阅读说明：本技术 一种矾根丛生芽超低温保存及恢复培养方法 (Ultralow-temperature preservation and recovery culture method for alum root clump buds ) 是由 林田 殷丽青 腾小英 李天菲 韩静 周丽 杨华 刘鸿艳 龙萍 罗利军 于 2020-05-22 设计创作，主要内容包括：本发明公开了一种矾根丛生芽超低温保存及恢复培养方法；包括如下步骤：1.无菌苗体系建立；2.叶片丛生芽的诱导；3.丛生芽的预培养；4.丛生芽的机械固定；5.丛生芽的装载及玻璃化的处理；6.冷冻及解冻洗涤；7.恢复培养。本发明首次提出对矾根叶片诱导丛生芽成功进行超低温保存,保存后矾根恢复生长状况良好。本发明将丛生芽带叶基盘整体作为冻存材料,省去了茎尖剥取的繁琐技术手段,并通过机械固定批量处理,进一步简化操作步骤,最终得到矾根的再生植株；因此,对建立便捷稳定高效的矾根超低温保存体系,有利于矾根属资源规模保存,构建资源库,具有很好的参考价值。(The invention discloses a method for ultralow-temperature preservation and recovery culture of alum root clump buds; the method comprises the following steps: 1. establishing an aseptic seedling system; 2. inducing leaf cluster buds; 3. pre-culturing cluster buds; 4. mechanically fixing the cluster buds; 5. loading cluster buds and vitrifying; 6. freezing and unfreezing and washing; 7. and (5) recovering culture. The invention firstly proposes that the alum root leaves can successfully carry out ultralow temperature preservation on the induced cluster buds, and the recovery growth condition of the alum root after preservation is good. The method takes the cluster bud basal disc with leaves as the whole cryopreservation material, omits the complicated technical means of stem tip stripping, further simplifies the operation steps by mechanical fixation and batch processing, and finally obtains the regeneration plant of the alum root; therefore, the method has good reference value for establishing a convenient, stable and efficient alum root ultralow temperature preservation system, facilitating the scale preservation of alum root resources and establishing a resource library.)

1. The ultralow-temperature preservation method of the alum root clumped buds is characterized by comprising the following steps of:

s1, establishment and multiplication of sterile line: sterilizing basal tissue of alum rhizome, inoculating on MS culture medium containing BA and NAA, and forming cluster buds after 20-40 days; dividing the cluster buds into single buds, and transferring the single buds to an MS culture medium containing BA for enrichment culture;

s2, inducing leaf cluster buds: taking 4-7mm sterile tissue culture seedling leaves, putting the back side of the sterile tissue culture seedling leaves upwards into an MS culture medium containing BA and NAA, and inducing cluster buds with the thickness of 0.5-1.5mm within 15-30 days;

s3, pre-culturing cluster buds: transferring the cluster buds with the leaf basal disc into an MS culture medium containing cane sugar to culture for 1-3 days;

s4, mechanically fixing the cluster buds, namely stringing the leaf base discs with the cluster buds to sterilized 12-15mm pins by using forceps, and stringing 3-8 leaf base discs by each pin to form a leaf base disc string;

s5, cluster bud loading and vitrification treatment: placing the cluster bud leaf basal disc in series in the pretreatment solution, loading for 1hr, transferring into vitrification protectant PVS2, and performing ice bath treatment for 20-60 min.

S6, freezing: and (4) rapidly putting the cluster bud leaf basal disc string into liquid nitrogen for freezing and storing.

2. The method of claim 1, wherein in step S1, the MS medium containing BA and NAA contains 6-BA 2.0mg/L, NAA 0.1.1 mg/L; the MS culture medium containing BA contains 0.1mg/L of 6-BA.

3. The method for cryopreservation of a symplocos alum radicis bud as claimed in claim 1, wherein in step S1, the culture temperature of proliferation culture is 24 ± 2 ℃, and the light intensity is 30-35 μmol · m^-2·s^-1The illumination time is 10-12 h/d. Subculturing for 1 time every 20-40 d.

4. The method of claim 1, wherein in step S2, the MS medium containing BA and NAA contains 2.0mg/L of 6-BA and 0.1mg/L of NAA.

5. The method for ultralow-temperature storage of alum-rooted shoots according to claim 1, wherein in step S3, the sucrose-containing MS medium contains sucrose in an amount of 0.3 mol/L.

6. The method for cryopreservation of alum-rooted shoots as claimed in claim 1, wherein in step S4, the pretreatment solution is MS culture medium +1.2mol/L glycerol +0.4mol/L sucrose.

7. A method for restoring and culturing alum-rooted shoots after cryopreservation by the method of claim 1, comprising the steps of:

a1, thawing and washing: taking out the frozen fasciculate bud leaf basal disc string, unfreezing in water bath at 35-40 ℃ for 60-90S, and washing in a 1.2M sucrose MS liquid culture medium for 15-20 minutes;

a2, recovery culture: separating the leaf basal disc with the cluster buds from the leaf basal disc string by using tweezers, sucking the cluster buds and the basal tissues of the leaves one by one on sterile filter paper, peeling the cluster buds and the basal tissues of the leaves under a stereoscopic microscope, inoculating the cluster buds to a recovery culture medium, culturing in the dark for 7 days, and then turning to normal illumination for culture.

8. The recovery culture method of claim 7, wherein the 1.2M sucrose liquid medium in step A1 is 1/2MS medium +1.2mol/L sucrose.

9. The recovery culture method of claim 7, wherein the recovery medium is 1/2MS Medium +6-BA2mg/L + NAA0.1mg/L + GA in step A2₃0.5mg/L。

10. The recovery culture method according to claim 7, wherein the parameters of the dark culture in the step A2 are a culture temperature of 24. + -. 2 ℃ and a light intensity of 0. mu. mol. m^-2·s^-1(ii) a The parameters of the illumination culture are that the culture temperature is 24 +/-2 ℃, and the illumination intensity is 30-35 mu mol.m^-2·s^-1Illumination of lightThe time is 12 h/d.

Technical Field

The invention belongs to the technical field of plant cell engineering; relates to an ultralow-temperature preservation and recovery culture method of alum root clumped buds, in particular to an ultralow-temperature preservation and plant regeneration method of alum root leaves for inducing clumped buds.

Background

The alum root (Heuchera micrantha) is perennial colorful-leaf herbaceous flower of alum root of Saxifragaceae, has the characteristics of colorful and evergreen leaf color, cold resistance, yin resistance, drought resistance, salt and alkali resistance and the like, is widely applied in gardens and has good development prospect. The alum belongs to more than 40 varieties and a plurality of horticultural hybrid varieties, and the effective preservation of germplasm resources of the alum is very important for cultivating new varieties with independent intellectual property rights. The conventional alum root propagation method comprises sowing and plant division, most alum root varieties are difficult to fruit, and the sexual propagation method cannot keep the original variety characteristics of alum roots; the propagation coefficient of the plant is extremely low, and the large-scale production cannot be realized; the preservation in the nursery land is time-consuming and labor-consuming, and is easily affected by natural disasters to cause germplasm loss.

The culture of plant tissues and cells opens up a new way for the germ plasm preservation of plants. The tissue culture can propagate in large amount and the regenerated plant can maintain the original genetic characteristic. In recent years, researchers have increased research on alum root tissue culture technology, but tissue culture propagation cost is high, the process is complicated, the period is long, and a more preservation method needs to be established urgently. Under the condition of ultralow temperature preservation (-196 ℃), the biochemical activity of the plant is nearly stopped, the physiological and genetic changes in the storage process can be controlled to the minimum, the germplasm variation or destruction caused by the fact that the field preservation is easily affected by natural disasters and the risk of genetic character variation or pollution caused by multiple subcultures in the tissue culture preservation are avoided, and the method is considered to be the optimal selection for long-term preservation of the plant genetic resources. In the research on ultra-low temperature preservation of plants, progress has been made in diversification and simplification of preservation techniques, and the number of plant species that can be preserved has been increasing, but most of them have been focused on ultra-low temperature preservation of plant genetic resources including woody plants such as apples, cherries, oranges, bananas, cassava, and the like, and economic plants such as potatoes, sweet potatoes, strawberries, and the like. At present, the ultralow-temperature preservation of alum roots is not reported. The stem tip tissue is good in genetic stability and generally used as a cryopreservation material, but the stem tip tissue is small and tender, so that the requirement on the technical level of workers is high, and the stem tip tissue is easily damaged by machinery in the operation process of stripping and subsequent cryopreservation, and the survival rate is influenced. The method takes the integrated tissue of the cluster bud matrix with the leaf blade as the cryopreservation material, omits the complicated technical means of stem tip stripping, takes the ready-made easily-obtained pin as a fixture, and utilizes the material to mechanically fix and carry out batch treatment, thereby further simplifying the operation steps and finally obtaining the regeneration plant of the alum root; therefore, the method has good reference value for establishing a convenient, stable and efficient alum root ultralow temperature preservation system, facilitating the scale preservation of alum root resources and establishing a resource library.

Disclosure of Invention

The invention aims to provide an ultralow-temperature preservation method of alum root cluster buds and a recovery culture method of the alum root cluster buds after ultralow-temperature preservation.

The purpose of the invention is realized by the following technical scheme:

the invention relates to an ultralow-temperature preservation method of alum root clump buds, which comprises the following steps:

s1, establishment and multiplication of sterile line: sterilizing basal tissue of alum rhizome, inoculating on MS culture medium containing BA and NAA, and forming cluster buds after 20-40 days; dividing the cluster buds into single buds, and transferring the single buds to an MS culture medium containing BA for enrichment culture;

s2, inducing leaf cluster buds: taking 4-7mm sterile tissue culture seedling leaves, putting the back side of the sterile tissue culture seedling leaves upwards into an MS culture medium containing BA and NAA, and inducing cluster buds with the thickness of 0.5-1.5mm within 15-30 days;

s3, pre-culturing cluster buds: transferring the cluster buds with the leaf basal disc into an MS culture medium containing cane sugar to culture for 1-3 days;

s4, mechanically fixing the cluster buds, namely stringing the leaf base discs with the cluster buds to sterilized 12-15mm pins by using forceps, and stringing 3-8 leaf base discs by each pin to form a leaf base disc string;

s5, cluster bud loading and vitrification treatment: placing the cluster bud leaf basal disc in series in the pretreatment solution, loading for 1hr, transferring into vitrification protectant PVS2, and performing ice bath treatment for 20-60 min.

S6, freezing: and (4) rapidly putting the cluster bud leaf basal disc string into liquid nitrogen for freezing and storing.

The clump bud leaf base plate string is used as the whole frozen material in the steps of loading, vitrification, freezing and subsequent unfreezing. In the recovery culture stage after thawing and washing, the cluster buds need to be peeled off from the basal tissues of the leaves for recovery culture.

As an embodiment of the present invention, in step S1, the MS medium containing BA and NAA contains 2.0mg/L, NAA 0.1.1 mg/L of 6-BA; the MS culture medium containing BA contains 0.1mg/L of 6-BA.

In one embodiment of the present invention, in step S1, the culture temperature for propagation culture is 24. + -.2 ℃ and the light intensity is 30 to 35. mu. mol. m^-2·s^-1The illumination time is 10-12 h/d. Subculturing for 1 time every 20-40 d.

As an embodiment of the present invention, in step S2, the MS medium containing BA and NAA contains 2.0mg/L of 6-BA and 0.1mg/L of NAA.

As an embodiment of the present invention, in step S3, the sucrose-containing MS medium contains sucrose at 0.3 mol/L.

As an embodiment of the present invention, in step S4, the pretreatment solution is MS medium +1.2mol/L glycerol +0.4mol/L sucrose.

The invention also relates to a recovery culture method of the alum root clumped buds preserved at ultralow temperature by adopting the method, which comprises the following steps:

a1, thawing and washing: taking out the frozen fasciculate bud leaf basal disc string, unfreezing in water bath at 35-40 ℃ for 60-90S, and washing in a 1.2M sucrose MS liquid culture medium for 15-20 minutes;

a2, recovery culture: separating the leaf basal disc with the cluster buds from the leaf basal disc string by using tweezers, sucking the cluster buds and the basal tissues of the leaves one by one on sterile filter paper, peeling the cluster buds and the basal tissues of the leaves under a stereoscopic microscope, inoculating the cluster buds to a recovery culture medium, culturing in the dark for 7 days, and then turning to normal illumination for culture.

As an embodiment of the present invention, in the step A1, the 1.2M sucrose liquid medium is 1/2MS medium +1.2mol/L sucrose.

As an embodiment of the present invention, in the step A2, the recovery medium is 1/2MS medium +6-BA2mg/L + NAA0.1mg/L + GA₃0.5mg/L。

As an embodiment of the present invention, in step A2, the parameters of dark culture are that the culture temperature is 24 + -2 deg.C, and the light intensity is 0 μmol. m^-2·s^-1(ii) a The parameters of the illumination culture are that the culture temperature is 24 +/-2 ℃, and the illumination intensity is 30-35 mu mol.m^-2·s^-1The illumination time is 12 h/d.

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

1) according to the invention, the alum root leaves are successfully subjected to ultralow temperature preservation on cluster buds induced by the alum root leaves for the first time, and the condition of recovery of growth of the alum root after preservation is good;

2) the invention takes the whole leaf basal disc with cluster buds as the cryopreservation material, thereby omitting the complicated technical means of stem tip stripping and finally obtaining the regeneration plant of the alum root;

3) through the method of mechanical fastening, assemble a plurality of leaf base discs of taking cluster bud and form the leaf base disc cluster, be favorable to batch processing, further simplify the operating procedure, specifically, the advantage lies in: 1. further batch processing of frozen material (stem tip-multiple shoots-leaf base disc string); 2. in the subsequent operation process, the direct contact of the tweezers with the material can be avoided through the treatment of the carrier (pins), so that the mechanical damage is avoided; 3. because the pin is directly used for mechanical fixation, the tedious operation procedure of embedding and fixing, the possible pollution and the trouble of embedding reagent preparation are avoided;

4) the method is favorable for establishing a convenient, stable and efficient alum root ultralow-temperature preservation system, is favorable for scale preservation of alum root resources, establishes a resource library, and has a good reference value.

Drawings

FIG. 1 is a schematic diagram of a sterile tissue culture seedling of alum root;

FIG. 2 is a schematic diagram of the secondary transfer of alum root clumped buds;

FIG. 3 is a schematic view of a leaf disk with multiple shoots;

FIG. 4 is a schematic view of a string of leaf disks;

FIG. 5 is a schematic representation of the recovery of cultured regenerated shoots after freezing; wherein the upper right figure is the regenerated bud detail.

Detailed Description

The present invention will be described in detail with reference to examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that it would be apparent to those skilled in the art that several modifications and improvements can be made without departing from the inventive concept. All falling within the scope of the present invention.