CN111657272B - Ultralow-temperature preservation method for rice callus - Google Patents

Abstract

The invention relates to a method for preserving rice callus at ultralow temperature; comprises selecting healthy and mature rice seeds, sterilizing, inoculating to NB culture medium containing 3 mg/L2, 4-D to induce and grow primary callus, transferring to NB culture medium containing 2 mg/L2, 4-D for subculture proliferation, screening embryogenic callus with screen after propagation, pre-culturing in NB culture medium containing 100g/L sucrose and 2 mg/L2, 4-D, and directly freezing and storing the pre-cultured callus at ultralow temperature. Taking out the frozen tube from liquid nitrogen during thawing, immediately placing into 39-41 deg.C water bath for rapid thawing for 2-4min, inoculating callus onto NB culture medium containing 2 mg/L2, 4-D for recovery culture, and after new callus grows out, selecting new callus for differentiation culture until green seedling grows out. The method for preserving the rice callus at the ultralow temperature is simple, convenient, feasible, stable and reliable, and has high survival rate of the callus and high differentiation and seedling rate after the rice is preserved at the ultralow temperature.

Description

Ultralow-temperature preservation method for rice callus

Technical Field

The invention belongs to the technical field of plant cell engineering, relates to a plant cryopreservation method, and particularly relates to a cryopreservation method for rice callus.

Background

Rice is one of the three most important grain crops in the world and is also the most important grain crop in Asia. Abundant rice genetic resources and continuously improved excellent rice varieties ensure the grain demand of about half of the population all over the world. However, with the rapidly expanding economic activities of modern mankind, the rapidly deteriorating natural environment and adverse climatic conditions in the global industrialized background lead to the decreasing of the rice planting area and the influence on the rice yield; meanwhile, the popularization of the mechanized large-scale planting mode and the breeding strategy taking the yield as the leading factor lead the applied rice varieties to be more and more simplified, the genetic basis of the resources becomes narrow, the genetic diversity is gradually lost, and the anti-pest, disease-resistant, drought-resistant, flooding-resistant and other stress-resistant genes in the original natural resources are gradually lost. Therefore, the protection of the genetic diversity of rice germplasm resources under the current situation is irresistible.

At present, the preservation mode of rice germplasm resources in China mainly takes a germplasm bank preserved by seeds as a main mode, but the construction cost of a low-temperature germplasm bank is high, the operation and maintenance cost is high, and few germplasm bank units which have conditional ability to be constructed are provided. With the development of biotechnology, the culture of plant tissues and cells opens up a new way for the preservation of plant germplasm, tissue cultures can be rapidly propagated in large quantities, and regenerated plants can keep the original genetic characteristics. 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 risk of genetic character variation or pollution is avoided, and the method is considered to be the optimal selection for long-term preservation of plant genetic resources.

Since the research on ultra-low temperature preservation of plants is developed, the ultra-low temperature preservation technology and the preservation efficiency are continuously improved, the variety of the plants subjected to ultra-low temperature preservation is continuously increased, but the ultra-low temperature preservation technology is mostly applied to woody plants such as apples, cherries, oranges, bananas, cassava and the like and economic plants such as potatoes, sweet potatoes, strawberries and the like. Cryopreservation of rice was first reported by Sala et al in 1979, and many studies have been focused on the use of suspension cell lines as cryopreservative materials. The cryopreservation research on rice germplasm resources has been few in the past, and the storage materials except suspension cells are few, so that only the professor dawn in 1996 carries out cryopreservation research on wild rice calluses through a programmed cooling instrument to obtain frozen regeneration plants. In 2000, Octopus macroteaching and the like adopt a programmed cooling method to carry out ultralow temperature preservation on adventitious buds induced by isolated culture of rice scions to obtain regenerated plants. The stem tip vitrification method for forest land and the like in agricultural biological gene center in Shanghai in 2016 preserves the common wild rice germplasm resources. However, the above preservation method is often complicated in steps, and has high requirements on equipment (such as a program cooling instrument) or materials (such as stem tips, young ears and other materials are fine, tender and difficult to obtain materials), so that the method is not beneficial to popularization and application, and therefore, the method is urgently needed and necessary for establishing a simpler, more efficient and low-cost rice ultra-low temperature preservation method, and tissue materials which are easy to reproduce are easily searched.

Disclosure of Invention

The existing rice ultra-low temperature preservation method has high requirements on equipment and operation technology, the process is more complicated, the pollution probability is high, and the method is not favorable for wide application. The invention aims to provide a simple, convenient and efficient ultralow-temperature preservation method for rice callus with low cost, provides a path with wide application and high efficiency for long-term safe preservation of rice germplasm resources, and has certain guiding significance on ultralow-temperature preservation methods of other gramineous plants.

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

the invention provides a method for preserving rice callus at ultralow temperature, which comprises the following steps:

step A1: taking healthy and mature rice seeds, sterilizing, and inoculating to an NB culture medium containing 2-3 mg/L2, 4-D to induce primary callus;

step A2: selecting primary callus, and transferring the primary callus to an NB culture medium containing 2 mg/L2, 4-D for subculture;

step A3: proliferating and propagating the callus, screening out callus particles with specific size by using a screen mesh, and pre-culturing the callus particles on an NB culture medium containing 100g/L of sucrose or maltose and 2mg/L of 2,4-D for 7-10 days;

step A4: directly filling the pre-cultured callus into freezing tubes, wherein the loading capacity of each freezing tube is not more than 1/2, and then quickly putting the freezing tubes into liquid nitrogen for storage.

Step A1 Rice mature embryos were used to induce primary callus on NB medium containing 2-3 mg/L2, 4-D.

As an embodiment of the present invention, in step A1, the induction culture condition is 30 ℃, the illumination is 12 h/day, and the illumination intensity is 1000-.

In an embodiment of the present invention, in step a1, the sterilization is performed by selecting mature, full and non-diseased rice seeds, hulling rice and then sterilizing. The sterilization method comprises sterilizing the surface with 70% ethanol for 1min, soaking in 10% sodium hypochlorite, shaking, sterilizing for 20min, and washing with sterilized water for 4-5 times.

As an embodiment of the present invention, in step A2, the subculture proliferation is carried out at 26 to 28 ℃ in the dark.

As an embodiment of the present invention, in step A3, embryogenic callus particles with a diameter of 1.5-3mm are screened out by a screen for pre-culture; the condition of the pre-culture is dark culture at 26-28 ℃.

As an embodiment of the present invention, in step A3, using different mesh sieves, the embryogenic callus particles with a compact texture and a size of about 1.5-3mm are selected out from the granules with a round shape and a light yellow color. The screening can be carried out by using 12-mesh and 8-mesh screens.

The invention also provides a recovery culture method of the rice callus after ultralow temperature preservation by adopting the method, which comprises the following steps:

Step B1: taking out the frozen tube from the liquid nitrogen, and immediately putting the tube into a water bath at 39-41 ℃ for fast thawing for 2-4 min;

step B2: inoculating the thawed callus to NB medium containing 2 mg/L2, 4-D, and culturing at 28-30 deg.C in dark for 10-14 days until new callus grows out.

In step B2, before the recovery culture, the thawed callus in the frozen tube is transferred to sterile filter paper in a clean bench work, air-dried for about 10min (8-12 min), and then transferred to a recovery culture medium for recovery culture.

As an embodiment of the present invention, in step B2, the recovery culture condition is dark culture at 28-30 ℃ and new callus grows out after 10-14 days of culture.

The invention also provides a culture method for differentiation and seedling formation of the rice callus after ultralow temperature preservation, which comprises the following steps:

the new callus growing after recovery culture by the method of the invention is picked and transferred to MS culture medium containing 2.0 mg/L6-BA, 2.0mg/L KT,0.2mg/L IAA and 0.2mg/L NAA for illumination differentiation culture, and green plantlets are differentiated after 20-30 days of culture.

As an embodiment of the invention, the illumination differentiation culture condition is 25 ℃, the illumination is 12 h/day, and the illumination intensity is 4000-.

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

1) the ultra-low temperature preservation material rice callus is convenient to obtain, and for most rice varieties, the induction and proliferation of mature embryo callus are easy, so that the high technical requirement that materials such as suspension cells or stem tips need cell culture or stem tip stripping is avoided;

2) the invention has simple and convenient operation, stability and reliability; after the cells are pre-cultured for a certain time by high sugar, the water content of the cells is reduced, and then the cells are directly put into liquid nitrogen for preservation, thereby omitting the most common vitrification treatment or programmed cooling treatment in ultralow temperature preservation and greatly reducing the complicated steps in the preservation process;

3) the rice callus after ultralow temperature preservation has good differentiation and seedling formation conditions; 1.5-3mm callus grains are screened out by a screen mesh in early culture and are used for ultralow temperature storage, the callus grains are embryogenic callus which has vigorous cell activity and most vitality, so the callus grains can quickly recover growth after recovery culture, the differentiation seedling rate is high, and the highest differentiation rate can reach more than 80%;

4) the invention realizes the high-efficiency ultralow temperature preservation of the rice callus for the first time through the special design of the preservation materials, the preservation mode and the like in the ultralow temperature preservation of the rice, provides a new effective way for the long-term safe preservation of the rice germplasm resources, plays a guiding role in establishing the ultralow temperature preservation method of other gramineous plants and has good reference value.

Drawings

FIG. 1 is a flow chart of the cryopreservation of rice calli according to the present invention;

FIG. 2 is a schematic diagram showing the state of embryogenic callus after being screened by a sieve in the present invention;

FIG. 3 is a schematic diagram showing the state of callus recovery culture after thawing in the present invention;

FIG. 4 is a diagram showing the state of callus differentiation and emergence after thawing in the present invention.

Detailed Description

The process of the ultra-low temperature preservation of the rice callus is shown in figure 1, and comprises the steps of inducing the primary callus by seeds, subculturing the callus, screening 1.5-3mm embryonic callus for pre-culture, directly freezing and preserving the callus after the pre-culture, thawing, recovering and culturing the callus, and differentiating and culturing the callus into seedlings. In order to more clearly describe the specific embodiments of the present invention, the present invention will be further described with reference to the following examples.

Example 1 obtaining of Rice embryogenic callus

The method comprises the following specific steps: 1) selecting mature and full rice seeds without disease spots, hulling, and sterilizing by sterilizing the surface with 70% alcohol for 1min, soaking in 10% sodium hypochlorite, shaking for 20min, and washing with sterilized water for 4-5 times. The disinfected seeds are inoculated to an NB culture medium containing 3 mg/L2, 4-D to induce primary callus, the induction culture condition is 30 ℃, the illumination is 12 h/day, and the illumination intensity is 1000-. 2) Subculture proliferation culture is carried out on the primary callus obtained by induction, wherein the subculture medium is an NB medium containing 2 mg/L2, 4-D, and the culture condition is dark culture at 28 ℃. 3) The NB solid medium used in the present invention contained 463mg (NH) in 1L of the medium ₄)₂·SO₄,125.33mg CaCl₂,90.37mg MgSO₄,400mg KH₂PO₄,2830mg KNO₃,3mg H₃BO₃,0.025mg CoCl₂·6H₂O,0.025CuSO4·5H₂O,27.8mg FeSO₄·7H₂O,37.26mg Na₂-EDTA·2H₂O,10mg MnSO₄·H₂O,0.25mg Na₂NoO₄.2H₂O,0.75mg KI,2mg ZnSO₄·7H₂O,100mg of inositol, 1mg of nicotinic acid, 1mg of pyridoxine hydrochloride, 10mg of thiamine nicotinate, 30g of sucrose, 7g of agar powder and the balance of water, and the pH value is adjusted to 5.9. 4) And screening the proliferated and propagated callus by using 12-mesh and 8-mesh screens to obtain embryogenic callus particles with the diameter of 1.5-3mm, wherein the embryogenic callus particles are round and granular in appearance, light yellow and compact in texture. The specific selected callus patterns are shown in FIG. 2.

Example 2 Effect of different carbon sources in Pre-culture Medium on callus cryopreservation efficiency

Respectively adding 100g/L sucrose and 100g/L maltose as carbon sources into a pre-culture medium, inoculating the subcultured callus onto the two media, pre-culturing for 7-10 days, directly filling the callus into a cryopreservation tube, storing for 3 days by using liquid nitrogen, taking out the cryopreservation tube from the liquid nitrogen when thawing, immediately putting the tube into a water bath at 39-41 ℃ for fast thawing for 2-4min, inoculating the thawed callus onto a recovery medium for recovery culture for about 14 days, performing dark culture at 28 ℃ under the recovery culture condition until new callus grows out, and counting the rate of the new callus growing out after the recovery culture.

TABLE 1 Effect of different C-derived pre-culture media on the callus rate of WDR48 after cryopreservation of calli

Group of	100g/L sucrose	Maltose 100g/L
			Average new wound healing rate	23.19％	22.26％

Test results show that the average new callus rate after 100g/L sucrose pre-culture and ultralow temperature storage is 23.19%; the average new callus rate after 100g/L maltose is pre-cultured and is 22.26% after being stored at ultralow temperature, and the two have no obvious difference through statistical analysis. Sucrose and maltose are both disaccharides, and maltose has an aldehyde group in its molecular structure, and is a reducing sugar with reducibility. In tissue culture, maltose is relatively easier to be absorbed and utilized by cells, and the effect of inhibiting tissue browning is better. But the price cost is about 60% higher than that of sucrose maltose. In the invention, the hypertonic treatment effect of maltose and sucrose on callus cells is not obviously different, so that the sucrose with higher cost performance is used as a carbon source of a pre-culture medium.

Example 3 Effect of vitrification treatment on the rate of neonatal callus

The effect of vitrification treatment and direct cryopreservation treatment on the new callus rate after recovery culture was compared. The vitrification treatment and thawing process were as follows: placing the pre-cultured callus into a freezing storage tube (no more than 1/2), adding Loading liquid LS (Loading Solution, Matsumoto et al, 1994), covering the callus, shaking for 20min, sucking off LS liquid in the tube, adding prepared vitrification protective liquid PVS2(Sakai, 1997), shaking for 40min in ice water, sucking off PVS2, adding fresh PVS2, and freezing and storing without callus. Thawing in 40 deg.C warm water for 2-3min, sucking dry PVS2 in the frozen tube, washing with washing solution US (unloading solution) under shaking for 10min for 2 times, pouring out the callus, sucking dry the callus on filter paper, and recovering culture. The direct cryopreservation treatment and thawing process was as follows: transferring the pre-cultured callus to sterilized filter paper in an ultra-clean bench, air drying for 0.5-1hr, placing into a freezing tube (no more than 1/2), directly freezing with liquid nitrogen, thawing in 40 deg.C warm water for 2-4min, and transferring the callus to recovery culture medium for recovery culture.

TABLE 2 Effect of vitrification on the rate of callus regeneration after cryopreservation of WDR48 callus

Group of	Vitrification treatment	Direct cryopreservation treatment
			Average new wound healing rate	0.53％	24.37％

Vitrification treatment is one of the most common means in ultralow temperature preservation, and adopts high concentration protecting liquid to treat cell or tissue and high viscosity to make the freezing liquid form solid state at ultralow temperature without crystallization so as to protect tissue cell. However, in the ultra-low temperature storage of rice callus, the vitrification treatment process is not only complicated, but also greatly reduces the new callus rate after recovery culture, so in the invention, the direct freezing storage method of callus is firstly adopted.

Example 4 Effect of callus size on the percentage of new callus after cryopreservation

In the process of rice callus culture, the tissue culture capability of the callus tissues with different sizes is different. In general, small calli (<1.5mm) grow vigorously, proliferate and differentiate faster, but are less tolerant to adverse culture conditions. While large calli (>3mm) are relatively more tolerant, survive more easily under unfavorable culture conditions, but are less viable, slow to grow, and relatively weak in proliferation and differentiation capacity. In order to verify the influence of calli of different sizes on the cryopreservation efficiency, the calli after multiplication and subculture are divided into 3 sizes (<1.5mm, 1.5-3mm, >3mm) by using screens with different meshes, and the new callus rates of the calli of different sizes after cryopreservation are compared through the steps of same pre-culture, freezing, thawing, recovery culture and the like.

TABLE 3 New callus rate of different size calli of Rheum L.3 after cryopreservation

Size of wound	New wound healing rate
		<1.5mm	20.9％
1.5-3mm	42.6％
		>3mm	30.4％

The callus with the size of 1.5-3mm is a kind of callus with better embryogenesis in tissue culture, the multiplication and differentiation capacity is stronger, the size is easy to operate, although the recovery growth time is slower than that of the callus with the size of less than 1.5mm, the tolerance to adverse culture conditions (such as ultra-low temperature freezing storage) is stronger than that of the small callus, and therefore the new callus rate after the ultra-low temperature storage with the size of 1.5-3mm is the highest.

Example 5 restoration culture of Rice callus and Green shoot differentiation

The callus in a dedifferentiated state can be transformed into an organ (root, stem, leaf, etc.) in a differentiated state by differentiation culture. However, the physiological activity of the callus immediately thawed from the ultralow temperature state is low, and the ability to respond to the "sharp" transition from callus to seedling in the differentiation culture is weak, thus resulting in a low differentiation rate. The invention adds the key step of recovery culture after thawing, can effectively improve the physiological activity of the frozen callus, so that the frozen callus can be proliferated to grow new callus, thereby finally improving the differentiation seedling rate. The method comprises the following specific steps: 1) when in unfreezing, the frozen tube is taken out from the liquid nitrogen and immediately put into warm water at 40 ℃ for fast unfreezing for 2-3 min. 2) Transferring the callus in the frozen tube to sterile filter paper in a super clean bench, naturally drying by 10 min or so, transferring the callus to an NB culture medium containing 2 mg/L2, 4-D, and performing recovery culture for 10-14 days under the condition of 28 ℃ dark culture until new callus grows out. The growth state of the newly-grown callus is shown in FIG. 3. 3) Selecting newly grown callus, selecting newly grown callus after recovery culture, transferring to MS culture medium containing 2.0 mg/L6-BA, 2.0mg/L K T,0.2mg/L IAA and 0.2mg/L NAA for illumination differentiation culture, and culturing for 20-30 days to differentiate green seedling. The state of the differentiation and emergence is shown in FIG. 4. The specific conditions of the illumination differentiation are as follows: culturing at 25 deg.C under 12 hr/day illumination with illumination intensity of 4500lux for 25 days to obtain green seedling with differentiation rate of over 80%.

TABLE 4 Green shoot differentiation Rate of callus with/without recovery culture

As can be seen from the above table, the differentiation culture of the newly-grown callus by recovery culture is significantly improved in the green seedling differentiation rate compared with the direct differentiation without recovery culture. Therefore, the ultra-low temperature preservation efficiency of the rice callus can be greatly improved by adopting the step of recovery culture after freezing.

The above-mentioned embodiments only express the embodiments of the present invention, and the description thereof is more detailed, and the modifications made by those skilled in the art after examining the embodiments of the present invention are within the scope of the present invention. The examples described herein are not to be construed as limiting the scope of the invention.

Claims (6)

1. A cryopreservation method of rice callus is characterized by comprising the following steps:

step A1: taking healthy and mature rice seeds, sterilizing, and inoculating to an NB culture medium containing 2-3 mg/L2, 4-D to induce primary callus;

step A2: selecting primary callus, and transferring the primary callus to an NB culture medium containing 2 mg/L2, 4-D for subculture;

step A3: proliferating and propagating the callus, screening out callus particles with specific sizes by using a screen mesh, and pre-culturing the callus particles on an NB culture medium containing 100g/L of sucrose and 2mg/L of 2,4-D for 7-10 days;

Step A4: directly filling the pre-cultured callus into freezing tubes, wherein the loading capacity of each freezing tube is not more than 1/2, and then quickly putting the freezing tubes into liquid nitrogen for storage;

NB medium 1L medium containing 463mg (NH)₄)₂· SO₄, 125.33mg CaCl₂, 90.37mg MgSO₄, 400mg KH₂PO₄, 2830mg KNO₃, 3mg H₃BO₃, 0.025mg CoCl₂·6H₂O, 0.025 CuSO4·5H₂O, 27.8mg FeSO₄·7H₂O, 37.26mg Na₂-EDTA·2H₂O, 10mg MnSO₄·H₂O, 0.25mg Na₂NoO₄.2H₂O, 0.75mg KI, 2mg ZnSO₄·7H₂O, 100mg of inositol, 1mg of nicotinic acid, 1mg of pyridoxine hydrochloride, 10mg of thiamine nicotinate, 30g of sucrose, 7g of agar powder and the balance of water, and the pH value is adjusted to 5.9;

in the step A3, screening embryogenic callus particles with the diameter of 1.5-3mm by a screen mesh for pre-culture; the condition of the pre-culture is dark culture at 26-28 ℃;

the method for restoring and culturing the rice callus after ultralow-temperature preservation comprises the following steps:

step B1: taking out the frozen tube from the liquid nitrogen, and immediately putting the tube into a water bath at 39-41 ℃ for fast thawing for 2-4 min;

step B2: inoculating the thawed callus to NB culture medium containing 2 mg/L2, 4-D, and performing recovery culture at 28-30 deg.C in dark until new callus grows out for 10-14 days; before recovery culture, transferring the thawed callus in the cryopreservation tube to sterile filter paper in a superclean bench work, naturally air-drying for 8-12 min, and then transferring to a recovery culture medium for recovery culture.

2. The method for cryopreservation of rice calli according to claim 1, wherein in step A1, the induction culture conditions are 30 ℃, 12 h/day of light irradiation, and 1000-1200lux of light irradiation intensity.

3. The method for cryopreservation of rice calli according to claim 1 wherein in step A2, the subculture multiplication culture conditions are 26-28 ℃ dark culture.

4. The cryopreservation method of rice callus as claimed in claim 1, wherein 1.5-3mm sized embryogenic callus particles with round granular shape, light yellow color, dense texture are screened out by using screens with different mesh numbers.

5. A culture method for differentiation and seedling formation of rice callus after cryopreservation is characterized by comprising the following steps: the first step is to store the callus of rice at ultra-low temperature according to the method of claim 1, the second step is to pick the new callus which grows out after the recovery culture of the method of claim 1, and transfer the new callus to MS culture medium containing 2.0 mg/L6-BA, 2.0mg/L KT, 0.2mg/L IAA and 0.2mg/L NAA for differentiation culture, and culture for 20-30 days to differentiate green seedling.

6. The method for culturing differentiation-grown seedlings of rice calli after cryopreservation as claimed in claim 5, wherein the differentiation culture conditions are 25 ℃, 12 h/day of illumination, and 4000 lux and 5000lux of illumination intensity.

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