Method for establishing a tissue culture regeneration system of Cardamine violacea leaves

Abstract

The invention discloses a method for establishing a tissue culture regeneration system of Cardamine violaceae leaves, and a tissue culture medium for Cardamine violaceae and its application. Cardamine violaceae leaves are used as explants, and the leaves are cleaned and sterilized and inoculated into an induction medium to induce for 15 to 20 days to obtain callus tissue, and then transferred to a differentiation medium for culture for 20 to 25 days, and buds with a height of 1 to 3 cm are transferred to a rooting medium for growth for 15 to 20 days to induce rooting to form a complete plant, which is then transplanted. The Cardamine violaceae leaf tissue culture regeneration system established by the above method can efficiently obtain Cardamine violaceae explant regeneration seedlings, can lay a foundation for constructing a genetic transformation system of Cardamine violaceae, and provide a technical reference for the cultivation and reproduction of excellent varieties of Cardamine violaceae, and is suitable for promotion and utilization in the field of biotechnology.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a method for establishing a tissue culture regeneration system of Cardamine violacea leaves. Background Art

[0002] Cardamine violifolia is an annual or biennial plant of the genus Cardamine in the family Cruciferae. It is also a super-selenium-accumulating vegetable unique to Enshi, my country. It has a strong ability to accumulate and tolerate selenium, and can accumulate more than 92% of organic selenium. The selenium content of Cardamine violifolia grown in the natural environment can reach 1365 g / kg, and the bioaccumulation coefficient of selenium in the aboveground part can reach up to about 43.8. As a super-selenium-accumulating plant with high nutritional value, it can be used as a raw material for health food and an excellent plant-based selenium-enriched feed.

[0003] Although the planting area of ​​Cardamine violacea has increased year by year, the breeding progress is slow due to the self-pollination of Cardamine violacea and the inability of seeds to be stored. With the development of molecular biology, many experiments involving gene function verification require genetic transformation through tissue culture. The tissue culture described in this patent uses Cardamine violacea leaves as explants to directly induce callus differentiation to obtain complete plants, providing a plant source for the study of plant selenium accumulation and selenium tolerance mechanisms, and can also be specifically applied in production practice and theoretical research. Through the leaf tissue culture regeneration system, a large number of high-quality Cardamine violacea seedlings can be quickly obtained, greatly reducing production costs. Summary of the invention

[0004] One of the purposes of the present invention is to provide a method for establishing a tissue culture regeneration system of Cardamine violacea leaves which can be used in production and experiments.

[0005] In order to solve the above problems, the present invention provides a tissue culture regeneration system of Cardamine violaceae leaves, which can quickly obtain a large number of high-quality Cardamine violaceae seedlings.

[0006] The technical solution adopted by the present invention is:

[0007] A method for establishing a tissue culture regeneration system of Cardamine violacea leaves mainly comprises the following steps:

[0008] (1) Explant disinfection:

[0009] Select young leaves of Cardamine violae as explants, rinse with tap water, disinfect with ethanol in a clean bench, rinse with sterile water, disinfect with sodium hypochlorite, and rinse with sterile water;

[0010] (2) Callus induction:

[0011] Cut the leaves treated in step (1) into small pieces of 1 cm × 1 cm using a sterile scalpel; inoculate the leaves with the front side facing upward into the induction medium and culture under light for 15 to 20 days to obtain callus tissue;

[0012] (3) Differentiation culture:

[0013] Transferring the callus obtained in step (2) to a differentiation medium and culturing under light for 20 to 25 days to culture adventitious buds of Cardamine violacea;

[0014] (4) Rooting culture:

[0015] The clustered adventitious buds obtained in step (3) are cut into individual plants, and when they grow to 1-3 cm in height and have three to five leaves, they are transferred to a rooting medium for light culture for 15-20 days to induce rooting, thereby culturing sterile regenerated seedlings of Cardamine violacea;

[0016] (5) Hardening and transplanting:

[0017] When the aseptic regenerated seedlings of Cardamine violacea in step (4) grow to 8-10 cm, they are transferred to a matrix composed of vermiculite and perlite and covered with plastic wrap to keep moisture. The plastic wrap is removed after 14 days, thus completing the establishment of the tissue culture regeneration system of Cardamine violacea leaves.

[0018] As an embodiment, the induction medium described in step (2) includes: MS medium 4.74 g / L, thidiazuron (TDZ) 0.4 mg / L, zeatin (ZT) 1.0 mg / L, naphthaleneacetic acid (NAA) 0.1 mg / L, sodium selenate (Na2SeO4) 1.0 mg / L, sucrose 30 g / L, and agar 7.5 g / L.

[0019] As an embodiment, the differentiation medium described in step (3) includes: MS medium 4.74 g / L, thidiazuron (TDZ) 0.4 mg / L, zeatin (ZT) 1.0 mg / L, naphthaleneacetic acid (NAA) 0.6 mg / L, sodium selenate (Na2SeO4) 1.0 mg / L, sucrose 30 g / L, and agar 7.5 g / L.

[0020] As an embodiment, the rooting medium described in step (4) includes: MS medium 4.74 g / L, NAA 1.0 mg / L, sucrose 30 g / L, sodium selenate (Na2SeO4) 1.0 mg / L, and agar 7.5 g / L.

[0021] As an embodiment, the pH value of the induction medium, differentiation medium and rooting medium is 5.8.

[0022] As an implementation method, 1 mg / L sodium selenate is added to the induction medium, differentiation medium and rooting medium to promote the induction and growth of Cardamine violacea leaves.

[0023] The technical effects of the present invention are:

[0024] The method of the present invention establishes an efficient tissue culture regeneration system for Cardamine violae leaves, selects different culture media and hormone ratios at different culture stages, and improves the dedifferentiation and redifferentiation ratios of Cardamine violae leaves. The method can realize the rapid propagation of Cardamine violae, and provides a technical reference for the construction of asexual propagation and genetic transformation systems of excellent varieties of Cardamine violae. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 a is a diagram showing the results of induction culture of Cardamine violacea leaves as explants;

[0026] Figure 1 b is a picture showing the callus formation results of Cardamine violacea leaves as explants for induction culture;

[0027] Figure 1 c and Figure 1 d is a picture showing the effect of obtaining adventitious buds by differentiation culture of Cardamine violacea leaves as explants;

[0028] Figure 1 e is a picture showing the complete regenerated plants formed after the rooting induction of Cardamine violaceum explants;

[0029] Figure 1 f is a diagram showing the root growth of regenerated plants of Cardamine violacea. DETAILED DESCRIPTION

[0030] In order to better understand the present invention, the present invention will be further described below in conjunction with specific implementation methods. It should be noted that the embodiments are only for the protection scope required for the present invention and are not limited to the scope represented by the embodiments.

[0031] Example 1

[0032] The present embodiment provides a method for constructing a tissue culture regeneration system of Cardamine violacea leaves, comprising the following steps:

[0033] 1. Explant selection and disinfection

[0034] The young leaves of Cardamine violacea were selected as explants. The leaves were placed in a mesh bag and rinsed under tap water for 30 minutes. Then they were transferred to a clean bench and disinfected with 75% ethanol solution for 10 seconds, washed with sterile water three times, disinfected with 3% NaClO for 9 minutes, washed with sterile water three times, placed on sterile filter paper to absorb moisture, and cut into small pieces of 1 cm × 1 cm with a sterile scalpel.

[0035] 2. Callus induction

[0036] The disinfected and cut leaves were inoculated into the induction medium with the front side facing up, and then placed in the tissue culture room for light culture for 15 to 20 days. The culture environment was a temperature of 25°C, a humidity of 75%, a light time of 12 h / d, and a light intensity of 500 lux to culture callus tissue. Figure 1 a and Figure 1 b is a picture showing the result of obtaining callus tissue by inducing culture using Cardamine violacea leaves as explants. From the picture, it can be seen that the callus tissue is in good growth condition.

[0037] The callus induction medium was based on MS medium, including 4.74 g / L MS medium, 0.4 mg / L thidiazuron (TDZ), 1.0 mg / L zeatin (ZT), 0.1 mg / L naphthaleneacetic acid (NAA), 1.0 mg / L sodium selenate (Na2SeO4), 30 g / L sucrose, and 7.5 g / L agar.

[0038] Table 1 lists the effects of MS medium, different hormone ratios and different selenium sources on callus induction of Cardamine violacea.

[0039] Table 1 Induction medium ratio table in this embodiment

[0040]

[0041] It can be concluded from Table 1 that callus tissue can be obtained from Cardamine violacea in both medium with and without sodium selenate, but adding 1.0 mg / L sodium selenate is helpful for callus induction. Therefore, the method for establishing a tissue culture regeneration system of Cardamine violacea leaves provided in an embodiment of the present invention preferably comprises 4.74 g / L MS medium, 0.4 mg / L thidiazuron (TDZ), 1.0 mg / L zeatin (ZT), 0.1 mg / L naphthaleneacetic acid (NAA), 1.0 mg / L sodium selenate (Na2SeO4), 30 g / L sucrose, and 7.5 g / L agar as the best embodiment, wherein the pH of the induction medium is 5.8.

[0042] 3. Callus induction to produce adventitious buds

[0043] The main function of differentiation culture is to make callus tissue differentiate into buds, which is conducive to the formation of a complete regeneration system. The formed callus tissue is cut into several small pieces and transferred to the differentiation medium, and then placed in the tissue culture room for 20-25 days. The culture environment is 25℃, 75% humidity, 12 h / d of light time, and 2000 lux of light intensity to cultivate adventitious buds of Cardamine violacea. Figure 1 c and Figure 1d is a graph showing the growth effects of adventitious buds at different growth times. It can be seen from the graph that the adventitious buds are in good growth condition.

[0044] The differentiation medium is based on MS medium, including 4.74 g / L MS medium, 0.4 mg / L thidiazuron (TDZ), 1.0 mg / L zeatin (ZT), 0.6 mg / L naphthaleneacetic acid (NAA), 1.0 mg / L sodium selenate (Na2SeO4), 30 g / L sucrose, and 7.5 g / L agar.

[0045] Table 2 shows the effects of MS medium and different concentrations of sodium selenate on the adventitious bud differentiation of Cardamine violacea.

[0046] Table 2 Differentiation medium ratio table in this example

[0047]

[0048] As can be seen from Table 2, when 4.74 g / L MS medium, 0.4 mg / L thidiazuron (TDZ), 1.0 mg / L zeatin (ZT), 0.6 mg / L naphthaleneacetic acid (NAA), 1.0 mg / L sodium selenate (Na2SeO4), 30 g / L sucrose, and 7.5 g / L agar, the proliferation rate of adventitious buds induced by explants is up to 500%. Therefore, the method for establishing the tissue culture regeneration system of Cardamine violacea leaves provided in the embodiment of the present invention adopts 4.74 g / L MS medium, 0.4 mg / L thidiazuron (TDZ), 1.0 mg / L zeatin (ZT), 0.6 mg / L naphthaleneacetic acid (NAA), 1.0 mg / L sodium selenate (Na2SeO4), 30 g / L sucrose, and 7.5 g / L agar as the best embodiment, wherein the pH of the culture medium is 5.8.

[0049] 4. Induce rooting of adventitious buds to obtain regenerated plants

[0050] The 1-3 cm high clustered adventitious buds were cut into individual plants and transferred to rooting medium. They were then placed in a tissue culture room for 20-25 days. The culture environment was a temperature of 25°C, a humidity of 75%, a light duration of 12 h / d, and a light intensity of 2000 lux to induce Cardamine violaceae to form adventitious roots. Figure 1 e and Figure 1 f is a diagram showing the effect of adventitious root growth. From the figure we can see that the adventitious roots are thick and in good growth condition.

[0051] The rooting medium used MS as the basic medium, including 4.74 g / L MS medium, 1.0 mg / L NAA, 30 g / L sucrose, 1.0 mg / L sodium selenate (Na2SeO4), and 7.5 g / L agar.

[0052] Table 3 shows the rooting rate of adventitious buds under different hormone concentration treatments.

[0053] Table 3 Rooting medium ratio table in this embodiment

[0054]

[0055] The results showed that 1 mg / L NAA treatment can shorten the average rooting time while ensuring a high rooting rate, and the incidence of adventitious roots can reach 100%. Therefore, the method for establishing the tissue culture regeneration system of Cardamine violacea leaves provided in the embodiment of the present invention adopts MS medium 4.74 g / L MS medium, NAA 1.0 mg / L, sucrose 30 g / L, sodium selenate (Na2SeO4) 1.0 mg / L, and agar 7.5 g / L as the best implementation, wherein the pH of the culture medium is 5.8.

[0056] 5. Hardening and transplanting of regenerated plants

[0057] Transplant the tissue culture seedlings with a height of 8 to 10 cm into the seedling medium, cover with plastic wrap to keep moisture, and remove the plastic wrap after 7 days to complete the establishment of the tissue culture regeneration system of Cardamine violacea leaves.

[0058] In addition, the MS used in this example contains potassium nitrate (KNO3) 1900 mg / L, ammonium nitrate (NH4NO3) 1650 mg / L, potassium dihydrogen phosphate (KH2PO4) 170 mg / L, magnesium sulfate (MgSO4·7H2O) 370 mg / L, calcium chloride (CaCl2·2H2O) 440 mg / L, manganese sulfate (MnSO4·4H2O) 22.3 mg / L, zinc sulfate (ZnSO4·7H2O) 8.6 mg / L, boric acid (H3BO3) 6.2 mg / L, potassium iodide (KI) 0.83 mg / L, sodium molybdate (NaMoO4·2H2O) 0.25 mg / L, copper sulfate (CuSO4·5H2O) 0.025 mg / L, cobalt chloride (CoCl2·6H2O) 0.025 mg / L, disodium ethylenediaminetetraacetic acid (Na2EDTA) 37.3 mg / L, iron sulfate (FeSO4·4H2O) 27.8 mg / L, inositol 100 mg / L, glycine 2.0 mg / L, pyridoxine hydrochloride 0.5 mg / L, niacin 0.5 mg / L, thiamine hydrochloride 0.1 mg / L.

[0059] The method for establishing a tissue culture regeneration system of Cardamine violacea leaves provided in the embodiment of the present invention can quickly and efficiently obtain in vitro regenerated seedlings of Cardamine violacea, and has high repeatability and excellent plant growth status. The different culture media and technical combinations set in different culture stages in the embodiment of the present invention can improve the dedifferentiation and redifferentiation efficiency of Cardamine violacea, and the generated regenerated seedlings can be used as raw materials for rapid propagation. The present invention provides a good foundation for genetic engineering, genetic improvement research, and the establishment of a regeneration system of Cardamine violacea.

[0060] In the present invention, d represents days, min represents minutes, s represents seconds, h / d represents hours per day, mg / L represents milligrams per liter, mm represents millimeters, and °C represents degrees Celsius.

Claims

1. A method for establishing a tissue culture regeneration system of Cardamine violacea leaves, characterized in that: The main steps include: (1) Explant disinfection: Select young leaves of Cardamine violacea as explants, rinse with tap water, and then disinfect with ethanol in a clean bench, then rinse with sterile water, disinfect with sodium hypochlorite, and rinse with sterile water; (2) Callus induction: Cut the leaves treated in step (1) into small pieces of 1 cm × 1 cm using a sterile scalpel; inoculate the leaves with the front side facing upward into an induction medium and culture under light for 15 to 20 days to obtain callus; The induction medium is: MS medium 4.74 g / L, thidiazuron (TDZ) 0.4 mg / L, zeatin (ZT) 1.0 mg / L, naphthaleneacetic acid (NAA) 0.1 mg / L, sodium selenate (Na2SeO4) 1.0 mg / L, sucrose 30 g / L, agar 7.5 g / L; (3) differentiation culture: transferring the callus obtained in step (2) to a differentiation medium and culturing under light for 20 to 25 days to culture adventitious buds of Cardamine violacea; The differentiation medium is: MS medium 4.74 g / L, thidiazuron (TDZ) 0.4 mg / L, zeatin (ZT) 1.0 mg / L, naphthaleneacetic acid (NAA) 0.6 mg / L, sodium selenate (Na2SeO4) 1.0 mg / L, sucrose 30 g / L, agar 7.5 g / L; (4) Rooting culture: The clustered adventitious buds obtained in step (3) are cut into individual plants, and when they grow to a height of 1 to 3 cm and have three to five leaves, they are transferred to a rooting medium for light culture for 15 to 20 days to induce rooting, thereby culturing sterile regenerated seedlings of Cardamine violacea; The rooting medium is: MS medium 4.74 g / L, NAA 1.0 mg / L, sucrose 30 g / L, sodium selenate (Na2SeO4) 1.0 mg / L, agar 7.5 g / L; (5) Hardening and transplanting: When the aseptic regenerated seedlings of Cardamine violacea in step (4) grow to 8 to 10 cm, they are transferred to a matrix composed of vermiculite and perlite and covered with plastic wrap to keep moisture. The plastic wrap is removed after 14 days, thus completing the establishment of the tissue culture regeneration system of Cardamine violacea leaves.

2. The method for establishing a tissue culture regeneration system of Cardamine violacea leaves according to claim 1, characterized in that: The pH values ​​of the induction medium, differentiation medium and rooting medium are all 5.8.

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