CN104542281B - Method of Tissue Culture Propagation of Mediterranean Viburnum - Google Patents

Abstract

The invention provides a method for tissue culture and propagation of Viburnum mediterranei, comprising: selection and treatment of explants: selecting the stem section of Viburnum mediterranei with axillary buds as a culture material, washing it with running water; Bacteria, rinsed with sterile water, then sterilized with mercuric chloride, then rinsed with sterile water, inoculated in induction medium; induction culture: use MS medium, 6‑benzylaminoadenine, naphthaleneacetic acid, sucrose, agar After mixing as an induction medium, the selected stem segments of Viburnum mediterranei with axillary buds were inoculated in the induction medium; proliferation culture: the explants grown on the induction medium were transferred to MS medium, 6-benzylamino Adenine, naphthalene acetic acid, sucrose, and agar were mixed as induction medium for continuous culture; rooting culture: choose 1/2MS medium, indole butyric acid, activated carbon, sucrose, and agar mixed as rooting medium; transplanting: put The rooted plants were taken out and transplanted to humus soil: charcoal residue: sawdust mixed substrate.

Description

Method of Tissue Culture Propagation of Mediterranean Viburnum

technical field

The invention relates to the technical field of agronomy, and more specifically relates to a method for tissue culture propagation of Mediterranean viburnum (Viburnum tinus).

Background technique

The crown of the Mediterranean viburnum is spherical, and the crown diameter can reach 2.5 to 3 meters. The leaves are oval, dark green, 10 cm long, cyme, single flower is small, only 0.6 cm, the flower buds are pink, the flower bud period is very long, up to more than 5 months, the flowers are white after blooming, and the diameter of the whole inflorescence reaches 10 cm, the flowering period is from November to April in the next spring in the place of origin. Tiny yellow-green flower buds can be seen in early October in the Shanghai area. As the inflorescences elongate, the flower buds become more and more densely covered on the top of the branches, and the color gradually deepens to bright red. Looking from a distance, they look like pieces of red clouds floating in the ink. The green canopy is particularly eye-catching, adding warmth and vitality to the winter. The full flowering period is in the middle and late March, when the red cloud-like flower buds bloom into a snow-white piece, shining brilliantly in the garden in spring. Fruit ovate, dark blue-black, 0.6 cm in diameter. Mediterranean Viburnum, it is easier to differentiate flower buds, and it is common for one to two-year-old young trees to bloom. If the vegetative growth is properly controlled, it can also make it bloom in summer or autumn, and group planting can make flowering plants common throughout the year. It grows fast, has luxuriant branches and leaves, and is resistant to pruning. It is suitable for hedgerows, and can also be planted in gardens for ornamental purposes. It is a rare evergreen shrub among winter flowering plants in the Yangtze River Delta region.

However, no effective rapid tissue culture propagation method of Viburnum mediterranei has been disclosed at present.

Contents of the invention

The present invention aims to solve at least one of the problems in the background art.

One of the objectives of the present invention is to provide a method for tissue culture and propagation of Viburnum mediterranei, which includes the following steps: Step 101: selection and treatment of explants: select the stem section of Viburnum mediterranei with axillary buds as the culture material, and wash it with running water; Sterilize the surface with alcohol on the ultra-clean bench, rinse with sterile water, then sterilize with mercuric chloride, then rinse with sterile water, and inoculate in the induction medium; step 102: induction culture: use MS medium, 6 -Benzylaminoadenine, naphthalene acetic acid, sucrose, and agar are mixed as an induction medium, and the selected stem segments of Viburnum mediterranei with axillary buds are inoculated in the induction medium; Step 103: Proliferation culture: grow on the induction medium The explants were transferred to MS medium, 6-benzylaminoadenine, naphthaleneacetic acid, sucrose, and agar mixed and then used as induction medium for continuous culture; step 104: rooting culture: select 1/2MS medium, indole Acid, activated carbon, sucrose, and agar are mixed as a rooting medium; Step 105: Transplanting: Take out the rooted plants and transplant them to humus: charcoal residue: sawdust mixed substrate.

Further, in the step 101, the washing time with water is 20-30 minutes, the alcohol ratio is 70%, the surface sterilization time is 30 seconds, the mercuric chloride ratio is 0.1%, and the mercuric chloride sterilization time is 4 -12 minutes.

Further, in the step 102, the ratio of 6-benzylaminoadenine is 0.5-2.0 mg/L, the ratio of naphthaleneacetic acid is 0.1-0.5 mg/L, the ratio of sucrose is 2%, the ratio of agar is 0.6%, and the induction culture The pH of the base is 5.8.

Further, in the step 103, the proportion of 6-benzylaminoadenine is 0.1-0.5mg/L, the proportion of naphthaleneacetic acid is 0.1-0.3mg/L, the proportion of sucrose is 2%, and the proportion of agar is 0.6%.

Further, the culture conditions of the culture medium in step 104: the light intensity is 1500-2000lx, the temperature is 23-27°C, the light time is 12h/d, the ratio of indolebutyric acid is 0.1-2.0mg/L, the ratio of activated carbon 0.1-0.5%, the proportion of sucrose is 2%, the proportion of agar is 0.6%, and the pH value of the rooting medium is 5.8.

Further, the ratio of humus soil:charcoal residue:sawdust in the step 105 is 3:1:1.

The beneficial effects of the present invention are: (1) the production of Viburnum mediterranei can be carried out under artificial control conditions without being affected by factors such as seasons, climate conditions, soil, diseases and insect pests and pesticides, and the quality of viburnum mediterranean seedlings can be strictly controlled. (2) The multiplication speed is fast, the production cycle is short, the equipment is simple, the floor space is small, the manpower and material resources are saved, and it is convenient for factory production. (3) The detoxification treatment was carried out in the tissue culture process, which improved the quality of Viburnum mediterranei seedlings. At the same time, this technical method solved the important technical links of rapid propagation and stable rooting of Viburnum mediterranei, and could be applied to industrial production. (4) The germplasm resources of Mediterranean viburnum can be preserved, and the tree species and resources thereof can be enriched for landscaping.

Description of drawings

FIG. 1 is a flowchart of a method for tissue culture propagation of Viburnum mediterranei according to an embodiment of the present invention.

detailed description

The present invention will be described in detail below in conjunction with specific embodiments. It should be noted that the technical features or combinations of technical features described in the following embodiments should not be regarded as isolated, and they can be combined with each other to achieve better technical effects.

FIG. 1 is a flowchart of a method for tissue culture propagation of Viburnum mediterranei according to an embodiment of the present invention.

The tissue culture propagation method of Viburnum mediterranei provided by the embodiments of the present invention comprises the following steps:

Step 101: Selection and treatment of explants: Select the stem section of Viburnum mediterranei with axillary buds as the culture material, rinse with running water; sterilize with alcohol on the ultra-clean bench, rinse with sterile water, and then sterilize with mercuric chloride , then rinsed with sterile water, and inoculated in the induction medium;

Step 102: Induction culture: using MS medium, 6-benzylaminoadenine, naphthaleneacetic acid, sucrose, and agar mixed as an induction medium, inoculating the selected stem segments of Viburnum mediterranei with axillary buds in the induction medium;

Step 103: Proliferation culture: transfer the explants grown on the induction medium to MS medium, 6-benzylaminoadenine, naphthaleneacetic acid, sucrose, and agar and mix them as induction medium for continuous culture;

Step 104: rooting culture: select 1/2MS medium, indole butyric acid, activated carbon, sucrose, and agar to mix as rooting medium;

Step 105: Transplanting: Take out the rooted plants and transplant them on the mixed substrate of humus soil: charcoal slag: sawdust.

Further, in the step 101, the washing time with water is 20-30 minutes, the alcohol ratio is 70%, the surface sterilization time is 30 seconds, the mercuric chloride ratio is 0.1%, and the mercuric chloride sterilization time is 4 -12 minutes.

In the present invention, the ratio of 6-benzylaminoadenine in step 102 is 0.5-2.0 mg/L, the ratio of naphthaleneacetic acid is 0.1-0.5 mg/L, the ratio of sucrose is 2%, and the ratio of agar is 0.6%, The pH of the induction medium was 5.8. In the step 103, the proportion of 6-benzylaminoadenine is 0.1-0.5mg/L, the proportion of naphthaleneacetic acid is 0.1-0.3mg/L, the proportion of sucrose is 2%, and the proportion of agar is 0.6%. The culture conditions of the culture medium in step 104: the light intensity is 1500-2000lx, the temperature is 23-27°C, the light time is 12h/d, the ratio of indolebutyric acid is 0.1-2.0mg/L, and the ratio of activated carbon is 0.1-2.0mg/L. 0.5%, the proportion of sucrose is 2%, the proportion of agar is 0.6%, and the pH value of the rooting medium is 5.8. In the step 105, the ratio of humus soil: charcoal residue: sawdust is 3:1:1.

The following will give specific examples to illustrate in detail:

First of all, the process of initiating and proliferating culture of Viburnum mediterranei in vitro is as follows:

(1) Selection and processing of explants

Cut the branches with axillary buds from the plants, cut them into about 1cm stems with axillary buds, and rinse them with running water for 20-30 minutes. Sterilize the surface with 70% alcohol for 30 seconds on the ultra-clean bench, rinse once with sterile water, then sterilize with 0.1% mercuric chloride, treat for 4, 6, 8, 10, 12 minutes respectively, and then rinse with sterile water for 5 minutes Once, inoculated in MS medium (containing 2% sucrose, 0.6% agar, pH 5.8), 50 bottles were inoculated for each treatment, and 1 explant was received in each bottle. After 30 days, the mortality rate, pollution rate and survival rate of each treatment were investigated. Mortality rate (%)=dead explant number/inoculation explant number×100%; Contamination rate (%)=contaminated explant number/inoculation explant number×100%; Survival rate (%)= The number of surviving explants/the number of inoculated explants×100%.

(2) Start training

The stem section with axillary buds of about 1 cm was selected as the test material. MS medium was used as the basic medium, and the following hormone concentrations (unit: mg/L) were added: 6-benzylaminoadenine 0.5 + naphthaleneacetic acid 0.1; 6-benzylaminoadenine 0.5 + naphthaleneacetic acid 0.3; 6-benzylaminoadenine Aminoadenine 0.5 + naphthaleneacetic acid 0.5; 6-benzylaminoadenine 1.0 + naphthaleneacetic acid 0.1; 6-benzylaminoadenine 1.0 + naphthaleneacetic acid 0.3; 6-benzylaminoadenine 1.0 + naphthaleneacetic acid 0.5; 6-benzylaminoadenine Purine 2.0 + naphthaleneacetic acid 0.1; 6-benzylaminoadenine 2.0 + naphthaleneacetic acid 0.3; 6-benzylaminoadenine 2.0 + naphthaleneacetic acid 0.5. Each medium was supplemented with 2% sucrose, 0.6% agar, pH 5.8. 50 bottles were inoculated for each treatment, and one explant was received in each bottle, and the germination rate of axillary buds and the growth of buds were counted after 30 days. Axillary bud germination rate = number of explants germinated from axillary buds/number of inoculated explants × 100%.

(3) Proliferation culture

The buds obtained from the priming culture were selected as the test material. MS medium was used as the basic medium, and the following hormone concentrations (unit: mg/L) were added: 6-benzylaminoadenine 0.1 + naphthaleneacetic acid 0.1; 6-benzylaminoadenine 0.1 + naphthaleneacetic acid 0.2; 6-benzylaminoadenine Aminoadenine 0.1+ naphthaleneacetic acid 0.3; 6-benzylaminoadenine 0.3+ naphthaleneacetic acid 0.1; 6-benzylaminoadenine 0.3+ naphthaleneacetic acid 0.2; 6-benzylaminoadenine 0.3+ naphthaleneacetic acid 0.3; 6-benzylaminoadenine Purine 0.5+ naphthaleneacetic acid 0.1; 6-benzylaminoadenine 0.5+ naphthaleneacetic acid 0.2; 6-benzylaminoadenine 0.5+ naphthaleneacetic acid 0.3. Each medium was supplemented with 2% sucrose, 0.6% agar, pH 5.8. 30 bottles were inoculated for each treatment, and one bottle was picked for each bottle. After 30 days, the proliferation coefficient was counted, and the growth of adventitious buds was observed. Proliferation coefficient = total number of adventitious buds/number of inoculated buds × 100%. The culture conditions of the above-mentioned medium: the light intensity is about 1500lx, the temperature is (25±2)°C, and the light time is 12h/d.

Experimental results on the above cultivation:

The results of the study showed that disinfection with 0.1% mercuric chloride for 8 minutes had the best effect, and its survival rate was 70%; the best starting medium for axillary buds was culture medium + 6-benzylaminoadenine 1.0 mg/L + naphthalene acetic acid 0.3 mg/L + sucrose 2% + agar 0.6%, the germination rate of axillary buds is 90%; the medium most conducive to the proliferation of adventitious buds is MS medium + 6-benzylaminoadenine 0.5mg/L + naphthaleneacetic acid 0.2mg/L + sucrose 2% + agar 0.6 %, cultured for 30 days, the proliferation coefficient was as high as 4.57.

Secondly, the experimental materials used in the present invention, experimental design, and experimental results are as follows:

(1) Experimental materials

The rootless tissue-cultured seedlings of Viburnum mediterranei are used as the material. The height of the seedlings is about 3cm, the growth is strong, and the leaves are dark green.

(2) Experimental design

Effect of culture medium on rooting of Viburnum mediterranean tissue culture seedlings: inoculate uniform viburnum mediterranean tissue culture plantlets on the medium, 1/2MS medium and 1/4MS medium respectively, and add 0.6% agar and 2% sucrose, pH 5.8, cultured at temperature (25±2)°C, light 12h/d, light intensity 1500-2000lx (the same below). 15 tissue culture seedlings were inoculated for each treatment, and 3 replicates were set. The rooting start time was recorded, and after cultivating for 25 days, the average rooting rate (number of rooted seedlings/number of inoculated seedlings), average rooting number (total number of roots/number of inoculated seedlings) and average length of roots were calculated for each treatment.

Effect of indole butyric acid concentration on rooting of Viburnum mediterranean tissue culture seedlings: 1/2MS medium was used as basic medium, and 0, 0.1, 0.5, 1.0, 1.5, 2.0 mg/L indole butyric acid were added to it , after sterilization, inoculate uniform viburnum tissue culture seedlings for cultivation. 15 tissue culture seedlings were inoculated for each treatment, and 3 replicates were set. The rooting start time was recorded, and after culturing for 25 days, the average rooting rate, the average number of roots and the average length of the roots were calculated for each treatment.

The effect of activated carbon on the rooting of Mediterranean viburnum tissue culture seedlings: 1/2MS medium was used as the basic medium, and 1.0mg/L indolebutyric acid solution was added to the medium, and 0, 0.1%, and 0.3% were added to it respectively And 0.5% activated carbon, after sterilization, inoculate uniform Viburnum plantlets for cultivation. 15 tissue culture seedlings were inoculated for each treatment, and 3 replicates were set. The rooting start time was recorded, and after culturing for 25 days, the average rooting rate, the average number of roots and the average length of the roots were calculated for each treatment.

(3) Test results

The research results show that the rooting effect of Viburnum mediterranei is the best when the 1/2MS medium is added with 1.0 mg/L indole butyric acid and 0.3% activated carbon, the average rooting rate is 95.6%, the average rooting number can reach 4.53, and the average rooting rate is 95.6%. The length is 2.67cm.

Finally, for the Viburnum mediterranei cultivated in the above-mentioned manner, the process of hardening and transplanting the test-tube plantlets is as follows:

Move the seedlings in the bottle directly from the tissue culture room to the greenhouse for 3 days, then uncover the bottle cap, and add a small amount of water to the bottle for 2 days. The strong seedlings after hardening are clamped out from the bottle, and after washing the medium, soak in 0.1% carbendazim solution for 1-2 hours, and then transplant them on the mixed substrate (60% humus soil + 20% charcoal residue) + Sawdust 20%), pour enough water, set up a small arch shed, cover with film, conduct regular ventilation, watering and disease prevention management, the survival rate is above 85%.

Beneficial effects of the present invention: (1) The production of Viburnum mediterranei can be carried out under artificial control conditions without being affected by factors such as seasons, climatic conditions, soil, diseases and insect pests and pesticides, and the quality of viburnum mediterranean seedlings can be strictly controlled. (2) The multiplication speed is fast, the production cycle is short, the equipment is simple, the floor space is small, the manpower and material resources are saved, and it is convenient for factory production. (3) The detoxification treatment was carried out in the tissue culture process, which improved the quality of Viburnum mediterranei seedlings. At the same time, this technical method solved the important technical links of rapid propagation and stable rooting of Viburnum mediterranei, and could be applied to industrial production. (4) The germplasm resources of Mediterranean viburnum can be preserved, and the tree species and resources thereof can be enriched for landscaping.

Although some embodiments of the present invention have been given herein, those skilled in the art should understand that the embodiments herein can be changed without departing from the spirit of the present invention. The above-mentioned embodiments are only exemplary, and the embodiments herein should not be used as limitations on the scope of rights of the present invention.

Claims (1)

1. a method for tissue culture propagation of Viburnum mediterranei, is characterized in that, comprises the steps: Step 101: Selection and treatment of explants: select the stem section of Viburnum mediterranei with axillary buds as the culture material, wash it with running water; sterilize it with alcohol on the ultra-clean bench, rinse it with sterile water, and then sterilize it with mercuric chloride , the mercuric chloride ratio is 0.1%, then rinsed with sterile water, and inoculated in the induction medium; Step 102: Induction culture: use MS medium, 6-benzylaminoadenine 1.0mg/L, naphthaleneacetic acid 0.3mg/L, sucrose 2% and agar 0.6% as the induction medium, and the selected Viburnum mediterranei The stem segment with axillary buds is inoculated in the induction medium; the pH value of the induction medium in the step 102 is 5.8; Step 103: Proliferation culture: transfer the explants grown on the induction medium to MS medium, mix 6-benzylaminoadenine 0.5mg/L, naphthaleneacetic acid 0.2mg/L, sucrose 2% and agar 0.6% Afterwards, it was used as an induction medium for continuous culture; Step 104: rooting culture: select 1/2MS medium, indole butyric acid 1.0mg/L, active carbon 0.3%, sucrose 2% and agar 0.6% to mix as rooting medium, the culture condition of the medium in step 104 : The light intensity is 1500-20001X, the temperature is 23 to 27°C, the light time is 12h/d, and the pH value of the rooting medium is 5.8; Step 105: Transplanting: Take out the rooted plants and transplant them on the mixed substrate of humus soil, charcoal residue and sawdust, wherein the ratio of humus soil: charcoal residue: sawdust is 3:1:1; Wherein, in the step 101, the washing time with water is 20-30 minutes, the alcohol ratio is 70%, the surface sterilization time is 30 seconds, and the mercuric chloride sterilization time is 4-12 minutes.