Method for preserving sugarcane germplasm resources in vitro

Abstract

The application discloses a method for preserving sugarcane germplasm resources in vitro, and belongs to the technical field of sugarcane germplasm preservation. The method comprises the following steps: taking a sugarcane tender leaf sheath as an explant, inducing and culturing callus, obtaining embryogenic cell clusters, and cryopreserving the obtained embryogenic cell clusters; the culture medium for the callus induction culture and the proliferation culture both contain mannitol and corn silk extract; the corn silk extract is an alcohol extract of corn silk. By adding mannitol and corn silk extract in the callus induction culture medium and the proliferation culture medium, the browning of the explant in the process of sugarcane in vitro tissue culture is effectively alleviated, the light condition is optimized, the activity of the embryogenic callus cell clusters is improved, the generation of sugarcane in vitro regenerated seedlings is promoted, and the survival rate of the regenerated seedlings after transplanting is improved.

Description

Technical Field

[0001] This invention relates to the field of sugarcane germplasm preservation technology, and in particular to a method for preserving sugarcane germplasm resources in vitro. Background Technology

[0002] Sugarcane (Saccharum officinarum L.) is a temperate and tropical crop, used as a raw material for sugar production, and can also be used to extract ethanol as an energy substitute. Sugarcane is rich in sugar and water, and also contains various vitamins, fats, proteins, organic acids, calcium, iron, and other substances that are very beneficial to human metabolism. It is mainly used for sugar production, and its skin is generally purple and green, but can also be red and brown.

[0003] The complex genetic background of sugarcane, along with its unclear origin and evolution, and the lag in genomics research, has hindered the optimal selection of parental materials in sugarcane breeding, severely restricting the development of the sugarcane industry. Germplasm resources are the genetic material directly passed from parents to offspring through reproductive cells or somatic cells, determining their traits. They are the material basis for biological genetic variation and biodiversity, and the genetic basis for the breeding and improvement of new varieties. The best method for preserving germplasm resources is through seed tuber preservation. With the development of tissue cell culture, in vitro preservation techniques for plant tissue cells have become increasingly mature.

[0004] In vitro preservation is a method of storing plant tissue cultures, such as single cells, protoplasts, callus, suspension cells, somatic embryos, and test-tube seedlings, under conditions that inhibit or eliminate growth, thereby achieving the purpose of preservation. Current research on sugarcane has found that the survival rate of sugarcane regenerated plants obtained through in vitro preservation is low, and multiple subcultures are required during the process. Frequent subcultures can easily lead to cross-contamination and genetic variation. Furthermore, factors such as endophytic contamination and browning of explants also restrict the induction of sugarcane callus and the differentiation efficiency of embryogenic cell clusters, resulting in unsatisfactory repeatability of sugarcane germplasm resource regeneration experiments using in vitro preservation. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preserving sugarcane germplasm resources in vitro, thereby solving the problems existing in the prior art, effectively alleviating the browning phenomenon of explants during sugarcane in vitro tissue culture, improving the vitality of embryogenic callus cell clusters, promoting the generation of sugarcane in vitro regenerated seedlings, and improving the transplant survival rate of regenerated seedlings.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a method for preserving sugarcane germplasm resources in vitro, comprising the following steps:

[0008] Using sugarcane leaf sheaths as explants, embryogenic cell clusters were obtained through callus induction culture and proliferation culture, and the embryogenic cell clusters were then preserved at ultra-low temperature.

[0009] The culture medium components for both the callus induction culture and the proliferation culture contain mannitol and corn silk extract.

[0010] The corn silk extract is an alcoholic extract of corn silk.

[0011] Furthermore, the culture medium for inducing callus induction consists of: 1 / 2 MS + 3 mg / L 2,4-D + 1.5 mg / L NAA + 0.2 mg / L 6-BA + 3 mg / L corn silk extract + 10 mg / L ascorbic acid + 8 mg / L mannitol + 20 g / L sucrose.

[0012] Furthermore, the culture medium for proliferation culture consists of: 1 / 2 MS + 3 mg / L 2,4-D + 3 mg / L NAA + 0.2 mg / L 6-BA + 3 mg / L corn silk extract + 10 mg / L ascorbic acid + 8 mg / L mannitol + 30 g / L sucrose.

[0013] Furthermore, the corn silk extract is obtained by heating and reflux extraction of corn silk and ethanol at a mass ratio of 1:5.

[0014] Furthermore, prior to the heating and reflux extraction, corn silk is soaked in water at a mass-volume ratio of 1g:10mL for 24 hours.

[0015] Furthermore, the heating and reflux extraction is followed by rotary evaporation to remove ethanol.

[0016] Furthermore, the callus induction culture is carried out by first culturing in the dark for 5 days, and then culturing in the light for 5 days; the intensity of the light is 1300 lx.

[0017] Furthermore, the proliferation culture is carried out under light conditions for 10 days; the light intensity is 1800 lx.

[0018] The present invention discloses the following technical effects:

[0019] This invention first cultured the explants in a dark environment, and then cultured them under light, which is more conducive to the formation of callus. By adding mannitol and corn silk extract to the callus induction medium and proliferation medium, the browning of explants during the culture of sugarcane tissue was effectively alleviated. Among them, mannitol, as an osmotic pressure regulator, works together with sterols and flavonoids in corn silk extract to enhance the inhibitory effect on browning of sugarcane tissue.

[0020] Optimized light conditions enhanced the ability of induced callus tissue to generate embryogenic cell clusters. The addition of mannitol and corn silk extract further promoted the formation of embryogenic cell clusters and improved their cell viability, thereby promoting the generation of sugarcane seedlings and increasing the transplant survival rate of the regenerated seedlings. Detailed Implementation

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0026] Example 1

[0027] (1) Obtaining explants

[0028] Select healthy, disease-free sugarcane plant tail sheaths from the field, sterilize their surfaces by wiping them with a 75% ethanol solution, peel off the surface leaves, wipe the surface again with a 75% ethanol solution, and then peel them off layer by layer. Take the tender leaf sheaths 0-5cm above the growing point and cut them into 1cm×0.5cm pieces with a scalpel as explants.

[0029] (2) Induction culture

[0030] The explants obtained in step (1) were inoculated into callus induction medium and cultured in the dark at 25°C for 5 days. Then, they were cultured under light for 12 hours / day with a light intensity of 1300 lx for another 5 days to obtain callus tissue.

[0031] The callus induction culture medium consisted of: 1 / 2 MS + 3 mg / L 2,4-D + 1.5 mg / L NAA + 0.2 mg / L 6-BA + 3 mg / L corn silk extract + 10 mg / L ascorbic acid + 8 mg / L mannitol + 20 g / L sucrose.

[0032] The preparation method of corn silk extract is as follows: 2000mL of water is added to 200g of corn silk, soaked for 24h and then dried. The mixture is then mixed with 70% ethanol at a mass ratio of 1:5 and heated under reflux for 5h to obtain the extract. The ethanol is removed by rotary evaporation to obtain the corn silk extract.

[0033] (3) Proliferation culture to induce embryonic cell mass

[0034] The callus obtained from the induction culture in step (2) was transferred to the proliferation medium and cultured at 30°C under light for 10 days with a light intensity of 1800 lx to obtain embryonic cell clusters.

[0035] The proliferation medium consisted of: 1 / 2 MS + 3 mg / L 2,4-D + 3 mg / L NAA + 0.2 mg / L 6-BA + 3 mg / L corn silk extract + 10 mg / L ascorbic acid + 8 mg / L mannitol + 30 g / L sucrose.

[0036] (4) Ultra-low temperature preservation

[0037] The embryonic cell mass obtained in step (3) was transferred into a 10 mL test tube, and then the test tube was placed in an ice bath. After the temperature was constant, a cryoprotectant (10% dimethyl sulfoxide + 0.5 mol / L sorbitol) pre-cooled to 0 °C was added. After pretreatment at 0 °C for 30 min, the cell mass was stored in liquid nitrogen at ultra-low temperature.

[0038] In this embodiment, sugarcane young leaf sheaths were used as explants to construct 40 tubes of sugarcane embryogenic cell clusters. Among them, one tube showed browning during the construction process and failed to successfully construct a sugarcane in vitro regenerated embryogenic cell cluster. The remaining 39 tubes were all successfully constructed. After thawing and recovery, they were further cultured and all regenerated seedlings were obtained. After hardening and transplanting, the survival rate was 97.5%.

[0039] Experimental Example 1

[0040] The callus induction and proliferation media were adjusted as follows: the callus induction medium consisted of 1 / 2 MS + 3 mg / L 2,4-D + 1.5 mg / L NAA + 0.2 mg / L 6-BA + 10 mg / L ascorbic acid + 20 g / L sucrose; the proliferation medium consisted of 1 / 2 MS + 3 mg / L 2,4-D + 3 mg / L NAA + 0.2 mg / L 6-BA + 10 mg / L ascorbic acid + 30 g / L sucrose. Based on this, different concentrations of mannitol were added as shown in Table 1, and the remaining procedures were the same as in Example 1. Forty sugarcane young leaf sheath tissues were subjected to in vitro regeneration culture. The browning of the callus and the formation of embryogenic cell clusters were statistically analyzed, and the results are shown in Table 1. As shown in Table 1, adding mannitol to the callus induction medium and proliferation medium helps to reduce the browning rate of callus and increase the embryogenic cell mass formation rate. When the concentration of mannitol added is 8 mg / L, the browning rate is the lowest and it is most conducive to the formation of embryogenic callus.

[0041] Table 1

[0042]

[0043] Experimental Example 2

[0044] The callus induction and proliferation media were adjusted as follows: the callus induction medium consisted of 1 / 2 MS + 3 mg / L 2,4-D + 1.5 mg / L NAA + 0.2 mg / L 6-BA + 10 mg / L ascorbic acid + 20 g / L sucrose; the proliferation medium consisted of 1 / 2 MS + 3 mg / L 2,4-D + 3 mg / L NAA + 0.2 mg / L 6-BA + 10 mg / L ascorbic acid + 30 g / L sucrose. Based on this, different concentrations of corn silk extract were added as shown in Table 2. The remaining procedures were the same as in Example 1. Forty sugarcane young leaf sheath tissues were subjected to in vitro regeneration culture. The browning of the callus and the formation of embryogenic cell clusters were statistically analyzed. The results are shown in Table 2. As shown in Table 2, adding corn silk extract to the callus induction medium and proliferation medium helps reduce the browning rate of callus and increase the embryogenic cell mass generation rate. When the concentration of corn silk extract is 3 mg / L, the browning rate is the lowest and it is most conducive to the generation of embryogenic callus.

[0045] Table 2

[0046]

[0047] Experimental Example 3

[0048] In the process of in vitro preservation of sugarcane germplasm resources using embryogenic cell clusters generated from callus tissue, although some embryogenic cell clusters were successfully obtained, it was difficult to obtain regenerated plants after thawing and subsequent tissue culture, or although regenerated plants were obtained, the transplant survival rate was low. Therefore, the activity of embryogenic cell clusters is crucial for the construction of regenerated plants. To further improve the activity of embryogenic cell clusters, this invention optimized other conditions in the induction culture. The effects of light conditions on the generation of embryogenic cell clusters during callus induction culture and proliferation culture were verified separately, as shown in Table 3. The remaining operations were the same as in Example 1. Based on this, 40 sugarcane young leaf sheath tissues were subjected to in vitro regeneration culture, and the generation of embryogenic cell clusters was statistically analyzed. At the same time, the TTC method combined with a Spector spectrophotometer was used to detect the absorbance at 485 nm to determine the activity of embryogenic cell clusters. The results are shown in Table 3. The results showed that light conditions have an important influence on the formation of embryogenic cell clusters. Although dark conditions are conducive to callus formation, the induction conditions of darkness followed by light are more conducive to callus formation. Optimizing light intensity can solve the problem of reduced embryogenic cell cluster activity caused by the addition of mannitol and corn silk extract. When the light intensity is adjusted to 1300 lx during the callus induction stage and 1800 lx during the proliferation culture stage, it is conducive to the formation of highly active embryogenic cell clusters.

[0049] Table 3

[0050]

[0051] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for in vitro cell preservation of sugarcane germplasm resources, characterized in that, Includes the following steps: Using sugarcane leaf sheaths as explants, embryogenic cell clusters were obtained through callus induction culture and proliferation culture, and the embryogenic cell clusters were then preserved at ultra-low temperature. The components of the culture medium for callus induction culture are: 1 / 2MS + 3 mg / L 2,4-D + 1.5 mg / L NAA + 0.2 mg / L 6-BA + 3 mg / L corn silk extract + 10 mg / L ascorbic acid + 8 mg / L mannitol + 20 g / L sucrose; The components of the culture medium for proliferation culture are: 1 / 2MS + 3mg / L 2,4-D + 3mg / L NAA + 0.2mg / L 6-BA + 3mg / L corn silk extract + 10mg / L ascorbic acid + 8mg / L mannitol + 30g / L sucrose; The preparation method of the corn silk extract is as follows: 2000mL of water is added to 200g of corn silk, soaked for 24h and then dried. The mixture is then mixed with 70% ethanol at a mass ratio of 1:5 and heated under reflux for 5h to obtain the extract. The ethanol is removed by rotary evaporation of the extract to obtain the corn silk extract. The callus induction culture was carried out by first culturing in the dark for 5 days, and then culturing in the light for 5 days.

2. The method according to claim 1, characterized in that, The intensity of the light is 1300 lx.

3. The method according to claim 1, characterized in that, The proliferation culture was carried out under light conditions for 10 days.

4. The method according to claim 3, characterized in that, The intensity of the light is 1800 lx.

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