CN116267592A - A kind of cold-resistant rose and its hybrid cultivation method - Google Patents

Abstract

The invention discloses a cold-resistant China rose and a hybridization cultivation method thereof, belonging to the technical field of China rose cultivation, and the method comprises the following steps: analyzing the rose genotype by a molecular marking technology, and selecting wild rose with cold resistance and cultivated rose as parents for hybridization; spraying an active spray before the blooming period of the wild rose, sticking pollen of cultivated rose on a stamen of the wild rose after the blooming period of the wild rose, and contacting the pollen and finishing pollination; after pollination, fertilizer is applied, cold-resistant varieties with stable phenotype and genotype are screened from each generation line of the hybridized China rose for breeding and reproduction, and the cold-resistant China rose is cultivated by genotype analysis and selective induction hybridization through a molecular marker technology, so that the breeding efficiency and quality can be improved, and the breeding period can be shortened. The cold-resistant China rose has the capability of adapting to low-temperature environment, can grow and bloom under cold climate conditions, and enlarges the cultivation area range of China rose.

Description

A kind of cold-resistant rose and its hybrid cultivation method

technical field

The invention relates to the technical field of rose cultivation, in particular to a cold-resistant rose and a hybrid cultivation method thereof.

Background technique

Rose is a plant of the genus Rosaceae, also known as rose. Its flowers are colorful and fragrant. Chinese rose is popular because of its rich colors, strong fragrance and long flowering period. It is one of the common plants for landscaping and ornamental. However, roses generally require warm climates to grow and reproduce. Once encountering cold weather, even if the temperature drops slightly, the rose flowers will stop growing or even die. Therefore, the climatic conditions in many areas are not suitable for the growth of rose flowers, and rose flowers are easily affected by cold weather, resulting in problems such as stunted growth and shortened flowering period.

In order to solve this problem, some researchers have improved the survival rate and growth rate of rose flowers in cold climates by breeding rose varieties adapted to cold climate conditions, such as 'Rose Fragrance'. However, these varieties are not yet hardy enough to cope with extreme cold conditions. Therefore, it is necessary to improve the cold resistance of rose flowers through other methods, so as to adapt to more extreme climatic conditions.

Cultivating cold-resistant rose varieties through breeding methods is one of the effective methods to solve the problem of growing rose flowers in cold climates. At home and abroad, a lot of research has been done on the breeding of rose flowers, especially some progress has been made in improving the disease resistance, drought resistance and flower color variation of rose flowers. For example, through cross breeding, new rose varieties with double flowers can be obtained.

However, there is no research report on the successful breeding of cold-resistant rose varieties adapted to extreme cold climate conditions through breeding methods. Therefore, the present invention provides a cold-resistant rose and its hybrid cultivation method to adapt to more extreme climatic conditions and improve the growth ability of rose flowers under various climatic conditions.

Contents of the invention

In view of this, the object of the present invention is to provide a kind of cold-resistant rose and its hybrid cultivation method, by selecting a cold-resistant rose variety for hybridization, a hybrid variety with stronger cold resistance is obtained, and the hybrid variety is asexually propagated. Obtain a large number of hybrid clones, screen the hybrid clones under low temperature conditions, screen out excellent hybrid clones that adapt to cold climate conditions, and breed cold-resistant rose varieties that are more suitable for cold climate conditions by breeding excellent hybrid clones , to solve the problems raised in the background art above.

To achieve the above object, the present invention provides the following technical solutions:

A cold-resistant rose, a rose variety with cold-resistant performance obtained through genotype analysis and selective induced hybridization by molecular marker technology.

A hybrid cultivation method for cold-resistant roses, used for cultivating cold-resistant rose varieties, comprising the following steps:

1. Analyze the genotypes of roses by molecular marker technology, and select wild roses with cold resistance and cultivated roses as parents for hybridization;

2. Spray the active spray before the flowering period of the wild rose, and paste the pollen of the cultivated rose on the pistil of the wild rose after the flowering period of the wild rose, and the pollen contacts and pollination is completed;

3. Apply fertilizer after pollination, select cold-resistant varieties with stable phenotype and genotype from each generation of hybrid roses for breeding and propagation, and conduct genotype analysis and selective induction hybridization cultivation through molecular marker technology A cold-resistant rose.

As a further solution of the present invention, carrying out genotype analysis and selectively inducing hybridization to cultivate cold-resistant roses through molecular marker technology includes the following steps:

1) Extract DNA from the selected rose parents and prepare DNA samples;

2) Select the primer sequence according to the target gene, and use PCR technology to amplify and isolate the PCR product;

3) The PCR products are separated by gel electrophoresis, analyzed and identified according to the size and composition of the target DNA fragments, and the data of the PCR products are counted, analyzed and compared;

4) According to the target genotype information and genetic diversity index, select breeding materials for selective induced hybridization to obtain rose varieties with cold resistance.

As a further solution of the present invention, the specific steps for preparing a DNA sample are as follows:

1. Take 3-5 g of young rose leaves, wash them and store them in a low-temperature environment of -80 °C;

2. Grind the tender rose leaves, add an appropriate amount of CTAB extract, and mix quickly;

3. Incubate in a water bath at 65°C for 30 min at room temperature until the tissue sample is dissolved, add an equal volume of chloroform-isoamylone mixed solvent to mix, and centrifuge until the two phases are separated;

4. Move the supernatant to a new centrifuge tube, add an equal volume of high-salt buffer prepared with isopropanol, mix and place at room temperature of 20 °C for precipitation;

5. After the precipitation, centrifuge until the precipitation solidifies, pour off the supernatant, wash the precipitation with 70% alcohol, and dry it at 55 ℃ for 45-60 min;

6. Add the precipitated sample to deionized water, dilute to the required concentration according to the preset ratio, and obtain the DNA sample.

As a further solution of the present invention, the CTAB extract is an extract composed of 2% w/v CTAB, 100 mM Tris-HCl and 20 mM EDTA.

Among them, w/v represents the mass concentration, and the unit is percentage (%). Specifically, 2 % w/v of CTAB means 2 grams of CTAB per 100 ml of extract.

mM stands for molar concentration, expressing the amount of substance of a solute per liter of solution, in moles. Specifically, 100mM Tris-HCl means 0.1 moles of Tris-HCl per liter of extract. Likewise, 20 mM EDTA means 0.02 moles of EDTA per liter of extract.

As a further solution of the present invention, the preparation of CTAB extract comprises the following steps:

1) Weigh the CTAB, Tris-HCl and EDTA reagents required to prepare the CTAB extract according to the ratio.

2) Weigh 1 g of CTAB, add 100 mL of deionized water, mix, and heat to 70 °C to dissolve;

3) Add 100mL Tris-HCl with a concentration of 1M (molar) preheated to 70°C, mix well and continue to heat to 70°C to dissolve;

4) Add 10 mL of EDTA with a concentration of 0.5 M (mole), mix well, then add an appropriate amount of deionized water to 500 mL, filter with a 0.2 μm filter to obtain CTAB extract, and refrigerate for later use.

As a further solution of the present invention, the young leaves of Chinese rose are ground, added to an appropriate amount of CTAB extract, and when mixed rapidly, it also includes adding TE buffer containing proteinase K to the CTAB extract; wherein, the TE buffer containing proteinase K The solution consisted of 10 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl, and 1 mg/mL proteinase K. TE buffer containing proteinase K was used to digest the protein in the CTAB extract.

As a further scheme of the present invention, the preparation method of the TE buffer solution containing proteinase K is:

Prepare Tris-HCl, EDTA, NaCl, and proteinase K reagents according to the composition of TE buffer containing proteinase K; add Tris-HCl, EDTA, and NaCl reagents to an appropriate amount of deionized water, mix well, and add 1 mg/mL of proteinase K , the TE buffer solution containing proteinase K can be obtained; wherein, the proteinase K is preheated to 70° C. before adding to the TE buffer solution.

As a further solution of the present invention, the chloroform-isoamylketone mixed solvent is a mixed solvent prepared by mixing chloroform and isoamylketone according to the same volume ratio of 24:1, that is: every 1 ml of isoamylketone is added with 24 ml chloroform was mixed evenly.

As a further solution of the present invention, the high-salt buffer is composed of 3M NaCl, 5M NH4Ac (ammonium acetate) and 1M Tris-HCl, with a pH value of 8.0.

As a further solution of the present invention, the active spray comprises the following components in mass percent:

0.1%～0.15% ethephon, 0.8%～1.2% amino acid, 1.2%～1.5% colchicine, 0.2%～0.35% keratin, 0.2%～0.35% ethanol, 2.0%～5% sucrose, the balance of purified water , wherein the amino acid contains proline.

As a further solution of the present invention, the preparation method of the active spray comprises the following steps:

Weigh ethephon, amino acid, colchicine, keratin, ethanol, sucrose and purified water by mass percentage;

Dissolve ethephon in purified water, stir evenly, and add amino acid, colchicine and keratin into the ethephon aqueous solution, stir evenly to obtain a mixed solution;

Add sucrose to the mixed solution, continue to stir until it is completely dissolved, and finally add ethanol and enough purified water to reach the required total volume.

Compared with the prior art, a cold-resistant rose and its hybrid cultivation method of the present invention have the following beneficial effects:

1. The cold-resistant rose of the present invention has the ability to adapt to a low temperature environment, can grow and bloom under cold climate conditions, and expands the cultivation area of rose.

2. In the present invention, by crossing wild roses with excellent genetics and cultivated roses, rose varieties with cold resistance can be obtained, and the germplasm resources of roses are increased.

3. The present invention uses molecular marker technology to perform genotype analysis and selective induction, which can improve breeding efficiency and quality, and shorten the breeding cycle.

4. The present invention uses specialized technology and equipment for planting and management, which can improve the quality and yield of roses, while reducing production costs and environmental pollution.

5. In the present invention, the cold-resistant rose is planted as a ground cover plant or a flower bed plant, which can beautify the urban environment and achieve the goals of energy saving, emission reduction and environmental protection.

The invention successfully breeds cold-resistant rose varieties adapting to cold climate conditions through the method of hybrid breeding and screening of excellent hybrid clones. This variety grows vigorously, has a long flowering period, has large flowers and rich colors, can adapt to growth under various climatic conditions, and has a wide range of application values.

Detailed ways

The technical solutions of the present invention will be further described in detail below in conjunction with specific embodiments.

Because the disease resistance, drought resistance and flower color variability of existing Chinese roses are poor in cold climates, the survival rate and growth speed of Chinese roses are hindered.

In order to solve the above technical problems, the present invention will be described in detail below in conjunction with specific solutions.

Example 1

A hybrid cultivation method for cold-resistant roses, used for cultivating cold-resistant rose varieties, comprising the following steps:

Step 1. Analyze the genotypes of roses by molecular marker technology, select wild roses with cold resistance and cultivated roses as parents for hybridization; this application selects wild roses with cold resistance 'Tianshan Xiaguang' as the male parent, and selects cultivated roses The rose 'Peaceful Beauty' was used as the female parent for hybridization, and all of them were produced in Xinjiang.

Step 2: Spray the active spray before the flowering period of the wild rose, and paste the pollen of the cultivated rose on the pistil of the wild rose after the flowering period of the wild rose, so that the pollen contacts and pollination is completed.

In this embodiment, the active spray comprises the following components by mass percentage: 0.1% ethephon, 0.8% amino acid, 1.2% colchicine, 0.2% keratin, 0.2% ethanol, 2.0% sucrose, purified water quantity.

Wherein, the preparation method of the active spray comprises the following steps: taking ethephon, amino acid, colchicine, cutanin, ethanol, sucrose and purified water by mass percentage; dissolving ethephon in purified water, stirring evenly, and Add amino acid, colchicine and keratin into the ethephon aqueous solution, stir evenly to obtain a mixed solution; add sucrose to the mixed solution, continue stirring until completely dissolved, finally add ethanol and add enough pure water to the required total capacity.

Wherein, the use method of active spray spraying is: during the growth period of rose, spray this active spray once every 7 days, spray this active spray once every 3 days during the flowering period of rose, before each use, spray active spray Shake the agent well, and adjust the spray angle and distance of the nozzle so that it can evenly cover the surface of the rose. After spraying, avoid watering or rainfall within 24 hours to ensure that the spray can be fully absorbed.

Among them, spraying active sprays can help roses grow in low temperature environments, thereby expanding their planting range. Among them, ethephon can promote the formation of cold-resistant enzymes in plants, and amino acids can enhance the stability of plant cell membranes. Adding sucrose to active sprays It can improve the cold resistance of plants, and the ethanol contained in it can further inhibit the bacteria of the large-flowered rose.

Step 3: Apply fertilizer after pollination, select cold-resistant varieties with stable phenotype and genotype from each generation of roses after hybridization for breeding and breeding, and perform genotype analysis and selective induction of hybridization through molecular marker technology Cultivate cold-resistant roses. Among them, the application of fertilizers after pollination mainly includes nitrogen fertilizer and potassium dihydrogen phosphate. Applying an appropriate amount of nitrogen fertilizer and potassium dihydrogen phosphate can enhance the immunity and cold resistance of roses, and help roses improve cold resistance and immunity.

In this example, genotype analysis and selective induction hybridization are carried out by molecular marker technology to cultivate cold-resistant roses, including the following steps:

1) Extract DNA from the selected rose parents and prepare DNA samples;

2) Select the primer sequence according to the target gene, and use PCR technology to amplify and isolate the PCR product;

3) The PCR products are separated by gel electrophoresis, analyzed and identified according to the size and composition of the target DNA fragments, and the data of the PCR products are counted, analyzed and compared;

4) According to the target genotype information and genetic diversity index, select breeding materials for selective induced hybridization to obtain rose varieties with cold resistance.

In this embodiment, the specific steps for preparing DNA samples are as follows:

1. Take 3-5 g of young rose leaves, wash them and store them in a low-temperature environment of -80 °C;

2. Grind the tender rose leaves, add an appropriate amount of CTAB extract, and mix quickly;

3. Incubate in a water bath at 65°C for 30 min at room temperature until the tissue sample is dissolved, add an equal volume of chloroform-isoamylone mixed solvent to mix, and centrifuge until the two phases are separated;

4. Move the supernatant to a new centrifuge tube, add an equal volume of high-salt buffer prepared with isopropanol, mix and place at room temperature of 20 °C for precipitation;

5. After the precipitation, centrifuge until the precipitation solidifies, pour off the supernatant, wash the precipitation with 70% alcohol, and dry it at 55 ℃ for 45-60 min;

6. Add the precipitated sample to deionized water, dilute to the required concentration according to the preset ratio, and obtain the DNA sample.

In this embodiment, the CTAB extract is an extract composed of 2% w/v CTAB, 100 mM Tris-HCl and 20 mM EDTA; wherein, w/v represents the mass concentration, and the unit is percentage ( % ). Specifically, 2 % w/v of CTAB means that every 100 ml of extract contains 2 grams of CTAB; mM means molar concentration, which means the amount of solute in each liter of solution, in moles. Specifically, 100 mM Tris-HCl means 0.1 moles of Tris-HCl per liter of extract. Likewise, 20 mM EDTA means 0.02 moles of EDTA per liter of extract.

In the present embodiment, the preparation of CTAB extract comprises the following steps:

1) Weigh the CTAB, Tris-HCl and EDTA reagents required to prepare the CTAB extract according to the ratio;

2) Weigh 1 g of CTAB, add 100 mL of deionized water, mix, and heat to 70 °C to dissolve;

3) Add 100mL Tris-HCl with a concentration of 1M (molar) preheated to 70°C, mix well and continue to heat to 70°C to dissolve;

4) Add 10 mL of EDTA with a concentration of 0.5 M (mole), mix well, then add an appropriate amount of deionized water to 500 mL, filter with a 0.2 μm filter to obtain CTAB extract, and refrigerate for later use.

In this example, the young leaves of Chinese rose were ground and added to an appropriate amount of CTAB extract. When mixing rapidly, it also included adding TE buffer solution containing proteinase K to the CTAB extract solution; wherein, the TE buffer solution containing proteinase K Consisting of 10 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl, and 1 mg/mL proteinase K, TE buffer containing proteinase K was used to digest the protein in the CTAB extract.

In this embodiment, the preparation method of the TE buffer solution containing proteinase K is:

Prepare Tris-HCl, EDTA, NaCl, and proteinase K reagents according to the composition of TE buffer containing proteinase K; add Tris-HCl, EDTA, and NaCl reagents to an appropriate amount of deionized water, mix well, and add 1 mg/mL of proteinase K , the TE buffer solution containing proteinase K can be obtained; wherein, the proteinase K is preheated to 70° C. before adding to the TE buffer solution.

In this example, the chloroform-isoamylone mixed solvent is a mixed solvent prepared by mixing chloroform and isoamylketone at the same volume ratio of 24:1, that is, for every 1 ml of isoamylketone, add 24 ml Prepared by mixing chloroform evenly.

In this embodiment, the high-salt buffer solution is composed of 3M NaCl, 5M NH4Ac and 1M Tris-HCl, with a pH value of 8.0.

Example 2

A hybrid cultivation method for cold-resistant roses, used for cultivating cold-resistant rose varieties, comprising the following steps:

Step 1. Analyze the genotypes of roses by molecular marker technology, select wild roses with cold resistance and cultivated roses as parents for hybridization; this application selects wild roses with cold resistance 'Tianshan Xiaguang' as the male parent, and selects cultivated roses The rose 'Peaceful Beauty' was used as the female parent for hybridization, and all of them were produced in Xinjiang.

Step 2: Spray the active spray before the flowering period of the wild rose, and paste the pollen of the cultivated rose on the pistil of the wild rose after the flowering period of the wild rose, so that the pollen contacts and pollination is completed.

In this embodiment, the active spray comprises the following components by mass percentage: 0.13% ethephon, 1.05% amino acid, 1.3% colchicine, 0.3% keratin, 0.3% ethanol, 3.5% sucrose, purified water quantity.

Wherein, the preparation method of the active spray comprises the following steps: taking ethephon, amino acid, colchicine, cutanin, ethanol, sucrose and purified water by mass percentage; dissolving ethephon in purified water, stirring evenly, and Add amino acid, colchicine and keratin into the ethephon aqueous solution, stir evenly to obtain a mixed solution; add sucrose to the mixed solution, continue stirring until completely dissolved, finally add ethanol and add enough pure water to the required total capacity.

Wherein, the use method of active spray spraying is: during the growth period of rose, spray this active spray once every 10 days, spray this active spray once every 5 days during the flowering period of rose, before each use, spray active spray Shake the agent well, and adjust the spray angle and distance of the nozzle so that it can evenly cover the surface of the rose. After spraying, avoid watering or rainfall within 24 hours to ensure that the spray can be fully absorbed.

Among them, spraying active sprays can help roses grow in low temperature environments, thereby expanding their planting range. Among them, ethephon can promote the formation of cold-resistant enzymes in plants, and amino acids can enhance the stability of plant cell membranes. Adding sucrose to active sprays It can improve the cold resistance of plants, and the ethanol contained in it can further inhibit the bacteria of the large-flowered rose.

Step 3: Apply fertilizer after pollination, select cold-resistant varieties with stable phenotype and genotype from each generation of roses after hybridization for breeding and breeding, and perform genotype analysis and selective induction of hybridization through molecular marker technology Cultivate cold-resistant roses. Among them, the application of fertilizers after pollination mainly includes nitrogen fertilizer and potassium dihydrogen phosphate. Applying an appropriate amount of nitrogen fertilizer and potassium dihydrogen phosphate can enhance the immunity and cold resistance of roses, and help roses improve cold resistance and immunity.

In this example, genotype analysis and selective induction hybridization are carried out by molecular marker technology to cultivate cold-resistant roses, including the following steps:

1) Extract DNA from the selected rose parents and prepare DNA samples;

2) Select the primer sequence according to the target gene, and use PCR technology to amplify and isolate the PCR product;

3) The PCR products are separated by gel electrophoresis, analyzed and identified according to the size and composition of the target DNA fragments, and the data of the PCR products are counted, analyzed and compared;

4) According to the target genotype information and genetic diversity index, select breeding materials for selective induced hybridization to obtain rose varieties with cold resistance.

Wherein, the steps for preparing DNA samples are the same as in Example 1.

Example 3

A hybrid cultivation method for cold-resistant roses, used for cultivating cold-resistant rose varieties, comprising the following steps:

Step 1. Analyze the genotypes of roses by molecular marker technology, select wild roses with cold resistance and cultivated roses as parents for hybridization; this application selects wild roses with cold resistance 'Tianshan Xiaguang' as the male parent, and selects cultivated roses The rose 'Peaceful Beauty' was used as the female parent for hybridization, and all of them were produced in Xinjiang.

Step 2: Spray the active spray before the flowering period of the wild rose, and paste the pollen of the cultivated rose on the pistil of the wild rose after the flowering period of the wild rose, so that the pollen contacts and pollination is completed.

In this embodiment, the active spray comprises the following components by mass percentage: 0.15% ethephon, 1.2% amino acid, 1.5% colchicine, 0.35% keratin, 0.35% ethanol, 5% sucrose, purified water quantity.

Wherein, the preparation method of the active spray comprises the following steps: taking ethephon, amino acid, colchicine, cutanin, ethanol, sucrose and purified water by mass percentage; dissolving ethephon in purified water, stirring evenly, and Add amino acid, colchicine and keratin into the ethephon aqueous solution, stir evenly to obtain a mixed solution; add sucrose to the mixed solution, continue stirring until completely dissolved, finally add ethanol and add enough pure water to the required total capacity.

Wherein, the usage method of active spray spraying is: during the growth period of rose, spray this active spray once every 15 days, spray this active spray once every 7 days during the flowering period of rose, before each use, spray active spray Shake the agent well, and adjust the spray angle and distance of the nozzle so that it can evenly cover the surface of the rose. After spraying, avoid watering or rainfall within 24 hours to ensure that the spray can be fully absorbed.

Among them, spraying active sprays can help roses grow in low temperature environments, thereby expanding their planting range. Among them, ethephon can promote the formation of cold-resistant enzymes in plants, and amino acids can enhance the stability of plant cell membranes. Adding sucrose to active sprays It can improve the cold resistance of plants, and the ethanol contained in it can further inhibit the bacteria of the large-flowered rose.

Step 3: Apply fertilizer after pollination, select cold-resistant varieties with stable phenotype and genotype from each generation of roses after hybridization for breeding and breeding, and perform genotype analysis and selective induction of hybridization through molecular marker technology Cultivate cold-resistant roses. Among them, the application of fertilizers after pollination mainly includes nitrogen fertilizer and potassium dihydrogen phosphate. Applying an appropriate amount of nitrogen fertilizer and potassium dihydrogen phosphate can enhance the immunity and cold resistance of roses, and help roses improve cold resistance and immunity.

In this example, genotype analysis and selective induction hybridization are carried out by molecular marker technology to cultivate cold-resistant roses, including the following steps:

1) Extract DNA from the selected rose parents and prepare DNA samples;

2) Select the primer sequence according to the target gene, and use PCR technology to amplify and isolate the PCR product;

3) The PCR products are separated by gel electrophoresis, analyzed and identified according to the size and composition of the target DNA fragments, and the data of the PCR products are counted, analyzed and compared;

4) According to the target genotype information and genetic diversity index, select breeding materials for selective induced hybridization to obtain rose varieties with cold resistance.

Wherein, the steps for preparing DNA samples are the same as in Example 1.

It should be noted that the Tris-HCl: Tris(hydroxymethyl)aminomethane buffer solution is used to adjust the pH value of the DNA extraction reaction system and keep the reaction system stable.

EDTA: Ethylenediaminetetraacetic acid, a chelating agent that can bind metal ions (such as magnesium, calcium, iron), etc., to prevent them from negatively affecting DNA.

CTAB: N-hexadecyl-N,N-dimethylaminoethanol hydrochloride, an anionic surfactant, can improve the solubility and stability of DNA by binding to the negative charge on the DNA surface.

The invention successfully breeds cold-resistant rose varieties adapting to cold climate conditions through methods such as hybrid breeding and screening of excellent hybrid clones. This variety grows vigorously, has a long flowering period, has large flowers and rich colors, can adapt to growth under various climatic conditions, and has a wide range of application values.

Cold resistance experiments were carried out on the cold-resistant roses of Examples 1 to 3 of the present invention, and the wild rose 'Tianshan Xiaguang' was selected as the control group 1, and the cultivated rose 'Peerless Beauty' was selected as the control group 2, and 30 plants were selected for each group of experiments In the winter, the overwintering experiment of cold-resistant roses is carried out at 0-30 degrees below zero, and the planting survival rate, flowering number, pollination success rate, and flowering time of each group of roses are counted. Among them, the pollination stage consists of flowers with rich aroma , to attract beneficial insects such as bees to pollinate, and the experimental results are shown in Table 1 below.

Table 1 Results of cold-resistant rose overwintering experiment

Rose Varieties Number of plants (strains) Planting survival number (strains) Number of blooms (flowers) Pollination success rate (%) Flowering time (days) Example 1 30 30 257 87.5 16 Example 2 30 30 264 88.2 16 Example 3 30 30 271 89.1 17 Control group 1 30 29 235 67.5 11 Control group 2 30 25 214 72.6 12

Experimental conclusion: the cold resistance cultivated by the present invention, compared with the control group, under the same cold temperature conditions, its flowering number increases significantly, the planting survival rate increases by 3% to 15%, the pollination success rate increases by 14.9 to 21.6%, and the flowering period prolongs 4 to 6 days.

The cold-resistant rose and its hybrid cultivation method of the present invention can make the rose better adapt to different environments, improve the adaptability of the rose, increase the growth period and productivity of the rose, increase the flowering amount and pollination success rate of the rose, and improve the yield of the rose. Disease resistance and stress resistance can reduce the loss of roses from diseases and insect pests. This method can efficiently breed cold-resistant rose varieties with excellent traits and strong adaptability, and can save time and cost.

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the above-mentioned embodiments, and can also be made without departing from the gist of the present invention within the scope of knowledge possessed by those of ordinary skill in the art. Variations.

Claims (10)

1. The hybridization cultivation method of the cold-resistant China rose is characterized by comprising the following steps of:

analyzing the rose genotype by a molecular marking technology, and selecting wild rose with cold resistance and cultivated rose as parents for hybridization;

spraying an active spray before the blooming period of the wild rose, sticking pollen of cultivated rose on a stamen of the wild rose after the blooming period of the wild rose, and contacting the pollen and finishing pollination;

and (3) applying fertilizer after pollination, screening cold-resistant varieties with stable phenotype and genotype from each generation line of the hybridized China rose, breeding and reproducing, and carrying out genotype analysis and selective induction hybridization by a molecular marking technology to obtain the cold-resistant China rose.

2. The hybridization cultivation method of cold-resistant China rose according to claim 1, wherein genotype analysis and selective induction hybridization cultivation are performed by a molecular marker technique to obtain the cold-resistant China rose, comprising the following steps:

1) Extracting DNA from the selected China rose parent, and preparing a DNA sample;

2) Selecting a primer sequence according to a target gene, and amplifying and separating a PCR product by adopting a PCR technology;

3) Separating the PCR product by gel electrophoresis, analyzing and identifying according to the size and the composition of the target DNA fragment, and carrying out statistics, analysis and comparison on the data of the PCR product;

4) According to the target genotype information and genetic diversity index, selecting a breeding material to carry out selective induction hybridization to obtain the China rose variety with cold resistance.

3. The method for hybrid cultivation of cold-resistant China rose according to claim 2, wherein in the step 1), the specific steps of preparing the DNA sample are as follows:

taking rose tender leaves 3-5 g, cleaning, and then storing in a low-temperature environment at-80 ℃;

grinding rose tender leaves, adding into a proper amount of CTAB extract, and rapidly mixing;

incubating for 30min at room temperature in a water bath at 65 ℃ until the tissue sample is dissolved, adding an equal volume of chloroform-isovalerone mixed solvent for mixing, and centrifuging until two phases are separated;

moving the supernatant into a new centrifuge tube, adding an equal volume of high-salt buffer solution prepared by isopropanol, mixing, and then placing the mixture at the room temperature of 20 ℃ for precipitation;

centrifuging the precipitate until the precipitate solidifies, pouring out supernatant, washing the precipitate with 70% alcohol, and drying at 55 ℃ for 45-60 min;

and adding the precipitate sample into deionized water, and diluting to the required concentration according to a preset proportion to obtain a DNA sample.

4. The method according to claim 3, wherein the CTAB extract is composed of 2% w/v CTAB, 100mM Tris-HCl and 20 mM EDTA.

5. The method for hybrid cultivation of cold-resistant China rose according to claim 4, wherein the preparation of the CTAB extract comprises the following steps:

weighing CTAB, tris-HCl and EDTA reagents required by preparing CTAB extract according to a proportion;

1 g of CTAB is weighed and added into 100mL deionized water, mixed and heated to 70 ℃ for dissolution;

adding 100mL of Tris-HCl with concentration of 1M which is preheated to 70 ℃, uniformly mixing, and continuously heating to 70 ℃ for dissolution;

adding EDTA with concentration of 0.5M of 10 mL, mixing, adding appropriate amount of deionized water to 500 mL, filtering with 0.2 μm filter to obtain CTAB extractive solution, and refrigerating.

6. The method according to claim 5, wherein the method comprises grinding rose tender leaves, adding into appropriate amount of CTAB extract, and adding TE buffer containing proteinase K into CTAB extract when mixing rapidly;

wherein, the TE buffer containing proteinase K consists of 10 mM Tris-HCl, 1mM EDTA, 100mM NaCl and 1 mg/mL proteinase K, and the TE buffer containing proteinase K is used for digesting protein in CTAB extract.

7. The method for hybrid cultivation of cold-resistant China rose according to claim 6, wherein the preparation method of the TE buffer containing proteinase K comprises the following steps:

preparing Tris-HCl, EDTA, naCl and proteinase K reagent according to the composition of TE buffer containing proteinase K; adding Tris-HCl, EDTA, naCl reagent into proper amount of deionized water, uniformly mixing, and adding 1m g/mL proteinase K to obtain TE buffer solution containing proteinase K; wherein proteinase K was added to TE buffer after being preheated to 70℃before addition.

8. The hybrid cultivation method of cold-resistant China rose according to claim 3, wherein the chloroform-isopentanone mixed solvent is prepared by mixing chloroform and isovalernone according to the same volume ratio of 24:1.

9. The hybridization cultivation method of cold-resistant China rose according to claim 1, wherein the active spray comprises the following components in percentage by mass: 0.1 0.15 percent of ethephon, 0.8 percent to 1.2 percent of amino acid, 1.2 percent to 1.5 percent of colchicine, 0.2 percent to 0.35 percent of keratan, 0.2 percent to 0.35 percent of ethanol, 2.0 percent to 5 percent of sucrose and the balance of purified water, wherein the amino acid contains proline.

10. A cold-resistant rose cultivated by the hybrid cultivation method of the cold-resistant rose according to any one of claims 1 to 9.