CN113545291A - Oyster leaf tissue culture bud propagation method - Google Patents

Abstract

The invention relates to the culture and reproduction capacity of oyster leaves, and discloses a method for propagating oyster leaves by tissue culture buds, which comprises the following steps: sequentially carrying out vibration rinsing on the explant in 75% alcohol, soaking and shaking in 0.1% mercuric chloride solution, and shaking and soaking in sterile water, and then inoculating the explant to a sterile bud culture medium for culture to obtain oyster leaf sterile buds; (2) inoculating oyster leaf sterile buds obtained by culture in a superclean workbench to a multiplication culture medium to obtain sterile seedlings for subculture; (3) taking out sterile seedlings subjected to subculture in a superclean bench, performing plant division treatment, and respectively inoculating to a rooting culture medium to obtain oyster leaf seedlings with roots; (4) the method can rapidly propagate high-quality oyster leaves, so that the oyster leaf variety and the excellent characteristics can be preserved.

Description

Oyster leaf tissue culture bud propagation method

Technical Field

The invention relates to the technical field of oyster leaf culture and propagation, and discloses an oyster leaf tissue culture bud propagation method.

Background

Oyster leaf (Mertensia maritime) also known as oyster leaf (oyster leaf) is a coastal littoral of the family Boraginaceae, originally produced in coastal gravels such as Norway and iceland in the northern hemisphere to the frigid zone, coastal gravels such as Russian and Japanese northern sea-island, or cobblestone beach alpine-resistant perennial plants, and a very unique species is gold beach pebble beach beside the sea. Its leaves are juicy, white frost, bluish green leaves and small, sharp, bell-shaped, pink to blue flowers, the growth habit is creeping, and flowers bloom from 6 months to 8 months. Since the oyster leaves have oyster-like flavor, they can be used as a substitute for oyster, and are often used in salad or pickles of French dishes. However, the fresh oyster leaves have the characteristic of high zinc content besides special flavor.

At present, fewer oyster leaves are planted in China, and outdoor cultivation is mainly used. The outdoor planting has the growth period limited by the external climatic conditions, more plant diseases and insect pests and poor cleanliness. The oyster leaves cultivated in the plant factory well play the advantages of the factory, namely annual production, extremely clean and no influence of diseases, insect pests and the like. But the propagation of the hybrid seeds is also a big problem, artificial pollination is needed, the germination rate of the seeds is low, and the hybrid seeds are not durable to store.

Disclosure of Invention

Therefore, the method for propagating the oyster leaves by tissue culture buds needs to be provided, and the problems of difficult planting and propagation, artificial planting and low planting germination rate of the existing domestic oyster leaves are solved.

In order to achieve the purpose, the invention provides a method for propagating oyster leaf tissue culture buds, which comprises the following steps:

(1) selecting an explant of oyster leaves subjected to tissue culture, cleaning the explant of the oyster leaves, sequentially carrying out vibration rinsing in 75% alcohol, soaking and shaking in 0.1% mercuric chloride solution, shaking and soaking in sterile water in a clean bench, and then inoculating the explant to a sterile bud culture medium for culture to obtain aseptic buds of the oyster leaves, wherein the sterile bud culture medium comprises the following components: b5 culture medium, 0.5-1.5mg/L6-BA, 0.1-0.5mg/L NAA, 7g/L agar and 30g/L sucrose;

(2) inoculating oyster leaf sterile buds obtained by culture in a superclean bench to a multiplication culture medium to obtain a subculture sterile seedling, wherein the multiplication culture medium comprises the following components: b5 culture medium, 0.5mg/L6-BA, 0-0.1mg/L NAA, 7g/L agar, 30g/L sucrose;

(3) taking out sterile seedlings for subculture in a superclean bench, cutting and separating the sterile seedlings for division, and respectively inoculating the sterile seedlings to a rooting culture medium to obtain oyster leaf seedlings with roots, wherein the rooting culture medium comprises: 1/4B5 medium or 1/2B5 medium or B5 medium, 0.2-1.0mg/L IBA, 7g/L agar, 30g/L sucrose;

further, the explant of the fresh oyster leaves is a tillering bud of the fresh oyster leaves.

Further, in the step (1), the explants are sequentially washed by shaking in 75% alcohol for 45s, soaked in mercuric chloride solution for 45s, and soaked in sterile water for 30 s.

Further, the sterile bud medium contains: 1.5mg/L6-BA, and 0.2mg/L NAA.

Further, the propagation medium contained 0.05mg/L NAA.

Further, the rooting medium comprises the following components: 1/4B5 medium, 0.5mg/L IBA, 7g/L agar, 30g/L sucrose.

Further, the culture conditions of the steps 1-3 are as follows: placing in a culture chamber with illumination intensity of 2000lx and photoperiod of 12h at 18-24 ℃.

The technical scheme has the following beneficial effects:

according to the invention, the oyster leaf tissue culture propagation is carried out in an indoor simulated climate and illumination environment, the problem that the oyster leaf growth is limited by seasonal climate is solved, annual uninterrupted propagation can be realized, and the culture process is as follows: sterile bud culture → bud multiplication culture → rooting culture → complete plant, can rapidly propagate high-quality sterile seedlings, can rapidly grow seedlings, and can overcome the problems of low germination rate and difficult propagation of oyster leaf seeds and long-time vegetative growth plant aging, so that oyster leaf varieties and excellent characteristics can be preserved, and annual production of oyster leaves can be realized.

Drawings

FIGS. 1 to 9 show the culture conditions of test groups 1 to 9 in example 1 according to the present embodiment.

FIG. 10 shows the rooting of test group 5 in example 3 according to the present embodiment.

Fig. 11 shows oyster leaves after the divided seedling culture in example 4 according to the specific embodiment.

Detailed Description

To explain technical contents, structural features, and objects and effects of the technical solutions in detail, the following detailed description is given with reference to the accompanying drawings in conjunction with the embodiments.

Example 1: culturing oyster sterile buds, optimizing an explant,

(1) four-factor three-level orthogonal assays were performed with explants, media, 6-BA, NAA.

Four factors: explant, culture medium, 6-BA and NAA.

And (3) three levels: selecting 1 (tillering bud), 2 (leaf blade) and 3 (seed) different parts of the explant; the culture medium addresses 1(MS), 2 (improved MS) and 3(B5) of 3 different culture media; 6-BA selecting 1(0.5mg/L), 2(1.0mg/L) and 3(1.5mg/L) of 3 different concentrations; NAA was selected at 3 different concentrations of 1(0.1mg/L), 2(0.2mg/L) and 3(0.5 mg/L). The proper explants, culture media, 6-BA and NAA are preliminarily screened through the orthogonal design. One treatment 3 bottles were repeated 3 times.

(2) Cleaning oyster leaves, seeds, leaves and tillering buds, placing the oyster leaves, seeds, leaves and tillering buds into 75% alcohol, shaking and rinsing for 45s, soaking in 0.1% mercuric chloride solution for 45s, shaking and soaking in sterile water for 60s, and then respectively inoculating the oyster leaves, seeds, leaves and tillering buds into different culture medium formulas, 7g/L agar, 30g/L sucrose and 0.1g/L inositol, wherein the specific test design is shown in table 1. (3) As shown in table 2-1, for a total of 9 treatments), 1 explant per vial was inoculated. Culturing at 24 deg.C in a culture room with illumination intensity of 2000lx and photoperiod of 12 h/d.

(3) FIGS. 1 to 9 are photographs of aseptic buds cultured in the above test groups 1 to 9, and from the test results of the growth of tillering buds, leaves and seeds of oyster leaves, it can be seen that the tillering buds of oyster leaves are a good explant for culturing the oyster leaf buds. The test group 7 of bud culture of oyster leaves has good growth state and certain proliferation effect. Therefore, the better formula for culturing the oyster leaf aseptic buds is as follows: b5 culture medium +1.5mg/L6-BA +0.2mg/L NAA +7g/L agar +30g/L sucrose

Therefore, in the invention, preferably, the explant for the tissue culture of oyster leaves is tillering buds, and the 6-BA dosage in the culture medium is 1.5 mg/L; the dosage of NAA is 0.2 mg/L.

Table 1 experimental setup for explant selection

Example 2: bud multiplication culture

(1) The oyster leaf sterile buds obtained from the test group 7 in the example 1 are taken, the oyster leaf sterile buds are subjected to the strain division treatment, then the oyster leaf sterile buds are respectively inoculated into different culture medium formulas +7g/L agar +30g/L cane sugar, 1 group culture bottle is inoculated with 1 bud, the specific test design is shown in table 3, the oyster leaf sterile buds are cultured in a culture room with the temperature of 18 ℃, the illumination intensity of 2000lx and the photoperiod of 12h/d, and the growth condition is regularly observed.

TABLE 2 bud growth culture conditions

Test group	Nutrient solution	6-BAmg/L	NAAmg/L	The culture temperature is lower
					YB1	B5	0.5	0.05	24
YB2	B5	0.5	0.1	24
					YB3	B5	0.5	0	24
YB4	B5	0.5	0.05	18

(2) Results of the experiment

After 30 days of inoculation, the number of the proliferated buds of each test group is counted, the plants are divided, and the multiplication times and the number of the effective buds of each test group are counted (if the buds with weaker growth in bud proliferation are easily damaged in the plant division process, the buds which cannot be used for next culture are ineffective buds because oyster leaves belong to basal leaves), which is detailed in table 3.

Table 3, experimental results of example 2.

As is clear from Table 3, YB4 showed the highest proliferation fold and the highest number of active shoots among 4 test groups, and no vitrification occurred. The division of the plants is easier. In conclusion, the preferable formula and temperature conditions for inducing the oyster leaf bud proliferation culture are as follows: b5+0.5mg/L6-BA +0.05mg/L NAA +7g/L agar +30g/L sucrose +18 ℃ culture.

Example 3: rooting culture

Table 4, rooting culture treatment table.

Test group	Nutrient solution	IBA(mg/L)
			1	1/2B5	0.2
2	1/2B5	0.5
			3	1/2B5	1.0
4	1/4B5	0.2
			5	1/4B5	0.5
6	1/4B5	1.0
			7	B5	0.2
8	B5	0.5
			9	B5	1.0

Sterile seedlings obtained from the YB4 test group in example 2 are taken, the sterile seedlings are subjected to division treatment in an ultra-clean workbench, then the sterile seedlings are respectively inoculated in different culture medium formulas, 7g/L agar and 30g/L sucrose, 1 tissue culture bottle is inoculated with 1 bud, the specific test design is shown in table 4, the sterile seedlings are cultured in a culture room with the illumination intensity of 2000lx, the photoperiod of 12h/d and the temperature of 18 ℃, and the growth condition is regularly observed.

The experimental results are shown in table 5, in the aspect of inducing oyster leafbud to root, the vain growth condition, the root length and the rooting rate condition of the bud are comprehensively considered, and a better culture medium formula and temperature conditions are obtained: 1/4B5+0.2-0.5mg/L IBA +7g/L agar +30g/L sucrose +18 ℃ culture, as shown in FIG. 10, showing rooting in test group 5.

TABLE 5 rooting treatment of oyster leaf buds.

Experimental group	Plant height/cm	Root length/cm	Rooting percentage/%)
				1	5.96±0.96	4.83±3.41	50
2	7.2±1.03	7.16±2.54	41.16
				3	7±1.61	6.06±1.5	66.67
4	5.7±1.31	4±0.89	75
				5	6.56±0.45	5.16±1.12	66.67
6	5.9±1.14	5.83±1.12	58.33
				7	8.33±0.93	6±0.77	58.33
8	7.8±0.47	7.5±3.49	50
				9	7.33±1.8	5.83±3.35	41.16

Example 4

Taking out oyster leaf seedlings with roots, washing the roots, transplanting the seedlings on a cultivation plate, hardening the seedlings, raising the seedlings, and separately cultivating the seedlings (as shown in figure 10), thereby obtaining oyster leaf products.

In the invention, tillering buds (lateral buds or axillary buds) of oyster leaves are preferably used as materials, the optimal growth conditions of each stage of tissue culture are explored, a tissue culture rapid propagation system of the oyster leaves is established, high-quality aseptic seedlings with good growth can be obtained in multiple times in aseptic seedling and bud propagation steps by the tissue culture method of the method, and plants with high rooting rate and longer root length can be obtained by rooting culture, so that the success rate of subsequent culture is facilitated.

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or terminal that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or terminal. Without further limitation, an element defined by the phrases "comprising … …" or "comprising … …" does not exclude the presence of additional elements in a process, method, article, or terminal that comprises the element. Further, herein, "greater than," "less than," "more than," and the like are understood to exclude the present numbers; the terms "above", "below", "within" and the like are to be understood as including the number.

Although the embodiments have been described, once the basic inventive concept is obtained, other variations and modifications of these embodiments can be made by those skilled in the art, so that the above embodiments are only examples of the present invention, and not intended to limit the scope of the present invention, and all equivalent structures or equivalent processes using the contents of the present specification and drawings, or any other related technical fields, which are directly or indirectly applied thereto, are included in the scope of the present invention.

Claims (7)

1. A method for propagating oyster leaf tissue culture buds is characterized by comprising the following steps:

(1) selecting an explant of oyster leaves subjected to tissue culture, cleaning the explant of the oyster leaves, sequentially carrying out vibration rinsing in 75% alcohol, soaking and shaking in 0.1% mercuric chloride solution, shaking and soaking in sterile water in a clean bench, and then inoculating the explant to a sterile bud culture medium for culture to obtain aseptic buds of the oyster leaves, wherein the sterile bud culture medium comprises the following components: b5 culture medium, 0.5-1.5mg/L6-BA, 0.1-0.5mg/L NAA, 7g/L agar, 30g/L sucrose;

(2) inoculating oyster leaf sterile buds obtained by culture in a superclean bench to a multiplication culture medium to obtain a subculture sterile seedling, wherein the multiplication culture medium comprises the following components: b5 culture medium, 0.5mg/L6-BA, 0-0.1mg/L NAA, 7g/L agar, 30g/L sucrose;

(3) taking out sterile seedlings for subculture in a superclean bench, cutting and separating the sterile seedlings for division, and respectively inoculating the sterile seedlings to a rooting culture medium to obtain oyster leaf seedlings with roots, wherein the rooting culture medium comprises: 1/4B5 culture medium or 1/2B5 culture medium or B5 culture medium, 0.2-1.0mg/L IBA, 7g/L agar, 30g/L sucrose.

2. The method for propagating tissue culture buds of oyster leaves according to claim 1, wherein the explant of the oyster leaves is oyster leaf tillering buds.

3. The method for propagating oyster leaf tissue culture buds as claimed in claim 1, wherein in the step (1), the explants are washed by shaking in 75% alcohol for 45s, soaked in mercuric chloride solution for 45s, and soaked in sterile water for 30 s.

4. The method for propagating oyster leaf tissue culture buds according to claim 1, wherein the sterile bud culture medium contains: 1.5mg/L6-BA, and 0.2mg/L NAA.

5. The method for propagating oyster leaf tissue culture buds according to claim 1, wherein the multiplication medium contains 0.05mg/L NAA.

6. The method for propagating oyster leaf tissue culture buds according to claim 1, wherein the rooting medium comprises the following components: 1/4B5 medium, 0.5mg/L IBA, 7g/L agar, 30g/L sucrose.

7. The method for propagating oyster leaf tissue culture buds according to claim 1, wherein the culture conditions in the steps 1-3 are as follows: placing in a culture chamber with illumination intensity of 2000lx and photoperiod of 12h at 18-24 ℃.

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