CN116034871A - Tissue culture rapid propagation method of glabrous tarragon - Google Patents

Abstract

The invention provides a method for tissue culture and rapid propagation of Artemisia Helanensis. Firstly, the branches of Artemisia Helanescens which grow vigorously and are free from diseases and insect pests are selected for hydroponics, and the newly grown axillary buds and stem sections on the branches are taken as explants, and the surface After sterilization and inoculation in the primary medium to induce clustered shoots, transfer them to the secondary proliferation medium to induce the clustered shoots to proliferate in large quantities, then inoculate the clustered shoots in the rooting medium for rooting culture, and finally the rooted shoots obtained Seedling hardening, transplanting and cultivation. The method of the invention is simple and easy to operate, can effectively reduce the pollution rate of the primary culture of Artemisia helanensis, shorten the growth cycle, and provide technical support and guarantee for the development and utilization of Artemisia helanensis and the protection of wild germplasm resources in the future.

Description

A tissue culture and rapid propagation method for Artemisia helanensis

Technical Field

The invention belongs to the technical field of tissue culture, and particularly relates to a tissue culture and rapid propagation method of Artemisia helanensis.

Background Art

Hippolytia alashanensis is a plant of the genus Hippolytia of the Asteraceae family. It is drought-resistant, cold-resistant and barren-resistant. It is endemic to China and is mainly distributed in Gansu, Inner Mongolia, Ningxia and other places. It grows in areas with an altitude of 1,900 to 2,250 meters. It mostly grows in stone crevices, grasslands, hillsides or desert grasslands. When its stems and leaves are green and fresh, sheep and camels like to eat them, horses eat them happily, and cattle eat them a little. After it is fruitful, sheep like to eat it the most, and camels and horses also like to eat it. Hippolytia alashanensis has the effect of accumulating fat and is a medium-grade forage plant. Its branches are well preserved in winter. It is an important forage plant on winter desert grassland pastures. Its crude protein content is high during the flowering period, its crude fiber content is low, and its fluoride-free extract is also high. At present, Hippolytia alashanensis has not been artificially introduced and cultivated, and lacks large-scale breeding and cultivation. Its market development in China is still in a blank stage. Therefore, it is necessary to carry out research on its breeding technology to effectively protect the wild germplasm resources of Artemisia argyi in Helan Mountain, my country, and at the same time promote the expansion and sustainable development and utilization of its wild populations.

Summary of the invention

The main purpose of the invention is to provide a method for rapid tissue culture and seedling propagation of Artemisia helanensis.

The present invention is achieved through the following technical solutions:

1. Explant selection

Select healthy and pest-free Artemisia argyi branches for hydroponics, take the newly grown axillary buds and stem segments on the branches as explants, and remove some leaves.

2. Explant disinfection

Place the axillary buds and stem segments of Helanshan Artemisia in a glass bottle filled with detergent solution, cover the bottle mouth with gauze, and rinse under running water for 30-40 minutes. Then move the washed explants to a clean bench, soak them in 75% alcohol for 30 seconds, rinse them with sterile water 3-4 times, then shake and disinfect them with 4% sodium hypochlorite solution for 10 minutes, and rinse them with sterile water 3-4 times.

3. Primary Culture

Use sterile paper to absorb the moisture on the surface of the explant, then place it in a sterile dish, use high-temperature sterilized tweezers and scissors to cut off the incision that has come into contact with the drug solution, and inoculate it in the primary culture medium. The pH is adjusted to 5.8~6.0, the culture temperature is 25±2℃, the light time is 12 h/d, the light intensity is 1500~2000 lx, and the culture period is 30 days.

4. Subculture

The sterile seedling stem segments of Artemisia helanensis obtained in the primary culture were cut into pieces of about 1.5-2 cm and transferred to the subculture proliferation medium for proliferation culture. The pH was adjusted to 5.8-6.0, the culture temperature was 25±2℃, the light time was 12 h/d, the light intensity was 1500-2000 lx, and the culture period was 30 days.

5. Rooting culture

Select Helanshan Artemisia cluster shoot plants of uniform size and strong growth, inoculate them into rooting medium for rooting culture, adjust the pH to 5.80~6.00, the culture temperature to 25±2℃, the light time to 12 h/d, the light intensity to 1500~2000 lx, and the culture period to 30 days.

6. Hardening and transplanting

Open the cap of the rooted tissue culture bottle in the tissue culture room, harden the seedlings in the tissue culture room for 3 days, then transfer them to the greenhouse for 2 days, then rinse the culture medium on the roots with tap water, and transplant them to the seedling medium of peat: perlite: vermiculite = 1:1:1 for cultivation.

A tissue culture and rapid propagation method for Helanshan Artemisia, characterized in that healthy and pest-free Helanshan Artemisia branches are selected for hydroponics, axillary buds and stem segments newly grown on the branches are taken as explants, and sterilized, primary culture, subculture, rooting culture and seedling hardening and transplanting are performed on them to finally obtain regenerated plants. The pH of the primary culture medium, subculture proliferation culture medium and rooting culture medium is 5.80-6.00, the culture temperature is 25±2°C, the illumination time is 12 h/d, the light intensity is 1500-2000 lx, and the culture time is 30 d.

The specific method of explant disinfection and sterilization is as follows: put the new stem segments and axillary buds of Helanshan Artemisia into a glass bottle filled with detergent solution, wrap the bottle mouth with gauze, and rinse under running water for 30-40 minutes. Then move the washed explants to the clean bench, soak them in 75% alcohol for 30 seconds, rinse them with sterile water for 3-4 times, and then shake and disinfect them with 4% sodium hypochlorite solution for 10 minutes, and rinse them with sterile water for 3-4 times.

The primary generation clustered bud induction sterile seedling culture medium is: MS+1.0 mg/L 6-BA+0.01 mg/L NAA+30 g/L sucrose+7 g/L agar.

The subculture proliferation medium is: MS+0.3 mg/L 6-BA+0.1 mg/L NAA+30 g/L sucrose+7 g/L agar.

The rooting medium is: 1/2 MS+0.3 mg/L NAA+30 g/L sucrose+7 g/L agar.

The substrate used for transplanting is sterilized with potassium permanganate, and the ratio of various substrates is peat: perlite: vermiculite = 1:1:1.

The beneficial effects of the present invention are as follows: the present invention provides a tissue culture and rapid propagation method of Helanshan Artemisia, by collecting new shoots and stem segments as explants to induce sterile seedlings, without going through the callus tissue approach, directly inducing bud differentiation of explants, and through primary culture, subculture, rooting culture and light substrate seedling cultivation, a large number of regenerated plants can be quickly obtained in a short period of time. The regenerated plants obtained by the method have stable results, high survival rate of tissue culture seedlings, stable genetic traits, can maintain the excellent genetic traits of the mother plant, and are not prone to mutation. The present invention plays an important role in protecting the germplasm resources of wild Helanshan Artemisia in my country, promoting the expansion of its wild population and sustainable development and utilization. The method of the present invention is simple, easy to operate, can effectively reduce the pollution rate of the primary culture of Helanshan Artemisia, shorten the growth cycle, and provide technical support and guarantee for the future development and utilization of Helanshan Artemisia and the protection of wild germplasm resources.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a state diagram of primary culture in the technical solution of Example 1;

Figures 2, 3 and 4 are diagrams showing the growth of clustered buds in the technical solution of Example 1;

FIG5 is a diagram showing the growth state of the rooted plants in the technical solution of Example 1;

FIG. 6 is a diagram showing the growth status of transplanted plants in the technical solution of Example 1.

DETAILED DESCRIPTION

In order to better understand the technical content of the present invention, specific examples are provided below to further illustrate the present invention. The terms involved in the present invention: 6-BA is 6-benzyladenine; NAA is naphthylacetic acid; MS basic medium is 4.43 g/L; 1/2MS medium is based on MS medium, with the macroelements and calcium salts reduced to 1/2 of the full amount; the reagents used in the examples of the present invention, etc., can all be obtained from commercial channels unless otherwise specified.

Embodiment 1:

A tissue culture and rapid propagation method for Helan Artemisia argyi comprises the following steps: selecting robust and disease- and insect-free Helan Artemisia argyi branches for hydroponics, taking newly grown axillary buds and stem segments on the branches as explants, and sequentially performing explant sterilization, primary culture, subculture, rooting culture and transplanting and hardening to finally obtain regenerated plants; wherein the pH of the primary culture medium, the subculture proliferation culture medium and the rooting culture medium is 5.80-6.00, the culture temperature is 25±2°C, the illumination time is 12 h/d, the light intensity is 1500-2000 lx, and the culture time is 30 d.

1) Explant selection: Select healthy and pest-free Artemisia argyi branches for hydroponics. Then, use the axillary buds and stem segments grown on the branches as explants. Remove some leaves and put them in a glass bottle filled with detergent solution. Wrap the bottle mouth with gauze and rinse under tap water for 30-40 minutes.

2) Disinfection of explants: Move the washed explants to a clean bench, soak them in 75% alcohol for 30 seconds, rinse them with sterile water 3 to 4 times, then use 4% sodium hypochlorite solution for 10 minutes for shock disinfection, rinse them with sterile water 3 to 4 times, finally put the washed explants into a sterile inoculation plate, dry them with sterile paper, and inoculate them into the primary culture medium;

3) Induction of primary cluster buds: The sterilized explants were moved onto sterile filter paper to absorb surface moisture, the wounds at both ends were cut off, and the explants were inoculated in a culture medium with MS as the basic culture medium and 6 BA (0.25, 0.5, 1.0 mg/L) and NAA (0.01 mg/L) as plant growth regulators for cluster bud induction. Thirty explants were inoculated in each treatment and repeated three times. Finally, they were placed in a tissue culture room with a light intensity of 1500-2000 lx, a photoperiod of 12 h/d, a temperature of (25±2)℃, and a humidity of 60%. After 30 days, the initiation rate and budding index were counted. At the same time, the effects of different culture media on budding and growth conditions were observed. As can be seen from Table 1, when the culture medium of Helanshan Artemisia was A1 (MS+0.25 mg/L 6-BA+0.01 mg/L NAA), the initiation rate was the highest, which was 80%; however, the germination index was the lowest, and the buds differentiated from the base showed vitrification; while in A3 (MS+1.0 mg/L6-BA+0.01 mg/L NAA) culture medium, the initiation rate was the lowest, which was 57.14%, but the germination index was the highest, which was 4.00, and the degree of vitrification of the differentiated buds was relatively mild; therefore, among these three primary culture media, A3 (MS+1.0 mg/L6-BA+0.01 mg/L NAA) had a relatively better effect, and the state diagram of primary culture is shown in Figure 1.

4) Subculture proliferation culture: The clustered buds of Artemisia helanensis that grew well in the primary culture were used as explants, and the axillary buds of the sterile seedlings were cut into pieces of about 1.5-2 cm, and transferred to the proliferation medium with MS as the basic culture medium and 6BA (0.3, 0.5, 1.0 mg/L) and NAA (0.1, 0.2 mg/L) as plant growth regulators to induce the massive reproduction of clustered buds; 30 explants were inoculated in each treatment, repeated 3 times, and the proliferation coefficient was counted after 30 days and the growth status of the clustered buds was observed. Finally, it was placed in a tissue culture room with a light intensity of 1500-2000 lx, a photoperiod of 12 h/d, a temperature of (25±2)℃, and a humidity of 60%. After 30 days, the proliferation coefficients of axillary buds and stem segments were calculated, and the effects of different culture media on the growth and development of proliferated buds were observed and recorded. According to the proliferation results in Table 2, the Helanshan Artemisia had the best proliferation effect in the medium of B5 (MS+1.0 mg/L 6-BA+0.05 mg/L NAA), with a proliferation coefficient of 9, and more callus tissue was induced at the base, but the buds differentiated from the callus showed a more serious vitrification phenomenon; in the medium of B2 (MS+0.3 mg/L 6-BA+0.1 mg/L NAA), the proliferation coefficient was lower, the callus tissue induced at the base was less, the differentiated buds were also less, and the degree of vitrification was lighter. Compared with other proliferation culture media, the bud growth state was the best, so it was more suitable as the subculture proliferation medium of Helanshan Artemisia. The proliferation situation is shown in Figures 2, 3, and 4.

5) Rooting culture: Select sterile seedlings of Helanshan Artemisia that are uniform in size and grow robustly and transfer them to a rooting medium with 1/2MS as the basic medium and NAA (0.1, 0.2, and 0.3 mg/L). Inoculate 30 explants for each treatment and repeat 3 times. One month later, the rooting rate and average number of roots of Helanshan Artemisia in different culture media were counted to observe the growth of the root system. As can be seen from Table 3, the rooting rate and average number of roots of Helanshan Artemisia in C2 (1/2 MS+0.2 mg/L NAA) medium were the highest, which were 81.81% and 6.36 respectively. The root system grew well and was relatively strong, which was suitable as a rooting medium for Helanshan Artemisia. At the same time, it can be seen that in the C1 (1/2 MS+0.1 mg/L NAA) medium, the rooting rate is high, but the number of roots is small, with an average of 4.91 roots, and the root system grows relatively weakly; in the C3 (1/2 MS+0.3 mg/L NAA) medium, the rooting rate of Helanshan Artemisia is 70%, producing more roots, the roots are relatively thick, and the growth state is also good. The rooting situation is shown in Figure 5.

6) Hardening and transplanting: Open the cap of the tissue culture bottle of the rooted Artemisia argyi in the tissue culture room, harden the seedlings for 3 days, then transfer to the greenhouse for 2 days, rinse the culture medium on the roots with tap water, and transplant them to a light substrate for cultivation. The substrate used is mixed in a ratio of peat: perlite: vermiculite = 1:1:1, and then sterilized with potassium permanganate; after transplanting, water thoroughly and spray Green Heng No. 2 to prevent root rot. Finally, place the transplanted seedlings in a cool place, cover with a moisturizing cover, spray water 2-3 times a day, and place them in the sun for growth after they grow stably. The growth after transplanting is shown in Figure 6.

The experimental data of Helanshan Artemisia selengensis is shown in the following table:

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Claims (7)

1. a tissue culture rapid propagation method of a glabrous tarragon is characterized in that a glabrous tarragon branch which grows robustly and has no plant diseases and insect pests is selected for water culture, newly grown axillary buds and stem sections on the branch are taken as explants, and the explants are sequentially subjected to sterilization, primary culture, secondary culture, rooting culture and transplanting seedling hardening, so that a regenerated plant is finally obtained; the pH value of the primary culture medium, the secondary proliferation culture medium and the rooting culture medium is 5.80-6.00, the culture temperature is 25+/-2 ℃, the illumination time is 12 h/d, the light intensity is 1500-2000 lx, and the culture time is 30 d.

2. The tissue culture rapid propagation method of the glabrous tarragon according to claim 1, wherein the disinfection and sterilization method of the explant is as follows: putting the newly-grown stem segments and axillary buds of the Artemisia rupestris L into a glass bottle containing a detergent solution, wrapping the bottle mouth with gauze, and flushing for 30-40 min under running water; and then, transferring the washed explant to an ultra-clean workbench, soaking the explant in 75% alcohol for 30s, washing the explant with sterile water for 3-4 times, and then oscillating and sterilizing the explant with 4% sodium hypochlorite solution for 10 minutes, and washing the explant with sterile water for 3-4 times.

3. The method for tissue culture and rapid propagation of tarragon in the glabra of claim 1, wherein the cluster bud induction sterile seedling culture medium used for the primary culture is: MS+1.0 mg/L6-BA+0.01 mg/L NAA+30 g/L sucrose+7 g/L agar.

4. The tissue culture rapid propagation method of the glabrous tarragon according to claim 1, wherein the proliferation culture medium adopted for the secondary culture is as follows: MS+0.3 mg/L6-BA+0.1 mg/L NAA+30 g/L sucrose+7 g/L agar.

5. The tissue culture rapid propagation method of the glabrous tarragon according to claim 1, wherein a rooting culture medium adopted for rooting culture is as follows: 1/2 MS+0.3 mg/L NAA+30 g/L sucrose+7 g/L agar.

6. The method for tissue culture and rapid propagation of tarragon according to claim 1, wherein the substrate used for transplanting and hardening seedlings is sterilized by potassium permanganate, and the ratio of each substrate is turf: perlite: vermiculite = 1:1:1.

7. A method for tissue culture and rapid propagation of tarragon according to any one of claims 1 to 6, wherein the method comprises the steps of:

1) Explant selection: selecting strong and pest-free glabra sweet wormwood branches for water culture, removing part of leaves by taking axillary buds and stem sections growing on the branches as explants, putting the explants into a glass bottle containing detergent solution, wrapping a bottle opening with gauze, and flushing the bottle opening under tap water for 30-40 min;

2) Sterilization of explants: transferring the washed explant to an ultra-clean workbench, soaking the washed explant in 75% alcohol for 30s, soaking the washed explant in sterile water for 3-4 times, then vibrating and sterilizing the washed explant in 4% sodium hypochlorite solution for 10 minutes, washing the washed explant in sterile water for 3-4 times, and finally placing the washed explant in a sterile inoculation tray, absorbing the water by using sterile paper, and inoculating the washed explant to a primary culture medium;

3) And (3) primary cluster bud induction: transferring the sterilized explants to sterile filter paper to absorb surface water, cutting off wounds at two ends, inoculating to a culture medium of 6BA and 0.01 mg/L NAA with MS as a basic culture medium, performing cluster bud induction on plant growth regulator of 1.0 mg/L, inoculating 30 explants to each treatment, repeating for 3 times, and finally, placing the explants in a tissue culture chamber with light intensity of 1500-2000 lx, photoperiod of 12 h/d, temperature of (25+/-2) DEG C and humidity of 60%, and culturing for 30 days, wherein the statistical start rate and bud index are counted;

4) And (3) subculturing and proliferation: the method comprises the steps of taking cluster buds of the glabra girl artemisia in primary culture as explants, shearing aseptic seedlings of the cluster buds to about 1.5-2 cm, transferring the cluster buds to a multiplication medium taking MS as a basic medium, and inducing the cluster buds to multiply in a large quantity, wherein the plant growth regulator is 0.3mg/L of 6BA and 0.1mg/L of NAA; inoculating 30 explants for each treatment, repeating for 3 times, counting proliferation coefficients after 30 days and observing the growth state of cluster buds; finally, placing the culture medium in a tissue culture room with light intensity of 1500-2000 lx, photoperiod of 12 h/d, temperature of 25+/-2 ℃ and humidity of 60%, culturing the culture medium, and calculating proliferation coefficients of axillary buds and stem segments after 30 days;

5) Rooting culture: selecting sterile seedlings of the glabrous greenbrier rhizome with consistent size and strong growth, transferring the sterile seedlings to a 0.3mg/L NAA rooting medium with 1/2MS as a basic medium, inoculating 30 explants for each treatment, and repeating for 3 times; counting rooting rates and average root numbers of the tarragon with different culture mediums after one month, and observing the growth condition of root systems;

6) Hardening and transplanting: opening a cover of a tissue culture bottle of the rooted glabra tarragon in a tissue culture room, hardening seedlings for 3 days, transferring the seedlings to a greenhouse for hardening seedlings for 2 days, washing a culture medium on a root system with tap water, transplanting the culture medium into a light matrix for culture, wherein the matrix is prepared from turf: perlite: mixing vermiculite=1:1:1, uniformly stirring, and sterilizing the substrate by adopting potassium permanganate; and (3) watering thoroughly after transplanting, spraying Lvheng No. 2 to prevent root rot, finally, placing the transplanted seedling in a shady place, covering a moisturizing cover, spraying water for 2-3 times per day, and placing the seedling in sunlight for growth after the seedling grows stably.

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