阅读说明：本技术 一种利用胚性愈伤组织进行钻喙兰人工快繁的方法 (Method for artificially and rapidly propagating rhynchophylla by utilizing embryogenic callus ) 是由 黄衡宇 徐福荣 于 2019-09-11 设计创作，主要内容包括：本发明公开了一种利用胚性愈伤组织进行钻喙兰人工快繁的方法,包括的步骤有：将消毒灭菌处理后的蒴果剖开,其内种子接种于萌发培养基中,待愈伤组织出现后,进行胚愈伤组织诱导和增殖,原球茎分化、生长和增殖,生根培养,炼苗移栽。本发明的核心在于钻喙兰种皮破裂后产生胚性愈伤组织,进而分化出原球茎；在此基础上,对培养基进行了优化调整,将胚性愈伤组织增殖、原球茎分化、萌发生长和增殖同步进行,简化了组培过程,3个培养过程同时在一个培养基中进行,大大提高了繁殖效率。此外,本发明成本低、周期短、种苗质量和成活率高。(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.)

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 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.

2. The method for artificially and rapidly propagating rhynchophorus according to claim 1, comprising the following steps of:

(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 scalpel, 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 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.

3. 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 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 no less than 3 min), and shaking the vessel completely during the whole sterilization process.

4. The method for artificially and rapidly propagating rhynchophylla by using embryogenic callus according to claim 1, wherein the culture medium A in the step 3 comprises the following raw materials:

1/2MS basic culture solution

5. The method for the artificial rapid propagation of rhynchophorus according to claim 4, wherein the pH value of the culture medium A is 5.4-5.6.

6. The method for artificially and rapidly propagating rhynchophylla by using embryogenic callus according to claim 1, wherein the culture medium B in the step 4 comprises the following raw materials:

1/2MS basic culture solution

7. The method for the artificial rapid propagation of rhynchophorus according to claim 6, wherein the pH value of the culture medium B is 5.4-5.6.

8. The method for artificially and rapidly propagating rhynchophorus according to claim 1, further comprising the steps of: cutting the cluster seedlings in the step 4 into 3-5 clusters, and transferring the callus with the partial basal part into a C culture medium, wherein the C culture medium 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.

9. The method for artificially and rapidly propagating rhynchophylla by using embryogenic callus according to claim 1, wherein the culture medium D in the step 5 comprises the following raw materials:

1/2MS basic culture solution

10. The method for the artificial rapid propagation of rhynchophorus according to claim 9, wherein the pH value of the culture medium D is 5.4-5.6.

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 scalpel, 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 no less than 3 min), and shaking the vessel completely during the whole sterilization process.

Further, the A culture medium in the step 3 comprises the following raw materials:

1/2MS basic culture solution

Further, the pH value of the A culture medium 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, and transferring the callus with the partial basal part into a C culture medium, wherein the C culture medium 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 D culture medium in the step 5 comprises the following raw materials:

1/2MS basic culture solution

Further, the pH value of the D culture medium 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 is a graph showing the growth and proliferation of the shoot of example 1 at various stages;

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.

FIG. 3 is a diagram of rejuvenation rooting and acclimatization and transplantation in example 1;

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.