Seedling breeding method for improving survival rate of turmeric

Abstract

The invention discloses a seedling breeding method for improving the survival rate of turmeric. Belongs to the technical field of tissue culture. The turmeric plant is obtained by using a plant tissue culture technology through explant sampling, disinfection, in-vitro culture and out-of-bottle transplanting. According to the method, seedlings can be rapidly propagated, the seedlings are robust, root systems are developed, the survival rate is high, the content of effective components is high, and an effective way is provided for meeting the market requirement of turmeric.

Description

Technical Field

[0001] The invention relates to the technical field of tissue culture, and more particularly to a seedling breeding method for improving the survival rate of turmeric. Background Art

[0002] Turmeric is a perennial herbaceous plant of the ginger family. Turmeric has a wide range of medical uses, with the effects of breaking blood and promoting menstruation, promoting qi and relieving pain. It is used for chest and flank pain, amenorrhea, rheumatic shoulder and arm pain, and swelling and pain caused by falls. The curcumin components contained in turmeric have a wide range of physiological activities and have an inhibitory effect on a variety of cancer cells. It has become a research hotspot in recent years.

[0003] The current mainstream tuber division propagation method of turmeric faces a series of problems such as poor controllability of propagation quality and slow speed, which limits the scale of its market development. Tissue culture rapid propagation technology of turmeric plants can provide an efficient propagation method for them, mass-produce high-quality and controllable seedlings, and provide important technical support for meeting market demand.

[0004] Although the existing technology has studied the tissue culture technology of turmeric, most of them remain at the optimization of the culture medium. There are still many shortcomings in the actual application of the tissue culture technology of turmeric, such as low survival rate and low content of effective ingredients, which limits the efficient and high-quality production of turmeric.

[0005] In summary, how to provide a seedling breeding method for improving the survival rate and active ingredient content of turmeric is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0006] In view of this, the present invention provides a seedling breeding method for improving the survival rate of turmeric.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] A seedling breeding method for improving the survival rate of turmeric comprises the following steps:

[0009] (1) Explant disinfection

[0010] (2) Cluster bud induction

[0011] The sterilized explants were inoculated on MS medium supplemented with 6-BA and NAA for culture;

[0012] (3) Bud proliferation

[0013] The buds induced in step (2) are cut and subcultured in a proliferation medium under the following culture conditions: culture temperature 25-30° C., light intensity 1800-2000 lx, and light duration 12 h / day;

[0014] (4) Rooting induction

[0015] The clustered buds that have grown to 3-5 cm are transferred to the rooting medium for seedling rooting culture. The culture temperature is 25-30°C, the light intensity is 1800-2000lx, the light time is 12h / day, and the culture time is 3 weeks.

[0016] (5) Hardening and transplanting

[0017] When the plants grow to more than 6 cm, they are hardened and transplanted, and then watered thoroughly and cultured at 25-30℃.

[0018] The transplanting medium includes peat soil, coconut bran, vermiculite, perlite and rice husk powder, and the mass ratio of the five is 1:1:1:1:1.

[0019] Furthermore, the specific operation of step (1) is as follows: the turmeric bulb buds are sequentially rinsed with clean water, soaked in 75% alcohol for 1 minute, and sterilized with 0.1% mercuric chloride for 5 minutes to obtain a sterilized explant.

[0020] Beneficial effects achieved: Explant selection is the basis of tissue culture technology. Different plant parts have an important impact on subsequent results. Improper explant material will affect the subsequent experiments. Different explant materials have their corresponding explant disinfection systems. Explant disinfection is the first step in tissue culture. Curcuma is a rhizome vegetative propagation plant. Explants are often attached to a large number of microorganisms. Effective sterilization and disinfection steps are particularly important.

[0021] Furthermore, in step (2), the culture temperature is 25-30° C., the light intensity is 1000-1300 lx, the light time is 12 h / day, and the culture time is 20-25 days.

[0022] Furthermore, in step (3), the proliferation culture medium includes: MS+TDZ 0.3 mg / L+sucrose 30 g / L+agar 5 g / L.

[0023] Furthermore, in step (4), the rooting medium includes: 1 / 2MS+NAA 0.03 mg / L+BA 0.2 mg / L+sucrose 10 g / L+agar 6 g / L.

[0024] Furthermore, in step (5), the specific operation is: when the plant grows to more than 6 cm, take out the test tube seedling with tweezers, wash off the culture medium on the root, and move it to the transplanting medium for culture.

[0025] It can be seen from the above technical solution that, compared with the prior art, the beneficial effects achieved by the present invention are:

[0026] The invention utilizes plant tissue culture technology, obtains turmeric plants through explant collection, disinfection, in vitro culture and bottle transplantation. The method can quickly propagate seedlings, the seedlings are strong, the root system is developed, the survival rate is high, and the content of effective ingredients is high, providing an effective way to meet the market demand for turmeric. DETAILED DESCRIPTION

[0027] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] The drugs required for the present invention are conventional experimental drugs, which are purchased from commercial channels; the experimental methods not mentioned are conventional experimental methods and will not be described in detail here.

[0029] Example 1

[0030] A turmeric seedling breeding method comprises the following steps:

[0031] (1) Explant disinfection

[0032] Turmeric plants with healthy growth and no diseases or insect pests were selected as the objects for collecting explants. The explant collection site was the Turmeric Garden of the Institute of Tropical Crops Variety Resources, Chinese Academy of Tropical Agricultural Sciences.

[0033] Remove part of the matrix of the turmeric corm, cut the sprouts on the turmeric corm, retain about 3 cm, rinse with clean water, soak in 75% alcohol for 1 minute, disinfect with 0.1% mercuric chloride for 5 minutes, rinse with sterile water 3 to 4 times, cut into 0.5 cm long segments, and obtain the disinfected explants.

[0034] (2) Cluster bud induction

[0035] The sterilized explants were inoculated on MS medium supplemented with 6-BA and NAA for culture;

[0036] The culture temperature is 25℃, the light intensity is 1000lx, the lighting time is 12h / day, and the culture time is 20 days.

[0037] (3) Bud proliferation

[0038] The buds induced in step (2) are cut and subcultured in a proliferation medium under the following culture conditions: culture temperature 25°C, light intensity 1800 lx, and light duration 12 h / day;

[0039] The proliferation culture medium includes: MS+TDZ 0.3 mg / L+sucrose 30 g / L+agar 5 g / L.

[0040] (4) Rooting induction

[0041] The clustered buds that grew to 3-5 cm were transferred to the rooting medium for seedling rooting culture. The culture temperature was 25°C, the light intensity was 1800 lx, the light time was 12 h / day, and the culture time was 3 weeks.

[0042] The rooting medium includes: 1 / 2MS+NAA 0.03mg / L+BA 0.2mg / L+sucrose 10g / L+agar 6g / L.

[0043] (5) Hardening and transplanting

[0044] When the plant grows to more than 6 cm, take out the test tube seedling with tweezers, wash off the culture medium on the root, transfer to the transplanting medium for cultivation, water thoroughly and cultivate at 25℃;

[0045] The transplanting medium includes peat soil, coconut bran, vermiculite, perlite and rice husk powder, and the mass ratio of the five is 1:1:1:1:1.

[0046] Example 2

[0047] A turmeric seedling breeding method comprises the following steps:

[0048] (1) Explant disinfection

[0049] Turmeric plants with healthy growth and no diseases or insect pests were selected as the objects for collecting explants. The explant collection site was the Turmeric Garden of the Institute of Tropical Crops Variety Resources, Chinese Academy of Tropical Agricultural Sciences.

[0050] Remove part of the matrix of the turmeric corm, cut the sprouts on the turmeric corm, retain about 3 cm, rinse with clean water, soak in 75% alcohol for 1 minute, disinfect with 0.1% mercuric chloride for 5 minutes, rinse with sterile water 3 to 4 times, cut into 1 cm long segments, and obtain the disinfected explants.

[0051] (2) Cluster bud induction

[0052] The sterilized explants were inoculated on MS medium supplemented with 6-BA and NAA for culture;

[0053] The culture temperature is 30℃, the light intensity is 1300lx, the lighting time is 12h / day, and the culture time is 25 days.

[0054] (3) Bud proliferation

[0055] The buds induced in step (2) are cut and subcultured in a proliferation medium under the following culture conditions: culture temperature 30°C, light intensity 2000lx, and light time 12h / day;

[0056] The proliferation culture medium includes: MS+TDZ 0.3 mg / L+sucrose 30 g / L+agar 5 g / L.

[0057] (4) Rooting induction

[0058] The clustered buds that grew to 3-5 cm were transferred to the rooting medium for seedling rooting culture at a temperature of 30°C, a light intensity of 2000 lx, a light duration of 12 h / day, and a culture time of 3 weeks;

[0059] The rooting medium includes: 1 / 2MS+NAA 0.03mg / L+BA 0.2mg / L+sucrose 10g / L+agar 6g / L.

[0060] (5) Hardening and transplanting

[0061] When the plant grows to more than 6 cm, take out the test tube seedling with tweezers, wash off the culture medium on the root, transfer to the transplanting medium for cultivation, water thoroughly and cultivate at 30℃;

[0062] The transplanting medium includes peat soil, coconut bran, vermiculite, perlite and rice husk powder, and the mass ratio of the five is 1:1:1:1:1.

[0063] Example 3

[0064] A turmeric seedling breeding method comprises the following steps:

[0065] (1) Explant disinfection

[0066] Turmeric plants with healthy growth and no diseases or insect pests were selected as the objects for collecting explants. The explant collection site was the Turmeric Garden of the Institute of Tropical Crops Variety Resources, Chinese Academy of Tropical Agricultural Sciences.

[0067] Remove part of the matrix of the turmeric corm, cut the buds that germinate on the turmeric corm, retain about 3 cm, rinse with clean water, soak in 75% alcohol for 1 minute, disinfect with 0.1% mercuric chloride for 5 minutes, rinse with sterile water 3 to 4 times, cut into 0.7 cm long segments, and obtain disinfected explants.

[0068] (2) Cluster bud induction

[0069] The sterilized explants were inoculated on MS medium supplemented with 6-BA and NAA for culture;

[0070] The culture temperature was 28°C, the light intensity was 1100 lx, the light time was 12 h / day, and the culture time was 22 days.

[0071] (3) Bud proliferation

[0072] The buds induced in step (2) are cut and subcultured in a proliferation medium under the following culture conditions: culture temperature 28°C, light intensity 1900 lx, and light duration 12 h / day;

[0073] The proliferation culture medium includes: MS+TDZ 0.3 mg / L+sucrose 30 g / L+agar 5 g / L.

[0074] (4) Rooting induction

[0075] The clustered buds that grew to 3-5 cm were transferred to the rooting medium for seedling rooting culture. The culture temperature was 28°C, the light intensity was 1900 lx, the light time was 12 h / day, and the culture time was 3 weeks.

[0076] The rooting medium includes: 1 / 2MS+NAA 0.03mg / L+BA 0.2mg / L+sucrose 10g / L+agar 6g / L.

[0077] (5) Hardening and transplanting

[0078] When the plant grows to more than 6 cm, take out the test tube seedling with tweezers, wash off the culture medium on the root, transfer to the transplanting medium for cultivation, water thoroughly and cultivate at 28℃;

[0079] The transplanting medium includes peat soil, coconut bran, vermiculite, perlite and rice husk powder, and the mass ratio of the five is 1:1:1:1:1.

[0080] Comparative Example 1

[0081] A turmeric seedling breeding method comprises the following steps:

[0082] (1) Explant disinfection

[0083] Turmeric plants with healthy growth and no diseases or insect pests were selected as the objects for collecting explants. The explant collection site was the Turmeric Garden of the Institute of Tropical Crops Variety Resources, Chinese Academy of Tropical Agricultural Sciences.

[0084] Cut the sprouts on the turmeric corm, retain about 3 cm, rinse with running water for 4 to 6 hours, dry and rinse with 75% alcohol for 30 seconds on a clean bench, disinfect with 3% bleaching powder tablet extract for 15 minutes, rinse with sterile water 3 to 4 times, cut off the outer leaves, rinse with 75% alcohol for 30 seconds, disinfect with 3% bleaching powder tablet extract for 10 minutes, rinse with sterile water 3 to 4 times, peel off the internal buds, and cut into 0.5 to 1 cm long sections.

[0085] (2) Callus induction

[0086] The material was vertically inoculated on callus induction medium. After one week, the buds sprouted and the base began to swell to form callus. After 30 days, the base of the buds further swelled, the callus turned green and buds emerged. The callus was cut into several pieces and inoculated on bud differentiation and proliferation medium for proliferation culture.

[0087] Callus induction medium: MS + 6-BA 1.0 mg / L + NAA 0.1 mg / L + sucrose 3% + agar 0.8%;

[0088] Bud differentiation and proliferation medium: MS + 6-BA 1.0 mg / L + IAA 0.1 mg / L + sucrose 3% + agar 0.8%.

[0089] (3) Bud differentiation and proliferation culture

[0090] The small piece of callus tissue inoculated into the bud differentiation and proliferation medium will enlarge after about 30 days, and buds and bud eyes of varying sizes will be differentiated on it; buds higher than 2 cm will be used for rooting culture.

[0091] (4) Transfer the new shoots with a height of 2 cm to the rooting medium. After 2 weeks, roots will grow. When the seedlings grow to 3-4 cm, remove them and transplant them. When transplanting, wash the root medium and plant them in a mixed medium of perlite + peat (1:1) that has been sterilized at high temperature. Harden the seedlings for 2 weeks.

[0092] Rooting medium: 1 / 2MS + NAA 0.2 mg / L + sucrose 3% + agar 0.8%.

[0093] Experiment 1

[0094] (1) The tissue culture seedlings obtained in the embodiment / comparative example were planted in a 128-hole tray, with one plant planted in each hole. The cultivation medium in the hole was peat soil: vermiculite = 3:1. After 1 to 2 months, turmeric seedlings with a plant height of 15 to 20 cm were obtained.

[0095] (2) Dig planting holes with a diameter of 14 to 16 cm and a depth of 9 to 12 cm, and move the turmeric seedlings and the cultivation medium into the planting holes. During the growth period of turmeric, conventional fertilization management is carried out according to the soil fertility, and conventional field management such as water, weeds, diseased plants and insect pests is also carried out.

[0096] The transplant survival rate was counted and the curcumin content in turmeric plants was determined.

[0097] The results are shown in Table 1.

[0098] Table 1 Survival rate and curcumin content results

[0099] Survival rate (%) Curcumin content (%) Example 1 97 4.5 Example 2 98 4.2 Example 3 100 4.8 Comparative Example 1 82 3.6

[0100] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0101] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A seedling breeding method for improving the survival rate of turmeric, characterized in that: The steps include: (1) Explant disinfection (2) Cluster bud induction The sterilized explants were inoculated on MS medium supplemented with 6-BA and NAA for culture; (3) Bud proliferation The buds induced in step (2) are cut and subcultured in a proliferation medium under the following culture conditions: culture temperature 25-30° C., light intensity 1800-2000 lx, and light duration 12 h / day; (4) Rooting induction The clustered buds that have grown to 3-5 cm are transferred to the rooting medium for seedling rooting culture. The culture temperature is 25-30°C, the light intensity is 1800-2000lx, the light time is 12h / day, and the culture time is 3 weeks. (5) Hardening and transplanting When the plants grow to more than 6 cm, they are hardened and transplanted, and then watered thoroughly and cultured at 25-30℃. The transplanting medium includes peat soil, coconut bran, vermiculite, perlite and rice husk powder, and the mass ratio of the five is 1:1:1:1:

1.

2. The method according to claim 1, characterized in that The specific operation of step (1) is as follows: the turmeric bulb buds are washed with clean water, soaked in 75% alcohol for 1 minute, and sterilized with 0.1% mercuric chloride for 5 minutes to obtain a sterilized explant.

3. The method according to claim 1, characterized in that In the step (2), the culture temperature is 25-30° C., the light intensity is 1000-1300 lx, the light time is 12 h / day, and the culture time is 20-25 days.

4. The method according to claim 1, characterized in that In the step (3), the proliferation culture medium includes: MS+TDZ0.3mg / L+sucrose 30g / L+agar 5g / L.

5. The method according to claim 1, characterized in that In the step (4), the rooting medium includes: 1 / 2MS+NAA0.03mg / L+BA 0.2mg / L+sucrose 10g / L+agar 6g / L.

6. The method according to claim 1, characterized in that In the step (5), the specific operation is as follows: when the plant grows to more than 6 cm, the test tube seedling is taken out with tweezers, the culture medium on the root is washed off, and the seedling is transferred to the transplanting medium for culture.