephyranthes omeiensis capsules and a rapid propagation method. According to the method, the healthy coerulea anthriscus sylvestris capsules are obtained by obtaining an artificial pollination mode, and the seeds of the capsules are utilized to carry out propagation research, so that a rapid propagation technical system is established, and the method has important practical significance for protecting precious wild resources of coerulea anthriscus sylvestris.
In order to achieve the purpose, the invention adopts the following technical scheme:
the rapid propagation method of the Emei glauca comprises the following steps:
a obtaining of capsule: selecting plants with strong adaptability, vigorous growth and capability of smoothly completing a life cycle, and performing artificial pollination on parents for pollination by adopting plants from different populations in a flowering period to obtain capsules, wherein the capsules are picked when the capsules are not cracked after 380 plus 520 days after pollination;
b, processing capsules: washing picked zephyranthes omeiensis capsules under tap water for 5-10 min, and then sucking water by using filter paper; then, respectively treating the raw materials with 75% alcohol by mass for 30-35 s and 0.1% mercuric chloride solution by mass for sterilization for 15-18 min, respectively washing the raw materials with sterile water for 4-6 times, and finally, absorbing the water on the surface with sterile filter paper; shearing capsule under aseptic condition to obtain seed;
c, induction culture: b, uniformly broadcasting the seeds obtained in the step b on an induction culture medium to induce protocorms;
d, proliferation and differentiation culture: c, inoculating the protocorm with the obviously visible green leaf primordium in the step c to a proliferation and differentiation culture medium for proliferation and differentiation culture; in the proliferation and differentiation culture, dark culture for 10-15 days is firstly carried out; then the illumination intensity is 1500-2000Lx, the illumination time is 12h/d, the culture time is 90-100d, and the culture temperature is 20 +/-1 ℃;
e, rooting culture: d, differentiating and growing the protocorm in the step d into a tissue culture seedling with 4-6 formed leaves and a height of 1-2cm, cutting off the root system of the tissue culture seedling, shallowly inserting the tissue culture seedling into a rooting culture medium with the depth of 1-2mm, transferring the tissue culture seedling into the rooting culture medium, firstly carrying out dark culture for 10-15d, and then carrying out 1500-year illumination 2000Lx illumination intensity, 12h/d illumination time and 120-year illumination 130d illumination time; the culture temperature is 20 +/-1 ℃;
f, hardening seedling cultivation: when the seedlings grow to have 6-8 leaves, 4-5cm high and 4-8 roots in the step e, moving to the natural light for hardening for 10-15d, opening the bottle cap, continuing hardening for 4-6d, taking out the seedlings from the tissue culture bottle, completely cleaning the culture medium on the plants, and carrying out shallow planting in rotten pine barks for wet cultivation after air drying; spraying water on the leaves of the tissue culture seedlings on the same day after planting, and not spraying water for 4-5 days later; then 1-2 times of water is sprayed every 1d within 15-20d, then 1-2 times of water is sprayed, the culture time is 80-90d, and after the new roots of the tissue culture seedlings grow out, the tissue culture seedlings are transferred to a greenhouse for conventional management.
Further, the seed broadcasting mode in the step c is as follows: and after the sterile capsule is cut in a sterile culture dish, sucking sterile water into the culture dish by using a sterile sucker to uniformly mix the seeds in the sterile water, and then sucking the sterile water mixed with the seeds to uniformly spread on an induction culture medium.
Further, the composition of the induction medium in step c is: MS basal medium, sucrose 20g/L, agar 5.5g/L, banana puree 60g/L, and pH 5.3-5.4.
Further, in the step c, the culture temperature is 20 +/-1 ℃, the illumination intensity is 1000-1200Lx, the illumination time is 12h/d, and the culture time is 60-68 d.
Further, the proliferation and differentiation medium in step d consists of: 1/2MS + 6-BA 0.1mg/L + IBA 0.2-0.4 mg/L + PVP 1-4g/L + sucrose 20g/L + agar 5.5g/L + coconut milk 40ml/L, pH 5.3-5.4.
Further, 15-20 protocorms are inoculated in each bottle during the inoculation in the step d.
Further, the rooting medium in step e consists of: 1/2MS + 6-BA 0.1mg/L + IBA 0.5-0.7 mg/L + PVP 1-4g/L + sucrose 20g/L + agar 5.5g/L + banana mud 60g/L, pH 5.3-5.4.
Further, in the transplanting culture in the step f, a culture substrate is pine bark with the thickness of 10-15cm and the diameter of 3-5mm, and the humidity of the substrate is kept between 85% and 90%.
Compared with the prior art, the invention has the beneficial effects that:
the invention discloses a method for obtaining a zephyr omeiensis capsule and breeding seeds of the zephyr omeiensis capsule to obtain seedlings. In order to fully protect the coeruleus anthriscus plant resources and solve the problems of few wild plants, extremely low natural germination rate, extremely rare wild capsules, lack of a breeding method and the like of the current coeruleus anthriscus, the invention selects plants with different introduction points, strong adaptability and vigorous growth after the near-field protection and the growth adaptability research for many years, and utilizes the plants from different populations to carry out artificial pollination to obtain healthy capsules; the seeds are utilized to carry out breeding research in the maturation stage of capsules, and the problems of serious browning and the like are overcome through the processes of aseptic seeding germination, proliferation differentiation, strong seedling rooting, seedling hardening cultivation and the like, so that high-quality healthy seedlings of the zephyranthes omeiensis are obtained. The invention adopts a mode of artificial pollination among plants with certain genetic distance to obtain capsules, and the maturing rate is 100%; a rapid propagation system of the zephyranthes omeiensis is established, the propagation coefficient of the system reaches more than 7 times, the rooting and seedling rate reaches more than 90%, and a scientific and technical basis is laid for protecting wild resources of the species; the obtained excellent coerulea sylvestris seedling provides resource foundation and technical support for sustainable utilization of the coerulea sylvestris seedling, actively promotes the industrial development of plant resources, and has extremely important practical significance and economic benefit. The method overcomes the difficulty that capsules cannot be collected because the capsules are rare under natural conditions, breaks through the limitations of high requirements on natural germination conditions and extremely low germination rate of the coerulea anthriscus seed, and opens up a way for rapid propagation of coerulea anthriscus. According to the invention, firstly, artificial pollination is carried out on plants with relatively long genetic distance to obtain capsules, and then, the cultivation is carried out at each stage of seed cultivation by selecting conditions such as proper temperature and illumination, so that the seed germination rate is greatly improved, and the tissue culture seedling grows most rapidly; meanwhile, the browning phenomenon in the rapid propagation test process of the zephyranthes omeiensis seeds is particularly obvious, the culture mode of dark culture and light culture is adopted in the proliferation differentiation culture and rooting culture, and a proper amount of adsorbent is added, so that the browning phenomenon of tissue culture seedlings is greatly reduced. According to the method, the coerulea anthriscus sylvestris capsules are obtained, the seeds are utilized to carry out propagation research, a rapid propagation technical system is established, a large amount of artificial propagation of the coerulea anthriscus sylvestris is realized, a resource foundation is laid for the population recovery of the extremely small population, the specific rare endangered plant of the coerulea anthriscus, and a foundation is laid for the future industrial production of the plant.
Detailed Description
The present invention will be further described with reference to the following examples, which are intended to illustrate only some, but not all, of the embodiments of the present invention. All other embodiments that can be obtained by a person skilled in the art based on the embodiments of the present invention without any creative effort belong to the protection scope of the present invention.
Example 1
a obtaining of capsule: after the field protection and the growth adaptability research of many years, selecting plants with strong adaptability and vigorous growth, selecting plants from different populations in the flowering period, carrying out artificial pollination, wherein the maturing rate is 100%, obtaining healthy capsules, marking the hybridization combination and pollination time during pollination, and picking capsules when 385d after pollination is not cracked;
b, processing capsules: picking uncracked capsules of zephyranthes omeiensis, washing the capsules for 8min in tap water, putting the capsules on an ultra-clean workbench for later use, sequentially treating the capsules for 35s with alcohol with the mass concentration of 75% and sterilizing the capsules for 18min with mercuric chloride solution with the mass concentration of 0.1% under the aseptic condition, washing the capsules for 6 times with sterile water respectively, and then sucking the water on the surfaces of the capsules by sterile filter paper; after a sterile capsule is cut off in a sterile culture dish, a sterile straw is used for sucking sterile water into the culture dish, so that seeds are uniformly mixed in the sterile water;
c, induction culture: sucking sterile water mixed with seeds, and uniformly scattering the sterile water on an induction culture medium, wherein the seeds are brown powder and mature; the contamination rate of the culture bottle is 0 percent, and the contamination phenomenon does not occur; the induction culture medium takes MS as a basic culture medium, 20g of sucrose, 5.5g of agar and 60g of banana puree are added into each liter, the pH value is between 5.3 and 5.4, the culture temperature is 20 +/-1 ℃, the illumination time is 12h/d, and the illumination intensity is between 1000-; inducing protocorm after culturing for 27 days, and selecting the protocorm when the leaf primordium can be seen after culturing for 60 days, and inoculating the protocorm to a proliferation and differentiation culture medium; the seed inductivity is more than 90 percent;
d, proliferation and differentiation culture: the proliferation and differentiation culture medium takes 1/2MS as a basic culture medium, 0.1mg of 6-BA, 0.2mg of IBA, 2g of PVP, 20g of cane sugar, 5.5g of agar and 40ml of coconut juice are added into each liter, the pH value is between 5.3 and 5.4, and the culture temperature is 20 +/-1 ℃; after the transfer, dark culture is carried out for 10d, then the illumination time is 12h/d, the illumination intensity is 1500-; the browning condition is obviously improved, the browning rate is less than 10%, the originally formed transfer protocorm can grow into a robust tissue culture seedling in 90 days, the tissue culture seedling with at least 4 new leaves and 1-2cm high is taken out, and the old root is cut off and transferred to a rooting culture medium;
e, rooting culture: the rooting culture medium takes 1/2MS as a basic culture medium, 0.1mg of 6-BA, 0.5mg of IBA, 2g of PVP, 20g of cane sugar, 5.5g of agar and 60g of banana puree are added into each liter, the pH value is 5.3-5.4, and the culture temperature is 20 +/-1 ℃; after the transfer, dark culture is carried out for 10 days, then the illumination time is 12h/d, the illumination intensity is 1500-2000Lx, the transferred tissue culture seedling is shallowly inserted into a rooting culture medium, and the depth is 1-2 mm; after 120 days of culture, the new root generation rate reaches 100%, the new roots are strong and healthy, and the browning rate is less than 5%;
f, hardening seedling cultivation: when the tissue culture seedling grows to have 6 or more leaves, 4-5cm high and 4-8 root systems, moving the tissue culture seedling to natural light for hardening the seedling for 10 days, opening a bottle cap, and continuing hardening the seedling for 4 days; carefully taking out the tissue culture seedlings without contamination after hardening the tissue culture seedlings, washing the culture medium at the root with tap water, airing, planting the tissue culture seedlings into wet and thoroughly decomposed pine barks with the diameter of 3-5mm, and not spraying water after 4 days of transplantation; then spraying water every 1d for 2 times in 20d, and then spraying water for 1-2d, wherein the culture time is 90 d; the new root generation rate of the tissue culture seedlings is more than 90 percent, and then the tissue culture seedlings are transferred to a greenhouse for conventional management.
Example 2
a obtaining of capsule: after the ground protection and the growth adaptability research for many years, selecting plants with strong adaptability and vigorous growth, selecting plants from different populations in the flowering period, carrying out artificial pollination, and obtaining healthy capsules with the maturing rate of 100%; and (3) marking hybridization combination and pollination time during pollination, and picking capsules 520d after pollination when the capsules are not cracked.
b, processing capsules: picking uncracked capsules of zephyranthes omeiensis, washing with tap water for 8min, and placing on an ultra-clean workbench for later use. Sequentially treating with 75% alcohol and 0.1% mercuric chloride solution under aseptic condition for 35s, sterilizing for 18min, washing with aseptic water for 6 times, drying with aseptic filter paper, and cutting with a sterilizing knife;
c, induction culture: inoculating the seeds onto an induction culture medium, wherein the seeds are brown powder and are completely mature, the bacteria contamination rate of a culture bottle is 0%, and the bacteria contamination phenomenon does not occur; the induction culture medium takes MS as a basic culture medium, 20g of sucrose, 5.5g of agar and 60g of banana puree are added into each liter, the pH value is between 5.3 and 5.4, the culture temperature is 20 +/-1 ℃, the illumination time is 12h/d, and the illumination intensity is between 1000-1200 Lx; inducing protocorm after culturing for 31d, culturing for 68d until leaf primordium can be seen, selecting protocorm group, and inoculating to proliferation and differentiation culture medium; the seed inductivity is more than 90 percent;
d, proliferation and differentiation culture: the proliferation and differentiation culture medium takes 1/2MS as a basic culture medium, 0.1mg of 6-BA, 0.4mg of IBA, 4g of PVP, 20g of cane sugar, 5.5g of agar and 40ml of coconut juice are added into each liter, the pH is between 5.3 and 5.4, and the culture temperature is 20 +/-1 ℃; dark culture is carried out for 14d after the transfer, then the illumination time is 12h/d, and the illumination intensity is between 1500-2000 Lx; when in inoculation, 15 protocorms are inoculated in each bottle and cultured for 38 days, the protocorms are proliferated, the proliferation coefficient is 5, and about 50 percent of tissue culture seedlings can be browned; the originally formed transfer protocorm can grow into a robust tissue culture seedling at 100 days. Taking out the tissue culture seedling with more than 4 new leaves and height of 1-2cm, cutting off old root, and transferring to rooting culture medium.
e, rooting culture: the rooting culture medium takes 1/2MS as a basic culture medium, 0.1mg of 6-BA, 0.7mg of IBA, 2g of PVP, 20g of cane sugar, 5.5g of agar and 60g of banana puree are added into each liter, the pH value is between 5.3 and 5.4, and the culture temperature is 20 +/-1 ℃; after the transfer, dark culture is carried out for 14d, then the illumination time is 12h/d, and the illumination intensity is between 1500-2000 Lx; the transferred tissue culture seedling is shallowly inserted into a rooting culture medium, and the depth is 1-2 mm; after 120 days of culture, the generation rate of new roots reaches 100%, but the growth condition of the roots is general; the browning rate is enough and is less than 10 percent;
f, hardening seedling cultivation: when the tissue culture seedling grows to have 6 or more leaves, 4-5cm high and 4-8 root systems, moving to the natural light to train the seedling for 14d, opening the bottle cap, and continuing to train the seedling for 6 d; about 10% of the tissue culture seedlings are infected with bacteria; carefully taking out the uninfected tissue culture seedlings, washing the culture medium on the roots with tap water, airing, and planting the seedlings into wet and thoroughly decomposed pine barks with the diameter of 3-5 mm. 5d after transplanting, no water is sprayed; then every 1d of water is sprinkled 1-2 times in 20d, and then every 1-2d of water is sprinkled. The culture time is 90 d; the new root generation rate of the tissue culture seedlings is more than 90 percent, and the tissue culture seedlings are transferred to a greenhouse for conventional management.
Example 3
a obtaining of capsule: after the ground protection and the growth adaptability research of many years, selecting plants with strong adaptability and vigorous growth, selecting plants from different populations in the flowering period, carrying out artificial pollination, wherein the maturing rate is 100%, obtaining healthy capsules, marking the hybridization combination and pollination time during pollination, and picking capsules 500d after pollination when not cracking;
b, processing capsules: picking uncracked capsules of zephyranthes omeiensis, washing the capsules for 5min in tap water, putting the capsules on an ultra-clean workbench for standby, sequentially treating the capsules for 30s with alcohol with the mass concentration of 75% and sterilizing the capsules for 15min with mercuric chloride solution with the mass concentration of 0.1% under the aseptic condition, washing the capsules for 4 times with sterile water respectively, and then sucking the water on the surfaces of the capsules with sterile filter paper; after a sterile capsule is cut off in a sterile culture dish, a sterile straw is used for sucking sterile water into the culture dish, so that seeds are uniformly mixed in the sterile water;
c, induction culture: sucking sterile water mixed with seeds, and uniformly scattering the sterile water on an induction culture medium, wherein the seeds are brown powder and mature; the contamination rate of the culture bottle is 0 percent, and the contamination phenomenon does not occur; the induction culture medium takes MS as a basic culture medium, 20g of sucrose, 5.5g of agar and 60g of banana puree are added into each liter, the pH value is between 5.3 and 5.4, the culture temperature is 20 +/-1 ℃, the illumination time is 12h/d, and the illumination intensity is between 1000-; culturing for 28 days to induce protocorm, culturing for 65 days until leaf primordium is visible, selecting protocorm, and inoculating to proliferation and differentiation culture medium; the seed inductivity is more than 90 percent;
d, proliferation and differentiation culture: the proliferation and differentiation culture medium takes 1/2MS as a basic culture medium, 0.1mg of 6-BA, 0.4mg of IBA, 1g of PVP, 20g of cane sugar, 5.5g of agar and 40ml of coconut juice are added into each liter, the pH value is between 5.3 and 5.4, and the culture temperature is 20 +/-1 ℃; after the transfer, dark culture is carried out for 13d, then the illumination time is 12h/d, the illumination intensity is 1500-; the browning condition is obviously improved, the browning rate is less than 30%, the originally formed transfer protocorm can grow into a robust tissue culture seedling in 94 days, the tissue culture seedling with at least 4 new leaves and 1-2cm high is taken out, and the old root is cut off and transferred to a rooting culture medium;
e, rooting culture: the rooting culture medium takes 1/2MS as a basic culture medium, 0.1mg of 6-BA, 0.6mg of IBA, 4g of PVP, 20g of cane sugar, 5.5g of agar and 60g of banana puree are added into each liter, the pH value is 5.3-5.4, and the culture temperature is 20 +/-1 ℃; after the transfer, dark culture is carried out for 15d, then the illumination time is 12h/d, the illumination intensity is 1500-2000Lx, the transferred tissue culture seedling is inserted into a rooting culture medium in a shallow depth of 1-2 mm; after 120 days of culture, the new root generation rate reaches 100%, the new roots are strong and healthy, and the browning rate is less than 35%;
f, hardening seedling cultivation: when the tissue culture seedling grows to have 6 or more leaves, 4-5cm high and 4-8 root systems, moving the tissue culture seedling to natural light for hardening the seedling for 13d, opening a bottle cap, and continuing hardening the seedling for 5 d; carefully taking out the tissue culture seedlings without contamination after hardening the tissue culture seedlings, washing the culture medium at the root with tap water, airing, planting the tissue culture seedlings into wet and thoroughly decomposed pine barks with the diameter of 3-5mm, and not spraying water after 4 days of transplantation; then spraying water every 1d for 2 times in 20d, and then spraying water for 1-2d, wherein the culture time is 90 d; the new root generation rate of the tissue culture seedlings is more than 90 percent, and then the tissue culture seedlings are transferred to a greenhouse for conventional management.
Example 4
a obtaining of capsule: after the ground protection and the growth adaptability research of many years, selecting plants with strong adaptability and vigorous growth, selecting plants from different populations in the flowering period, carrying out artificial pollination, wherein the maturing rate is 100%, obtaining healthy capsules, marking the hybridization combination and pollination time during pollination, and picking capsules when the capsules are not cracked at 380 days after pollination;
b, processing capsules: picking uncracked capsules of zephyranthes omeiensis, washing the capsules for 10min in tap water, putting the capsules on an ultra-clean workbench for later use, sequentially treating the capsules for 32s with alcohol with the mass concentration of 75% and sterilizing the capsules for 16min with mercuric chloride solution with the mass concentration of 0.1% under the aseptic condition, washing the capsules for 5 times with sterile water respectively, and then sucking the water on the surfaces of the capsules by sterile filter paper; after a sterile capsule is cut off in a sterile culture dish, a sterile straw is used for sucking sterile water into the culture dish, so that seeds are uniformly mixed in the sterile water;
c, induction culture: sucking sterile water mixed with seeds, and uniformly scattering the sterile water on an induction culture medium, wherein the seeds are brown powder and mature; the contamination rate of the culture bottle is 0 percent, and the contamination phenomenon does not occur; the induction culture medium takes MS as a basic culture medium, 20g of sucrose, 5.5g of agar and 60g of banana puree are added into each liter, the pH value is between 5.3 and 5.4, the culture temperature is 20 +/-1 ℃, the illumination time is 12h/d, and the illumination intensity is between 1000-; inducing protocorm after culturing for 29d, and selecting the protocorm when the leaf primordium can be seen after culturing for 66d, and inoculating the protocorm to a proliferation and differentiation culture medium; the seed inductivity is more than 90 percent;
d, proliferation and differentiation culture: the proliferation and differentiation culture medium takes 1/2MS as a basic culture medium, 0.1mg of 6-BA, 0.2mg of IBA, 3g of PVP, 20g of cane sugar, 5.5g of agar and 40ml of coconut juice are added into each liter, the pH value is between 5.3 and 5.4, and the culture temperature is 20 +/-1 ℃; after the transfer, dark culture is carried out for 15d, then the illumination time is 12h/d, the illumination intensity is 1500-; the browning condition is obviously improved, the browning rate is less than 35%, the originally formed transfer protocorm can grow into a robust tissue culture seedling in 92 days, the tissue culture seedling with at least 4 new leaves and 1-2cm high is taken out, and the old root is cut off and transferred to a rooting culture medium;
e, rooting culture: the rooting culture medium takes 1/2MS as a basic culture medium, 0.1mg of 6-BA, 0.7mg of IBA, 1g of PVP, 20g of cane sugar, 5.5g of agar and 60g of banana puree are added into each liter, the pH value is between 5.3 and 5.4, and the culture temperature is 20 +/-1 ℃; after the transfer, dark culture is carried out for 13d, then the illumination time is 12h/d, the illumination intensity is 1500-2000Lx, the transferred tissue culture seedling is shallowly inserted into a rooting culture medium, and the depth is 1-2 mm; after 120 days of culture, the new root generation rate reaches 100%, the new roots are strong and healthy, and the browning rate is less than 25%;
f, hardening seedling cultivation: when the tissue culture seedling grows to have 6 or more leaves, 4-5cm high and 4-8 root systems, moving the tissue culture seedling to natural light for hardening the seedling for 12d, opening a bottle cap, and continuing hardening the seedling for 4 d; carefully taking out the tissue culture seedlings without contamination after hardening the tissue culture seedlings, washing the culture medium at the root with tap water, airing, planting the tissue culture seedlings into wet and thoroughly decomposed pine barks with the diameter of 3-5mm, and not spraying water after 4 days of transplantation; then spraying water every 1d for 2 times in 20d, and then spraying water for 1-2d, wherein the culture time is 90 d; the new root generation rate of the tissue culture seedlings is more than 90 percent, and then the tissue culture seedlings are transferred to a greenhouse for conventional management.
Example 5
a obtaining of capsule: after the field protection and the growth adaptability research of many years, selecting plants with strong adaptability and vigorous growth, selecting plants from different populations in the flowering period, carrying out artificial pollination, wherein the maturing rate is 100%, obtaining healthy capsules, marking the hybridization combination and pollination time during pollination, and picking capsules when the capsules are not cracked at 450d after pollination;
b, processing capsules: picking uncracked capsules of zephyranthes omeiensis, washing the capsules for 7min in tap water, putting the capsules on an ultra-clean workbench for later use, treating the capsules for 33s with alcohol with the mass concentration of 75% and sterilizing the capsules for 17min with mercuric chloride solution with the mass concentration of 0.1% under the aseptic condition, washing the capsules for 6 times by using sterile water respectively, and then drying the water on the surfaces of the capsules by using sterile filter paper; after a sterile capsule is cut off in a sterile culture dish, a sterile straw is used for sucking sterile water into the culture dish, so that seeds are uniformly mixed in the sterile water;
c, induction culture: sucking sterile water mixed with seeds, and uniformly scattering the sterile water on an induction culture medium, wherein the seeds are brown powder and mature; the contamination rate of the culture bottle is 0 percent, and the contamination phenomenon does not occur; the induction culture medium takes MS as a basic culture medium, 20g of sucrose, 5.5g of agar and 60g of banana puree are added into each liter, the pH value is between 5.3 and 5.4, the culture temperature is 20 +/-1 ℃, the illumination time is 12h/d, and the illumination intensity is between 1000-; inducing protocorm after culturing for 30d, and selecting protocorm when leaf primordium can be seen after culturing for 67d, and inoculating to proliferation and differentiation culture medium; the seed inductivity is more than 90 percent;
d, proliferation and differentiation culture: the proliferation and differentiation culture medium takes 1/2MS as a basic culture medium, 0.1mg of 6-BA, 0.3mg of IBA, 2g of PVP, 20g of cane sugar, 5.5g of agar and 40ml of coconut juice are added into each liter, the pH value is between 5.3 and 5.4, and the culture temperature is 20 +/-1 ℃; after the transfer, dark culture is carried out for 12d, then the illumination time is 12h/d, the illumination intensity is 1500-; the browning condition is obviously improved, the browning rate is less than 10%, the originally formed transfer protocorm can grow into a robust tissue culture seedling in 96 days, the tissue culture seedling with at least 4 new leaves and 1-2cm high is taken out, and the old root is cut off and transferred to a rooting culture medium;
e, rooting culture: the rooting culture medium takes 1/2MS as a basic culture medium, 0.1mg of 6-BA, 0.5mg of IBA, 3g of PVP, 20g of cane sugar, 5.5g of agar and 60g of banana puree are added into each liter, the pH value is 5.3-5.4, and the culture temperature is 20 +/-1 ℃; after the transfer, dark culture is carried out for 12d, then the illumination time is 12h/d, the illumination intensity is 1500-2000Lx, the transferred tissue culture seedling is inserted into a rooting culture medium in a shallow depth of 1-2 mm; after 120 days of culture, the new root generation rate reaches 100%, the new roots are strong and healthy, and the browning rate is less than 30%;
f, hardening seedling cultivation: when the tissue culture seedling grows to have 6 or more leaves, 4-5cm high and 4-8 root systems, moving the tissue culture seedling to natural light for hardening the seedling for 15d, opening a bottle cap, and continuing hardening the seedling for 4 d; carefully taking out the tissue culture seedlings without contamination after hardening the tissue culture seedlings, washing the culture medium at the root with tap water, airing, planting the tissue culture seedlings into wet and thoroughly decomposed pine barks with the diameter of 3-5mm, and not spraying water after 4 days of transplantation; then spraying water every 1d for 2 times in 20d, and then spraying water for 1-2d, wherein the culture time is 90 d; the new root generation rate of the tissue culture seedlings is more than 90 percent, and then the tissue culture seedlings are transferred to a greenhouse for conventional management.
Comparative example 1
a. After the ground protection and the growth adaptability research for many years, selecting plants with strong adaptability and vigorous growth, selecting plants from different populations in the flowering period, carrying out artificial pollination, and obtaining capsules with the maturing rate of 100%; and (3) marking hybridization combination and pollination time during pollination, and picking capsules at 370d after pollination but not cracking.
b. Picking uncracked capsules of zephyranthes omeiensis, washing with tap water for 5min, and placing on an ultra-clean workbench for later use. Sequentially treating with 75% alcohol for 30s and 0.1% mercuric chloride solution under aseptic condition, sterilizing for 15min, washing with sterile water for 4 times, and drying with sterile filter paper; after a sterile capsule is cut in a sterile culture dish, sterile water is sucked into the culture dish by a sterile sucker, so that the seeds are uniformly mixed in the sterile water, and then the sterile water mixed with the seeds is sucked and uniformly spread on an induction culture medium. At this point the seeds were partially white and partially brown, and not yet fully mature. The contamination rate of the culture bottle is 0 percent, and the contamination phenomenon does not occur.
c. The induction culture medium takes MS as a basic culture medium, 20g of cane sugar, 5.5g of agar and 60g of banana puree are added into each liter, the pH value is between 5.3 and 5.4, the culture temperature is 20 +/-1 ℃, the illumination time is 12h/d, and the illumination intensity is between 1500-2000 Lx. The protocorm can be induced after 37 days of culture. And culturing for 68d until the leaf primordium can be seen, selecting protocorms, and inoculating the protocorms to a proliferation and differentiation culture medium. The seed inductivity is more than 30%.
d. The proliferation and differentiation culture medium takes 1/2MS as a basic culture medium, 0.1mg of 6-BA, 0.2mg of IBA, 20g of cane sugar, 5.5g of agar, 60g of banana puree and 2g of active carbon are added into each liter, the pH value is between 5.3 and 5.4, the culture temperature is 20 +/-1 ℃, the illumination time is 12h/d, and the illumination intensity is between 1500 ion sources and 2000 Lx. When inoculating, 15 protocorms are inoculated in each bottle for culture. The browning is very severe, and the white roots gradually blacken and then rot.
Comparative example 2
a. After the ground protection and the growth adaptability research for many years, selecting plants with strong adaptability and vigorous growth, selecting plants from different populations in the flowering period, carrying out artificial pollination, and obtaining capsules with the maturing rate of 100%; and (3) marking hybridization combination and pollination time during pollination, and picking capsules at 370d after pollination but not cracking.
b. Picking uncracked capsules of zephyranthes omeiensis, washing with tap water for 5min, and placing on an ultra-clean workbench for later use. Sequentially treating with 75% alcohol for 30s and 0.1% mercuric chloride solution under aseptic condition, sterilizing for 15min, washing with sterile water for 4 times, and drying with sterile filter paper; after a sterile capsule is cut in a sterile culture dish, sterile water is sucked into the culture dish by a sterile sucker, so that the seeds are uniformly mixed in the sterile water, and then the sterile water mixed with the seeds is sucked and uniformly spread on an induction culture medium. At this point the seeds were partially white and partially brown, and not yet fully mature. The contamination rate of the culture bottle is 0 percent, and the contamination phenomenon does not occur.
c. The induction culture medium takes MS as a basic culture medium, 20g of cane sugar, 5.5g of agar and 60g of banana puree are added into each liter, the pH value is between 5.3 and 5.4, the culture temperature is 20 +/-1 ℃, the illumination time is 12h/d, and the illumination intensity is between 1500-; the protocorm can be induced after being cultured for 37 days; and culturing for 68d until the leaf primordium can be seen, selecting protocorms, and inoculating the protocorms to a proliferation and differentiation culture medium. The seed inductivity is more than 30 percent;
d. the proliferation and differentiation culture medium takes 1/2MS as a basic culture medium, 0.1mg of 6-BA, 0.2mg of IBA, 2g of PVP, 20g of cane sugar, 5.5g of agar and 40ml of coconut juice are added into each liter, the pH value is between 5.3 and 5.4, and the culture temperature is 20 +/-1 ℃; the illumination time is 12h/d, and the illumination intensity is between 1500-2000 Lx; inoculating 15 protocorms in each bottle during inoculation and culturing; about 80% of tissue culture seedlings will be browned.
As is clear from comparative example 1, the seed induction rate was extremely low in late-stage induction culture when the capsule was picked up without being completely matured. In comparative example 1, the proliferation and differentiation culture was performed by full light and activated carbon in the culture medium, and from the culture results, the browning was very severe, and the white roots were gradually blackened, so the PVP of the present application was greatly superior to activated carbon in the effect of inhibiting browning. As can be seen from example 1 and comparative example 2, the browning phenomenon can be effectively suppressed by dark culture followed by light culture in the step of proliferation and differentiation culture; as can be seen from comparative example 2 and example 2, the proliferation and differentiation culture of the present application adopts a culture mode of first dark culture and then light culture, and a proper amount of proper adsorbent is added, so that the browning phenomenon of the tissue culture seedling is greatly reduced. As can be seen from example 2, the present invention also adopts a cultivation method of first dark cultivation and then light cultivation in the rooting cultivation, and adds a proper amount of adsorbent, thereby further reducing the browning phenomenon of the tissue culture seedling.
The above disclosure is only for the purpose of illustrating the preferred embodiments of the present invention, and it is therefore to be understood that the invention is not limited by the scope of the appended claims.
Claims (5)
1. The rapid propagation method of the Emei schoenleinii is characterized in that: the method comprises the following steps:
a obtaining of capsule: selecting plants with strong adaptability, vigorous growth and capability of smoothly completing a life cycle, and performing artificial pollination on parents for pollination by adopting plants from different populations in a flowering period to obtain capsules, wherein the capsules are picked when the capsules are not cracked after 380 plus 520 days after pollination;
b, processing capsules: washing picked zephyranthes omeiensis capsules under tap water for 5-10 min, and then sucking water by using filter paper; then, respectively treating the raw materials with 75% alcohol by mass for 30-35 s and 0.1% mercuric chloride solution by mass for sterilization for 15-18 min, respectively washing the raw materials with sterile water for 4-6 times, and finally, absorbing the water on the surface with sterile filter paper; shearing capsule under aseptic condition to obtain seed;
c, induction culture: b, uniformly broadcasting the seeds obtained in the step b on an induction culture medium to induce protocorms; the composition of the induction medium is as follows: MS basal medium, sucrose 20g/L, agar 5.5g/L, banana puree 60g/L, and pH 5.3-5.4;
d, proliferation and differentiation culture: c, inoculating the protocorm with the obviously visible green leaf primordium in the step c to a proliferation and differentiation culture medium for proliferation and differentiation culture; in the proliferation and differentiation culture, dark culture for 10-15 days is firstly carried out; then the illumination intensity is 1500-2000Lx, the illumination time is 12h/d, the culture time is 90-100d, and the culture temperature is 20 +/-1 ℃; the proliferation and differentiation culture medium comprises the following components: 1/2MS + 6-BA 0.1mg/L + IBA 0.2-0.4 mg/L + PVP 1-4g/L + sucrose 20g/L + agar 5.5g/L + coconut milk 40ml/L, pH 5.3-5.4;
e, rooting culture: d, differentiating and growing the protocorm in the step d into a tissue culture seedling with 4-6 formed leaves and a height of 1-2cm, cutting off the root system of the tissue culture seedling, shallowly inserting the tissue culture seedling into a rooting culture medium with the depth of 1-2mm, transferring the tissue culture seedling into the rooting culture medium, firstly carrying out dark culture for 10-15d, and then carrying out 1500-year illumination 2000Lx illumination intensity, 12h/d illumination time and 120-year illumination 130d illumination time; the culture temperature is 20 +/-1 ℃; the rooting medium comprises the following components: 1/2MS + 6-BA 0.1mg/L + IBA 0.5-0.7 mg/L + PVP 1-4g/L + sucrose 20g/L + agar 5.5g/L + banana mud 60g/L, pH 5.3-5.4;
f, hardening seedling cultivation: when the seedlings grow to have 6-8 leaves, 4-5cm high and 4-8 roots in the step e, moving to the natural light for hardening for 10-15d, opening the bottle cap, continuing hardening for 4-6d, taking out the seedlings from the tissue culture bottle, completely cleaning the culture medium on the plants, and carrying out shallow planting in rotten pine barks for wet cultivation after air drying; spraying water on the leaves of the tissue culture seedlings on the same day after planting, and not spraying water for 4-5 days later; then 1-2 times of water is sprayed every 1d within 15-20d, then 1-2 times of water is sprayed, the culture time is 80-90d, and after the new roots of the tissue culture seedlings grow out, the tissue culture seedlings are transferred to a greenhouse for conventional management.
2. The rapid propagation method of Emei gomphor rutilus according to claim 1, wherein: the seed broadcasting mode in the step c is as follows: and after the sterile capsule is cut in a sterile culture dish, sucking sterile water into the culture dish by using a sterile sucker to uniformly mix the seeds in the sterile water, and then sucking the sterile water mixed with the seeds to uniformly spread on an induction culture medium.
3. The rapid propagation method of Emei gomphor rutilus according to claim 1, wherein: in the step c, the culture temperature is 20 +/-1 ℃, the illumination intensity is 1000-1200Lx, the illumination time is 12h/d, and the culture time is 60-68 d.
4. The rapid propagation method of Emei gomphor rutilus according to claim 1, wherein: and d, inoculating 15-20 protocorms in each bottle during the inoculation in the step d.
5. The rapid propagation method of Emei gomphor rutilus according to claim 1, wherein: in the transplanting culture in the step f, a culture substrate is pine bark with the thickness of 10-15cm and the diameter of 3-5mm, and the humidity of the substrate is kept between 85% and 90%.
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