Ultralow-temperature preservation method for root tips of wild rice

Abstract

The invention relates to an ultralow-temperature preservation method for root tips of wild rice, and belongs to the technical field of plant cell engineering. The ultralow-temperature preservation method comprises the following steps: S1, taking robust sterile tissue culture seedlings of wild rice subjected to subculture for 20-60 days, cutting off root tissues, inoculating the seedlings into a sucrose-containing MS culture medium, and inducing new roots; s2, transferring the sterile tissue culture seedlings with new roots into a culture medium containing cane sugar and ABA for pre-culture; and S3, cutting the root tips of the sterile tissue culture seedlings after pre-culture, and carrying out ultralow-temperature preservation. According to the method, the wild rice root tips are successfully preserved at the ultralow temperature for the first time, and the preserved root tips form calluses and can be differentiated into seedlings, so that the growth condition is good. The reference value is realized on the ultra-low temperature preservation of the wild rice and the gramineous plants.

Description

Technical Field

[0001] The present invention relates to the technical field of plant cell engineering, in particular to a method for ultra-low temperature preservation of wild rice root tips, and in particular to a method for tissue culture, ultra-low temperature preservation and plant regeneration of wild rice root tips. Background Art

[0002] Wild rice is an important genetic resource for rice breeding and improvement, and has extremely high scientific research value. The current rapidly deteriorating environment and unfavorable climatic conditions and the rapid expansion of modern human economic activities have led to the loss and deterioration of wild rice habitats and a sharp reduction in habitats; in addition, wild rice has biological characteristics such as exposed stigmas, high cross-pollination rate, strong seed dormancy, strong grain shedding, and low germination rate. These factors put wild rice species at risk of extinction, and the protection of wild rice genetic diversity is urgent.

[0003] At present, the main protection measures for wild rice are in situ protection and ex situ protection (i.e., germplasm banks preserved by seeds, germplasm nurseries preserved by seed stems). Under ultra-low temperature storage (-196°C), the biochemical activity of the plant is almost stopped, and the physiological and genetic changes during storage can be controlled to a minimum, avoiding the risk of germplasm mutation or destruction caused by natural disasters in field storage, and the risk of genetic trait mutation or contamination caused by multiple subcultures in tissue culture storage. It is considered to be the best choice for long-term preservation of plant genetic resources.

[0004] The genus Oryza includes cultivated rice (Oryza sativa) and wild rice, with a total of 24 species, and there are great differences between species. Among them, since Sala et al. first reported the cryopreservation of rice suspension cells in 1979, related research has focused on tissue culture such as suspension cell lines, callus, protoplasts and embryoids. There are fewer reports on wild rice, especially the cryopreservation technology of intact organs, and they are concentrated in China. For example, Tan Guangxuan et al. used a programmed cooling method to culture wild rice panicles in vitro to induce plants and directly cryopreserved them, but the green buds produced turned white and died after one week. Lin Tian et al. established a cryopreservation system for the stem tips of common wild rice through the vitrification freezing method of wild rice stem tips (patent application number: 201610131944.1). However, (1) wild rice is a typical difficult-to-cultivate species compared to cultivated rice, with a long cultivation cycle, and it is difficult to obtain a large amount of uniform material in a short period of time; and (2) the stem tip stripping step has high technical requirements and is difficult, and it is easy to damage the growth point during operation, thus affecting the survival rate; (3) the corresponding supporting technical means in the process are too cumbersome and require high technical skills from the operator, and a more convenient and stable operation method needs to be found. Compared with stem tip tissue, root tip tissue can obtain a large amount of uniform material in a short period of time due to the vigorous growth of wild rice roots, and because there are no tightly wrapped leaves around the root tip, the cutting operation is convenient, making it an ideal cryopreservation material. Summary of the invention

[0005] In view of the shortcomings of the prior art, the object of the present invention is to provide a method for ultra-low temperature preservation of wild rice root tips.

[0006] The objective of the present invention is achieved through the following technical solutions:

[0007] In a first aspect, the present invention provides a method for ultra-low temperature preservation of wild rice root tips, comprising the following steps:

[0008] S1. Root tip induction: Take the robust sterile tissue culture seedlings of wild rice that have been subcultured for 20 to 60 days, cut off the root tissue, inoculate it into MS medium containing sucrose, and induce new roots of 1 to 5 mm;

[0009] S2. Root tip pre-culture: transfer the sterile tissue culture seedlings with new roots to MS medium containing sucrose and ABA for pre-culture for 1 to 3 days;

[0010] S3. Root tip freezing: Cut the root tips of the sterile tissue culture seedlings after pre-culture and store them at ultra-low temperatures.

[0011] As a preferred embodiment, in step S1, the wild rice is selected from any one of long-stamen wild rice and Dongxiang wild rice.

[0012] As a preferred embodiment, in step S1, the concentration of sucrose in the sucrose-containing MS medium is 0.2-0.4 mol·L -1 .

[0013] As a preferred embodiment, in step S1, the induction time is 1 to 3 days.

[0014] As a preferred embodiment, in step S2, the concentration of sucrose in the medium containing sucrose and ABA is 0.6-0.8 mol·L -1 , the concentration of ABA is 0.5~5mg·L -1 .

[0015] As a preferred embodiment, before the ultra-low temperature preservation in step S3, the step of loading and vitrification of the root tips of the sterile tissue culture seedlings cut after pre-culture is also included.

[0016] The steps of loading and vitrification treatment are specifically as follows: cutting the root tips of the sterile tissue culture seedlings after pre-culture and placing them in a pre-treatment solution for treatment, then transferring them into a vitrification protective agent for ice bath treatment, and then removing the vitrification protective agent;

[0017] When the root apex that has been loaded and vitrified is ultra-low temperature preserved, fresh vitrification protective agent needs to be added to the root apex.

[0018] As a preferred embodiment, the length of the cut root tip is 1 to 5 mm.

[0019] As a preferred solution, the pretreatment solution contains 2 mol·L -1 Glycerol and 0.4 mol·L -1 Liquid MS medium containing sucrose.

[0020] As a preferred solution, the vitrification protective agent is PVS2.

[0021] As a preferred solution, the treatment time in the pretreatment solution is 0.5 to 1.5 hours.

[0022] As a preferred embodiment, the time for transferring to the vitrification protective agent and performing ice bath treatment is 1 to 3 hours.

[0023] As a preferred embodiment, in step S3, the ultra-low temperature storage is performed by low temperature liquid nitrogen storage.

[0024] In a second aspect, the present invention provides a method for cultivating regenerated plants based on wild rice root tips, comprising the following steps:

[0025] A. Thawing and washing: Take out the wild rice root tips preserved according to the above method and immediately put them into a 38-42℃ water bath for rapid thawing for 1-3 minutes;

[0026] B. Recovery culture: transfer the thawed root tips to MS semi-solid medium containing 2,4-D, 6-BA and NAA or MS semi-solid medium containing 2,4-D for dark culture; by combining different growth hormones, the callus can be controlled to grow into roots, buds or embryonic callus;

[0027] C. Callus subculture and differentiation into seedlings: After 15 to 20 days of dark culture, the root tips from which callus tissue has been induced are transferred to MS solid medium containing 2,4-D for dark culture. After being transferred to the same medium for subculture 1 to 3 times every 20 to 30 days, the obtained embryonic callus tissue is transferred to differentiation medium for culture and differentiated into seedlings.

[0028] As a preferred embodiment, in step A, the concentration of sucrose in the sucrose-containing MS culture medium is 1 to 1.5 mol·L -1 .

[0029] As a preferred embodiment, step A further comprises the step of washing the root tip with MS culture medium containing sucrose at room temperature for 2 to 3 times, each time for 5 to 10 minutes, after rapid thawing.

[0030] As a preferred embodiment, in step B, the concentration of 2,4-D in the MS semi-solid medium containing 2,4-D, 6-BA and NAA is 2 to 5 mg·L -1, the concentration of 6-BA is 0.4~0.6mg·L -1 , the concentration of NAA is 0.1~0.2mg·L -1 .

[0031] As a preferred embodiment, in steps B and C, the concentration of 2,4-D in the MS solid medium containing 2,4-D is 1 to 3 mg·L -1 .

[0032] As a preferred embodiment, in step C, the culture time in the differentiation medium is 30 to 60 days.

[0033] As a preferred embodiment, in step C, the differentiation medium contains 2.0 mg·L -1 6-BA, 0.5mg·L -1 MS medium with NAA;

[0034] The differentiation culture conditions are 25° C., 12 h / day of illumination, and 4000-5000 lux of illumination intensity.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] (1) The present invention utilizes the habit of vigorous root growth of wild rice to obtain a large number of uniform root tips in a short period of time as cryopreservation materials.

[0037] (2) Since there are no leaves tightly wrapped around the root tip, the cutting operation is convenient. The method of the present invention eliminates the cumbersome technical means of stripping the wild rice stem tip, avoids the damage to the growth point during mechanical operation in the operation process, thereby improving the survival rate and finally obtaining the regenerated plants of wild rice. The green seedling differentiation rate of the wild rice root tip based on ultra-low temperature preservation of the present invention is 30-60%.

[0038] (3) The ultra-low temperature preservation method of the present invention further simplifies the process and reduces the technical requirements of the operator. This method has a good reference value for establishing a convenient, stable and efficient ultra-low temperature preservation system for wild rice, which is beneficial to the large-scale preservation of wild rice resources and the construction of a wild resource library. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0040] Figure 1 This is a photo of the sterile tissue culture seedlings of long-stamen wild rice with new roots induced in Example 1;

[0041] Figure 2This is a photo of the wild rice root tip after freezing recovery and dark culture in Example 2; the upper right corner is a partial enlarged view;

[0042] Figure 3 These are photos of wild rice callus differentiation into green seedlings in Example 2; wherein the upper photo is a typical example photo of 20 days of differentiation culture, and the lower photo is a typical example photo of 40 days of differentiation culture. DETAILED DESCRIPTION

[0043] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several variations and improvements may be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0044] Compared with cultivated rice, wild rice is a typical species that is difficult to cultivate. The difficulties in its cultivation are: (1) Field sampling is easily contaminated, and the vitality of seeds is unstable when germinated, requiring dormancy-breaking treatment. Therefore, it takes too long to obtain materials suitable for freezing, and it is difficult to obtain a large amount of uniform materials, which is not conducive to the quantity and quality control of freezing. (2) The step of stem tip stripping requires high technical requirements and is difficult. It is easy to damage the growth point during operation, thus affecting the survival rate. (3) In order to fully protect the growth point of the stem tip, the corresponding technical means in the process are too cumbersome and require high technical skills of the operator.

[0045] Based on this, the present invention successfully ultra-low temperature preservation of wild rice root tips (such as long-stamen wild rice and Dongxiang wild rice) for the first time, and the wild rice recovered and grew well after preservation. By using the induced wild rice root tips as cryopreservation materials, the operation difficulty is significantly reduced, the operation process and processing time are simplified, which is convenient and efficient, conducive to large-scale preservation, and provides technical reserves for the establishment of a wild rice cryopreservation bank.

[0046] In a specific embodiment of the present invention, a method for ultra-low temperature preservation of wild rice root tips is provided, comprising the following steps:

[0047] 1) Root tip induction: Take the robust sterile tissue culture seedlings of wild rice that have been subcultured for 20 to 60 days, excise the root tissue, and inoculate it into MS medium containing sucrose for 1 to 3 days to induce new roots of 1 to 5 mm;

[0048] 2) Root tip pre-culture: transfer the sterile tissue culture seedlings with new roots to MS medium containing sucrose and ABA for pre-culture for 1 to 3 days;

[0049] 3) Root tip loading and vitrification treatment: Cut the 1-5 mm root tip of the sterile tissue culture seedlings after pre-culture, place it in the pre-treatment solution for 0.5-1.5 hours, and then transfer it to the vitrification protective agent for ice bath treatment for 1-3 hours;

[0050] 4) Root apex freezing: The vitrified protective agent is removed, and then fresh vitrified protective agent is added to the root apex for ultra-low temperature preservation.

[0051] In a specific embodiment, when the wild rice is Dongxiang wild rice, since it is the most frost-resistant wild rice, the vitrification treatment in step 3) is not required, and ultra-low temperature preservation can be achieved by directly freezing the root tips of the sterile tissue culture seedlings after pre-culture.

[0052] The aforementioned methods can all be used to achieve ultra-low temperature preservation of wild rice root tips.

[0053] The technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0054] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available or prepared by conventional methods unless otherwise specified.

[0055] Example 1

[0056] This embodiment provides a method for ultra-low temperature preservation of wild rice root tips, and the specific steps are as follows:

[0057] 1) Establishment of sterile seedling system and subculture: Take the stem segment with sprouted buds of long-stamen wild rice underground stems, carefully wash away the mud and sand attached to the roots, soak in 1% carbendazim for 1 hour, and culture in clean water for 10-20 days. Take the newly sprouted buds, rinse them repeatedly under running water for about 1 hour, disinfect them with 70% alcohol for 1 minute, disinfect them with 0.1% mercuric chloride for 15 minutes, rinse them with sterile water several times, and peel off the stem tip of about 2 mm under a dissecting microscope and inoculate them in a solution containing 4 mg·L -1 BA and 0.5mg·L -1 The buds were induced on MS medium containing NAA. The culture temperature was 25±2℃ and the light intensity was 12h·d. -1 , light intensity 36μmol-2 -1

[0058] ms;

[0059] 2) Take the sterile tissue culture seedlings of long-stamen wild rice that have been subcultured for 30 days, remove the root tissue, and inoculate it into a medium containing 0.3 mol·L sucrose. -1 In MS medium, 3 mm new roots were induced after about 3 days of cultivation;

[0060] 3) The long-stamen wild rice sterile tissue culture seedlings (such as Figure 1 0.7 mol·L sucrose -1 and ABA 5mg·L -1 Pre-cultured on the medium for 3 days, cut the root tip of 3mm, put it in a 2ml cryotube, load it in the pre-treatment solution for 1 hour, and then transfer it to the vitrification protective agent PVS2 for 3 hours of ice bath treatment;

[0061] 4) After the PVS2 is completely aspirated, add 0.5 ml of fresh PVS2 and quickly place the cryovial in liquid nitrogen for storage.

[0062] Example 2

[0063] This embodiment provides a method for cultivating regenerated plants based on wild rice root tips, and the specific steps are as follows:

[0064] 1) After taking out the cryovials from Example 1 that had been frozen in liquid nitrogen for more than 24 hours, they were immediately placed in a 40°C water bath for rapid thawing for 2 minutes. The thawed root tips were then heated to room temperature with 1.2 mol·L -1 The root tips were washed 2-3 times with sucrose MS medium, each time for 5-10 min. The washed root tips were blotted dry on filter paper and then transferred to a 2,4-D medium containing 3 mg·L -1 +6-BA 0.5mg·L -1 +NAA0.1mg·L -1 The cells were cultured in dark in MS semi-solid medium.

[0065] 2) After about 20 days of dark culture of the root tip, a small amount of white callus (such as Figure 2 As shown), transfer it to a 2,4-D 2mg·L -1 The cells were cultured in dark on MS solid medium and subcultured on the same medium every 30 days. After subculture for 2 times, the pale yellow embryonic calli were transferred to 6-BA 2 mg·L -1 +NAA 0.5mg·L -1 The differentiation culture was carried out on MS medium under light conditions (25°C, 12h / day, light intensity 4000-5000lux) (photos of different culture time are shown in Figure 2). Figure 3 The seedlings differentiated into green shoots in about 30-60 days. The green shoot differentiation rate was 30-60%.

[0066] Example 3

[0067] This embodiment provides a method for ultra-low temperature preservation of wild rice root tips, and the specific steps are as follows:

[0068] 1) Establishment of sterile seedling system and subculture: Take the stem segments with sprouts from underground stems of Dongxiang wild rice, carefully wash away the mud and sand attached to the roots, soak them in 1% carbendazim for 1 hour, and culture them in clean water for 10-20 days. Take the newly sprouted buds, rinse them repeatedly under running water for about 1 hour, disinfect them with 70% alcohol for 1 minute, disinfect them with 0.1% mercuric chloride for 15 minutes, rinse them with sterile water several times, and peel off the stem tips of about 2 mm under a dissecting microscope and inoculate them in a solution containing 4 mg·L -1 BA and 0.5mg·L -1 The buds were induced on MS medium containing NAA. The culture temperature was 25±2℃ and the light intensity was 12h·d. -1 , light intensity 36μmol-2 -1

[0069] ms;

[0070] 2) Take the sterile tissue culture seedlings of Dongxiang wild rice that have been subcultured for 30 days, remove the root tissue, and inoculate it into a medium containing 0.3 mol·L sucrose -1 In MS medium, 3 mm new roots were induced after about 3 days of cultivation;

[0071] 3) The long-stamen wild rice sterile tissue culture seedlings with new roots were transferred into a sucrose solution containing 0.7 mol·L -1 and ABA 5mg·L -1 Pre-culture on culture medium for 3 days, cut 3 mm root tips, put them in 2 ml cryovials, and quickly put the cryovials into liquid nitrogen for storage.

[0072] Example 4

[0073] This embodiment provides a method for cultivating regenerated plants based on wild rice root tips, and the specific steps are as follows:

[0074] 1) After taking out the cryovials stored in liquid nitrogen for more than 24 hours in Example 3, immediately put them in a 40°C water bath for rapid thawing for 2 minutes. After blotting dry the root tips on filter paper, transfer them to a 2,4-D 2 mg·L -1 The cells were cultured in dark in MS semi-solid medium for 15-20 days;

[0075] 2) After dark culture, a small amount of white callus tissue was produced and transferred to a medium containing 2 mg·L 2,4-D. -1 The cells were cultured in dark on MS solid medium and subcultured every 30 days on the same medium. After subculture for 2 times, the pale yellow embryonic calli were transferred to 6-BA 2 mg·L -1 +NAA 0.5mg·L -1 The cells were cultured on MS medium under illumination conditions (25°C, 12 h / day, 4000-5000 lux) and differentiated into green seedlings in about 30-60 days. The green seedling differentiation rate was 30-60%.

[0076] The present invention has many specific application paths, and the above is only a preferred embodiment of the present invention. It should be pointed out that the above embodiments are only used to illustrate the present invention, and are not used to limit the protection scope of the present invention. For those of ordinary skill in the art, several improvements can be made without departing from the principle of the present invention, and these improvements should also be regarded as the protection scope of the present invention.

Claims

1. A method for ultra-low temperature preservation of wild rice root tips, characterized in that: The steps include: S1. Root tip induction: Take the robust sterile tissue culture seedlings of wild rice that have been subcultured for 20 to 60 days, cut off the root tissue, inoculate it into MS medium containing sucrose, and induce new roots of 1 to 5 mm; S2. Root tip pre-culture: transfer the sterile tissue culture seedlings with new roots to MS medium containing sucrose and ABA for pre-culture for 1 to 3 days; S3. Root tip freezing: Cut the root tips of the sterile tissue culture seedlings after pre-culture and store them at ultra-low temperatures.

2. The method for cryopreservation of wild rice root tips according to claim 1, characterized in that: In step S1, the wild rice is selected from any one of long-stamen wild rice and Dongxiang wild rice.

3. The method for cryopreservation of wild rice root tips according to claim 1, characterized in that: In step S1, the concentration of sucrose in the sucrose-containing MS medium is 0.2-0.4 mol·L -1 ; The induction time is 1 to 3 days; In step S2, the concentration of sucrose in the medium containing sucrose and ABA is 0.6-0.8 mol·L -1 , the concentration of ABA is 0.5~5mg·L -1 .

4. The method for cryopreservation of wild rice root tips according to claim 1 or 2, characterized in that: Before the ultra-low temperature preservation in step S3, the step of loading and vitrification of the root tips of the sterile tissue culture seedlings cut after pre-culture is also included.

5. The method for cryopreservation of wild rice root tips according to claim 4, characterized in that: The steps of loading and vitrification treatment are specifically as follows: cutting the root tips of the sterile tissue culture seedlings after pre-culture and placing them in a pre-treatment solution for treatment, then transferring them into a vitrification protective agent for ice bath treatment, and then removing the vitrification protective agent; When the root apex that has been loaded and vitrified is ultra-low temperature preserved, fresh vitrification protective agent needs to be added to the root apex.

6. The method for cryopreservation of wild rice root tips according to claim 5, characterized in that: The length of the cut root tip is 1 to 5 mm; The pretreatment solution contains 2 mol·L -1 Glycerol and 0.4 mol·L -1 Liquid MS medium with sucrose; The vitrification protective agent is PVS2.

7. The method for cryopreservation of wild rice root tips according to claim 5, characterized in that: The treatment time in the pretreatment solution is 0.5 to 1.5 hours; The time for transferring the mixture into the vitrification protective agent and performing ice bath treatment is 1 to 3 hours.

8. The method for cryopreservation of wild rice root tips according to claim 1, characterized in that: In step S3, the ultra-low temperature storage adopts low temperature liquid nitrogen storage.

9. A method for cultivating regenerated plants based on wild rice root tips, characterized in that: The following steps are involved: A. Thawing and washing: taking out the wild rice root tip preserved by the method according to any one of claims 1 to 8, and immediately placing it in a 38-42° C. water bath for rapid thawing for 1-3 minutes; B. Recovery culture: transfer the thawed root tips to MS semisolid medium containing 2,4-D, 6-BA and NAA or MS semisolid medium containing 2,4-D for dark culture; C. Callus subculture and differentiation into seedlings: After 15 to 20 days of dark culture, the root tips from which callus tissue has been induced are transferred to MS solid medium containing 2,4-D for dark culture. After being transferred to the same medium for subculture 1 to 3 times every 20 to 30 days, the obtained embryonic callus tissue is transferred to differentiation medium for culture and differentiated into seedlings.

10. The method for cultivating wild rice root tips according to claim 9, characterized in that: In step A, the concentration of sucrose in the MS culture medium containing sucrose is 1 to 1.5 mol·L -1 . In step B, the concentration of 2,4-D in the MS semi-solid medium containing 2,4-D, 6-BA and NAA is 2 to 5 mg·L -1 , the concentration of 6-BA is 0.4~0.6mg·L -1 , the concentration of NAA is 0.1~0.2mg·L -1 . In steps B and C, the concentration of 2,4-D in the MS solid medium containing 2,4-D is 1 to 3 mg·L -1 ; In the step, the culture time on the differentiation medium is 30 to 60 days.

Images