阅读说明：本技术 石榴种质化学离体保存方法 (Chemical in-vitro preservation method for pomegranate germplasm ) 是由 钱晶晶 王宁 于 2021-07-26 设计创作，主要内容包括：本发明公开了石榴种质化学离体保存方法,方法步骤如下：S11：将石榴组培苗切段,每段包含2-3个生长点；S12：配置WPM+IBA 0.5-0.7mg·L～(-1)+琼脂6g·L～(-1)+蔗糖20-30g·L～(-1)+多效唑5-9mg·L～(-1)培养基；S13：将S11中切段后的石榴组培苗接种到S2中的培养基,并置于培养室内变温保存。本发明石榴种质离体保存打破了常规石榴保存因自然灾害而出现的品种或种质丢失的困境,也可以在实际工业化大规模生产上,将不繁殖品种进行储存备用,在需要时直接扩繁,极适合企业工厂化育苗及科学研究,此外本发明打破木本果树由于褐化、极易木质化等问题带来的种质离体保存为零的研究状态,很好的克服了在离体保存过程中出现的褐化问题,显著地提高了离体保存时间,在保存180d时仍可保证组培苗100％的存活率。(The invention discloses a chemical in-vitro preservation method of pomegranate germplasm, which comprises the following steps: s11: cutting pomegranate tissue culture seedlings into sections, wherein each section comprises 2-3 growing points; s12: the WPM + IBA is prepared into 0.5-0.7 mg.L ‑1 + agar 6 g. L ‑1 + sucrose 20-30 g.L ‑1 + paclobutrazol 5-9 mg. L ‑1 A culture medium; s13: and inoculating the pomegranate tissue culture seedlings cut into sections in the S11 to a culture medium in the S2, and placing the pomegranate tissue culture seedlings in a culture room for temperature-changing storage. The method breaks the dilemma of variety or germplasm loss caused by natural disasters in the conventional pomegranate germplasm in vitro preservation, can also store the non-propagated variety for later use in actual industrialized large-scale production, directly expands and propagates the non-propagated variety as required, is extremely suitable for industrial seedling culture and scientific research of enterprises, breaks the research state that the germplasm in vitro preservation of woody fruit trees is zero due to the problems of browning, easy lignification and the like, well overcomes the browning problem in the in vitro preservation process, remarkably improves the in vitro preservation time, and can still ensure the survival rate of 100 percent of tissue culture seedlings when preserved for 180 days.)

1. The culture medium for chemically preserving the pomegranate germplasm in vitro is characterized by comprising the following components in parts by weight: WPM + IBA0.5-0.7 mg.L^-1+ agar 6 g. L^-1+ sucrose 20-30 g.L^-1+ paclobutrazol 5-9 mg. L^-1。

2. The chemical in vitro preservation method of pomegranate germplasm is characterized by comprising the following steps:

s11: cutting pomegranate tissue culture seedlings into sections, wherein each section comprises 2-3 growing points;

s12: configuring the culture medium of claim 1;

s13: and inoculating the pomegranate tissue culture seedlings cut into sections in the S11 to a culture medium in the S12, and placing the pomegranate tissue culture seedlings in a culture room for temperature-changing storage.

3. The method for chemically preserving pomegranate seed substance in vitro according to claim 2, wherein the pH of the medium is 6.3 to 6.7.

4. The method for chemically preserving pomegranate seed substance in vitro as claimed in claim 2, wherein the medium is autoclaved at 122 ℃ for 15-25min before inoculation.

5. The chemical in vitro preservation method for pomegranate germplasm of claim 2, wherein the conditions for temperature preservation in S13 are as follows: humidity is 50-80%, illumination intensity is 2000-.

6. The method for chemically preserving pomegranate seed substance in vitro according to claim 2, wherein the tissue culture seedlings of pomegranate in S11 are acclimatized so as not to generate browning phenomenon during the in vitro preservation.

7. The pomegranate germplasm chemical in vitro preservation method according to claim 6, wherein the method for domesticating pomegranate tissue culture seedlings comprises the following steps:

s21: preparing a culture medium containing activated carbon for pomegranate tissue culture;

s22: selecting disease-free and insect-free pomegranate branches, and performing stem tip detoxification and inoculation on the pomegranate branches to a culture medium in S21;

s23: subculturing every 30-40d for 1-2 times to obtain domesticated pomegranate tissue culture seedling without browning.

8. The method for chemically preserving pomegranate germplasm in vitro according to claim 7, wherein the culture medium in the S21 is WPM + IBA0.5-0.7 mg-L^-1+ agar 6 g. L^-1+ sucrose 20-30 g.L^-1+ 0.6-1.2 g.L of active carbon^-1。

9. The method for chemically preserving pomegranate germplasm in vitro according to claim 7, wherein the addition amount of activated carbon in the new culture medium for each subculture is reduced by 0.2-0.4 g-L^-1。

Technical Field

The invention relates to the technical field of plant tissue culture, in particular to a chemical in-vitro preservation method for pomegranate germplasm.

Background

In vitro preservation is a germplasm resource preservation method established on the basis of a tissue culture technology, is a new approach for genetic diversity of plant germplasm resources, can prolong the preservation period of tissue culture seedlings, avoids excessive and frequent subculture, saves the cost of land, labor, financial resources and the like, is not influenced by various plant diseases and insect pests and natural disasters, and maintains the excellent characteristics of excellent quality and difficult degradation of tissue culture seedlings. Is one of the important research means for germplasm collection, breeding and industrial production.

At present, the biggest problem of in vitro preservation of pomegranate is that the content of polyphenol is high, and the phenomenon of browning is easy to occur in the preservation process, so that the survival rate of in vitro preservation of pomegranate germplasm is influenced.

Disclosure of Invention

Based on the technical problems in the background art, the invention provides a chemical in-vitro preservation method for pomegranate germplasm, which effectively inhibits the browning problem in the in-vitro preservation process of the pomegranate germplasm and prolongs the preservation period.

The invention provides a culture medium for chemical in-vitro preservation of pomegranate germplasm, which comprises the following components in parts by weight: WPM + IBA0.5-0.7 mg.L^-1+ agar 6 g. L^-1+ sucrose 20-30 g.L^-1+ paclobutrazol 5-9 mg. L^-1。

The invention provides a chemical in-vitro preservation method of pomegranate germplasm, which comprises the following steps:

s11: cutting pomegranate tissue culture seedlings into sections, wherein each section comprises 2-3 growing points;

s12: preparing the culture medium;

s13: and inoculating the pomegranate tissue culture seedlings cut into sections in the S11 to a culture medium in the S12, and placing the pomegranate tissue culture seedlings in a culture room for temperature-changing storage.

Preferably, the pH of the medium is 6.3-6.7.

Preferably, the medium is autoclaved at a temperature of 120 ℃ and 122 ℃ for 15-25min before inoculation.

Preferably, the conditions for temperature preservation in S13 are as follows: humidity is 50-80%, illumination intensity is 2000-.

Preferably, the pomegranate tissue culture seedlings in S11 are acclimatized so that no browning occurs in the ex vivo storage.

Preferably, the method for domesticating the pomegranate tissue culture seedlings comprises the following steps:

s21: preparing a culture medium containing activated carbon for pomegranate tissue culture;

s22: selecting disease-free and insect-free pomegranate branches, and performing stem tip detoxification and inoculation on the pomegranate branches to a culture medium in S21;

s23: subculturing every 30-40d for 1-2 times to obtain domesticated pomegranate tissue culture seedling without browning.

Preferably, the culture medium in the S21 is WPM + IBA0.5-0.7 mg.L^-1+ agar 6 g. L^-1+ sucrose 20-30 g.L^-1+ 0.6-1.2 g.L of active carbon^-1。

Preferably, the addition amount of activated carbon in the new culture medium for each subculture is reduced by 0.2-0.4 g.L^-1。

The action mechanism is as follows:

in tissue culture, browning is caused by the fact that phenolic acid substances form quinones under the catalytic action of various oxidases, and the quinones generate colored substances through polymerization so as to cause the tissue to be brown; this phenomenon inhibits plant growth or poisons the entire explant tissue, and has a serious impact on inducing dedifferentiation of the explant and redifferentiation of the culture, and thus is crucial to the success of tissue culture of certain plants. Particularly, woody plants, especially fruit trees, contain classified compounds much higher than those of herbaceous or vine plants, so that how to reduce and inhibit browning of these plants becomes one of the main difficulties in overcoming the tissue culture of fruit trees.

Especially for pomegranate, its polyphenol content is high, the problem that browning appears in the tissue culture in-process very easily, and this application is through the selection to adsorbent and plant growth regulator to carry out the separation preservation through the alternating temperature, fine solution the browning problem that appears in the separation preservation of pomegranate.

Compared with the prior art, the invention has the beneficial technical effects that:

(1) the in vitro culture of the pomegranate is one of the steps of the industrialized growth of pomegranate seedlings, the good detoxification effect can ensure the high yield of the pomegranate seedlings, the seedlings with consistent growth vigor can ensure the unified management of pomegranate trees in production, the labor and the material resources are saved, and particularly, the dilemma that the varieties of the pomegranate are degraded by means of cutting propagation can be broken.

(2) The preservation in vitro of the pomegranate germplasm breaks the dilemma of variety or germplasm loss caused by natural disasters in the conventional preservation of the pomegranate, can also store the variety which is not bred for later use in actual industrialized large-scale production, directly expands and breeds when needed, and is extremely suitable for industrial seedling culture and scientific research of enterprises.

(3) The pomegranate is used as a research sample, the research state that germplasm in vitro preservation brought by problems of browning, very easy lignification and the like of woody fruit trees is zero is broken, the browning problem occurring in the in vitro preservation process is well overcome, the in vitro preservation time is remarkably prolonged, and the survival rate of 100 percent of tissue culture seedlings can still be ensured when 180d is preserved.

Drawings

FIG. 1 shows tissue culture seedlings after 180 days of in vitro preservation according to example 1 of the present invention;

FIG. 2 shows a tissue culture seedling of comparative example 1 in vitro after 180 days of storage;

FIG. 3 shows a tissue culture seedling of comparative example 2 according to the present invention after being preserved in vitro for 180 days.

Detailed Description

The WPM is a culture medium for woody plants, and is purchased from Qingdao Haibo organisms; IBA (indoleacetic acid), agar, sucrose and paclobutrazol were all analytical grade and purchased from national reagent (Hu test).

Example 1

The invention provides a chemical in-vitro preservation method of pomegranate germplasm, which comprises the following steps:

s11: cutting pomegranate tissue culture seedlings into sections, wherein each section comprises 2 growing points;

s12: the WPM + IBA is 0.5 mg.L^-1+ agar 6 g. L^-1+ sucrose 20 g.L^-1+ paclobutrazol 5 mg.L^-1The culture medium has pH of 6.3, and is autoclaved at 120 deg.C for 15min before inoculation;

s13: inoculating the pomegranate tissue culture seedlings cut into sections in the S11 to a culture medium in the S12, and placing the pomegranate tissue culture seedlings in a culture room for temperature change storage, wherein the specific storage conditions are that the humidity is 50%, the illumination intensity is 2000Lx, the illumination period is 16L:8D, the temperature under the illumination condition is 23 ℃ and the temperature under the dark condition is 14 ℃.

Before in vitro preservation, pomegranate tissue culture seedlings need to be acclimatized so as not to generate browning phenomenon in the in vitro preservation, and the acclimatization treatment method comprises the following steps:

s21: the WPM + IBA is 0.5 mg.L^-1+ agar 6 g. L^-1+ sucrose 20 g.L^-1+ 0.6 g.L of activated carbon^-1A culture medium;

s22: selecting disease-free and insect-free pomegranate branches, and performing stem tip detoxification and inoculation on the pomegranate branches to a culture medium in S21;

s23: subculturing every 30d for 1 time to obtain domesticated non-browning tissue culture seedling of fructus Punicae Granati, and reducing the addition amount of active carbon in new culture medium by 0.2 g.L^-1。

After the tissue culture seedlings are preserved in vitro for 180 days by adopting the scheme, the survival rate is still 100 percent.

Example 2

The invention provides a chemical in-vitro preservation method of pomegranate germplasm, which comprises the following steps:

s11: cutting pomegranate tissue culture seedlings into sections, wherein each section comprises 3 growing points;

s12: the WPM + IBA is 0.7 mg.L^-1+ agar 6 g. L^-1+ sucrose 30 g.L^-1+ paclobutrazol 9 mg.L^-1The culture medium has pH of 6.7, and is autoclaved at 122 deg.C for 25min before inoculation;

s13: inoculating the pomegranate tissue culture seedlings cut into sections in the S11 to a culture medium in the S12, and placing the pomegranate tissue culture seedlings in a culture room for temperature change storage, wherein the specific storage conditions are that the humidity is 80%, the illumination intensity is 5000Lx, the illumination period is 16L:8D, the temperature under the illumination condition is 27 ℃ and the temperature under the dark condition is 18 ℃.

Before in vitro preservation, pomegranate tissue culture seedlings need to be acclimatized so as not to generate browning phenomenon in the in vitro preservation, and the acclimatization treatment method comprises the following steps:

s21: the WPM + IBA is 0.7 mg.L^-1+ agar 6 g. L^-1+ sucrose 30 g.L^-1+ activated carbon 1.2 g.L^-1A culture medium;

s22: selecting disease-free and insect-free pomegranate branches, and performing stem tip detoxification and inoculation on the pomegranate branches to a culture medium in S21;

s23: subculturing every 40d for 2 times to obtain domesticated non-browning tissue culture seedling of fructus Punicae Granati, and reducing the addition amount of active carbon in new culture medium by 0.4 g.L^-1。

After the tissue culture seedlings are preserved in vitro for 180 days by adopting the scheme, the survival rate is still 100 percent.

Example 3

The invention provides a chemical in-vitro preservation method of pomegranate germplasm, which comprises the following steps:

s11: cutting pomegranate tissue culture seedlings into sections, wherein each section comprises 2 growing points;

s12: the WPM + IBA is 0.6 mg.L^-1+ agar 6 g. L^-1+ sucrose 25 g.L^-1+ paclobutrazol 7 mg.L^-1The culture medium has pH of 6.5, and is autoclaved at 121 deg.C for 20min before inoculation;

s13: inoculating the pomegranate tissue culture seedlings cut into sections in the S11 to a culture medium in the S12, and placing the pomegranate tissue culture seedlings in a culture room for temperature-changing storage, wherein the specific storage conditions are that the humidity is 65%, the illumination intensity is 3500Lx, the illumination period is 16L:8D, the temperature under the illumination condition is 25 ℃ and the temperature under the dark condition is 16 ℃.

Before in vitro preservation, pomegranate tissue culture seedlings need to be acclimatized so as not to generate browning phenomenon in the in vitro preservation, and the acclimatization treatment method comprises the following steps:

s21: the WPM + IBA is 0.6 mg.L^-1+ agar 6 g. L^-1+ sucrose 25 g.L^-1+ 1.0 g.L of activated carbon^-1A culture medium;

s22: selecting disease-free and insect-free pomegranate branches, and performing stem tip detoxification and inoculation on the pomegranate branches to a culture medium in S21;

s23: subculturing every 35d for 2 times to obtain domesticated non-browning tissue culture seedling of fructus Punicae Granati, and reducing the addition amount of active carbon in new culture medium by 0.3 g.L^-1。

After the tissue culture seedlings are preserved in vitro for 180 days by adopting the scheme, the survival rate of the tissue culture seedlings is still 100 percent, and the tissue culture seedlings are shown in a figure 1.

Comparative example 1

The difference between this protocol and example 3 is that the storage temperature of S13 is kept at 25 deg.C, and the other conditions are the same as example 3.

The survival rate of the tissue culture seedlings after 180 days of in vitro preservation by adopting the scheme is only 81.25 percent, and the tissue culture seedlings are shown in figure 2.

Comparative example 2

The difference between the scheme and the embodiment 3 is that the domestication treatment is not carried out before the tissue culture, the other conditions are the same as the embodiment 3, and the tissue culture seedling is shown in a figure 3.

After the scheme is adopted, the survival rate of the tissue culture seedlings is 0% after the tissue culture seedlings are preserved for 180 days in vitro, and the problem of browning is caused in the tissue culture process.

In addition, in order to demonstrate the preservation effect of the chemical in vitro preservation method for pomegranate seeds, the growth amount and rooting condition of each tissue culture seedling after 180d of in vitro preservation and 90d of transplantation of the acclimated tissue culture seedling were analyzed, and the results are shown in tables 1 and 2.

TABLE 1 tissue culture seedling transplanting 90d growth rate

Group of	Net height (cm)	Ground diameter (cm)
			Tissue culture seedling after 180 days of in vitro preservation	80.3±2.85a	15.6±0.41a
Domesticated tissue culture seedling	80.9±2.93a	16.2±0.35a

Note: mean ± standard deviation in the table, different lower case letters indicate significant difference (p < 0.05).

TABLE 2 transplanting of tissue culture seedlings for 90 days to take root

Group of	Net height (cm)	Ground diameter (cm)
			Tissue culture seedling after 180 days of in vitro preservation	80.3±2.85a	15.6±0.41a
Domesticated tissue culture seedling	80.9±2.93a	16.2±0.35a

Note: mean ± standard deviation in the table, different lower case letters indicate significant difference (p < 0.05).

As can be seen from tables 1 and 2, the net growth and rooting state of the tissue culture seedlings after hardening-seedling and transplanting and the tissue culture seedlings after direct transplanting and domestication which are preserved for 180 days by adopting the in vitro preservation method are not obvious in difference, which shows that the in vitro preservation method has good preservation effect, breaks through the dilemma of variety or germplasm loss caused by natural disasters in the preservation of the conventional pomegranate, can also store the non-propagated varieties for later use in actual industrial large-scale production, directly expands propagation when needed, and is very suitable for industrial seedling culture and scientific research of enterprises.

The present invention is not limited to the above preferred embodiments, and any modifications, equivalent substitutions, improvements, etc. within the spirit and principle of the present invention should be included in the protection scope of the present invention.