CN110100740B

CN110100740B - Tissue culture rapid propagation method of platycerium giganteum

Info

Publication number: CN110100740B
Application number: CN201910558487.8A
Authority: CN
Inventors: 谢松林; 黄良伟; 杨小霞; 罗雪梅
Original assignee: Sichuan Qicai Forestry Co ltd
Current assignee: Sichuan Lide Seedling Technology Co.,Ltd.
Priority date: 2019-06-26
Filing date: 2019-06-26
Publication date: 2020-10-20
Anticipated expiration: 2039-06-26
Also published as: CN110100740A

Abstract

The invention discloses a tissue culture and rapid propagation method of platycerium giganteum. The method mainly comprises the following steps: firstly, collecting mature spores of robust growing bifidobacterium platycerium leaf quilt without plant diseases and insect pests, and sterilizing. Inoculating the sterilized spores into a starting culture medium for culture, inoculating the spores into an enrichment culture medium after the spores germinate to turn green, culturing for 4-6 weeks in a culture chamber, generating cluster buds, selecting the robust cluster buds, cutting the cluster buds into single buds, transferring the single buds into a rooting culture medium, and culturing for 3-5 weeks in the culture chamber to obtain the rooted aseptic seedlings of the platycerium bifidum. Washing the robust aseptic seedlings of the bifidobacterium aquilegia, and directly transplanting the seedlings into a seedbed of a greenhouse for cultivation after a culture medium is washed off. The method has the advantages of simple culture medium formula, simple tissue culture process, shortened tissue culture time, high propagation coefficient, and high survival rate, and can provide technical support for industrialized seedling culture and deep processing of Deerhorn.

Description

Tissue culture rapid propagation method of platycerium giganteum

Technical Field

The invention belongs to the technical field of breeding and cultivating of platycerium giganteum, and particularly relates to a tissue culture rapid propagation method of platycerium giganteum.

Background

Deerhorn (scientific name: Platycerium bifurcatum (Cav.) C. Chr.) Roxb is a species of genus Deerhorn of family Deerhorn. Leaves growing like twigs of deer horn, drooping, with deep and concave branches at the top. The plant height is 40-50 cm. The leaves include nutritive leaves (sterile leaves, naked leaves) and sporophyll (fertile leaves, solid leaves). The sterile leaves are round and convex, the edges are wavy, the new leaves are green-white, and the old leaves are brown. The main function is to collect the chippings falling from the tree, decompose them into nutrients, wrap around the roots, protect the fern roots and store water, besides holding the support. The cultivated leaves are bushy and grey green. Bifurcating into narrow lobes. The sporangia begin to extend up to the top of the lobe below the fovea.

The platycerium giganteum has a fantastic plant shape and a beautiful posture, and can grow in dark and dark places of the hall. It is the most peculiar in posture in ornamental fern, belonging to epiphytic ornamental fern. The platycerium can be arranged on the decorations of parks, plantations, shops, rooms, windowsills and the like, is attached to ancient withered wood or decorated in a hanging basin, is arranged in a hanging or embedding way, and is a good material for indoor three-dimensional greening.

At present, the platycerium giganteum is mainly propagated through division and spores, but the survival rate is not very high, and the demand of the market can not be met. The tissue culture technology is adopted for propagation, the propagation coefficient of the platycerium bifidus can be effectively improved, although some research reports on the platycerium bifidus exist at present, such as patent CN101133704A, the process is complex, the tissue culture takes over 28 weeks, the time and the labor are consumed, and the operability is not strong.

Disclosure of Invention

In order to solve the technical problems, the invention provides a tissue culture and rapid propagation method of platycerium giganteum.

In order to achieve the purpose, the invention adopts the following technical scheme:

a tissue culture and rapid propagation method of Ceratopteris dichotoma comprises the following steps:

(1) and (3) explant sterilization: collecting mature spores of robust growing Bifidobacterium Ceratopteris Linn leaf quilt, subpackaging with weighing paper into small paper bags, and standingOn a clean bench, sterile GA is used₃Soaking in the solution, sterilizing with 75% ethanol and 0.1% mercuric chloride, and washing with sterile water;

(2) starting culture: inoculating the sterilized spores in the step (1) into a starting culture medium, and after culturing for 4-7 weeks, germinating the spores to turn green;

(3) and (3) proliferation culture: inoculating the spores germinated and turned green in the step (2) into a proliferation culture medium, culturing for 4-6 weeks in a culture chamber, generating cluster buds, and performing the next operation when the cluster buds reach a certain plant height;

(4) rooting culture: selecting the cluster buds growing vigorously in the step (3), cutting the cluster buds into single buds, transferring the single buds into a rooting culture medium for culture, and obtaining rooted aseptic seedlings of the platycerium bifidum;

(5) hardening and transplanting seedlings: and (4) washing the healthy and strong aseptic seedlings of the platycerium bifidus grown in the step (4) to remove the culture medium, and directly transplanting the seedlings into a seedling bed substrate of a greenhouse for cultivation.

Further, the sterile GA in the step (1)₃Soaking the solution for 5 min; sterilizing with 75% alcohol for 40-60 s; the sterilizing time of 0.1 percent mercuric chloride is 6-8 min, and the times of washing with sterile water are 4-6.

Further, the culture time in the step (2) is 4-7 weeks; the culture conditions in the culture room are as follows: the illumination intensity is 2000-3000 LX, the illumination time is 12-16 h/d, and the temperature is 20-24 ℃.

Furthermore, the components of the start culture medium in the step (2) are MS, 1.5mg/L, NAA 0.2.2 mg/L of 6-BA, 30g/L of sucrose, 5.5g/L of agar and pH of 5.8-6.2.

Further, the culture time in the step (3) is 4-6 weeks; the plant height of the cluster buds needs to reach 1-3 cm; the culture conditions in the culture room are as follows: the illumination intensity is 2000-3000 LX, the illumination time is 12-16 h/d, and the temperature is 20-24 ℃.

Furthermore, the proliferation medium in the step (3) comprises MS, 0.5mg/L, NAA 0.2.2 mg/L of 6-BA, 30g/L of sucrose, 5.5g/L of agar and pH of 5.8-6.2.

Further, the culture time in the step (4) is 3-5 weeks; the culture conditions in the culture room are as follows: the illumination intensity is 2000-3000 LX, the illumination time is 12-16 h/d, and the temperature is 20-24 ℃.

Furthermore, the rooting medium in the step (4) comprises 1/2MS, 0.2mg/L IBA, 20g/L sucrose, 5.5g/L agar and pH 5.8-6.2.

Further, the temperature in the greenhouse in the step (5) is 18-25 ℃; the seedbed matrix is humus mixed perlite, and the mixing ratio is 2: 1.

Compared with the prior art, the invention has the following beneficial effects:

(1) the tissue culture and rapid propagation method of the bifidobacterium aquilegia provided by the invention has the advantages of simple formula of the used culture medium, low cost of the culture medium, simple and convenient culture process and short time consumption of tissue culture, and can rapidly obtain the bifidobacterium aquilegia aseptic seedlings with consistent genetic characters.

(2) The tissue culture and rapid propagation method of the bifidobacterium aquilegia has the propagation coefficient of 6-10, and improves the propagation efficiency of the bifidobacterium aquilegia.

Drawings

FIG. 1 shows the cluster buds generated in the multiplication medium;

FIG. 2 shows aseptic seedlings of Bifidobacterium brevifilis in rooting medium;

FIG. 3 shows the root system of aseptic seedlings of Bifidobacterium brevifilis in rooting medium;

FIG. 4 shows the mature seedling after hardening off and transplanting.

Detailed Description

Example 1

Collecting mature spores of robust growing Bifidobacterium Ceratopteris Linnaeus without diseases and insect pests, subpackaging with weighing paper into small paper bags, placing on super clean bench, and packaging with sterile GA₃Soaking in the solution for 5min, sterilizing with 75% alcohol for 40-60 s, sterilizing with 0.1% mercuric chloride for 6-8 min, and washing with sterile water for 4-6 times. Inoculating the sterilized spores into a starting culture medium, and after culturing for 4-7 weeks in a culture room, the spores germinate to turn green. Inoculating the germinated and green spores into a proliferation culture medium, culturing for 4-6 weeks in a culture room, and generating cluster buds, wherein the plant height is 1-3 cm, and the proliferation coefficient is 6-10 as shown in figure 1. Selecting strongly growing cluster buds, cutting the cluster buds into single buds, transferring the single buds to a rooting culture medium,culturing in culture room for 3-5 weeks to obtain rooted aseptic seedling of Bifidobacterium brevifolius as shown in FIG. 2 and FIG. 3. The vigorous aseptic seedlings of the platycerium bifidus are directly transplanted into a seedbed of a greenhouse after a culture medium is washed off, the temperature in the greenhouse is controlled to be 18-25 ℃ by paying attention to shading, cooling and moisturizing, the survival rate can reach 90% as shown in figure 4.

The medium components, the transplanting medium components and the culture chamber culture conditions used in this example were as follows:

the components of the start culture medium are MS, 1.5mg/L, NAA 0.2.2 mg/L of 6-BA, 30g/L of cane sugar, 5.5g/L of agar and pH value is 5.8-6.2.

The proliferation medium comprises MS, 6-BA 0.5mg/L, NAA 0.2.2 mg/L, sucrose 30g/L, agar 5.5g/L, and pH5.8-6.2.

The rooting medium comprises 1/2MS, IBA 0.2mg/L, sucrose 20g/L, agar 5.5g/L and pH 5.8-6.2.

The culture conditions in the culture room are as follows: the illumination intensity is 2000-3000 LX, the illumination time is 12-16 h/d, and the temperature is 20-24 ℃.

The transplanting matrix is humus mixed perlite with the mixing ratio of 2: 1.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Claims

1. A tissue culture and rapid propagation method of Ceratopteris dichotoma is characterized in that: the method comprises the following steps:

(1) and (3) explant sterilization: collecting mature spores of robust growing Bifidobacterium Ceratopteris Linnaeus without diseases and insect pests, subpackaging with weighing paper into small paper bags, placing on super clean bench, and packaging with sterile GA₃Soaking in the solution, sterilizing with 75% ethanol and 0.1% mercuric chloride, and washing with sterile water;

(2) starting culture: inoculating the sterilized spores in the step (1) into a start-up culture medium, and after culturing for 4-7 weeks in a culture room, enabling the spores to germinate green, wherein the components of the start-up culture medium comprise MS, 6-BA 1.5mg/L, NAA 0.2.2 mg/L, sucrose 30g/L, agar 5.5g/L and pH is 5.8-6.2; the culture conditions in the culture room are as follows: the illumination intensity is 2000-3000 LX, the illumination time is 12-16 h/d, and the temperature is 20-24 ℃;

(3) and (3) proliferation culture: inoculating the spores germinated and turned green in the step (2) into a multiplication culture medium, culturing in a culture room for 4-6 weeks, generating cluster buds, and carrying out the next operation when the plant height of the cluster buds reaches 1-3 cm, wherein the multiplication culture medium comprises the components of MS, 0.5mg/L, NAA 0.2.2 mg/L of 6-BA, 30g/L of sucrose, 5.5g/L of agar and pH 5.8-6.2; the culture conditions in the culture room are as follows: the illumination intensity is 2000-3000 LX, the illumination time is 12-16 h/d, and the temperature is 20-24 ℃;

(4) rooting culture: selecting the cluster buds growing vigorously in the step (3), cutting the cluster buds into single buds, transferring the single buds into a rooting culture medium, and culturing in a culture room for 3-5 weeks to obtain rooted aseptic seedlings of the platycerium bifidum, wherein the rooting culture medium comprises 1/2MS, IBA 0.2mg/L, sucrose 20g/L, agar 5.5g/L and pH 5.8-6.2; the culture conditions in the culture room are as follows: the illumination intensity is 2000-3000 LX, the illumination time is 12-16 h/d, and the temperature is 20-24 ℃;

2. The tissue culture rapid propagation method of Ceratopteris dichotoma as claimed in claim 1, which comprises: the sterile GA in the step (1)₃Soaking the solution for 5 min; sterilizing with 75% alcohol for 40-60 s; the sterilizing time of 0.1 percent mercuric chloride is 6-8 min, and the times of washing with sterile water are 4-6.

3. The tissue culture rapid propagation method of Ceratopteris dichotoma as claimed in claim 1, which comprises: the temperature in the greenhouse in the step (5) is 18-25 ℃; the seedbed matrix is humus mixed perlite, and the mixing ratio is 2: 1.

4. The application of the tissue culture rapid propagation method of Ceratopteris dichotoma as claimed in any one of claims 1-3 in the technical field of Ceratopteris dichotoma propagation and cultivation.