CN111448985A

CN111448985A - Tissue culture method of rosa tenuifolia

Info

Publication number: CN111448985A
Application number: CN201910060366.0A
Authority: CN
Inventors: 李惠玲; 章漳
Original assignee: Jialan Group Co ltd
Current assignee: Jialan Group Co ltd
Priority date: 2019-01-22
Filing date: 2019-01-22
Publication date: 2020-07-28

Abstract

The invention relates to plant tissue culture, and discloses a tissue culture method of rosa multiflora, which comprises the following steps: (1) axillary bud induction: taking annual stem segments with stem nodes of the rosa minutissima as explants to be inoculated into a primary culture medium for axillary bud induction culture; (2) inducing and proliferating cluster buds: transplanting the germinated axillary buds into a proliferation culture medium for cluster bud differentiation and proliferation; (3) rooting culture: and (3) transplanting the seedlings obtained in the step (2) into a rooting culture medium, and performing rooting culture for 14-30 days. The culture method of the invention realizes the artificial culture of the rosa tenuifolia for the first time, obtains higher seed emergence rate, axillary bud germination rate, multiplication coefficient and rooting rate, is beneficial to the rapid mass propagation of the rosa tenuifolia, and has good application prospect.

Description

Tissue culture method of rosa tenuifolia

Technical Field

The invention relates to a plant cultivation and plant tissue culture method, in particular to a method for obtaining a regeneration plant by taking a stem section of rosa multiflora thunb as an explant to carry out tissue culture.

Background

Rosa (Rosa L.) is one of the world famous ornamental plants, the courtyard cultivation is common, more than 200 plants are in the world, wherein about 95 Chinese native products are one of the important distribution centers of Rosa resources, Rosa tenuifolia (Rosa graciliflora Rehd. et Wils), also called spine chestnut, are mostly grown in a hillside with elevation 3300 meters, under spruce forest or forest side shrubs, and are distributed only in parts of Yunnan, Sichuan, Tibet and Shaanxi.

The rosa multiflora has bright and beautiful color and pleasant fragrance, is an excellent landscaping material, and roots and fruits of the rosa multiflora can be used as medicines, so that the rosa multiflora not only can improve the immunity, but also has an indispensable effect on certain diseases, and is a rosa multiflora which is worthy of attention and deep development. The seeds of wild roses have deep dormancy, low germination rate and irregular germination, which is one of the reasons that the wild roses are difficult to popularize and apply.

At present, reports about the germination of wild rose seeds are few, the rosa tenuifolia is completely a wild species, and no relevant research report about seed germination and tissue culture is found. The research carries out a germination test on the rosa tenuifolia seeds to obtain a complete plant, selects annual stem segments of the rosa tenuifolia seeds as explants on the basis, develops the research of a plant tissue culture rapid propagation system, and lays a foundation for mass propagation, popularization and application of the rosa tenuifolia.

Disclosure of Invention

The invention aims to provide a tissue culture method of rosa minutissima.

The technical scheme is that the tissue culture method of the rosa minutissima comprises the following steps:

(1) axillary bud induction: taking an annual stem segment of the rosa tenuifolia as an explant, washing and disinfecting, and vertically and upwardly inoculating the stem segment into a primary culture medium for axillary bud induction culture;

the primary culture medium takes an MS culture medium as a basic culture medium and contains 25-35 g/L of cane sugar, 6-12 g/L of agar, 0.5-1.5 mg/L of 6-benzylamino adenine and 0.05-0.15 mg/L of naphthylacetic acid, preferably, the primary culture medium takes an MS culture medium as a basic culture medium and contains 27-32 g/L of cane sugar, 8-10 g/L of agar and 0.8-1.2 mg/L of 6-benzylamino adenine and 0.08-0.12 mg/L of naphthylacetic acid;

the length of the stem section of the explant is 1.5-4 cm, and the stem section of the explant comprises at least 1 stem node; preferably, the length is 2-3cm and comprises 1 node.

(2) Inducing and proliferating cluster buds: taking the axillary buds cultured for 30-40 days in the step (1), longitudinally cutting and transplanting the axillary buds into a proliferation culture medium, and culturing for 25-30 days;

the proliferation culture medium takes 1/2MS culture medium as basic culture medium and contains 25-35 g/L of cane sugar and 6-12 g/L of agar, 1.5-2.5 mg/L of 6-benzylamino adenine (6-BA) and 0.1-0.25 mg/L of naphthylacetic acid, and preferably takes 1/2MS culture medium as basic culture medium and contains 27-32 g/L of cane sugar and 8-10 g/L of agar, and 0.15-0.25 mg/L of 6-benzylamino adenine (6-BA) and 1.5-2.0 mg/L of naphthylacetic acid.

(3) Rooting culture:

transferring the seedlings obtained in the step (2) into a rooting culture medium, and culturing for 14-30 days;

the rooting culture medium takes the WPM culture medium as a basic culture medium for rooting, contains 25-35 g/L of sucrose, 5-10 g/L of agar and 0.2-0.5 mg/L of indolebutyric acid (IBA), and preferably takes the WPM culture medium for rooting, and contains 27-32 g/L of sucrose, 6-8 g/L of agar and 0.2-0.4 mg/L of indolebutyric acid.

The pH value of the primary culture medium, the pH value of the proliferation culture medium and the pH value of the rooting culture medium are 5.7-5.8.

In the step (1), the axillary bud induction culture conditions comprise the temperature of 22 +/-1 ℃, the humidity of 60-75%, the illumination intensity of 1500L x-2000L x, the illumination time of 10-13 hours/day, preferably the illumination time of 12 hours/day, and preferably the culture time of 30-40 days.

In the step (2), the conditions for inducing and proliferating the cluster buds comprise that the temperature is 22 +/-1 ℃, the humidity is 60-75%, the illumination intensity is 1500L x-2000L x, and the illumination time is 10-13 hours/day, preferably 12 hours/day.

In the step (3), the rooting culture conditions comprise the temperature of 20 +/-1 ℃, the humidity of 60-75%, the illumination intensity of 1500L x-2000L x, and the illumination time of 6-10 hours/day, preferably 8 hours/day.

In the steps (1) to (3), the medium is replaced every 14 to 42 days, and more preferably every 30 days.

Preferably, the primary culture medium in step (1) contains sucrose 30 g/L, 6-benzylamino adenine 1 mg/L and α -naphthylacetic acid 0.1 mg/L, more preferably, the primary culture medium in step (1) is MS culture medium added with sucrose 30 g/L, 6-benzylamino adenine 1 mg/L and α -naphthylacetic acid 0.1 mg/L.

Preferably, the proliferation culture medium in the step (2) contains 30 g/L of sucrose, 9 g/L of agar, 2 mg/L of 6-benzylamino adenine and 0.2 mg/L of α -naphthalene acetic acid, and more preferably, the proliferation culture medium in the step (2) is 1/2MS culture medium added with 30 g/L of sucrose, 9 g/L of agar, 2 mg/L of 6-benzylamino adenine and 0.2 mg/L of α -naphthalene acetic acid.

Preferably, the rooting medium in step (3) contains 30 g/L of sucrose, 7 g/L of agar and 0.3 mg/L of indolebutyric acid, and more preferably, the rooting medium in step (3) is WPM medium added with 30 g/L of sucrose, 7 g/L of agar and 0.3 mg/L of indolebutyric acid.

The explant of the rosa multiflora in the step (1) is obtained by the following method:

and (3) picking mature rosa tenuifolia fruits at 11 months, taking out the seeds, cleaning and airing the seeds, and then mixing the seeds with wet sand according to a ratio of 1: 4-8 volume percent, and carrying out low-temperature sand storage treatment at the humidity of 70-80% and the temperature of 5 +/-1 ℃. Preferably, the volume ratio of the seeds to the wet sand is 1: 5. and in the next 9 months, taking out the sand-stored seeds for sowing, cultivating after seedling emergence, taking annual branches to remove leaves and leaf stalks, and cutting stem sections which are 1.5-4 cm long and have at least one stem node after washing.

Preferably, a stem section with a stem node is cut, wherein the length of the stem section is 3-3 cm.

The matrix used for sowing is 2-4 by volume: 1 peat and perlite mixture. Preferably, the volume ratio of peat to perlite is 3: 1.

the explant disinfection method comprises the following steps: soaking and disinfecting the mixture for 20 to 40 seconds by using 65 to 75 percent ethanol, and soaking the mixture in hypochlorite solution with the effective chlorine content of 0.9 to 1.2 percent for disinfection for 8 to 12min after being washed by sterile water; and (3) soaking the glass fiber in 0.04-0.08% mercuric chloride solution for disinfection for 4-8 min after sterile water washing, and washing with sterile water. The hypochlorite solution contains 0.01-0.5 wt% of surfactant, preferably 0.01-0.5 wt% of Tween 20.

The tissue culture seedling obtained by the method can be used for subsequent cultivation or grafting.

According to the culture method, the proper seed germination conditions and tissue culture conditions are selected, so that the high seed emergence rate, the high axillary bud germination rate, the high multiplication coefficient and the high rooting rate are obtained, and the artificial culture of the rosa multiflora is realized for the first time.

The invention carries out germination test from the rosa tenuifolia seeds to obtain a complete plant, selects annual stem segments as explants to carry out tissue culture on the basis, does not need stem segments with axillary buds, can also induce the generation of the axillary buds and can quickly obtain a large amount of tissue culture seedlings of the rosa tenuifolia, is beneficial to the quick mass propagation of the rosa tenuifolia, and has good application prospect.

Drawings

FIG. 1 is a photograph of a wild rosa tenuifolia plant, wherein A is a flower of the wild rosa tenuifolia, B is a wild fruit, C is a wild seed, and D is a seed sprouting.

FIG. 2 is a photograph of a wild rosa tenuifolia tissue cultured according to the present invention, wherein A' is a cultivated plant; b ' is axillary bud induced by tissue culture stem segment, C ' is induction and proliferation of cluster bud, and D ' is rooting of tissue culture seedling.

Detailed Description

The technical solution of the present invention will be described below with reference to specific examples.

Basic culture medium: MS culture medium, 1/2MS culture medium, WPM culture medium;

α -naphthylacetic acid (α -NAA), 6-benzylamino adenine (6-BA), indolebutyric acid (IBA), sucrose, agar, KT (kinetin);

preparation of a culture medium: after a basic culture medium, cane sugar and plant growth hormone are prepared into a culture medium according to a certain proportion, the pH value is adjusted to 5.7-5.8, and the mixture is sterilized in a sterilization pot for 20min at 121 ℃ for later use.

Example 1 seed Germination treatment

The flower of wild fine-stalk rose is shown in fig. 1A, and in 11 months, mature fine-stalk rose fruits (shown in fig. 1B) are picked, seeds are cut off in time and cleaned, and the seeds are dried for standby (shown in fig. 1C), and then the seeds are mixed with wet sand 1: 5 volume ratio, keeping the humidity at 70-80%, and carrying out low-temperature sand storage (low-temperature lamination) treatment at 5 ℃. In the next 9 months, the seeds stored in the sand are taken out for sowing, and the matrix adopts peat: and (3) perlite is added to the soil in a ratio of 3:1 (volume ratio), the soil in the pot is kept moist, and normal cultivation management is carried out after seedling emergence. The rate of emergence was counted as 35% 1 month after sowing (see fig. 1D).

The germination rates of the seeds treated at different low-temperature sand storage times are shown in table 1.

TABLE 1 Effect of different Low temperature stratification time on seed germination percentage

Serial number	Treatment of	Time of sowing	Number of seeds sowed	Number of sprouts	The germination percentage is%
						1	Control	Month 12	100	0	0
2	Storing in sand at low temperature for 3 months	Month 3	100	7	7.0
						3	Storing in sand at low temperature for 5 months	Month 5	100	19	19.0
4	Storing in sand at low temperature for 7 months	Month 7	100	22	22.0
						5	Storing in sand at low temperature for 9 months	9 months	100	35	35.0

As can be seen from the table 1, the germination of the seeds of the rosa tenuifolia needs low-temperature stratification for a certain time, and the germination rate of the seeds is continuously improved along with the prolonging of the low-temperature stratification time, wherein the germination rate of the seeds in 9 months of low-temperature sand storage is the highest and is 35 percent; the germination rate of the seeds stored in sand at low temperature for 3 months is the lowest, and is only 7.0 percent; the germination rate of seeds which are not subjected to low-temperature sand storage is 0 percent. In general, the germination rate of the rosa tenuifolia seeds is low, the average germination rate is 20.75%, and the optimal low-temperature sand storage time of the seeds is 9 months.

EXAMPLE 2 Stem tissue culture

(1) Explant sterilization

After the germination and emergence of one year (the cultivated plants are shown in FIG. 2A'), the annual stem of Rosa strigosa with good growth is taken, the leaves and petioles are removed, and the stem is washed under running water for 30 min. Intercepting 2-3cm of stem section containing one nodule (stem node), soaking and disinfecting with 70% ethanol for 30s, and washing with sterile water for 3-4 times; then using sodium hypochlorite solution (about 1 percent of available chlorine) containing about 0.1 percent (wt) of Tween 20 and 10 percent of sodium hypochlorite solution to disinfect for 10min, and washing with sterile water for 3-4 times; sterilizing with 0.05% mercuric chloride for 5min, and washing with sterile water for 3-4 times.

(2) Axillary bud induction

Inoculating the sterilized stem segments of the fine-stalk roses vertically upwards into a primary culture medium, placing 5 stem segments in each bottle into a culture box with the temperature of 22 +/-1 ℃, the humidity of 60-75% and the illumination intensity of 1500L x-2000L x for culture and observation, illuminating for 12 hours every day, and replacing the culture medium every 30 days.

The formula of the primary culture medium comprises MS, cane sugar 30 g/L, agar 9 g/L, 6-BA 1.0 mg/L and NAA 0.1 mg/L, and the pH value is 5.7-5.8.

After 30 days of culture, the axillary bud germination rate is counted to be 60.8%. As in fig. 2B'.

(3) Induction and proliferation of multiple shoots

The axillary buds cultured for about 30-40 days are longitudinally cut and transplanted to a proliferation culture medium, one axillary bud is inoculated in each bottle, the culture condition temperature is 22 +/-1 ℃, the humidity is 60-75%, the illumination intensity is 1500L x-2000L x, the illumination is 12 hours every day, and the culture medium is replaced every 30 days.

The formula of the proliferation culture medium is 1/2MS + sucrose 30 g/L + agar 9 g/L +6-BA 2.0 mg/L + NAA 0.2 mg/L, after 25 days, the maximum proliferation multiple can reach 4 times, and the average proliferation coefficient is 2.1, as shown in figure 2C'.

The effect of different hormone types and concentration combinations on the induction and proliferation of multiple shoots is shown in table 2. KT is kinetin, namely 6-furfuryl amino purine.

TABLE 2 Effect of different hormone combinations on Cluster bud Induction and proliferation

(4) Rooting

And inoculating the well-grown plants after the propagation culture for 25-30 days into a rooting culture medium for rooting culture, inoculating an explant into each bottle of culture medium, and irradiating for 8 hours every day under the conditions that the temperature is 20 +/-1 ℃, the humidity is 60-75%, and the illumination intensity is 1500L-2000L x.

The rooting medium comprises WPM, sucrose 30 g/L, agar 7 g/L and IBA 0.3 mg/L.

After 20 days, the rooting rate was counted. The rosa tenuifolia roots 3-9, the average root length is 4.5 mm. The rooting rate reaches more than 90%, as shown in fig. 2D'.

And cleaning the rooted rosa tenuifolia tissue body to remove the culture medium, and using the culture medium for subsequent cultivation or grafting.

The rooting results are shown in table 3 using different basal media and phytohormone types and concentrations:

TABLE 3 Effect of different media and hormone combinations on Cluster bud rooting

Claims

1. A tissue culture method of rosa minutissima is characterized by comprising the following steps:

(1) axillary bud induction: taking annual stem segments of rosa tenuifolia as explants, washing and disinfecting, and vertically and upwardly inoculating the stem segments into a primary culture medium for axillary bud induction culture;

the primary culture medium takes an MS culture medium as a basic culture medium and contains 25-35 g/L of sucrose, 6-12 g/L of agar and 0.5-1.5 mg/L of 6-benzylamino adenine and 0.05-0.15 mg/L of naphthylacetic acid;

the length of the stem section of the explant is 1.5-4 cm, and the stem section of the explant comprises at least 1 stem node;

the proliferation culture medium takes 1/2MS culture medium as basic culture medium and contains 25-35 g/L of sucrose, 6-12 g/L of agar and 0.1-0.25 mg/L of 6-benzylamino adenine and 1.5-2.5 mg/L of naphthylacetic acid;

(3) rooting culture: transferring the seedlings obtained in the step (2) into a rooting culture medium, and culturing for 14-30 days;

the rooting culture medium takes a WPM culture medium as a basic culture medium for rooting, and contains 25-35 g/L g of sucrose, 5-10 g/L g of agar and 0.2-0.5 mg/L of indolebutyric acid.

2. The tissue culture method of rosa multiflora of claim 1, wherein in the step (1), the axillary bud induction culture conditions comprise a temperature of 22 ± 1 ℃, a humidity of 60% to 75%, a light intensity of 1500L x to 2000L x, and a light time of 10 to 13 hours/day;

in the step (2), the conditions for inducing and proliferating the cluster buds comprise that the temperature is 22 +/-1 ℃, the humidity is 60-75%, the illumination intensity is 1500L x-2000L x, and the illumination time is 10-13 hours/day;

in the step (3), the rooting culture conditions comprise the temperature of 20 +/-1 ℃, the humidity of 60-75%, the illumination intensity of 1500L x-2000L x and the illumination time of 6-10 hours/day.

3. The method for tissue culture of a rosa multiflora thunb according to claim 1, wherein the pH of the primary medium, the propagation medium and the rooting medium is 5.7 to 5.8.

4. The method for tissue culture of a rosa multiflora of claim 1, wherein the primary culture medium of step (1) contains 27 to 32 g/L of sucrose, 8 to 10 g/L of agar, 0.8 to 1.2 mg/L of 6-benzylamino adenine, 0.08 to 0.12 mg/L of naphthylacetic acid;

the multiplication culture medium in the step (2) contains 27-32 g/L of sucrose, 8-10 g/L of agar, 1.5-2.0 mg/L of 6-benzylamino adenine (6-BA) and 0.15-0.25 mg/L of naphthylacetic acid;

the rooting medium in the step (3) contains 27-32 g/L of sucrose, 6-8 g/L of agar and 0.2-0.4 mg/L of indolebutyric acid.

5. The method for tissue culture of a rose stem according to claim 1, wherein the primary culture medium in the step (1) contains sucrose 30 g/L and 6-benzylamino adenine 1 mg/L-naphthaleneacetic acid 0.1 mg/L.

6. The method for tissue culture of a rose plug as claimed in claim 1, wherein the multiplication medium in step (2) contains sucrose 30 g/L, agar 9 g/L, 6-benzylamino adenine 2 mg/L-naphthylacetic acid 0.1 mg/L.

7. The method for tissue culture of rosa multiflora of claim 1, wherein the rooting medium in step (3) comprises sucrose 30 g/L, agar 7 g/L, and indolebutyric acid 0.3 mg/L.

8. The method for tissue culture of rosa multiflora of claim 1, wherein the length of the stem of the explant is 2-3cm and comprises 1 stem node.

9. The method for tissue culture of rosa capillata according to claim 1, wherein the explant of rosa capillata of step (1) is obtained by:

and (3) picking mature rosa tenuifolia fruits at 11 months, taking out the seeds, cleaning and airing the seeds, and then mixing the seeds with wet sand according to a ratio of 1: uniformly mixing 4-8 volume percent, and carrying out low-temperature sand storage treatment at the humidity of 70-80% and the temperature of 5 +/-1 ℃; and in the next 9 months, taking out the sand-stored seeds for sowing, cultivating after seedling emergence, taking annual branches to remove leaves and leaf stalks, and cutting stem sections with stem nodes after washing.

10. The tissue culture method of rosa multiflora of claim 1, wherein the explant sterilization method comprises: soaking and disinfecting the mixture for 20 to 40 seconds by using 65 to 75 percent ethanol, and soaking the mixture in hypochlorite solution with the effective chlorine content of 0.9 to 1.2 percent for disinfection for 8 to 12min after being washed by sterile water; and (3) soaking the glass fiber in 0.04-0.08% mercuric chloride solution for disinfection for 4-8 min after sterile water washing, and washing with sterile water.