CN115530075A - Artificial efficient propagation method of dendrobium nobile - Google Patents
Artificial efficient propagation method of dendrobium nobile Download PDFInfo
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- A—HUMAN NECESSITIES
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Abstract
The invention discloses an artificial efficient propagation method of dendrobium nobile, and relates to the technical field of biotechnology. The invention comprises the following steps: (1) obtaining stem segments as explants; (2) regenerating and culturing the explant in the A culture medium; (3) inducing the shoot clusters and protocorms of the basal stems; (4) rejuvenating and rooting culture; and (5) hardening and transplanting the seedlings. The invention optimizes and adjusts the artificial rapid propagation of the dendrobium primordium, the regeneration of stem segments, the induction of clustered buds of basal stems, the generation, the proliferation and the development of protocorms are finished in one culture medium, the rejuvenation and the rooting are also finished in the same culture medium, the whole culture process can be finished by only 1 culture medium, the artificial rapid propagation process is simplified, the propagation efficiency is high, the cost is low, the time is short, the survival rate of the test-tube seedling hardening is improved, and the seedling culture period is greatly shortened.
Description
Technical Field
The invention relates to the technical field of biotechnology, and particularly relates to an artificial efficient propagation method of dendrobium nobile.
Background
The dendrobium is a traditional Chinese herbal medicine and has extremely high medicinal value; the medicine part is fresh or dry stem of Dendrobium (Dendrobium); in addition to Chinese countries, india, australia and Nepal are also used as traditional medicines. The dendrobe plants are various in types, the effective ingredients of the dendrobe plants are different, and the main active ingredients of the dendrobe plants are compounds such as dendrobe polysaccharide, sesquiterpene, flavonoid, fluorenone, coumarin, bibenzyl, phenanthrene, lignan, steroid, phenol, alkaloid and the like. According to the record of traditional Chinese medicine, dendrobe has the functions of benefiting stomach and promoting fluid production, nourishing yin and clearing heat, and is commonly used for treating diseases such as fever body fluid injury, dry mouth, polydipsia, flaccidity of bones and muscles and the like. Along with the intensive research of modern medicine, the dendrobium also has the effects of enhancing the immunity of the immune system, resisting aging, expanding blood vessels, treating eye diseases such as cataract and the like; in addition, it also has certain therapeutic effect on diabetes and dermatosis. The dendrobium plants have strict requirements on the growth environment, mainly grow on cliffs, rock seams or trunks of high mountains, and are obviously influenced by microclimate environments (temperature, humidity and the like); most fruits are capsules, contain extremely fine seeds, mostly have no endosperm, and cannot provide nutrient substances for the growth and development of embryos. Researches indicate that the germination of dendrobium plant seeds is closely related to certain fungi, and the dendrobium plant seeds need to be symbiotic with the fungi in the later period to normally grow; the growth and development are slow in the natural environment, and the growth period is long (usually 2-3 years); the fruit setting rate is only 17.3 percent through sexual propagation, the seed germination rate is as low as 2 to 5 percent, the yield is low, the period is long, a large amount of labor cost is required, and the economic effect is not ideal. In addition, with serious damage to ecological environment and severe change of global climate, the dendrobium plant lacks suitable habitat and the population quantity is reduced. Since the 50 s of the last century, people began to engage in large scale predatory mining of dendrobium plants driven by interest. At present, wild germplasm resources of dendrobium are greatly reduced, and various kinds of wild germplasm resources are endangered to be extinct, and are listed in the famous rare or endangered protective plant directory of China.
Among the huge dendrobium groups, there are some kinds of dendrobium officinale (d.officinale Kimura et Migo), dendrobium nobile (d.nobile lindl.), dendrobium chrysotoxum (d.chrysotoxum lindl.), dendrobium fimbriatum (d.fimbriatum hook), dendrobium huoshanense (d.huoshanense c.z.tang et s.j.cheng), and the like which have very high medicinal value, and also some kinds of dendrobium officinale (d.primula lindl.), dendrobium candidum (d.william lindl.), dendrobium candidum (d.williamsonii Day rchb.f.), dendrobium bibracteatum (d.bracteatum Warner), and the like which have both medicinal and ornamental values and are commonly used as gardening. The primula is a perennial epiphytic herb plant which grows on a trunk in a mountain sparse forest at the altitude of 700-1800m and is fond of warm, humid, semi-yin and semi-yang environments; mainly produced from southeast to southwest of Yunnan province of China, and distributed in other countries such as Nipol, india, thailand, laos, vietnam, etc. The primrose dendrobium is a very special existence in dendrobium, beautiful flowers enable the dendrobium to become a rare species in ornamental plants, compared with other types of dendrobium, the dendrobium has huge volume (25-45 cm higher), stem nodes cluster and droop; fragrant flowers bloom from the leafless stem nodes. Due to beautiful posture, gorgeous flower color, abnormal fragrance and splendid feeling in the open, the dendrobium nobile is widely favored by consumers in the potted flower cultivation market and is an orchid variety with commercial prospect after the Phalaenopsis amabilis (Phalaenopsis ahrodite H.G. Reichenbach) and the Cymbidium faberi x hybridum.
Similar to other dendrobium plants, the propagation method of the primula seedling comprises sexual propagation and asexual propagation. Sexual propagation, i.e. seed propagation, is not commonly used because of low field propagation rate and no endosperm in seeds; the asexual propagation comprises plant division propagation, cutting propagation, tissue culture and the like; cutting propagation is adopted, so that the seed properties are not favorably preserved; the traditional plant division propagation requires a large number of stock plants and extremely low propagation efficiency; tissue culture is an effective method for large-scale propagation and protection of Orchidaceae (Orchidaceae) plants. At present, the main types of dendrobe research are medicinal dendrobe such as dendrobium officinale, dendrobium nobile and dendrobium huoshanense, and the research mainly focuses on artificial breeding, chemical components, clinical application, plant physiology and other aspects. Relatively few research reports of ornamental dendrobium exist, but few researches on artificial breeding of the dendrobium primula are available. In-vitro rapid propagation of dendrobium plants is mainly realized by obtaining protocorms through non-symbiotic germination of seeds and further inducing and differentiating the protocorms into seedlings; although the propagation coefficient is greatly improved by the method, researches find that the clonal variation of somatic cells is easily generated by aseptic seeding to cause certain character separation; under the stimulation of 'dendrobium heat', a plurality of tissue culture units blindly pursue the yield of tissue culture seedlings in order to meet the increased seedling consumption of the market, so that the quality of the seedlings is reduced, and the medicinal and ornamental values of the seedlings are seriously influenced; the stem section of the dendrobium is used as the explant to carry out in vitro rapid propagation, so that the defects can be avoided to a certain extent, and the excellent characters can be stably inherited.
At present, the primrose dendrobium depends on the digging of wild resources; the consequence is that the wild population is reduced sharply and the habitat is fragmented, thereby increasing the probability of genetic drift and inbreeding and being difficult to recover after the wild population is damaged due to the obstacle of sexual reproduction. The plant division propagation period in the artificial cultivation is long, the efficiency is low, the breeding process is slow, and the planting scale of the dendrobium nobile is extremely limited. In order to protect endangered wild orchid germplasm resources and provide resource guarantee for researches on various aspects such as biological diversity, gardening and pharmacology, a novel asexual propagation method which is low in cost, short in time, high in quality and survival rate and capable of fixing excellent characters is urgently needed to be found to expand the propagation quantity of the primula forbesii germchits and carry out industrial production of the high-quality germchits, and the method becomes an inevitable choice for protecting and sustainably utilizing the primula forbesii germplasm resources.
Disclosure of Invention
The invention aims to provide an artificial high-efficiency propagation method of dendrobium nobile, which solves the following technical problems:
the dendrobium primrose wild resources are deficient, the plant division propagation period in artificial cultivation is long, the efficiency is low, the breeding process is slow, and the planting scale is extremely limited.
The purpose of the invention can be realized by the following technical scheme:
an artificial high-efficiency propagation method of dendrobium nobile comprises the following steps: cutting the disinfected and sterilized stem into proper size for regeneration culture; then, inducing the shoot tip with the node to carry out the induction of the shoot cluster buds and the protocorm; the propagation and the development are seedling culture; rejuvenation and rooting culture; hardening and transplanting the seedlings.
As a further scheme of the invention: an artificial high-efficiency propagation method of dendrobium nobile comprises the following steps:
(1) Obtaining an explant: taking stem segments of the dendrobium nobile plants as explants;
(2) Stem section regeneration: sterilizing the stem segments obtained in the step (1), cutting the stem segments by using a scalpel according to the number of nodes, putting the stem segments into a culture medium A, and controlling the illumination intensity, illumination time and temperature to perform stem segment regeneration culture;
(3) Shoot clumpy shoot initiation and protocorm induction: transferring the stem section with the 1-2 node stem tip in the step (2) to a culture medium A, controlling the illumination intensity, illumination time and temperature, and performing the generation of the basal stem cluster buds and the induction of protocorm;
(4) Protocorm proliferation and development culture: dividing the protocorm mass in the step (3) into blocks with the diameter of 1.5-2.0cm, transferring the blocks to a culture medium A, controlling the illumination intensity, illumination time and temperature, and synchronously performing protocorm proliferation, development and regeneration;
(5) Rejuvenation and rooting culture: transferring the cluster buds of the basal stem in the step (3) and the seedlings developed from the protocorm in the step (4) to a culture medium A according to 2-3 plants as a cluster, and controlling the illumination intensity, illumination time and temperature to perform rejuvenation and rooting culture of the test-tube seedlings;
(6) Hardening and transplanting seedlings: taking the rooting bottle in the step (5), placing at room temperature, hardening for 3 days, and then opening the bottle cap to harden for 2 days; and taking out the seedlings, cleaning the residual culture medium on the surfaces of the seedlings, soaking the seedlings in a carbendazim solution, and transplanting the seedlings into the disinfected crushed pine barks for culture to obtain the transplanted seedlings.
As a further scheme of the invention: the stem segment disinfection and sterilization of the step (2) comprises the following steps: cleaning dust on the surface of the stem section by using tap water, soaking by using a washing powder solution, vibrating, stirring, washing by using running water, and placing on a workbench; treating with ethanol solution, sterilizing with mercuric chloride water solution, and washing to obtain sterilized stem material.
As a further scheme of the invention: the stem section disinfection and sterilization of the step (2) specifically comprises the following steps: cleaning 6-8cm stem segments with 5-6 joints in length in the step (1), cleaning dust and impurities on the surface by using tap water, soaking for 10min by using a washing powder solution with the mass concentration of 10%, slightly shaking and stirring, washing for 20min by using running water, sterilizing for 10s by using alcohol with the volume ratio of 70% on a super-clean workbench, sterilizing for 12-15min by using mercury bichloride with the mass percentage of 0.1%, finally washing for 4-5 times by using sterile water, wherein each time is not less than 3min, fully shaking a vessel in the whole sterilization process, and finally shearing to 1-2 joints with the length of 1.5-2.0cm.
As a further scheme of the invention: the culture medium A comprises the following raw materials in parts by weight:
as a further scheme of the invention: the pH value of the A culture medium is 5.4-5.6.
As a further scheme of the invention: the mass concentration of the carbendazim solution in the step (6) is 0.1-0.5%.
As a further scheme of the invention: in the step (6), the size of the crushed pine bark is 1.5 multiplied by 1.5cm, and the disinfection method is boiling in boiling water for 2-3h.
As a further scheme of the invention: in the step (6), the temperature is 23-27 ℃, and the humidity is 60-80%.
The invention has the beneficial effects that:
(1) The stem section of the dendrobium nobile is selected as the explant, so that the stable genetic characteristic of the dendrobium nobile is ensured, and the problem of germplasm decline caused by genetic drift and inbreeding is solved. The invention induces embryonic callus with strong meristematic ability at the wound after cutting the stem tip regenerated from stem segments of the dendrobium nobile, thereby generating protocorm; the protocorm can proliferate and develop with high efficiency; although a large number of seedlings can be obtained through artificial non-symbiotic germination of seeds, the test-tube plantlet loses the reproductive capacity along with the increase of the transfer times and the proliferation algebra and finally falls into a dead cycle; and the seedlings obtained by seed tissue culture have uneven genotypes. On the premise of ensuring high propagation coefficient, the invention creates the most effective propagation mode for keeping the excellent seed properties of the dendrobium nobile: i.e., the protocorm is directly proliferated, thereby overcoming the above-mentioned problems of the aseptic seedlings.
(2) The invention simplifies the artificial rapid propagation process of the dendrobium nobile, and integrally cultures stem regeneration, induction, proliferation and seedling formation of protocorm from the section with the node stem tip, one culture period for proliferation and seedling formation only needs about 60d, the proliferation coefficient reaches more than 20.0, and the culture period is greatly reduced; the regenerated plants after rooting by rejuvenation are robust, have developed root systems and are particularly easy to domesticate and survive. The method can realize the regeneration culture of the test-tube plantlets only within 120 days at most, has low cost, and is easy for standardization and industrial operation, thereby solving the problems of long plant division and propagation period, low efficiency, slow breeding process and extreme limitation on the planting scale of the dendrobium primordium in artificial cultivation.
The invention reduces the times of transfer by the integrated culture of induction, proliferation and seedling formation of the protocorm, and solves the problems that the sterile seedlings of the dendrobium plant lose the reproductive capacity and the genotypes of the sterile seedlings are uneven along with the increase of the times of transfer and proliferation algebra. The invention can keep the same genotype background for all the seedlings, is easy for standardization and industrial operation, effectively improves the quality of the seedlings, can provide uniform and standard excellent seedlings for germplasm resource preservation, and solves the problems of more virus accumulation, unstable characters and limited planting scale of the dendrobium plants caused by long-term artificial plant division propagation.
(3) The invention solves the problems of female parent character separation, long period, weak regeneration seedling win, difficult survival and the like caused by the non-symbiotic germination of the dendrobium plants by utilizing seeds. Because the test-tube plantlet is from a direct organ generation way, the regenerated plant plantlet has strong root and thick root, and the survival rate of hardening and transplanting the plantlet can reach 100 percent.
(4) The invention can realize annual production in the culture room by using the tissue culture technology, thereby saving land resources, improving economic benefits and overcoming the difficulty that the traditional propagation mode can not carry out annual production.
(5) The invention only needs one culture medium to solve the problems of proliferation, rejuvenation and rooting, and is beneficial to the arrangement of production plans.
Drawings
The invention is further described below with reference to the accompanying drawings.
FIG. 1 is a diagram of stem segment regeneration in example 1 of the present invention;
FIG. 2 is a graph showing protocorm proliferation and multiple shoot initiation in example 1 of the present invention;
FIG. 3 is a diagram of hardening-seedling transplantation in example 1 of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
An artificial high-efficiency propagation method for stem segments of dendrobium nobile comprises the following steps:
(1) Obtaining an explant: selecting a dendrobium nobile plant with excellent phenotypic character, and taking a stem section of the dendrobium nobile plant as an explant;
(2) Cleaning the stem segments of 5-6 sections with length of about 6-8cm in step (1), cleaning surface dust and impurities with tap water, soaking in 10% washing powder solution for 10min, slightly shaking and stirring, washing with running water for 20min, sterilizing with 70% alcohol on a clean bench for 10s, and sterilizing with 0.1% mercury mercuric chloride (HgCl) 2 ) Sterilizing for 12min, washing with sterile water for 3min for 4-5 times, and shaking the vessel during the whole sterilizing process. Finally shearing to 1-2 sections with length of 1.5-2.0cm.
(3) Stem regeneration culture: cutting the stem sections sterilized in the step (2) into 1.5cm by a scalpel according to a section, and inoculating the stem sections into the culture medium A for culture.
The culture conditions are as follows: culturing for 10 days under the conditions of illumination intensity of 1800-2500lx, illumination time of 10h/d and temperature controlled at 22 +/-1 ℃, and allowing axillary buds of stem segments to start to germinate and to generate light green bud spots; culturing for 20d, leading axillary buds to germinate in large quantity and further differentiate into leaves, and leading the base parts to have adventitious roots; culturing for 35d, thickening axillary buds, and developing strongly and initially having plant morphology; after the culture is carried out for 60 days, the stem segments grow rapidly, the leaves are full and spread, the main stems are thick, the root systems are developed, and the multiplication coefficient can reach about 8.4 at the moment. In the process, the high-position buds and the basic buds of different stem sections have different numbers, and the number of the regenerated buds can reach 6 at most; the height of each regenerated bud increases as the root system grows further.
(4) Shoot development and induction of protocorm in clumpy buds: and (4) transferring the stem tip with 1-2 nodes in the step (3) into a fresh A culture medium, and performing basal stem cluster bud generation and protocorm induction.
The culture conditions are as follows: culturing under the conditions of illumination intensity of 1800-2500lx, illumination time of 10h/d and temperature controlled at 22 + -1 deg.C for 20d, wherein about 5% of stem tip cuts have embryogenic callus clusters; after being transferred into the culture medium A, the cell mass with loose structure, light color and transparency is changed into a light yellow spherical cell mass with green spots on the top; after 30d, the protocorm is proliferated and simultaneously develops into seedlings, and after 60d, the mixed culture of the protocorm and the seedlings covers the whole culture medium surface; at the moment, 2-3 plants are connected to the culture medium A in a grouped manner, obvious growth can be seen after 30 days, and the seedlings can grow into regenerated seedlings with strong seedlings and thick roots after 60 days; this pathway can occur repeatedly with a proliferation factor of 20.65. While about 95% of the stem tips did not have protocorm, but rather, shoot clusters appeared at the basal nodes, with a proliferation factor of 7.36.
(5) Hardening and transplanting seedlings: and (3) taking a rooting bottle which is obtained in the step (4) and has 4 blades, 3 nodes and 3.0cm root length and is grown into a whole plant, hardening the seedling for 3d at room temperature, opening a bottle cover to harden the seedling for 2d, taking out the seedling from the culture medium, cleaning the residual culture medium, putting the residual culture medium into a carbendazim solution with the mass concentration of 0.1% to sterilize for 3min, then transplanting the seedling into a flowerpot which takes crushed pine bark (1.5 multiplied by 1.5 cm) boiled in boiling water for 2h as a matrix, preserving the temperature (25 ℃), preserving the moisture (70%) and culturing for 60d to obtain a transplanted seedling, wherein the survival rate is 100%.
Wherein, the culture medium A in the step (3) and the step (4) comprises the following raw materials in parts by weight:
the pH of the A medium was 5.6.
Example 2
An artificial high-efficiency propagation method for stem segments of dendrobium nobile comprises the following steps:
(1) Obtaining an explant: selecting primula pseudolaris plants with excellent phenotypic characters, and taking stem segments of the primula pseudolaris plants as explants.
(2) Cleaning the stem segments with length of about 6-8cm with 5-6 segments in step 1 with tap water to remove dust and impurities on the surface, soaking in 10% washing powder solution for 10min, slightly shaking and stirring, washing with running water for 20min, sterilizing with 70% alcohol on a clean bench for 10s, and adding 0.1% mercury mercuric chloride (HgCl) 2 ) Sterilizing for 14min, washing with sterile water for 4-5 times (each time for 3min or more), and shaking the vessel during the whole sterilizing process. Finally shearing to 1-2 sections with length of 1.5-2.0cm.
(3) Stem regeneration culture: cutting the stem sections sterilized in the step (2) into 1.8cm by a scalpel according to a section, and inoculating the stem sections into the culture medium A for culture.
The culture conditions are as follows: culturing for 10 days under the conditions of illumination intensity of 1800-2500lx, illumination time of 10h/d and temperature controlled at 22 +/-1 ℃, and allowing axillary buds of stem segments to start to germinate and to generate light green bud spots; culturing for 20 days, leading axillary buds to germinate in large quantity and further differentiate into leaves, and leading the base parts to have adventitious roots; culturing for 35d, thickening axillary buds, and developing strongly and initially having plant morphology; after the culture is carried out for 60 days, the stem segments grow rapidly, the leaves are full and spread, the main stems are thick, the root systems are developed, and the multiplication coefficient can reach about 8.4 at the moment. In the process, the high-position buds and the basal buds of different stem sections have different generation numbers, and the maximum number of the regenerated buds can reach 5; the height of each regenerated bud increases as the root grows further.
(4) Shoot clumpy shoot initiation and protocorm induction: and (4) transferring the stem tips with the 1-2 nodes in the step (3) to a culture medium A, and performing basal stem clustered shoot generation and protocorm induction. The culture conditions are as follows: culturing under the conditions of illumination intensity of 1800-2500lx, illumination time of 10h/d and temperature controlled at 22 + -1 deg.C for 20d, wherein about 5% of stem tip cuts have embryogenic callus clusters; transferring the cells into a fresh culture medium, and changing the loose-structure, light-color and transparent cell masses into light yellow spherical cell masses with green spots on the tops; after 30d, the protocorm is proliferated and simultaneously develops into seedlings, and after 60d, the mixed culture of the protocorm and the seedlings covers the whole culture medium surface; at the moment, 2-3 plants are connected to the culture medium A in a grouped manner, obvious growth can be seen after 30 days, and the seedlings can grow into regenerated seedlings with strong seedlings and thick roots after 60 days; this pathway occurs repeatedly with a proliferation factor of 20.55. While about 95% of the stem tips did not have protocorm, but rather, shoot clusters appeared at the basal nodes, with a proliferation factor of 7.5.
(5) Hardening and transplanting seedlings: taking a rooting bottle with 5 leaves, 4 nodes and a root length of about 4.0cm, which is grown in the test-tube plantlet in the step 4, putting the rooting bottle at room temperature for hardening for 3d, opening a bottle cover for hardening for 2d, taking out the seedling from a culture medium, cleaning the residual culture medium, putting the residual culture medium into a carbendazim solution with the mass concentration of 0.3% for disinfection for 4min, then transplanting the seedling into a flowerpot which takes crushed pine bark (1.5 multiplied by 1.5 cm) boiled in boiling water for 2h as a matrix, preserving heat (25 ℃) and preserving moisture (60%) for culturing for 60d, and obtaining the transplanted seedling with the survival rate of 100%.
Wherein, the culture medium A in the step (3) and the step (4) comprises the following raw materials in parts by weight:
the pH of the A medium was 5.5.
Example 3
An artificial high-efficiency propagation method for stem segments of dendrobium nobile comprises the following steps:
(1) Obtaining an explant: selecting a dendrobium nobile plant with excellent phenotypic character, and taking a stem section of the dendrobium nobile plant as an explant.
(2) Cleaning 5-6 stem segments with length of about 6-8cm in the step (1) with tap water to remove dust and impurities on the surface, soaking in 10% washing powder solution for 10min, slightly shaking and stirring, washing with running water for 20min, sterilizing with 70% alcohol on a clean bench for 10s, and adding 0.1% mercury mercuric chloride (HgCl) 2 ) Sterilizing for 15min, washing with sterile water for 4-5 times (each time for 3min or more), and shaking the vessel completely. Finally shearing the mixture to 1-2 sections of the belt, wherein the length of the belt is about 1.5-2.0cm.
(3) Stem regeneration culture: cutting the stem sections sterilized in the step (2) into 2.0cm by a scalpel according to one section, and inoculating the cut stem sections into the culture medium A for culture.
The culture conditions are as follows: culturing for 10 days under the conditions of illumination intensity of 1800-2500lx, illumination time of 10h/d and temperature controlled at 22 +/-1 ℃, and allowing axillary buds of stem segments to start to germinate and to generate light green bud spots; culturing for 20d, leading axillary buds to germinate in large quantity and further differentiate into leaves, and leading the base parts to have adventitious roots; culturing for 35d, thickening axillary buds, and developing strongly and initially having plant morphology; after the culture is carried out for 60 days, the stem segments grow rapidly, the leaves are full and spread, the main stems are thick and strong, the root systems are developed, and the multiplication coefficient can reach 8.5 at the moment. In the process, the high-position buds and the basic buds of different stem sections have different numbers, and the number of the regenerated buds can reach 6 at most; the height of each regenerated bud increases as the root system grows further.
(4) Shoot clumpy shoot initiation and protocorm induction: and (4) transferring the stem tip with the 1-2 nodes in the step (3) to a culture medium A, and performing basal stem cluster bud generation and protocorm induction.
The culture conditions are as follows: culturing under the conditions of illumination intensity of 1800-2500lx, illumination time of 10h/d and temperature controlled at 22 + -1 deg.C for 20d, wherein about 5% of stem tip cuts have embryogenic callus clusters; transferring the cells into a fresh culture medium, and changing the loose-structure, light-color and transparent cell masses into light yellow spherical cell masses with green spots on the tops; after 30d, the protocorm is proliferated and simultaneously develops into seedlings, and after 60d, the mixed culture of the protocorm and the seedlings covers the whole culture medium surface; at the moment, 2-3 plants are connected to the culture medium A in a cluster, obvious growth can be seen after 30 days, and the regenerated seedlings with strong seedlings and thick roots can grow after 60 days; this pathway occurs repeatedly with a proliferation factor of 20.85. About 95% of the stem tips have no protocorm, but have clustered shoot at the base node, and the multiplication coefficient is 7.3.
(5) Hardening and transplanting seedlings: and (5) taking a rooting bottle which is used for hardening the seedling in the test tube in the step (4) until the whole seedling has 6 leaves, 5 nodes and the root length is about 5.0cm, placing the rooting bottle at room temperature for 3d, opening a bottle cover for hardening the seedling for 2d, taking out the seedling from the culture medium, cleaning the residual culture medium, putting the seedling into a carbendazim solution with the mass concentration of 0.5% for disinfection for 5min, then transplanting the seedling into a flowerpot which takes pine bark (1.5 multiplied by 1.5 cm) boiled in boiling water as a matrix, preserving heat (25 ℃) and preserving moisture (80%) for culture for 60d, and obtaining the transplanted seedling with the survival rate of 100%.
Wherein, the culture medium A in the step (3) and the step (4) comprises the following raw materials in parts by weight:
the pH of the A medium was 5.6.
Referring to FIG. 1, FIG. 1-A shows that axillary buds of stem segments begin to germinate and appear in a light green bud dot pattern after culturing for 10 d; FIG. 1-B shows that after 20 days of culture, axillary buds germinate in large numbers and further differentiate into leaves, and root maps appear at the base; FIG. 1-C is the diagram of the plant morphology after the axillary buds are thickened and the development is strong for 35 d; FIG. 1-D shows that after 60 days of culture, the stem segment grows rapidly, the leaves are full and spread, the main stem is thick and strong, the root system is developed, and the proliferation coefficient can reach about 8.4; FIGS. 1-E, F and G show that the high-position buds and the basal buds of different stem segments have different numbers, and the number of the regenerated buds can reach 6 at most; FIGS. 1-H are graphs showing that the height of each regenerated bud increases as the root system grows further.
Referring to fig. 2, scale: panel a =0.5cm, panel B, C =0.1cm, the remainder =1.0cm; wherein FIG. 2-A shows the regenerated embryogenic callus at the cut of the shoot tip; carrying out structure observation on the protocorm under a stereoscope; FIG. 2-B is a diagram showing the differentiation period of the stem and leaf of protocorm; FIG. 2-C is a drawing showing the stem and leaf elongation period; FIG. 2D is a diagram showing the collective culture of protocorms in the stem-leaf differentiation stage; FIG. 2-E is a diagram of a mixed culture of seedlings and protocorms obtained by culturing for 30 d; FIG. 2-F shows the whole culture flask being densely packed for 60 d; protocorm differentiation map; FIG. 2-G is a diagram showing the obvious growth of seedlings after culturing strong clumpy seedlings of protocorm for 30 d; FIG. 2-H shows that the plants are obviously thickened and strengthened after being cultured for 60 days.
Referring to FIG. 3, FIG. 3-A is a diagram of test-tube plantlets after 10d transplanting; FIG. 3-B is a diagram of a 60d transplanted Dendrobium primula plant; FIG. 3-C is a diagram showing that after 120d of transplantation, the leaves of the plants are spread, the leaves are dark green, and the plants grow normally.
While one embodiment of the present invention has been described in detail, the description is only a preferred embodiment of the present invention and should not be taken as limiting the scope of the invention. All equivalent changes and modifications made within the scope of the present invention shall fall within the scope of the present invention.
Claims (8)
1. An artificial high-efficiency propagation method of Dendrobium primula is characterized by comprising the following steps:
(1) Obtaining an explant: taking stem segments of the dendrobium nobile plants as explants;
(2) Stem section regeneration: sterilizing the stem segments obtained in the step (1), cutting the stem segments by using a scalpel according to the number of nodes, and putting the cut stem segments into a culture medium A for culture;
(3) Shoot clumpy shoot initiation and protocorm induction: transferring the stem section with the 1-2 node stem tip in the step (2) to a culture medium A for culture;
(4) And (3) carrying out proliferation and development culture on protocorms: dividing the protocorm mass in the step (3) into blocks with the diameter of 1.5-2.0cm, and transferring the blocks into a culture medium A for culture;
(5) Rejuvenation and rooting culture: transferring the shoot clusters of the basal stem in the step (3) and the seedlings developed from the protocorm in the step (4) into a culture medium A for culturing according to 2-3 plants as a cluster;
(6) Hardening and transplanting seedlings: taking the rooting bottle in the step (5), placing at room temperature, hardening seedlings for 3 days, and opening a bottle cap to harden seedlings for 2 days; and taking out the seedlings, cleaning the residual culture medium on the surfaces of the seedlings, soaking the seedlings in a carbendazim solution, and transplanting the seedlings into the disinfected crushed pine barks for culture to obtain transplanted seedlings.
2. The artificial efficient propagation method of primula pseudodenticulata according to claim 1, wherein the stem segment disinfection and sterilization of step (2) comprises the following steps: firstly, washing dust on the surface of the stem section by using tap water, then soaking by using a washing powder solution, vibrating and stirring, then washing by running water, and placing on a workbench; treating with ethanol solution, sterilizing with mercuric chloride aqueous solution, and washing to obtain sterilized stem segment material.
3. The artificial efficient propagation method of Dendrobium primula according to claim 2, wherein the stem disinfection and sterilization of step (2) comprises the following steps: cleaning 6-8cm stem segments with 5-6 joints in length in the step (1), cleaning dust and impurities on the surface by using tap water, soaking for 10min by using a washing powder solution with the mass concentration of 10%, slightly shaking and stirring, washing for 20min by using running water, sterilizing for 10s by using alcohol with the volume ratio of 70% on a super-clean workbench, sterilizing for 12-15min by using mercury bichloride with the mass percentage of 0.1%, finally washing for 4-5 times by using sterile water, wherein each time is not less than 3min, fully shaking a vessel in the whole sterilization process, and finally shearing to 1-2 joints with the length of 1.5-2.0cm.
4. The artificial efficient propagation method of dendrobium nobile according to claim 1, wherein the culture medium A comprises the following raw materials in parts by weight:
5. the method for artificial efficient propagation of Dendrobium primula according to claim 4, wherein the pH value of the culture medium A is 5.4-5.6.
6. The artificial efficient propagation method of primula dendrobium according to claim 1, wherein the mass concentration of the carbendazim solution in the step (6) is 0.1-0.5%.
7. The artificial efficient propagation method of primula dendrobium according to claim 1, wherein the size of the crushed pine bark in step (6) is 1.5 x 1.5cm, and the disinfection method is boiling in boiling water for 2-3h.
8. The artificial efficient propagation method of Dendrobium primula according to claim 1, wherein the temperature in step (6) is 23-27 ℃ and the humidity is 60-80%.
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