CN104938340A - Method for conducting rose tissue culturing rapid propagation on explant by using autumn and winter dormant bud - Google Patents

Abstract

The invention discloses a method for tissue culture and rapid propagation of roses by using dormant buds in autumn and winter as explants, and belongs to the technical field of asexual propagation of ornamental plant seedlings. The present invention aims to establish a rose tissue rapid propagation system using dormant buds in autumn and winter as explants, including four stages of explant disinfection treatment, primary culture, proliferation culture and rooting culture. The invention has the advantages of low pollution rate, high reproduction coefficient, short cultivation period, breaks the limitation of autumn and winter reproduction, well solves the problems of low efficiency and long period of the traditional breeding method of roses, and can be widely used in actual production.

Description

A method for tissue culture and rapid propagation of roses using autumn and winter dormant buds as explants

technical field

The invention relates to a method for tissue culture and rapid propagation of roses by using dormant buds in autumn and winter as explants, and belongs to the technical field of asexual propagation of ornamental plant seedlings.

Background technique

Rose (Rosa rugosa Thunb.) is a deciduous shrub of the genus Rosa in the family Rosaceae. It has various varieties, rich colors and strong aroma. It is not only an excellent plant material for landscaping, but also one of the oldest natural spice plants in the world. It is a source of spices for chemical products and an important additive raw material for the food and beverage industries. The rose essential oil extracted from its flowers is a world-famous and precious essential oil, which is extremely expensive and is known as "liquid gold". It is mainly used in high-end perfume, cosmetics and health care industries. It has the functions of relieving mental stress, regulating human endocrine, delaying aging, and beauty. and other effects. Because of its wide application in the fields of medicine, food, and daily chemical industry, the demand for rose seedlings in the international and domestic markets is increasing day by day. The market demand is large and the prospect is broad.

The traditional rose propagation method has a long propagation cycle and a low survival rate of seedlings, making it difficult to achieve rapid factory production. In order to meet market demand, the establishment of a rapid rose propagation system method has become a current hot spot. Rose tissue culture technology can be used to achieve rapid propagation of roses. Based on the existing research reports, leaves and axillary buds of tender stems are mostly selected as explants, and the materials are collected in spring and summer plant growth seasons. There is no direct selection of dormant bud tissues in autumn and winter. Content related to rapid propagation of roses. Therefore, the research and establishment of a rose micropropagation technology using dormant buds as explants can break the reproductive restrictions of roses in autumn and winter and improve the reproductive efficiency of roses.

After literature search, there is no published content about the use of rose dormant buds for tissue culture and rapid propagation.

Contents of the invention

The purpose of the present invention is to break the restriction on the reproduction of roses in autumn and winter, and to invent a method for rapid micropropagation by using dormant buds of roses in autumn and winter.

The present invention is achieved through the following technical proposals: a method of using dormant buds in autumn and winter as explants to carry out rapid propagation of rose tissue culture, which is characterized in that it comprises the following steps:

A. Disinfection treatment of explants: Cut the annual growth and vigorous branches without pests and diseases in sunny weather in winter, brush them lightly with a soft brush, cut them into small sections about 4cm in length, containing 3-4 dormant axillary buds, and wrap them in clean gauze Afterwards, rinse with running water for 3 hours, and then transfer to the aseptic workbench for disinfection;

Soak in 75% alcohol and stir at a constant speed for 60 seconds, rinse with sterile water for 3-5 times, then soak with 3% NaClO and stir for 10 minutes, rinse with sterile water for 3-5 times, take it out and dry the surface with sterile filter paper , use a scalpel to cut it into single axillary bud stem segments about 1cm long for later use;

B primary culture: the formula of the primary medium is MS+1mg/LBA+0.05-0.1mg/LNAA+30g/L sucrose+7g/L agar, the pH value is 5.8; the culture conditions are light intensity 2000-3000Lx, light duration 14h/ d, temperature 25±2°C; peel off the scales of the axillary buds from the sterilized single axillary bud stems with a scalpel (be careful not to damage the axillary buds), then insert them horizontally on the primary culture medium, and culture them in the dark for 1 week. After 20 days, the dormant buds germinate. After 30 days, the dormant buds that germinated and grew well were cut and inserted into the proliferation medium to continue culturing;

C Proliferation culture: Proliferation medium formula is MS+1-2mg/LBA+0.05mg/LNAA+0.6mg/L GA ₃ +30g/L sucrose+7g/L agar, pH value is 5.8; culture condition is light intensity 2000 -3000Lx, light duration 14h/d, temperature 25±2°C; the dormant buds transferred from the primary medium to the proliferation medium can differentiate into a large number of clustered buds after 30 days;

D rooting culture: rooting medium formula is 1/2MS+0.2-0.5mg/LIBA+0.2-0.5mg/LNAA+1-1.5g activated carbon+30g/L sucrose+7g/L agar, pH value is 5.8; Adventitious single buds growing robustly in buds with a stem height of 1-2cm are inserted into the rooting medium; the culture conditions are light intensity 2000-3000Lx, light duration 14h/d, temperature 25±2°C; start after 2-3 weeks of cultivation Germinate adventitious roots.

The disinfection method of rose dormant buds in autumn and winter in the step (A) is: 75% alcohol 60s+3%NaClO 10min.

In the step (B), the formulation of the first-generation culture medium for dormant buds of roses in autumn and winter is MS+1mg/LBA+0.05mg/LNAA+30g/L sucrose+7g/L agar, and the pH value is 5.8;

In the step (B), the fall and winter dormant buds of roses are inoculated on the primary culture medium after peeling off the axillary bud scales (paying attention to avoid injuring the axillary buds).

In the step (C), the rose autumn and winter dormant bud proliferation medium formula is MS+2mg/LBA+0.05mg/LNAA+0.6mg/L GA ₃ +30g/L sucrose+7g/L agar, and the pH value is 5.8; In the step (D), the rose autumn and winter dormant bud rooting medium formula is 1/2MS+0.2mg/LIBA+0.2mg/LNAA+1g activated carbon+30g/L sucrose+7g/L agar, and the pH value is 5.8.

The invention successfully selects the dormant buds of roses in autumn and winter as explants, and combines a series of tissue culture techniques such as disinfection, primary culture, proliferation culture, and rooting culture to realize rapid propagation of roses by using dormant buds in autumn and winter.

The invention has the advantages of low pollution rate, high reproduction coefficient, short cultivation period, breaks the limitation of reproduction in autumn and winter, well solves the problems of low efficiency and long period of the traditional breeding method of roses, and can be applied to actual production.

Detailed ways

The method of the invention selects dormant buds of roses in autumn and winter, and realizes rapid propagation of dormant bud tissues of roses in autumn and winter by regulating four main steps of disinfection, primary culture, proliferation culture and rooting culture. Specifically include the following steps:

A. Disinfection treatment of explants: Cut the annual growth and vigorous branches without pests and diseases in sunny weather in winter, brush them lightly with a soft brush, cut them into small sections about 4cm in length, containing 3-4 dormant axillary buds, and wrap them in clean gauze After washing with running water for 3 hours, move it to a sterile workbench for disinfection treatment; first soak in 75% alcohol and stir at a constant speed for 60 seconds, rinse with sterile water for 3-5 times, then soak with 3% NaClO and stir for 10 minutes at a constant speed, rinse with sterile water for 3 -5 times, after taking it out, blot the surface moisture with sterile filter paper, and cut it into single axillary bud stems with a length of about 1cm with a scalpel for later use;

B primary culture: the formula of the primary medium is MS+1mg/LBA+0.05mg/LNAA+30g/L sucrose+7g/L agar, the pH value is 5.8; the culture conditions are light intensity 2000-3000Lx, light duration 14h/d, The temperature is 25±2°C; the sterilized single axillary bud stem section is peeled off the scales of the axillary buds with a scalpel (be careful not to damage the axillary buds), and then placed horizontally on the primary culture medium, cultured in the dark for 1 week, dormant buds germinate after 20 days, and after 30 days Cut out the dormant buds that germinate and grow well and insert them into the proliferation medium to continue culturing;

C Proliferation culture: Proliferation medium formula is MS+2mg/LBA+0.05mg/LNAA+0.6mg/L GA ₃ +30g/L sucrose+7g/L agar, pH value is 5.8; culture condition is light intensity 2000-3000Lx , the light duration is 14h/d, and the temperature is 25±2°C; the dormant buds transferred from the primary culture medium to the proliferation medium can differentiate into a large number of clustered buds after 30 days;

D rooting culture: rooting medium formula is 1/2MS+0.2mg/LIBA+0.2mg/LNAA+1g activated carbon+30g/L sucrose+7g/L agar, pH value is 5.8; Single adventitious buds with a height of 1-2cm are inserted into the rooting medium; the culture conditions are light intensity 2000-3000Lx, light duration 14h/d, and temperature 25±2°C; adventitious roots begin to germinate after 2-3 weeks of cultivation.

The present invention uses the explant processing and disinfection involved in the following examples, primary culture, proliferation culture, rooting culture technology research to fully illustrate the technical solutions of the present invention, to prove the authenticity, correctness and operability of the research results .

a. Study on the optimal disinfection treatment method for explants

The rose variety "Purple Branch Rose" was selected as the test material. After the retrieved explants were pretreated by the above-mentioned method in A, the single dormant bud stem section was sterilized on the aseptic workbench with three disinfection methods. The scales were inoculated on the first-generation culture medium and cultured, 20 for each treatment, repeated 3 times, the culture conditions were the same as in B, and the pollution rate, browning rate and start-up rate were counted after 20 days.

The test found that with the prolongation of the disinfection time, the pollution rate gradually decreased, the browning rate gradually increased, and the start-up rate gradually decreased; among them, the pollution rate of treatment 2 was significantly lower than that of treatment 1, and the decline was greater than 30%, and the start-up rate could still be maintained. More than 90%; treatment 3 pollution rate is less than 10% lower than treatment 2, and the start-up rate is as low as 77.8%; comprehensive consideration, determine that 75% alcohol 60s+3%NaClO 10min is the best for the winter dormant bud tissue culture of Zizhi rose The best way to disinfect.

Table 1 Effects of different disinfection methods on explants

deal with Disinfection method Pollution rate% Browning rate% Start rate% 1 75% alcohol 60s+3% NaClO 5min 50 0 100 2 75% alcohol 60s+3% NaClO 10min 17 6.25 93.75 3 75% alcohol 60s+3% NaClO 15min 9.8 22.2 77.8

b. Research on the best primary culture medium

Plant growth regulator is an essential substance for inducing the regeneration of winter dormant buds of rose rose. In order to determine the optimal concentration combination, 4 treatments were set up, and 20 explants were inoculated in each treatment, repeated 3 times, and statistics were started after 20 days rate, observe and record its growth.

The test found that BA played a key role in inducing the regeneration of dormant buds in winter in Roses purple branch, and the increase of BA concentration was beneficial to the germination of dormant buds; under the same concentration of BA, the activation rate of 0.05mg/LNAA was higher than that of 0.1mg/LNAA. The dormant buds of treatment 3 and treatment 4 germinated into adventitious buds and had callus formation at the same time, which was conducive to further proliferation and culture, and the initiation rate of treatment 3 was the highest, so it was determined that MS+1mg/LBA+0.05mg/LNAA+30g/L sucrose +7g/L agar is the best primary medium.

Table 2 Effects of different concentrations of plant growth regulators on adventitious bud germination of explants

deal with BA mg/L NAA mg/L Start rate% Growth Status 1 0.5 0.05 83.8 No callus, large spread of adventitious bud leaves 2 0.5 0.1 78.4 No callus, poor growth of adventitious buds 3 1 0.05 93 There is callus formation, the adventitious buds grow well, and the color is green 4 1 0.1 86 There is callus formation, adventitious buds grow well

c. Research on optimal proliferation medium

In order to realize the rapid propagation of rose rose, the adventitious buds germinated in the primary culture should be multiplied and cultured, and four treatments of different concentration combinations of BA, NAA and GA ₃ three kinds of plant growth regulator substances should be set up, and the germination buds on the best primary medium should be cultured. The dormant bud explants were excised and transferred to it, 20 per treatment, repeated 3 times, the growth status was observed and recorded, and the proliferation coefficient was counted after 30 days.

The experiment found that the addition of GA ₃ was helpful for the proliferation of adventitious buds, and the proliferation coefficient showed an upward trend with the increase of GA ₃ concentration; increasing the concentration of BA, the proliferation coefficient of adventitious buds increased significantly, clustered buds had more tillers, and the growth was good, so it was determined that the proliferation coefficient was the highest Treatment 4 formula MS+2mg/LBA+0.05mg/LNAA+0.6mg/L GA ₃ +30g/L sucrose+7g/L agar is the best proliferation medium.

Table 3 Effects of different concentrations of plant growth regulators on the proliferation of adventitious buds of explants

deal with BA mg/L NAA mg/L GA ₃ mg/L Multiplication coefficient Growth Status 1 1 0.05 0.3 2.0 Few cluster buds, most leaves spread out 2 2 0.05 0.3 3.0 There are many clusters of buds, and the color of adventitious buds is light green 3 1 0.05 0.6 2.8 Few buds, good growth 4 2 0.05 0.6 4.4 There are many clusters of buds, the growth is vigorous, and the color is green

d. Research on the best rooting medium

Using 1/2MS medium as the basic medium, adding different concentrations of NAA, IBA, and activated carbon, cutting out a single adventitious bud with robust growth and a stem height of 1-2 cm, and inserting it into the rooting medium, a total of 4 treatments, each treatment 20 Adventitious buds, repeated 3 times, observed and recorded the rooting condition, and counted the rooting rate after 30 days.

The test found that except for treatment 4, the rooting rate of the other three treatments reached more than 90%, and the rooting rate of treatment 2 was the highest. However, the rooting status and growth of adventitious buds were observed, and it was found that the adventitious roots of treatment 1 were short and thick, and the growth of parts of the plant was fast, and the adventitious roots of treatment 2 were thin. Long, part of the plant grows slowly, part of the leaves turn yellow, the adventitious root of treatment 3 is short and thick, the aboveground part grows slowly, and the plant is short. Based on the above, it is determined that 1/2MS+0.2mg/LIBA+0.2mg/LNAA+1g activated carbon+30g/L sucrose+7g/L agar is the best rooting medium.

Table 4 Effects of different concentrations of plant growth regulators on rooting of adventitious shoots

deal with NAA mg/L IBA mg/L Activated carbon g Rooting rate% 1 0.2 0.2 1 90.9 2 0.2 0.5 1.5 95.4 3 0.5 0.2 1.5 90.9 4 0.5 0.5 1 86.3

The invention has the advantages of low pollution rate, high reproduction coefficient, short cultivation period, breaking the limitation of reproduction in autumn and winter, and well solves the problems of low efficiency and long period of the traditional breeding method of roses, and can be widely used in actual production.

The invention uses the dormant buds of roses in autumn and winter as explants, and realizes rapid propagation of roses through tissue culture technology. The invention has great significance for improving the reproduction coefficient of roses, shortening the cultivation cycle, breaking the reproduction restriction in autumn and winter, and realizing the large-scale production of rose seedlings.

Claims (6)

1. a kind of method that utilizes dormant bud in autumn and winter to carry out rose tissue culture rapid propagation for explant, it is characterized in that comprising the following steps: (A) Disinfection treatment of explants: in fine weather after the fall of leaves in autumn and winter, cut annual growth and healthy branches without pests and diseases, lightly brush them with a soft brush, cut them into small sections containing 3-4 dormant axillary buds, and clean them with clean After being wrapped in gauze and rinsed with running water, move it into a sterile workbench for disinfection; Soak in 75% alcohol and stir at a constant speed for 60 seconds, rinse with sterile water for 3-5 times, then soak with 3% NaClO and stir for 5-10 minutes, rinse with sterile water for 3-5 times, take it out and dry it with sterile filter paper surface moisture, use a scalpel to cut it into single axillary bud stem segments for later use; (B) Primary culture: The formula of the primary medium is MS+1mg/LBA+0.05mg/LNAA+30g/L sucrose+7g/L agar, the pH value is 5.8; the culture conditions are light intensity 2000-3000Lx, light duration 14h/ d, temperature 25±2°C; peel off the scales of the sterilized single axillary bud stems with a scalpel, then insert them horizontally on the primary culture medium, and culture them in the dark for 1 week. After 20 days, the dormant buds germinate, and after 30 days, they are cut and germinated and grow well. The dormant buds were inserted into the proliferation medium to continue culturing; (C) Proliferation culture: the formula of the proliferation medium is MS+2mg/LBA+0.05mg/LNAA+0.6mg/L GA ₃ +30g/L sucrose+7g/L agar, the pH value is 5.8; the culture condition is light intensity 2000 -3000Lx, light duration 14h/d, temperature 25±2°C; the dormant buds transferred from the primary medium to the proliferation medium can differentiate into a large number of clustered buds after 30 days; (D) rooting culture: rooting medium formula is 1/2MS+0.2mg/LIBA+0.2mg/LNAA+1-1.5g gac+30g/L sucrose+7g/L agar, pH value is 5.8; Adventitious single buds with strong growth and stem height of 1-2cm are inserted into the rooting medium; the culture conditions are light intensity 2000-3000Lx, light duration 14h/d, temperature 25±2°C; adventitious roots will start to germinate after 2-3 weeks of cultivation . 2. The method of using autumn and winter dormant buds as explants for rapid propagation of rose tissue culture according to claim 1, characterized in that the disinfection method for rose autumn and winter dormant buds in the step (A) is: 75% alcohol for 60 seconds +3%NaClO 10min. 3. A method of using autumn and winter dormant buds as explants for tissue culture and rapid propagation of roses according to claim 1, characterized in that the formula of the primary culture medium for rose autumn and winter dormant buds in the step (B) is MS +1mg/LBA+0.05mg/LNAA+30g/L sucrose+7g/L agar, the pH value is 5.8. 4. The method of using autumn and winter dormant buds as explants for rapid propagation of rose tissue culture according to claim 1, characterized in that in the step (B), the axillary bud scales are peeled off from the autumn and winter dormant buds of roses (note that avoiding damage to Axillary buds) were inoculated on the primary medium. 5. The method of using autumn and winter dormant buds as explants for rapid propagation of rose tissue culture according to claim 1, characterized in that the formulation of the growth medium for rose autumn and winter dormant buds in the step (C) is MS+2mg/ LBA+0.05mg/LNAA+0.6mg/L GA ₃ +30g/L sucrose+7g/L agar, the pH value is 5.8. 6. A method of using autumn and winter dormant buds as explants for rapid propagation of rose tissue culture according to claim 1, characterized in that in the step (D), the formulation of the rooting medium for rose autumn and winter dormant buds is 1 /2MS+0.2mg/LIBA+0.2mg/LNAA+1g activated carbon+30g/L sucrose+7g/L agar, the pH value is 5.8.