CN103355174A - Low-cost high-efficiency industrialized seedling production method for cold-resistant Chinese rose - Google Patents

Abstract

The invention relates to a low-cost high-efficiency industrialized seedling production method for cold-resistant Chinese rose. The method comprises the following steps: (1) preparation of an MS medium and 6-BA and NAA mother liquors; (2) preparation of a medium; (3) preparation of an explant; (4) multiplication of cluster buds; (5) rooting of tissue culture seedlings; (6) preparation before transplanting; and (7) transplanting. According to the invention, instead of propagation of cold-resistant Chinese rose by using a traditional cutting means, the method employs plant tissue culture technology to rapidly propagate cold-resistant Chinese rose, realizing industrialized seedling production.

Description

A low-cost and high-efficiency factory seedling raising method for cold-resistant rose

technical field

The invention relates to a low-cost and high-efficiency industrial seedling raising technology for cold-resistant roses, in particular to a rapid seedling-raising method for industrial production using cold-resistant roses that can overwinter in the open field in alpine regions as materials through tissue culture technology.

Background technique

The invention is a technology aimed at industrialized production of high-quality cold-resistant rose high-quality seedlings in the technical field of agricultural production. As a traditional famous flower in my country, rose is a perennial Rosaceae plant with rich colors and long flowering period. It occupies an important position in the field of landscaping and beautification and has extremely high commercial value. However, due to the limitation of cold resistance, the resources of flowering woody plants available for viewing in urban landscaping in cold northern regions such as the three provinces in Northeast China, Inner Mongolia, and Xinjiang have always been scarce.

The successful breeding of cold-resistant roses has added new varieties of perennial green flowers in cold regions, and made it possible to change the investment of green flowers from repeated investment every year to annual investment to benefit for many years. Propagating a large number of excellent cold-resistant rose seedlings can reduce labor, seedlings and other inputs in the process of urban greening, save money, and meet the overall goal of developing a low-carbon society.

The propagation of rose can be sown, layered, grafted and cut. Seed propagation, layering, and grafting are time-consuming and labor-intensive, and the propagation speed is relatively slow. In order to successfully propagate Chinese roses by cuttings, two conditions must be met. One is the internal condition, that is, the choice of cuttings, that is, the cuttings should be selected from branches that are fully developed in the lower part after flowering, and the amount of seedlings is limited by the number of cuttings; the other is external conditions. The factor is to create suitable rooting conditions for cuttings such as certain temperature, humidity and light. Due to the above two reasons, the survival rate of cutting rose seedlings is low, and the scale of seedling cultivation is limited, which restricts the propagation speed of cold-resistant rose seedlings. , which in turn affects its application in urban gardens in northern cold regions.

Plant tissue culture technology is a method of propagating plants under sterile conditions using a part of the plant body, including cells, tissues or organs, under artificially controlled nutritional and environmental conditions. Compared with conventional vegetative propagation such as cuttings, it has many obvious advantages: (1) There are very few materials for propagation. It only needs to use a small amount of plant materials, such as stem cuttings, leaves, stem tips or other tissues and organs, to establish a repeated multiplication system in a culture bottle, which greatly saves a large amount of female parent consumption in conventional asexual reproduction. plant material. (2) The breeding speed is fast and the breeding line is very high. On the artificial culture medium, under good light and temperature conditions throughout the year, the daily temperature is 25-28°C and the night temperature is 20°C; 16 hours of light and 8 hours of darkness in 24 hours. Each culture, "explant," produces either a large number of shoots or a large number of somatic embryos. The cycle of micropropagation is only 1-2 months, which is much shorter than conventional asexual reproduction. In addition, it is not affected by seasons and climatic conditions. It reproduces and produces every year, so the reproduction speed is extremely fast and the reproduction coefficient is high. Often up to tens of thousands of times. (3) Space and land are greatly saved. Due to the miniaturization of explants in the culture bottle and the use of multi-layer culture racks, a small amount of space and a small amount of land can be used to produce a large number of plants.

At present, the propagation of anti-cold rose is mainly through cuttings, but cutting propagation is limited by the season and the number of cuttings, and it is difficult to carry out large-scale production, which limits its application in urban gardens in northern cold regions.

Contents of the invention

The purpose of the present invention is to overcome the disadvantages in the above-mentioned technologies, and provide a low-cost and high-efficiency industrial seedling raising method for cold-resistant roses.

In order to achieve the above object, the technical scheme adopted in the present invention is:

(1) Preparation of MS medium and 6-BA, NAA mother solution

According to the general formula of MS medium, the mother liquors of macroelements, trace elements, iron salts, and organic substances were respectively prepared. To prevent precipitation, when preparing the mother liquors of macroelements, the calcium nitrate in the formula should be prepared separately and stored separately. into a 100-fold concentrated solution,

The preparation method of 6-BA mother liquor:

Preparation of 6-BA and NAA mother liquor Weigh 0.1g of 6-BA with a balance of one ten-thousandth accuracy, put it into a clean 200ml beaker, pour a little hydrochloric acid of 1 mole per liter into the beaker, and dissolve 6-BA After fully dissolving, pour the solution in the beaker into a 100ml volumetric flask, rinse the beaker with a small amount of distilled water for 1-2 times, pour the rinsed distilled water into the volumetric flask, set the volume to 100ml, and pour the solution in the volumetric flask After fully mixing, put it in a brown bottle, label it, and mark the name of the mother solution, the date of preparation and the concentration on the label, and set it aside for later use.

The preparation method of NAA mother liquor:

Weigh 0.1g of NAA with a balance with an accuracy of one ten-thousandth, put it into a clean 200ml beaker, pour a little 0.1 mol per liter of sodium hydroxide into the beaker, fully dissolve the NAA, and pour the solution in the beaker Put it into a 100ml volumetric flask, rinse the beaker with a small amount of distilled water for 1-2 times, pour the rinsed distilled water into the volumetric flask, and set the volume to 100ml, mix the solution in the volumetric flask thoroughly and put it in a brown bottle , affix a label, and after indicating the name of the mother solution, the date of preparation and the concentration on the label, set aside,

(2) Preparation of culture medium: boil tap water in a rice cooker, then cool naturally. Pour out the clear cold boiled water on the top, pour off the part with more sediment at the bottom, and then prepare the culture medium with the clear cold boiled water after cooling, the formula is MS+6-BA0.5-2.5mg/L+NAA0.01-0.2mg /L+carrageenan with a strength of 1200 6.5g/L+white sugar 30g/L, when the medium is boiled and the volume is constant, add 1 mole per liter of hydrochloric acid or sodium hydroxide to each liter of medium with a rubber dropper , adjust the pH value of the medium to 5.8-6.5, adjust the acidity of the medium, and divide it into 250 ml glass culture bottles before it solidifies, cover it and sterilize it under high pressure, and sterilize it at room temperature and pressure for 20 minutes Finally, take out the culture medium, after cooling, place it in a cool place in the storage room for 3-7 days, and use it for subsequent rose inoculation after observing that there is no pollution.

(3) Explant preparation: Select the young and tender branches with axillary buds of the cold-resistant rose that are free from diseases and insect pests, cut off the leaves and petioles of the selected explants, cut off 1-2cm stems with axillary buds, and rinse with tap water 15min, first disinfect with 75% alcohol for 30s, then disinfect with 0.1% mercury liter for 3-5min, rinse with sterile water for 3 times, inoculate the sterilized explants on MS blank medium, and culture in the cultivation room under light. The light time is 10-12h/d, the light intensity is 20001x, and the temperature of the culture room is 25°C±2°C.

(4) Proliferation of clustered buds: the above-mentioned aseptic young and tender stems were cultivated for about 10-15 days, and axillary buds on the stems would grow out. The shoots on the top were cut off and inoculated on the medium of MS+6-BA0.5-2.5mg/L+NAA0.01-0.2mg/L+carrageenan 6.5g/L+white sugar 30g/L with a strength of 1200, Continue to place in the cultivation room for proliferation and cultivation, the light time is 10-12h/d, the light intensity is 20001x, the temperature is 25°C±2°C,

(5) rooting of tissue culture seedlings

Cut out the 2-4 cm high Chinese rose buds in the culture bottle from the base of each individual plant under aseptic conditions, and inoculate them in the rooting medium, the rooting medium formula is 1/4-1/2MS +NAA0.1-1.0mg/L+carrageenan with a strength of 1200 6.5g/L+white sugar 20g/L, placed in the cultivation room for 15-20 days under light cultivation, and transplanted when the root grows to 1.0-1.5 cm.

(6) Preparation before transplanting

Spread waste newspapers of appropriate size on the bottom of a plastic flower pot with a diameter of 15 cm or a plastic seedling tray with a height of 5 cm, moisten the newspaper with a watering can, and then add vermiculite to the flower pot or seedling tray, and remove it from the flowerpot or seedling tray. When the edge of the plate is 1 cm, use a ruler to scrape the vermiculite, then use a watering can to pour the vermiculite in the flower pot or seedling plate, drain the water, and set aside.

(7) Transplanting

When the roots of rose seedlings in the bottle grow to 1.0-1.5 cm, open the bottle cap, pinch out the seedlings with tweezers, wash off the root medium in clean water, and plant the seedlings into the flower tray or seedling tray prepared above Then use a watering can to pour a suitable concentration of rooting agent, fill up the loosened vermiculite when planting seedlings, drain the water, cover the flower pot with a small and thin plastic shopping bag, and use 2 The roots are supported by thin iron wires, and then the seedling tray is covered with plastic film. The contact between the plastic film and the seedling tray is sealed with transparent tape. Use scissors to open a small hole with a diameter of 1.0-1.5 cm on the plastic film, gradually reduce the air humidity in the flowerpot or seedling tray, and remove all the film 6-8 days after opening the small hole, and the seedlings can be removed after 15 days. The seedlings were transplanted into the pre-prepared nutrient soil.

The advantages of the present invention are:

The invention breaks through the traditional method of propagating cold-resistant roses by cuttings, and utilizes plant tissue culture technology to rapidly propagate cold-resistant roses to realize industrialized seedling cultivation. When using this method to quickly propagate roses, use cheap clean cold boiled water instead of relatively expensive distilled water, use carrageenan instead of relatively expensive agar, use cheap white granulated sugar instead of sucrose, and use vermiculite as a cultivation substrate. Fungal contamination leads to the problem of low survival rate. Covering flower trays or seedling trays with plastic bags or mulch can ensure the air humidity requirements of seedlings during acclimation. The seedlings will generally send out new roots after 10 days under the condition of 90% relative humidity.

The present invention uses the culture medium formula suitable for the rapid proliferation of the cold-resistant rose to carry out the rapid proliferation and rooting in the culture room. In the multiplication process, use cold boiled water instead of distilled water, carrageenan instead of agar, and white sugar instead of sucrose. Compared with normal tissue culture rapid propagation, the cost can be reduced by 50%. Quantitative acid adjustment with hydrochloric acid is convenient and avoids the use of expensive acidity meters. , using vermiculite covering film instead of peat soil as the cultivation substrate can prevent the seedlings from being polluted and die, and can also improve the survival rate of domesticated seedlings. Compared with the traditional method of propagating rose seedlings by cuttings, the cold-resistant rose seedlings produced by tissue culture technology have a high survival rate, fast rooting and growth speed, shorten the cultivation time by 10% to 20%, and maintain excellent quality.

Detailed ways

Embodiments of the present invention are further described in detail below.

Embodiment 1,

(1) Preparation of MS medium and 6-BA, NAA mother solution

According to the general formula of MS medium, the mother liquors of macroelements, trace elements, iron salts, and organic substances were prepared respectively. In order to prevent precipitation, when preparing the mother liquors of macroelements, the calcium nitrate in the formula should be prepared separately and stored separately. Each mother liquor is made into 100 times concentrated solution,

The preparation method of 6-BA mother liquor:

Preparation of 6-BA and NAA mother liquor Weigh 0.1g of 6-BA with a balance of one ten-thousandth accuracy, put it into a clean 200ml beaker, pour a little hydrochloric acid of 1 mole per liter into the beaker, and dissolve 6-BA After fully dissolving, pour the solution in the beaker into a 100ml volumetric flask, rinse the beaker with a small amount of distilled water for 1-2 times, pour the rinsed distilled water into the volumetric flask, set the volume to 100ml, and pour the solution in the volumetric flask After fully mixing, put it in a brown bottle, label it, and mark the name of the mother solution, the date of preparation and the concentration on the label, and set it aside for later use.

The preparation method of NAA mother liquor:

Weigh 0.1g of NAA with a balance with an accuracy of one ten-thousandth, put it into a clean 200ml beaker, pour a little 0.1 mol per liter of sodium hydroxide into the beaker, fully dissolve the NAA, and pour the solution in the beaker Put it into a 100ml volumetric flask, rinse the beaker with a small amount of distilled water for 1-2 times, pour the rinsed distilled water into the volumetric flask, and set the volume to 100ml, mix the solution in the volumetric flask thoroughly and put it in a brown bottle , affix a label, and after indicating the name of the mother solution, the date of preparation and the concentration on the label, set aside,

(2) Preparation of medium

Boil the tap water in an electric rice cooker, then cool it naturally, pour out the clear cold boiled water on the top, pour out the part with more sediment at the bottom, and prepare the culture medium with the clear cold boiled water after cooling,

The following takes the preparation of 1 liter of cold-resistant rose medium as an example to illustrate the specific preparation method:

a. Use a syringe to sequentially extract 10 ml of a large amount of element mother liquid, calcium nitrate mother liquid, trace element mother liquid, iron salt and organic matter mother liquid into a 500 ml beaker, and add 6-BA to the mixed mother liquid with a pipette. 2.5 ml, NAA stock solution 0.01 ml.

b. Use a beaker to measure 600 ml of cold boiled water and pour it into the rice cooker, add 6.5 grams of carrageenan with a strength of 1200, stir well with a glass rod, then add 30 grams of white sugar, stir to melt,

c. Pour the solution containing mother liquor and plant hormones in the above beaker into the rice cooker, rinse the beaker with a small amount of cold boiled water, then pour the water into the rice cooker, stir evenly, disconnect the power supply, and put the Pour the hot culture medium into a 1L beaker, add cold boiled water to make the volume to 1L, then add 1 mole per liter of hydrochloric acid to the beaker with a rubber dropper, and adjust the pH of the culture medium to 6.5 by monitoring with a pH meter.

d. The above-mentioned acid-adjusted culture medium is divided into 250 ml glass culture bottles before solidification, and the height of each bottle of culture medium is preferably 1.5 cm, and then the lid containing the sterile vent hole is covered.

e. Sterilization of medium

Sterilize the prepared medium at normal temperature and pressure for 20 minutes. When the pressure drops to 0, take out the medium and place it in a cool place for observation for 5 days. If no bacteria appear on the medium, it can be used for the next rose inoculation. ,

(3) Preparation of explants

Select the young and tender branches with axillary buds that were born in the year of the cold-resistant rose without diseases and insect pests, cut off the leaves and petioles of the selected explants, cut off the 3cm stems with axillary buds, and rinse them with tap water for 15 minutes; % alcohol for 30s, then 0.1% mercuric liter for 3 minutes, rinsed with sterile water for 3 times, set aside,

(4) Proliferation of clustered buds

Use sterile tweezers and a scalpel to remove 0.5 cm from the bottom of the cold-resistant rose stem section sterilized in the above step (3) in the ultra-clean workbench, inoculate the cut upper stem section on the medium, cover the lid, and put Proliferate and cultivate in the culture room,

After 30 days, when the clustered buds are full of the bottle, cut the clustered buds with a sterile scalpel and tweezers in the ultra-clean workbench, and continue to inoculate them on the proliferation medium for proliferation and culture until the required number is multiplied.

(5) Preparation of rooting medium

Take the preparation of 1 liter of Chinese rose rooting medium as an example to illustrate the specific preparation method:

Repeat the four steps of a, b, c, and d in the above (1), except that 10 milliliters of mother liquor in step a is replaced by 2.5 milliliters; 0.1 milliliters of NAA mother liquor is added to the mixed mother liquor without adding 6- BA; What added in step b is 20 grams of white granulated sugar, then repeat the step sterilization of (e) in the above-mentioned (2),

When the rose cluster buds in the culture bottle are 2.5cm high, inoculate each bud on the rooting medium under aseptic conditions, and then place it in the culture room for about 15 days of light cultivation, and each bud will produce 3-5 roots of 1.0- 1.5 cm white root,

(6) Preparation before transplanting

Spread waste newspapers of appropriate size on the bottom of a plastic flower pot with a diameter of 15 cm or a plastic seedling tray with a height of 5 cm, moisten the newspaper with a watering can, and then add vermiculite to the flower pot or seedling tray, and remove it from the flowerpot or seedling tray. When the edge of the plate is 1 cm, use a ruler to scrape the vermiculite, then use a watering can to pour the vermiculite in the flower pot or seedling plate, drain the water, and set aside.

(7) Transplanting

When the roots of the rose seedlings in the culture bottle grow to 1.0-1.5 cm, carefully clamp the seedlings out with tweezers, put the root medium in clean water to wash off, and place the seedlings in flowerpots or seedling trays soaked in water. Then pour it again with a suitable concentration of rooting agent, fill the vermiculite, drain the water, bend it into a bracket with iron wire, then cover it with plastic film, seal the edges tightly, and place it in the cultivation room for light cultivation. After 10 days, it will grow on the film Open holes on the top to reduce humidity, remove the plastic film after one week, and continue to cultivate for 15 days before transplanting into the soil and entering normal management.

Embodiment 2,

(1) Preparation of MS medium and 6-BA, NAA mother solution

According to the general formula of MS medium, the mother liquors of macroelements, trace elements, iron salts, and organic substances were prepared respectively. In order to prevent precipitation, when preparing the mother liquors of macroelements, the calcium nitrate in the formula should be prepared separately and stored separately. Each mother liquor is made into 100 times concentrated solution,

The preparation method of 6-BA mother liquor:

Preparation of 6-BA and NAA mother liquor Weigh 0.1g of 6-BA with a balance of one ten-thousandth accuracy, put it into a clean 200ml beaker, pour a little hydrochloric acid of 1 mole per liter into the beaker, and dissolve 6-BA After fully dissolving, pour the solution in the beaker into a 100ml volumetric flask, rinse the beaker with a small amount of distilled water for 1-2 times, pour the rinsed distilled water into the volumetric flask, set the volume to 100ml, and pour the solution in the volumetric flask After fully mixing, put it in a brown bottle, label it, and mark the name of the mother solution, the date of preparation and the concentration on the label, and set it aside for later use.

The preparation method of NAA mother liquor:

Weigh 0.1g of NAA with a balance with an accuracy of one ten-thousandth, put it into a clean 200ml beaker, pour a little 0.1 mol per liter of sodium hydroxide into the beaker, fully dissolve the NAA, and pour the solution in the beaker Put it into a 100ml volumetric flask, rinse the beaker with a small amount of distilled water for 1-2 times, pour the rinsed distilled water into the volumetric flask, and set the volume to 100ml, mix the solution in the volumetric flask thoroughly and put it in a brown bottle , affix a label, and after indicating the name of the mother solution, the date of preparation and the concentration on the label, set aside,

(2) Preparation of medium

Boil the tap water in an electric rice cooker, then cool it naturally, pour out the clear cold boiled water on the top, pour out the part with more sediment at the bottom, and prepare the culture medium with the clear cold boiled water after cooling,

The following takes the preparation of 1 liter of cold-resistant rose medium as an example to illustrate the specific preparation method:

a. Use a syringe to sequentially extract 10 ml of a large amount of element mother liquid, calcium nitrate mother liquid, trace element mother liquid, iron salt and organic matter mother liquid into a 500 ml beaker, and add 6-BA to the mixed mother liquid with a pipette. 2.0 ml, NAA stock solution 0.05 ml,

b. Use a beaker to measure 600 ml of cold boiled water and pour it into the rice cooker, add 6.5 grams of carrageenan with a strength of 1200, stir well with a glass rod, then add 30 grams of white sugar, stir to melt,

c. Pour the solution containing mother liquor and plant hormones in the above beaker into the rice cooker, rinse the beaker with a small amount of cold boiled water, then pour the water into the rice cooker, stir evenly, disconnect the power supply, and put the Pour the hot culture medium into a 1L beaker, add cold boiled water to make the volume to 1L, then add 1 mole per liter of hydrochloric acid to the beaker with a rubber dropper, and adjust the pH of the culture medium to 6.5 by monitoring with a pH meter.

d. The above-mentioned acid-adjusted culture medium is divided into 250 ml glass culture bottles before solidification, and the height of each bottle of culture medium is preferably 1.5 cm, and then the lid containing the sterile vent hole is covered.

e. Sterilization of medium

Sterilize the prepared medium at normal temperature and pressure for 20 minutes. When the pressure drops to 0, take out the medium and place it in a cool place for observation for 7 days. If no bacteria appear on the medium, it can be used for the next rose inoculation. ,

(3) Preparation of explants

Select the young and tender branches with axillary buds of the cold-resistant rose that are free from diseases and insect pests, cut off the leaves and petioles of the selected explants, cut off the 2.5cm stems with axillary buds, and wash them with tap water for 20 minutes; Disinfect with 75% alcohol for 30 seconds, then disinfect with 0.1% mercury liter for 4 minutes, rinse with sterile water for 4 times, set aside,

(4) Proliferation of clustered buds

Use sterile tweezers and a scalpel to remove 0.6 cm from the bottom of the cold-resistant rose stem section sterilized in the above step (3) in the ultra-clean workbench, inoculate the cut upper stem section on the medium, cover the lid, and put Proliferate and cultivate in the culture room,

After 30 days, when the clustered buds are full of the bottle, cut the clustered buds with a sterile scalpel and tweezers in the ultra-clean workbench, and continue to inoculate them on the proliferation medium for proliferation and culture until the required number is multiplied.

(5) Preparation of rooting medium

Take the preparation of 1 liter of Chinese rose rooting medium as an example to illustrate the specific preparation method:

Repeat the four steps of a, b, c, and d in the above (1), except that 10 milliliters of mother liquor in step a is replaced by 3.3 milliliters; 0.2 milliliters of NAA mother liquor is added to the mixed mother liquor, without adding 6- BA; What added in step b is 20 grams of white granulated sugar, then repeat the step sterilization of (e) in the above-mentioned (2),

When the rose buds in the culture bottle are 3.0cm high, inoculate each bud on the rooting medium under aseptic conditions, and then place it in the culture room for about 20 days of light cultivation, and each bud will produce 3-5 roots of 1.0- 1.5 cm white root,

(6) Preparation before transplanting

Spread waste newspapers of appropriate size on the bottom of a plastic flower pot with a diameter of 15 cm or a plastic seedling tray with a height of 5 cm, moisten the newspaper with a watering can, and then add vermiculite to the flower pot or seedling tray, and remove it from the flowerpot or seedling tray. When the edge of the plate is 1 cm, use a ruler to scrape the vermiculite, then use a watering can to pour the vermiculite in the flower pot or seedling plate, drain the water, and set aside.

(7) Transplanting

When the roots of the rose seedlings in the culture bottle grow to 1.0-1.5 cm, carefully clamp the seedlings out with tweezers, put the root medium in clean water to wash off, and place the seedlings in flowerpots or seedling trays soaked in water. Then pour it again with a suitable concentration of rooting agent, fill the vermiculite, drain the water, bend it into a bracket with iron wire, then cover it with plastic film, seal the edges tightly, and place it in the cultivation room for light cultivation. After 8 days, it will grow on the film Open holes on the top to reduce humidity, remove the plastic film after one week, and continue to cultivate for 12 days before transplanting into the soil and entering normal management.

Embodiment 3,

(1) Preparation of MS medium and 6-BA, NAA mother solution

According to the general formula of MS medium, the mother liquors of macroelements, trace elements, iron salts, and organic substances were prepared respectively. In order to prevent precipitation, when preparing the mother liquors of macroelements, the calcium nitrate in the formula should be prepared separately and stored separately. Each mother liquor is made into 100 times concentrated solution,

The preparation method of 6-BA mother liquor:

Preparation of 6-BA and NAA mother liquor Weigh 0.1g of 6-BA with a balance of one ten-thousandth accuracy, put it into a clean 200ml beaker, pour a little hydrochloric acid of 1 mole per liter into the beaker, and dissolve 6-BA After fully dissolving, pour the solution in the beaker into a 100ml volumetric flask, rinse the beaker with a small amount of distilled water for 1-2 times, pour the rinsed distilled water into the volumetric flask, set the volume to 100ml, and pour the solution in the volumetric flask After fully mixing, put it in a brown bottle, label it, and mark the name of the mother solution, the date of preparation and the concentration on the label, and set it aside for later use.

The preparation method of NAA mother liquor:

Weigh 0.1g of NAA with a balance with an accuracy of one ten-thousandth, put it into a clean 200ml beaker, pour a little 0.1 mol per liter of sodium hydroxide into the beaker, fully dissolve the NAA, and pour the solution in the beaker Put it into a 100ml volumetric flask, rinse the beaker with a small amount of distilled water for 1-2 times, pour the rinsed distilled water into the volumetric flask, and set the volume to 100ml, mix the solution in the volumetric flask thoroughly and put it in a brown bottle , affix a label, and after indicating the name of the mother solution, the date of preparation and the concentration on the label, set aside,

(2) Preparation of medium

Boil the tap water in an electric rice cooker, then cool it naturally, pour out the clear cold boiled water on the top, pour out the part with more sediment at the bottom, and prepare the culture medium with the clear cold boiled water after cooling,

Take the preparation of 1 liter of cold-resistant rose medium as an example to illustrate the specific preparation method:

a. Use a syringe to sequentially extract 10 ml of a large amount of element mother liquid, calcium nitrate mother liquid, trace element mother liquid, iron salt and organic matter mother liquid into a 500 ml beaker, and add 6-BA to the mixed mother liquid with a pipette. 2.1 ml, NAA stock solution 0.08 ml,

b. Use a beaker to measure 600 ml of cold boiled water and pour it into the rice cooker, add 6.5 grams of carrageenan with a strength of 1200, stir well with a glass rod, then add 30 grams of white sugar, stir to melt,

c. Pour the solution containing mother liquor and plant hormones in the above beaker into the rice cooker, rinse the beaker with a small amount of cold boiled water, then pour the water into the rice cooker, stir evenly, disconnect the power supply, and put the Pour the hot culture medium into a 1L beaker, add cold boiled water to make the volume to 1L, then add 1 mole per liter of hydrochloric acid to the beaker with a rubber dropper, and adjust the pH of the culture medium to 5.8 by monitoring with a pH meter.

d. The above-mentioned acid-adjusted culture medium is divided into 250 milliliters of glass culture bottles before solidification, and the height of each bottle of culture medium is preferably 1.5 centimeters, and then the lid containing the sterile vent hole is covered.

e. Sterilization of medium

Sterilize the prepared medium at normal temperature and pressure for 20 minutes. When the pressure drops to 0, take out the medium and place it in a cool place for observation for 6 days. If no bacteria appear on the medium, it can be used for the next rose inoculation. ,

(3) Preparation of explants

Select the young and tender branches with axillary buds that were born in the cold-resistant rose season without diseases and insect pests, cut off the leaves and petioles of the selected explants, cut off the 2.6cm stems with axillary buds, and rinse them with tap water for 30 minutes; Disinfect with 75% alcohol for 30 seconds, then disinfect with 0.1% mercury liter for 5 minutes, rinse with sterile water for 5 times, set aside,

(4) Proliferation of clustered buds

Use sterile tweezers and a scalpel to remove 0.2 cm from the bottom of the cold-resistant rose stem section sterilized in the above step (3) in the ultra-clean workbench, inoculate the cut upper stem section on the medium, cover the lid, and put Proliferate and cultivate in the culture room,

After 30 days, when the clustered buds are full of the bottle, cut the clustered buds with a sterile scalpel and tweezers in the ultra-clean workbench, and continue to inoculate them on the proliferation medium for proliferation and culture until the required number is multiplied.

(5) Preparation of rooting medium Take the preparation of 1 liter of Chinese rose rooting medium as an example to illustrate the specific preparation method:

Repeat the four steps of a, b, c, and d in the above (1), except that 10 milliliters of mother liquor in step a is replaced by 5 milliliters; 0.15 milliliters of NAA mother liquor is added to the mixed mother liquor without adding 6- BA; What added in step b is 20 grams of white granulated sugar, then repeat the step sterilization of (e) in the above-mentioned (2),

When the rose buds in the culture bottle are 2.7cm high, inoculate each bud on the rooting medium under aseptic conditions, and then place it in the culture room for about 20 days of light cultivation, and each bud will produce 3-5 roots of 1.0- 1.5 cm white root,

(6) Preparation before transplanting

Spread waste newspapers of appropriate size on the bottom of a plastic flower pot with a diameter of 15 cm or a plastic seedling tray with a height of 5 cm, moisten the newspaper with a watering can, and then add vermiculite to the flower pot or seedling tray, and remove it from the flowerpot or seedling tray. When the edge of the plate is 1 cm, use a ruler to scrape the vermiculite, then use a watering can to pour the vermiculite in the flower pot or seedling plate, drain the water, and set aside.

(7) Transplanting

When the roots of the rose seedlings in the culture bottle grow to 1.0-1.5 cm, carefully clamp the seedlings out with tweezers, put the root medium in clean water to wash off, and place the seedlings in flowerpots or seedling trays soaked in water. Then pour it again with a suitable concentration of rooting agent, fill the vermiculite, drain the water, bend it into a bracket with iron wire, then cover it with plastic film, seal the edges tightly, and place it in the cultivation room for light cultivation. After 7 days, it will grow on the film Open holes on the top to reduce humidity, remove the plastic film after one week, and continue to cultivate for 20 days before transplanting into the soil and entering normal management.

Claims (1)

1. A low-cost and high-efficiency industrial seedling raising method for cold-resistant rose, characterized in that: (1) Preparation of MS medium and 6-BA, NAA mother solution According to the general formula of MS medium, the mother liquors of macroelements, trace elements, iron salts, and organic substances were respectively prepared. To prevent precipitation, when preparing the mother liquors of macroelements, the calcium nitrate in the formula should be prepared separately and stored separately. into a 100-fold concentrate, The preparation method of 6-BA mother liquor: Preparation of 6-BA and NAA mother liquor Weigh 0.1g of 6-BA with a balance of one ten-thousandth accuracy, put it into a clean 200ml beaker, pour a little hydrochloric acid of 1 mole per liter into the beaker, and dissolve 6-BA After fully dissolving, pour the solution in the beaker into a 100ml volumetric flask, rinse the beaker with a small amount of distilled water for 1-2 times, pour the rinsed distilled water into the volumetric flask, set the volume to 100ml, and pour the solution in the volumetric flask After fully mixing, put it in a brown bottle, label it, and mark the name of the mother solution, the date of preparation and the concentration on the label, and set it aside for later use. The preparation method of NAA mother liquor: Weigh 0.1g of NAA with a balance with an accuracy of one ten-thousandth, put it into a clean 200ml beaker, pour a little 0.1 mol per liter of sodium hydroxide into the beaker, fully dissolve the NAA, and pour the solution in the beaker Put it into a 100ml volumetric flask, rinse the beaker with a small amount of distilled water for 1-2 times, pour the rinsed distilled water into the volumetric flask, and set the volume to 100ml, mix the solution in the volumetric flask thoroughly and put it in a brown bottle , affix a label, and after indicating the name of the mother solution, the date of preparation and the concentration on the label, it is ready for use. (2) Preparation of culture medium: boil tap water in a rice cooker, then cool naturally. Pour out the clear cold boiled water on the top, pour off the part with more sediment at the bottom, and then prepare the culture medium with the clear cold boiled water after cooling, the formula is MS+6-BA0.5-2.5mg/L+NAA0.01-0.2mg /L+carrageenan with a strength of 1200 6.5g/L+white sugar 30g/L, when the medium is boiled and the volume is constant, add 1 mole per liter of hydrochloric acid or sodium hydroxide to each liter of medium with a rubber dropper , adjust the pH value of the medium to 5.8-6.5, adjust the acidity of the medium, and divide it into 250 ml glass culture bottles before it solidifies, cover it and sterilize it under high pressure, and sterilize it at room temperature and pressure for 20 minutes Finally, take out the culture medium, after cooling, place it in a cool place in the storage room for 3-7 days, and use it for subsequent rose inoculation after observing that there is no pollution. (3) Preparation of explants: Select the young and tender branches with axillary buds of the cold-resistant rose that year without diseases and insect pests, cut off the leaves and petioles of the selected explants, cut off 1-2cm stems with axillary buds, and rinse them with tap water for 15 minutes , first sterilized with 75% alcohol for 30s, then sterilized with 0.1% mercuric chloride for 3-5min, rinsed with sterile water for 3 times, inoculated the sterilized explants on MS blank medium, cultured in the cultivation room under light, and light The time is 10-12h/d, the light intensity is 20001x, the temperature of the culture room is 25℃±2℃, (4) Proliferation of clustered buds: the above-mentioned aseptic young and tender stems were cultivated for about 10-15 days, and axillary buds on the stems would grow out. The shoots on the top were cut off and inoculated on the medium of MS+6-BA0.5-2.5mg/L+NAA0.01-0.2mg/L+carrageenan 6.5g/L+white sugar 30g/L with a strength of 1200, Continue to place in the cultivation room for proliferation and cultivation, the light time is 10-12h/d, the light intensity is 20001x, the temperature is 25°C±2°C, (5) rooting of tissue culture seedlings Cut out the 2-4 cm high Chinese rose buds in the culture bottle from the base of each individual plant under aseptic conditions, and inoculate them in the rooting medium, the rooting medium formula is 1/4-1/2MS +NAA0.1-1.0mg/L+carrageenan with a strength of 1200 6.5g/L+white sugar 20g/L, placed in the cultivation room for 15-20 days under light cultivation, and transplanted when the root grows to 1.0-1.5 cm. (6) Preparation before transplanting Spread waste newspapers of appropriate size on the bottom of a plastic flower pot with a diameter of 15 cm or a plastic seedling tray with a height of 5 cm, moisten the newspaper with a watering can, and then add vermiculite to the flower pot or seedling tray, and remove it from the flowerpot or seedling tray. When the edge of the plate is 1 cm, use a ruler to scrape the vermiculite, then use a watering can to pour the vermiculite in the flower pot or seedling plate, drain the water, and set aside. (7) Transplanting When the roots of rose seedlings in the bottle grow to 1.0-1.5 cm, open the bottle cap, pinch out the seedlings with tweezers, wash off the root medium in clean water, and plant the seedlings into the flower tray or seedling tray prepared above Then use a watering can to pour a suitable concentration of rooting agent, fill up the loosened vermiculite when planting seedlings, drain the water, cover the flower pot with a small and thin plastic shopping bag, and use 2 The roots are supported by thin iron wires, and then the seedling tray is covered with plastic film. The contact between the plastic film and the seedling tray is sealed with transparent tape. Use scissors to open a small hole with a diameter of 1.0-1.5 cm on the plastic film, gradually reduce the air humidity in the flowerpot or seedling tray, and remove all the film 6-8 days after opening the small hole, and the seedlings can be removed after 15 days. The seedlings were transplanted into the pre-prepared nutrient soil.