CN110800609B - Method for artificially and rapidly propagating rhynchophylla by utilizing embryogenic callus - Google Patents
Method for artificially and rapidly propagating rhynchophylla by utilizing embryogenic callus Download PDFInfo
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Abstract
The invention discloses a method for carrying out artificial rapid propagation on rhynchophylla by utilizing embryonic callus, which comprises the following steps: splitting the disinfected capsule, inoculating the seeds in a germination culture medium, inducing and proliferating embryonic callus after the callus appears, differentiating, growing and proliferating protocorm, rooting culture, hardening off and transplanting. The core of the invention is that embryonic callus is generated after the seed coat of the coracocephalum rhynchophyllum is cracked, and then protocorm is differentiated; on the basis, the culture medium is optimized and adjusted, the embryogenic callus proliferation, the protocorm differentiation, the germination growth and the proliferation are synchronously carried out, the tissue culture process is simplified, 3 culture processes are simultaneously carried out in one culture medium, and the propagation efficiency is greatly improved. In addition, the invention has low cost, short period and high seedling quality and survival rate.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for artificially and rapidly propagating coracocephalum cochinchinensis by utilizing embryonic callus.
Background
Rhynchophylla (Rhynchostylis) is perennial epiphytic herb of Orchidaceae (Orchidaceae), about 6 species, mainly distributed in tropical asia, and 2 species in China, namely rhynchophylla in Hainan (r.gigantea (Lindl.) Ridl.) and rhynchophylla in Hainan (r.retusa (L.) Bl.), which are mainly produced in tropical regions in south. The rhynchophylla plant has elegant and fragrant flower color, plump inflorescence, drooping or upright, and is shaped like the tail of a hairy fox, so the rhynchophylla plant is commonly called foxtail orchid in a commodity and is epiphytic orchid with high ornamental value. The rhynchophylla is rich in colors such as red, pink, white, blue and orange, and is extremely popular with people due to the unique flower type, long flowering phase and excellent new spring greeting flowers before and after the traditional festival-spring festival in China.
The traditional propagation mode of the rhynchophorus is plant division propagation and seed propagation, although the characteristics of a female parent can be maintained by the plant division propagation, the efficiency is low, the multiplication times are only 1-3 times, the period is long, and the requirement of industrial production is difficult to meet. Most seeds of the orchid family are small and lack cotyledons and endosperms, so that embryos are incompletely developed and need to be germinated in cooperation with related symbiotic bacteria under natural conditions, so that the germination time is long, the germination rate is low (only 5%), the seedling rate is low, and the period from seed germination to flowering plants capable of being subjected to character identification is generally 4-5 years or more. Therefore, a new asexual propagation method with low cost, short time, considerable multiplication coefficient and high seedling quality and survival rate is needed to expand the propagation quantity of the cymbidium coracoides seedlings and carry out factory production of high-quality seedlings so as to meet the planting requirement.
Disclosure of Invention
The invention aims to solve the defects of the prior breeding technology and provides a method for carrying out the artificial rapid propagation of the Rhynchosia rhynchophylla by utilizing the embryogenic callus, and the method lays a technical foundation for the artificial selective breeding and the development of artificial planting of filial generations. The invention can also provide high-quality seedlings which are produced by a single protocorm and have consistent genotype background so as to meet the requirement of artificial planting.
In order to solve the technical problems, the invention adopts the following technical scheme:
a method for carrying out artificial rapid propagation of rhynchophylla by utilizing embryogenic callus comprises the following steps: splitting the disinfected capsule, inoculating the seeds in a germination culture medium, inducing and proliferating embryonic callus after the callus appears, differentiating, growing and proliferating protocorm, rooting culture, hardening off and transplanting.
Further, the method for carrying out artificial rapid propagation of rhynchophylla by utilizing the embryogenic callus comprises the following steps:
(1) obtaining an explant: selecting healthy and strong plants with good growth vigor and no plant diseases and insect pests, and taking mature and plump capsules after artificial pollination;
(2) sterilizing the capsule obtained in the step 1;
(3) seed germination, callus induction, protocorm generation: and (3) longitudinally splitting the capsule sterilized in the step (2) on a workbench by using a surgical knife, and uniformly scattering seeds in the capsule into the following culture medium A, wherein the culture medium A comprises the following raw materials:
1/2MS basic culture solution
6-benzylaminopurine (6-BA)
Activated Carbon (AC)
Banana mud
Sucrose
Agar powder
Seed germination, callus induction and protocorm generation are carried out under the conditions of controlling illumination intensity, temperature and illumination time;
(4) embryogenic callus proliferation, protocorm generation and proliferation, protocorm germination into seedlings: transferring the callus cultured in the step 3 into a culture medium B, wherein the culture medium B comprises the following raw materials:
1/2MS basic culture solution
6-benzylaminopurine (6-BA)
Naphthylacetic acid (NAA)
Kinetin (KT)
Activated Carbon (AC)
Banana mud
Sucrose
Agar powder
Performing embryogenic callus proliferation, protocorm generation and proliferation, and protocorm germination and seedling formation under the conditions of controlling illumination, temperature and illumination time;
(5) rejuvenation and rooting culture: and (4) separating individual buds in the cluster buds in the step (4), selecting leaves and seedlings with basically consistent growth vigor, cutting roots and inoculating the seedlings into a culture medium D, wherein the culture medium D comprises the following raw materials:
1/2MS basic culture solution
Naphthylacetic acid (NAA)
Activated Carbon (AC)
Coconut juice
Sucrose
Agar powder
pH
Performing rejuvenation rooting culture under the conditions of controlling illumination, temperature and illumination time;
(6) hardening and transplanting seedlings: and (3) putting the rooted plants in the step (5) at room temperature for hardening seedlings, taking out the seedlings from the culture medium D, washing the root culture medium D by using clear water, then putting the root culture medium D in a room, airing the root culture medium D until the roots are whitened, transplanting the roots into the crushed pine barks sterilized and disinfected by chlorothalonil, and keeping the humidity for a period of time to obtain the transplanted seedlings.
Further, the method for sterilizing the capsule in the step 2 comprises the following steps: cleaning the capsule 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 30min, sterilizing with 75% alcohol on a clean bench for 30s, and sterilizing with 0.1% HgCl2Sterilizing for 20min, washing with sterile water for 2-3 times (each time not less than 3 min)The vessel was shaken thoroughly during the sterilization process.
Further, the culture medium A in the step 3 comprises the following raw materials:
1/2MS basic culture solution
Further, the pH value of the culture medium A is 5.4-5.6.
Further, the culture medium B in the step 4 comprises the following raw materials:
1/2MS basic culture solution
Further, the pH value of the culture medium B is 5.4-5.6.
Further, the method also comprises the following steps: cutting the cluster seedlings in the step 4 into 3-5 clusters of primary callus tissues with partial basal part, and transferring the clusters of primary callus tissues into a culture medium C, wherein the culture medium C comprises the following raw materials:
1/2MS basic culture solution
And performing embryogenic callus proliferation, protocorm generation and proliferation, and protocorm germination and seedling formation under the conditions of controlling illumination, temperature and illumination time.
Further, the culture medium D in the step 5 comprises the following raw materials:
1/2MS basic culture solution
Further, the pH value of the medium D is 5.4-5.6.
The invention has the following beneficial effects:
(1) 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;
(2) the invention realizes the purpose of high-efficiency rapid propagation, 90d is a propagation culture period, and the propagation coefficient can reach more than 10.0;
(3) the invention solves the problems of low breeding efficiency, long period and the like of the traditional seeds, can obtain a great number of seedlings in a short time, is easy for standardization and industrial operation, effectively improves the quality of the seedlings, and can provide uniform and standard excellent seedlings for large-area popularization and planting;
(4) the invention optimizes the rhynchophylla pallidum rapid propagation system, can simultaneously carry out embryogenic callus proliferation, protocorm generation and proliferation and protocorm germination and seedling formation in the same culture medium, and simplifies the culture procedure; in the whole rapid propagation process, except for seed germination, embryogenic callus proliferation and protocorm generation in the initial stage, only 2 culture media are needed to solve the problems of embryogenic callus proliferation, protocorm generation and proliferation, protocorm germination and seedling formation and rooting, and the production plan is favorably arranged;
(5) the test-tube plantlet is from the protocorm, so the test-tube plantlet is strong in root and thick, and the survival rate of hardening and transplanting is high.
Drawings
FIG. 1 is a diagram showing callus proliferation and various growth stages of shoot initiation;
wherein FIG. 1-A is a diagram showing callus proliferation after 20d of transfer; FIG. 1-B is a photograph showing the germination trace of the differentiated protocorm on the surface of the callus; FIG. 1-C is a graph of embryogenic callus appearing on each material, and the resulting protocorm with pronounced pseudoroot hairs (rhizoids); FIG. 1-D is a graph showing the beginning of massive germination of protocorms, each protocorm showing false root hairs; FIGS. 1-E show that seedlings germinated from protocorms became "clumpy" with the proliferation of calli; FIG. 1-F is a diagram of the differentiated protocorm of embryogenic callus; FIG. 1-G is a diagram showing the germination of protocorm after its root; FIGS. 1-H are graphs of "clumps" initiated by numerous protocorms.
FIG. 2-A is a diagram showing the growth of shoots grafted about 20 d; FIG. 2-B, C shows the rapid callus proliferation at the base of the leaf of shoot extending for about 40 days; FIG. 2-D is a emerald green color of callus due to the massive occurrence of protocorms; FIGS. 2-E, F are graphs showing the "clumping" of protocorm sprouting into shoots; FIGS. 2-G, H are graphs showing growth after culturing for about 90 days.
FIGS. 3-A, B are graphs of rejuvenated rooted shoots cultured for 20 days; FIG. 3-C shows that after 50 days, the test-tube plantlet grows rapidly, new roots continuously grow, and the number of new roots reaches 4-5; FIGS. 3-E, F are diagrams showing the growth of the rooted seedlings after 90 d; FIGS. 3-G, H are diagrams of tube plantlets after acclimation for 90 d.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention are described below clearly and completely, and it is obvious that the described embodiments are some, not all embodiments of the present invention. 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
A method for carrying out artificial rapid propagation on rhynchophylla by utilizing embryonic callus comprises the following steps:
1. obtaining an explant: selecting strong plants with good growth vigor and no plant diseases and insect pests, and taking mature and plump capsules after artificial pollination.
2. Cleaning the capsule obtained in step 1 with tap water to remove dust and impurities on the surface, soaking in 10% washing powder solution (by mass) for 10min, slightly shaking and stirring, washing with running water for 30min, sterilizing with 75% alcohol on a clean bench for 30s, and sterilizing with 0.1% HgCl2Sterilizing for 20min, and washing with sterile water for 3min for 2 times. The vessel was shaken thoroughly throughout the sterilization process.
3. Seed germination, callus induction, protocorm generation: and (3) longitudinally splitting the capsule sterilized in the step (2) on a clean bench by using a surgical knife, and uniformly scattering seeds in the capsule into the following culture medium A:
1/2MS basic culture solution
The culture conditions are as follows: the seeds are germinated under the conditions of illumination intensity of 1500-; after 30 days, the green callus is gradually transformed into embryogenic, and granular protocorm is differentiated on the green callus; after 60 days, the callus (embryogenic callus) proliferated in large amounts, covering essentially the entire medium surface.
4. Embryogenic callus proliferation, protocorm generation and proliferation, protocorm germination into seedlings: the callus cultured in step 3 was cut into pieces of 0.5 × 0.5cm containing several protocorms, and transferred to medium B:
1/2MS basic culture solution
The culture conditions are as follows: culturing for 90 days under the conditions of illumination intensity of 2000-. Embryogenic callus appears in large numbers and produces an extremely large number of protocorms; as the protocorm germinated, clumpy shoots were generated on each callus piece, and obvious pseudoroot hairs (rhizoids) were visible. The callus proliferation coefficient of the step is 17.68, and the bud generation coefficient also reaches 20.16.
5. Cutting the cluster seedlings in the step 4 into 3 clusters, and transferring the callus with the partial basal band into a culture medium C:
1/2MS basic culture solution
The culture conditions are as follows: culturing the base part callus for about 20d to start proliferation under the conditions of illumination intensity of 2000-; the leaves of the sprouts extend for about 40 days, and the callus of the base parts of the sprouts is obviously increased; after 50 days, the sprouts have 4 new leaves, and the calluses at the base parts are turned into emerald green due to the massive appearance of protocorms; after 70d, the protocorms on the calluses germinate in a large quantity; after 90 days, the young buds grow into plantlets, the base parts of the plantlets are covered by new buds sprouted from protocorms, the bud generation coefficient is 17.87, and the callus multiplication coefficient can also reach 10.32.
6. Rejuvenation and rooting culture: separating the bigger individual plant in the cluster buds in the step 5, selecting seedlings with 3cm height, 3 leaves and basically consistent growth vigor, cutting off 2/3 roots, and inoculating into a culture medium D:
1/2MS basic culture solution
The culture conditions are as follows: culturing for 20 days under the conditions of illumination intensity of 2000-; after 50 days, new strong and rapidly growing roots can be seen; after 90 days, the test-tube plantlet leaves are completely unfolded and have 5 new roots with the root thickness of 14 mm. No callus and protocorm occurred during the entire rejuvenating rooting culture, and no "clumpy buds" appeared, indicating that the culture medium is very suitable for the cultivation of Rhynchophorus rhynchophyllus at this stage.
7. Hardening and transplanting seedlings: and (3) taking the rooting plant with 4 leaves, 4cm in root length and 5cm in plant height in the step (6), hardening the seedling for 3 days at room temperature, taking out the seedling from the culture medium D, carefully cleaning the root culture medium D with clear water, then placing the seedling in a room for airing water, transplanting the seedling to the crushed pine bark sterilized and disinfected by chlorothalonil with the mass concentration of 0.1% when the root of the seedling is whitened, and keeping the humidity at 62% and 90 days to obtain the transplanted seedling.
Example 2
A method for carrying out artificial rapid propagation on rhynchophylla by utilizing embryonic callus comprises the following steps:
1. obtaining an explant: selecting strong plants with good growth vigor and no plant diseases and insect pests, and taking mature and plump capsules after artificial pollination.
2. Cleaning the capsule obtained in step 1 with tap water to remove dust and impurities on the surface, soaking in 10% washing powder solution (by mass) for 10min, slightly shaking and stirring, washing with running water for 30min, sterilizing with 75% alcohol on a clean bench for 30s, and sterilizing with 0.1% HgCl2Sterilizing for 20min, and washing with sterile water for 2-3 times (each time no less than 3 min). The vessel was shaken thoroughly throughout the sterilization process.
3. Seed germination, callus induction, protocorm generation: and (3) longitudinally splitting the capsule sterilized in the step (2) on a clean bench by using a surgical knife, and uniformly scattering seeds in the capsule into the following culture medium A:
1/2MS basic culture solution
The culture conditions are as follows: the seeds are germinated under the conditions of illumination intensity of 1500-; after 30 days, the green callus is gradually transformed into embryogenic, and granular protocorm is differentiated on the green callus; after 60 days, the callus (embryogenic callus) proliferated in large amounts, covering essentially the entire medium surface.
4. Cutting the protocorm seedlings in the step 3 into 4 clusters, transferring the callus with partial basal part into a culture medium C:
1/2MS basic culture solution
The culture conditions are as follows: culturing the base part callus for about 20d to start proliferation under the conditions of illumination intensity of 2000-; the leaves of the sprouts extend for about 40 days, and the callus of the base parts of the sprouts is obviously increased; after 50 days, the buds have 5 new leaves, and the calluses at the base parts are changed into emerald green due to the massive appearance of protocorms; after 70d, the protocorms on the calluses germinate in a large quantity; after 90 days, the young buds grow into plantlets, the base parts of the plantlets are covered by new buds sprouted from protocorms, the bud generation coefficient is 17.37, and the callus multiplication coefficient can also reach 10.85.
From the actual situation of the rhynchophorus, the 'original step 4' is reduced and the seedling forming time is shortened during the culture in the step, and by adopting the culture medium (namely the culture medium C) in the step, the embryonic callus proliferation, protocorm generation and proliferation, protocorm germination and seedling forming and other stages can be completed at one time, and the culture time of 180d or even longer can be shortened to about 90 d.
5. Rejuvenation and rooting culture: separating the bigger individual plant in the cluster buds in the step 4, selecting seedlings with height of 3.1cm, 4 leaves and basically consistent growth vigor, cutting off 2/3 roots, and inoculating into a culture medium D:
1/2MS basic culture solution
The culture conditions are as follows: culturing for 20 days under the conditions of illumination intensity of 2000-; after 50 days, new strong and rapidly growing roots can be seen; after 90 days, the test-tube plantlet leaves are completely unfolded and have 6 new roots with the root thickness of 14.2 mm. No callus and protocorm occurred during the entire rejuvenating rooting culture, and no "clumpy buds" appeared, indicating that the culture medium is very suitable for the cultivation of Rhynchophorus rhynchophyllus at this stage.
6. Hardening and transplanting seedlings: and (3) taking the rooting plant with 5 leaves, the root length of which is 5cm and the plant height of which is 5.3cm in the step (6), hardening the seedling for 3 days at room temperature, taking out the seedling from the culture medium D, carefully cleaning the root culture medium D with clear water, then placing the seedling in a room for air drying, transplanting the seedling into the crushed pine bark sterilized and disinfected by chlorothalonil with the mass concentration of 0.1% when the root of the seedling turns white, and keeping the humidity at 73% and 90 days to obtain the transplanted seedling.
The technical principle of the invention is as follows:
the core of the invention is that the seed coat of the coracocephalum rhynchophyllum is cracked to generate embryogenic callus, so that protocorm is differentiated, and the fact that natural additional product coconut juice plays an important role in the in-vitro rapid propagation of the coracocephalum rhynchophyllum is proved; on the basis, the culture medium is optimized and adjusted, 3 exogenous hormones with the best synergistic effect are screened out through a 4-factor 3-level orthogonal test and are matched for use, embryogenic callus proliferation, protocorm differentiation, germination growth and proliferation are synchronously performed, the tissue culture process is simplified, 3 culture processes are simultaneously performed in one culture medium, the culture time of 180 days or even longer is shortened to about 90 days, and the propagation efficiency is greatly improved.
The efficient artificial propagation method of the rhynchophylla creeper through the embryogenic callus has the advantages of low cost, short time, high seedling quality and survival rate, and capability of fixing excellent characters; the method can be used for expanding the propagation quantity of the cymbidium coracoides seedlings, thereby carrying out the industrial production of high-quality seedlings to meet the planting requirement.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (2)
1. A method for carrying out artificial rapid propagation on rhynchophylla by utilizing embryogenic callus is characterized by comprising the following steps: splitting the disinfected and sterilized capsule, inoculating seeds in a germination culture medium, after callus appears, performing embryonic callus induction and proliferation, protocorm differentiation, growth and proliferation, rooting culture, hardening seedling and transplanting;
the method specifically comprises the following steps:
(1) obtaining an explant: selecting healthy and strong plants with good growth vigor and no plant diseases and insect pests, and taking mature and plump capsules after artificial pollination;
(2) sterilizing the capsule obtained in the step 1;
(3) seed germination, callus induction, protocorm generation: and (3) longitudinally splitting the capsule sterilized in the step (2) on a workbench by using a surgical knife, and uniformly scattering seeds in the capsule into the following culture medium A, wherein the culture medium A comprises the following raw materials:
1/2MS basic culture solution
Seed germination, callus induction and protocorm generation are carried out under the conditions of controlling illumination intensity, temperature and illumination time;
(4) embryogenic callus proliferation, protocorm generation and proliferation, protocorm germination into seedlings: transferring the callus cultured in the step 3 into a culture medium B, wherein the culture medium B comprises the following raw materials:
1/2MS basic culture solution
Performing embryogenic callus proliferation, protocorm generation and proliferation, and protocorm germination and seedling formation under the conditions of controlling illumination, temperature and illumination time;
(5) cutting the cluster seedlings in the step 4 into 3-5 clusters of primary callus tissues with partial basal part, and transferring the clusters of primary callus tissues into a culture medium C, wherein the culture medium C comprises the following raw materials:
1/2MS basic culture solution
Performing embryogenic callus proliferation, protocorm generation and proliferation, and protocorm germination and seedling formation under the conditions of controlling illumination, temperature and illumination time;
(6) rejuvenation and rooting culture: and (3) separating individual buds in the cluster buds in the step (5), selecting leaves and seedlings with basically consistent growth vigor, cutting roots and inoculating the seedlings into a culture medium D, wherein the culture medium D comprises the following raw materials:
1/2MS basic culture solution
Performing rejuvenation rooting culture under the conditions of controlling illumination, temperature and illumination time;
(7) hardening and transplanting seedlings: and (3) putting the rooted plants in the step (6) at room temperature for hardening seedlings, taking out the seedlings from the culture medium D, washing the root culture medium by using clear water, then putting the root culture medium in a room, airing the root culture medium, transplanting the root culture medium into the crushed pine barks sterilized and disinfected by chlorothalonil when the roots are whitened, and keeping the humidity for a period of time to obtain the transplanted seedlings.
2. The method for artificially and rapidly propagating rhynchophylla by utilizing embryogenic callus according to claim 1, wherein the method for disinfecting the capsule in the step 2 comprises the following steps: cleaning the capsule 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 30min, sterilizing with 75% alcohol on a clean bench for 30s,using HgCl with the mass percent of 0.1%2Sterilizing for 20min, washing with sterile water for 2-3 times (each time no less than 3 min), and shaking the vessel completely during the whole sterilization process.
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