Method for in-vitro preservation of common wild rice and Nwavalar wild rice

Abstract

The invention discloses a method for in-vitro preservation of common wild rice and Nwa wild rice, and belongs to the technical field of plant tissue culture. The method comprises the following steps: selecting a stem node with an axillary bud as an explant; soaking in 75% alcohol for 10-15 seconds, soaking in 10% 84 disinfectant for 15 minutes, performing vacuum treatment in 0.1% mercuric chloride solution under-0.1 Mpa for 5 minutes, and performing water bath at 40 DEG C for 20 minutes for combined sterilization; inserting the sterilized explant into a bud differentiation culture medium containing 2mg / L KT and 0.1 mg / L NAA, and culturing for 3-5 days under the conditions that the temperature is 23 DEG C and illumination is performed for 16 hours per day; cutting off buds growing to 3-5cm, and transferring the buds into a rooting culture medium; (5) carrying out test-tube plantlet propagation through stem node shearing or tillering cutting; the tissue culture seedlings with well developed root systems are hardened and then transplanted to a mixture of vermiculite and nutrient soil (v / v = 1 / 1). According to the method, the pollution rate is as low as 16.35%, the differentiation rate of the explant reaches 47.36%, the process is simple, convenient and efficient, the production period is short, the seed quality resources of common wild rice and Nilval wild rice can be effectively preserved, and the method has important significance on protection of precious wild rice resources.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, specifically, it relates to a method for in vitro preservation of common wild rice (…). Oryza rufipogon Griff.) and Nivara wild rice ( Oryza nivara Sharma & Shastry) resource methods. Background Technology

[0002] Wild rice has accumulated a variety of excellent genetic genes under the background of long-term natural selection, such as genes for specific traits such as disease resistance, insect resistance, stress tolerance, high quality, and high yield. It is a valuable gene pool for rice variety improvement.

[0003] Methods for preserving wild rice germplasm resources in China can be categorized into ex-situ and in-situ preservation based on their location, including germplasm nursery, germplasm bank, and native habitat preservation. However, these methods are time-consuming and labor-intensive, and there is a risk of resource contamination or extinction due to natural disasters or human factors, as well as a reduction in genetic diversity. Furthermore, rice germplasm is primarily preserved in the form of seeds. Due to genotypic or environmental influences, some wild rice varieties, as well as sterile lines, aneuploids, and other special resources, may produce few or no seeds, or their offspring may segregate, making seed preservation difficult. Therefore, it is necessary to research safe, reliable, and labor-saving methods as supplementary means of germplasm preservation.

[0004] With the advancement of plant tissue culture research, in vitro preservation techniques for germplasm resources have been applied to the preservation of various plant germplasm. Researchers have established a germplasm preservation method for Muscat Alexandria grapes by using shoot tips and stem segments as explants and inducing them to differentiate into buds and roots. Other studies have used potato shoots and stem segments as explants to cultivate buds and roots, which are then used to preserve germplasm materials of different ploidy levels. Similarly, in vitro culture of garlic, banana, and plantain shoot tips has been used to protect and store their related germplasm resources.

[0005] In the preservation of rice germplasm resources, existing studies have used axillary buds of sterile rice seedlings as explants to induce the production of shoots and roots, thereby propagating wild and hybrid rice materials. Other studies have induced callus formation in the roots of wild rice, which then differentiates into seedlings. However, these methods mostly obtain explants through seed culture of sterile seedlings, which is unsuitable for some special materials. Furthermore, they require 2-3 subcultures to obtain rootable shoots, making the process complex and inefficient. Moreover, current technologies suffer from high contamination rates and low differentiation rates, failing to meet the demands for efficient preservation.

[0006] The relative importance of cytokinins and auxins, two major classes of plant growth regulators, in the development of tissue-cultured plants has long been known. By altering the ratio of cytokinins to auxins in the culture medium, the regeneration of various differentiation patterns in in vitro cultured tissues can be induced, namely embryo formation, organ formation, or root growth. This invention addresses the above-mentioned problems by providing an in vitro preservation method that is simple to operate, has a low contamination rate, and a high differentiation rate. Oryza rufipogon Griff.) and Nivara wild rice ( Oryza nivara The method of Sharma & Shastry. Summary of the Invention

[0007] The purpose of this invention is to provide a method for in vitro preservation of common wild rice and Niwara wild rice. This method is simple to operate, has a low contamination rate and a high differentiation rate, and can effectively preserve the germplasm resources of common wild rice and Niwara wild rice.

[0008] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: This invention provides a method for in vitro preservation of common wild rice and Niwara wild rice, comprising the following steps: (1) Explant preparation: Select healthy, disease-free, and jointed wild rice seedlings, remove the leaves to expose the stem nodes and axillary buds, and cut them 0.5cm-1cm above and below the stem nodes to obtain stem node explants with axillary buds; (2) Sterilization of explants: The explants were subjected to the following sterilization treatments in sequence: immersion in 75% alcohol for 10-15 seconds; immersion in 10% 84 disinfectant for 15 minutes, shaking twice during the process; treatment in 0.1% mercuric chloride solution under -0.1 MPa vacuum for 5 minutes; treatment in a 40℃ water bath for 20 minutes; and rinsing with sterile water 5-7 times. (3) Explant differentiation: Sterilized explants were inserted into the bud differentiation medium in the direction of axillary bud growth, with the axillary bud in contact with the medium. One explant was inoculated per tube and cultured for 3-5 days at a temperature of 23℃ and a light duration of 16 hours / day. (4) Rooting culture: Cut off the buds that have grown to 3cm-5cm and insert them into the rooting culture medium. Inoculate one bud per tube and culture for 1-2 weeks under the conditions of 23℃ and 16 hours / day of light to start rooting; (5) Propagation of test-tube seedlings: Cut off the tillers of the test-tube seedlings that have been cultured for 2-3 months and place them in the rooting medium. At the same time, cut off the stem nodes for bud differentiation culture. (6) Transplanting: After hardening off, tissue culture seedlings with well-developed root systems and 6-8 roots are transplanted into culture pots containing vermiculite and nutrient soil in a volume ratio of 1:1.

[0009] Furthermore, in step (2), Tween 20 is added to both the 84 disinfectant and the 0.1% mercuric chloride solution.

[0010] Further, in step (3), the bud differentiation culture medium is: 4.4 g / L MS powder + 0.1 mg / L NAA + 2 mg / L KT + 300 mg / L Termetine + 400 mg / L Carbenicillin + 7 g / L plant gel, pH 5.6.

[0011] Further, in step (4), the rooting medium is: 2.2 g / L MS powder + 30 g / L sucrose + 4 g / L plant gel, pH 5.6.

[0012] Furthermore, in step (5), the propagation of the test-tube seedlings also includes: cutting off the buds and culturing them in a bud differentiation medium for 1-2 months to produce bud clusters, and then performing rooting culture.

[0013] Furthermore, the bud differentiation culture medium is: 4.4 g / L MS powder + 0.1 mg / L NAA + 2 mg / L KT + 2 mg / L 6-BA + 300 mg / L termethin + 400 mg / L carbenicillin + 30 g / L sucrose + 7 g / L plant gel, pH 5.6.

[0014] Further, in step (6), the seedling hardening process is as follows: the gel on the roots of the tissue culture seedlings is rinsed clean, and the seedlings are placed in a triangular flask containing distilled water and cultured for 3 days at 26°C and 16 hours / day of light. Beneficial effects

[0015] (1) This invention uses stem nodes as explants and collects materials directly from the field, avoiding the tedious process of obtaining explants through seed culture of sterile seedlings. It is particularly suitable for wild rice materials with low seed setting rate or sterility.

[0016] (2) The present invention optimizes the sterilization conditions by using a combination of soaking in 10% 84 disinfectant for 15 minutes, vacuum treatment with 0.1% mercuric chloride for 5 minutes, and treatment at 40°C for 20 minutes, which reduces the explant contamination rate to 16.35% while ensuring a growth rate of 46.15%.

[0017] (3) The present invention has determined the optimal hormone ratio, using a combination of 2 mg / L KT and 0.1 mg / L NAA, which resulted in an explant differentiation rate of 47.36%, significantly higher than other hormone combinations.

[0018] (4) By optimizing the bud differentiation culture medium, the present invention uses a combination of 2 mg / L KT + 0.1 mg / L NAA + 2 mg / L 6-BA, which can produce 3.5 buds after one month, thus improving the propagation efficiency.

[0019] (5) The process of this invention is simple and efficient, with a short production cycle. It can effectively preserve the germplasm resources of common wild rice and Niwara wild rice in vitro, which is of great significance for protecting precious wild rice germplasm resources. Attached Figure Description

[0020] Figure 1 This is a flowchart illustrating the in vitro preservation process of wild rice as provided in an embodiment of the present invention.

[0021] Figure 2 This is a flowchart illustrating the propagation process of test-tube seedlings provided in an embodiment of the present invention.

[0022] Figure 3 A statistical graph showing the differentiation rate of explants under different concentrations of KT and NAA.

[0023] Figure 4 This is a statistical chart showing the number of clustered shoots under different hormone combinations. Detailed Implementation

[0024] To enable those skilled in the art to better understand the technical solutions of this invention, the present application will be further described in detail below with reference to embodiments.

[0025] Example 1: In vitro preservation of common wild rice and Niwara wild rice 1. Explant preparation Select healthy, disease-free, and pest-free common wild rice and Niwara wild rice seedlings that have already jointed, and rinse off the surface soil with running water. Remove the leaves to expose the stem nodes and axillary buds, and cut the explants 0.5cm to 1cm above and below the stem nodes. Place the cut explants in distilled water for later use.

[0026] 2. Sterilization of explants In a clean bench, the explants were immersed in 75% alcohol for 10 to 15 seconds for surface sterilization; then, they were immersed in 10% 84 disinfectant solution (with one drop of Tween 20) for 15 minutes, shaking twice during the process, and the solution was discarded. The explants were then washed twice with sterile water; then, the explants were transferred to 0.1% mercuric chloride solution (with one drop of Tween 20) and treated under a vacuum of -0.1 MPa for 5 minutes; then, they were treated in a 40°C water bath for 20 minutes; next, they were rinsed 5 to 7 times with sterile water, and finally, the explants were placed on sterile filter paper to thoroughly absorb the moisture.

[0027] 3. Explant Differentiation Sterilized explants were inserted into the bud differentiation medium in the direction of axillary bud growth, ensuring that the axillary buds were in contact with the medium. One explant was inoculated per tube and cultured for 3 to 5 days at a temperature of 23°C and a light duration of 16 h / d.

[0028] The bud differentiation medium formula is as follows: 4.4 g / L MS powder + 0.1 mg / L NAA + 2 mg / L KT + 300 mg / L termethin + 400 mg / L carbenicillin + 7 g / L plant gel, with pH adjusted to 5.6 by 0.1 mol / L NaOH.

[0029] 4. Rooting culture Cut off buds that have grown to 3-5 cm and insert them into rooting medium, one bud per tube. Rooting will begin after 1-2 weeks of culture. Continue culture for 2-3 months for propagation. Culture conditions are 23℃ and 16h / d light. Seedlings with 6-8 roots are ready for hardening off and transplanting.

[0030] The rooting medium formula is: 2.2 g / L MS powder + 30 g / L sucrose + 4 g / L plant gel, with 0.1 mol / L NaOH adjusted to 5.6.

[0031] 5. Propagation of in vitro seedlings Method 1: Cut off the stem nodes of the test-tube seedlings using sterile scissors and culture them according to the explant differentiation steps. Cut off the buds with a sterile blade and culture them in a shoot differentiation medium for 1-2 months to produce shoot clusters. Separate the shoot clusters and perform rooting culture according to the rooting culture steps. Figure 2 ).

[0032] Method 2: Remove the test-tube seedlings that have been cultured for 2-3 months in a clean bench. Use a sterile blade to cut off the tillers and place them in rooting medium. Inoculate one tiller per tube and proceed with rooting culture according to the rooting culture steps. Simultaneously, use sterile scissors to cut off the stem nodes of the test-tube seedlings and culture them according to the explant differentiation steps. Cut off the buds and place them in rooting medium, then proceed with rooting culture according to the rooting culture steps. Figure 2 ).

[0033] The formula for the bud differentiation medium is as follows: 4.4 g / L MS powder + 0.1 mg / L NAA + 2 mg / L KT + 2 mg / L 6-BA + 300 mg / L termethin + 400 mg / L carbenicillin + 30 g / L sucrose + 7 g / L plant gel, with the pH adjusted to 5.6 by 0.1 mol / L NaOH.

[0034] 6. Hardening off seedlings and transplanting Select tissue culture seedlings with well-developed root systems, rinse the gel off the roots with tap water, and place the seedlings in Erlenmeyer flasks filled with distilled water. Culture them for 3 days at 26℃ with a light duration of 16 hours / day. Then transplant them into culture pots containing vermiculite and nutrient soil in a 1:1 ratio, water them, and place them in a greenhouse at 26℃ with a light duration of 16 hours / day for further growth.

[0035] See the flowchart for wild rice in vitro preservation. Figure 1 .

[0036] Example 2: Effects of disinfectant combination on explant contamination rate and differentiation rate This embodiment compares the effects of different concentrations of 84 disinfectant and mercuric chloride combined sterilization on the contamination rate and growth rate of explants. The specific methods are as follows: Explants were prepared according to the explant preparation method in Example 1, and then different combinations of disinfectant reagents were set up for treatment (Table 1). Each treatment group was set up with 3 replicates, and each replicate had 30 explants.

[0037] The disinfection method is the same as in Example 1, but the concentration of the disinfectant and the treatment time are changed.

[0038] After sterilization, the explants were inoculated into the shoot differentiation medium described in Example 1 and cultured at 23°C for 15 days under 16 hours of light per day. The contamination rate and differentiation rate of each treatment group were then calculated. Contamination rate = (number of contaminated explants / total number of inoculated explants) × 100%; Differentiation rate = (number of differentiated explants / total number of inoculated explants) × 100%.

[0039] The results showed that the optimal disinfection combination was 10% 84 disinfectant for 15 min, 0.1% mercuric chloride for 5 min under vacuum and 40℃ for 20 min. Under these disinfection conditions, the explant contamination rate was the lowest at 16.35%, and the growth rate was 46.15% (Table 1).

[0040] Table 1. Contamination rate and differentiation rate of explants under different concentrations of 84 disinfectant and HgCl2.

[0041] Example 3: Effects of Hormone Ratio on Explant Differentiation This example compares the effects of different concentrations of NAA and KT on explant differentiation rate, as detailed below: Explants were treated according to the explant preparation and sterilization method described in Example 1.

[0042] Different hormone combinations were set up for treatment: control group (no hormone added), 1 mg / L KT, 2 mg / L KT + 0.1 mg / L NAA, 4 mg / L KT + 0.1 mg / L NAA, and 4 mg / L KT + 0.2 mg / L NAA. Each treatment was set up in 3 replicates, and each replicate contained 30 explants.

[0043] The basic formulation of the shoot differentiation medium was the same as in Example 1, except that the concentrations of NAA and KT were changed. Sterilized explants were inoculated into shoot differentiation media with different hormone combinations and cultured at 23°C under 16 hours of light per day for 30 days. The differentiation rate of each treatment group was then calculated. Differentiation rate = (number of differentiated explants / total number of inoculated explants) × 100%.

[0044] The results showed that the differentiation rate was 9.77% without hormones in the culture medium; 13.64% with only 1 mg / L KT; and 47.36% with 2 mg / L KT and 0.1 mg / L NAA. Increasing the concentration of KT and NAA slightly decreased the differentiation rate, but there was no significant difference compared with the combination of 2 mg / L KT and 0.1 mg / L NAA. Figure 3 Therefore, 2 mg / L KT and 0.1 mg / L NAA is the optimal hormone combination.

[0045] Example 4: Effects of Hormone Combinations on Differentiation of Clustered Buds This example compares the effects of different hormone combinations on the differentiation of clustered shoots, as detailed below: Explants were treated according to the explant preparation and sterilization method in Example 1, and inoculated into the optimal bud differentiation medium (2 mg / L KT + 0.1 mg / L NAA) determined in Example 3 to obtain single buds.

[0046] Three hormone combination treatments were set up: (1) Combination 1: 2 mg / L KT + 0.1 mg / L NAA; (2) Combination 2: 2 mg / L KT + 0.1 mg / L NAA + 2 mg / L 6-BA; (3) Combination 3: 5 mg / L 6-BA + 0.4 mg / L NAA; Each treatment group was set up with 3 replicates, and each replicate had 20 buds.

[0047] The basic formulation of the shoot differentiation medium was the same as in Example 1, except that the hormone combination was changed. The obtained single shoots were inoculated into shoot differentiation mediums with different hormone combinations and cultured at 23°C for 30 days under 16 hours of light per day. The number of shoots in each treatment group was then counted.

[0048] The results showed that combinations 1 (2 mg / L KT + 0.1 mg / L NAA) and 3 (5 mg / L 6-BA + 0.4 mg / L NAA) were not ideal in inducing shoot clusters, producing only 0.75 and 1.25 shoot clusters, respectively. However, combination 2 (2 mg / L KT + 0.1 mg / L NAA + 2 mg / L 6-BA) produced significantly more shoot clusters (3.5) than combinations 1 and 3. Figure 4 Therefore, compared to the other two groups, combination 2 is the most suitable hormone combination for differentiating clustered buds.

[0049] In summary, this invention, by comparing the effects of different concentrations of disinfectant combinations on explant contamination and differentiation rates, determined that disinfection with 10% 84 disinfectant for 15 minutes, vacuum treatment with 0.1% mercuric chloride for 5 minutes, and treatment at 40℃ for 20 minutes were the optimal disinfection measures. These measures reduce endophytic bacterial contamination of explants while ensuring bud differentiation. Adding hormones further facilitated bud differentiation and the formation of clustered buds in explants, ultimately yielding in vitro seedlings of both common wild rice and Niwara wild rice. These seedlings showed good growth after hardening and transplanting. The in vitro preservation method for wild rice established in this invention provides valuable reference for the in vitro preservation of other types of wild rice germplasm resources.

Claims

1. A method for preserving Oryza rufipogon and Oryza sativa L. ssp. Niutaoensis in vitro, characterized by, The method comprises the following steps: (1) Preparation of explants: select wild rice seedlings which are healthy, free of pests and diseases, and have jointed, remove the leaves, expose the stem joints and axillary buds, and cut at a distance of 0.5-1 cm above and below the stem joints to obtain stem joint explants with axillary buds; (2) Sterilization of explants: The explants are subjected to the following sterilization treatments in sequence: 75% alcohol immersion for 10-15 seconds; 10% 84 disinfectant solution immersion for 15 minutes, with shaking for 2 times during the process; 0.1% mercury chloride solution treatment under-0.1 Mpa vacuum for 5 minutes; 40°C water bath treatment for 20 minutes; and sterile water rinsing for 5-7 times; (3) Differentiation of explants: insert the sterilized explants into bud differentiation medium according to the growth direction of the axillary buds, with the axillary buds contacting the medium, inoculate 1 explant per tube, and culture at a temperature of 23°C and a light time of 16 hours / day for 3-5 days; (4) Rooting culture: cut the buds which have grown to 3-5 cm and insert them into rooting medium, inoculate 1 bud per tube, and culture at a temperature of 23°C and a light time of 16 hours / day for 1-2 weeks to start rooting; (5) Test tube seedling propagation: cut the tillers of the test tube seedlings which have been cultured for 2-3 months and place them in rooting medium, and cut the stem joints for bud differentiation culture; (6) Transplanting of plants: after acclimatization, transplant the tissue culture seedlings with well-developed root systems and 6-8 roots into culture pots containing vermiculite and nutrient soil at a volume ratio of 1:

1.

2. The method for preserving O. officinalis and O. nervara in vitro according to claim 1, wherein, In step (2), Tween 20 is added to the 84 disinfectant solution and the 0.1% mercury chloride solution.

3. The method for preserving O. officinalis and O. nervara in vitro according to claim 1, wherein, In step (3), the bud differentiation medium is: 4.4 g / L MS powder + 0.1 mg / L NAA + 2 mg / L KT + 300 mg / L timentin + 400 mg / L carbenicillin + 7 g / L plant gel, with a pH value of 5.

6.

4. The method for preserving O. officinalis and O. nervara in vitro according to claim 1, wherein, In step (4), the rooting medium is: 2.2 g / L MS powder + 30 g / L sucrose + 4 g / L plant gel, with a pH value of 5.

6.

5. The method for preserving O. officinalis and O. nervara in vitro according to claim 1, wherein, In step (5), the test tube seedling propagation further comprises: cutting the buds and placing them in cluster bud differentiation medium for 1-2 months to produce cluster buds, and then performing rooting culture.

6. The method for preserving O. officinalis and O. nervara in vitro according to claim 5, wherein, The cluster bud differentiation medium is: 4.4 g / L MS powder + 0.1 mg / L NAA + 2 mg / L KT + 2 mg / L 6-BA + 300 mg / L timentin + 400 mg / L carbenicillin + 30 g / L sucrose + 7 g / L plant gel, with a pH value of 5.

6.

7. The method for preserving O. officinalis and O. nervara in vitro according to claim 1, wherein, In step (6), the acclimatization process is: rinse the gel on the root system of the tissue culture seedlings, place them in a flask containing distilled water, and culture at a temperature of 26°C and a light time of 16 hours / day for 3 days.

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