CN112493128A

CN112493128A - Tissue culture seedling breeding method for commercially producing red daphne

Info

Publication number: CN112493128A
Application number: CN202011339105.1A
Authority: CN
Inventors: 王婷婷; 胡春宏; 常苹; 季翔
Original assignee: Jiangsu Dongyu Plant Technology Co ltd
Current assignee: Jiangsu Dongyu Plant Technology Co ltd
Priority date: 2020-11-25
Filing date: 2020-11-25
Publication date: 2021-03-16

Abstract

The application relates to the field of red-daphne seedling breeding, and particularly discloses a tissue culture seedling breeding method for commercially producing red-daphne. The breeding method comprises the following steps: selecting an explant; sterilizing explants; starting culture: inoculating the explant into a starting culture medium, wherein the starting culture medium comprises an MS culture medium, BA, IBA, sucrose and agar; and (3) proliferation culture: inoculating the germinated plant into a proliferation culture medium, wherein the proliferation culture medium comprises an MS culture medium, BA, KT, IBA, sucrose and agar; rooting culture: inoculating the germinated plant into rooting culture medium comprising 1/2WPM culture medium, IBA, sucrose and agar. According to the method, the starting culture medium, the propagation culture medium and the rooting culture medium are prepared by selecting specific components and specific mixing amount, so that the possibility of producing callus by the plant can be effectively reduced, the possibility of character separation of the plant due to regeneration of the plant by the callus is reduced, and the bred seedling can keep excellent characters of the female parent so as to meet the product requirement of commercial seedling breeding.

Description

Tissue culture seedling breeding method for commercially producing red daphne

Technical Field

The application relates to the field of red daphne wood breeding, in particular to a tissue culture seedling breeding method for commercially producing red daphne wood.

Background

In gardens, the red raspberries are shrubs which can be used for enjoying red trunks in winter, are mostly planted on lawns, forest edges, river banks and lakesides, are bright red in autumn leaves and white in small fruits, and are red and gorgeous like corals after leaves fall, are rare ornamental plants and are good branch cutting materials. Due to the extremely high ornamental value of the rosewood, the demand for the rosewood in the garden construction is gradually increased in recent years.

At present, the common propagation method of the Reptilia is mainly focused on sowing, cuttage and layering propagation, the cuttage propagation mode generally comprises the steps of cutting off tender branches of the Reptilia, cutting into cuttings with specific lengths, removing residual branches and leaves, carrying out cuttage in the next 3-4 months after sand storage in autumn and winter, transplanting after the cuttings take roots, layering the branches can be annularly cut in 5 months and then buried in soil, and the branches can be separated from mother plants in the next spring after the branches take roots and then are separated.

In view of the above-mentioned related technologies, the inventors found that although the cutting mode can obtain the seedling, during the cutting propagation process, the section of the cutting strip is easy to generate callus, and the callus is easy to regenerate the plant, so that the characters of the cultivated seedling deviate from those of the female parent, and the excellent characters of the female parent are not inherited, so that the ornamental value of the cultivated seedling is not high.

Disclosure of Invention

In order to reduce the possibility of character separation or variation of seedlings in the propagation process, the application provides a tissue culture seedling propagation method for commercially producing the red raspberries.

The tissue culture seedling breeding method for the commercial production of the red daphne adopts the following technical scheme:

a tissue culture seedling breeding method for commercially producing Rhus red seedlings comprises the following steps:

s1, selecting explants;

s2, disinfecting explants;

s3, start culture: inoculating the explant in S2 into a starting culture medium, wherein the starting culture medium comprises an MS culture medium, 0.3-0.7mg/L BA, 0.08-0.12mg/L IBA, 27-33g/L sucrose and 6.5-7.0g/L agar, the pH value of the starting culture medium is 5.75-5.90, and culturing for 3-4 weeks to obtain a germinated plant;

s4, proliferation culture: inoculating the germinated plant in S3 into a multiplication culture medium, wherein the multiplication culture medium comprises an MS culture medium, 0.1-0.5mg/L BA, 0.1-0.4mg/L KT, 0.08-0.12mg/L IBA, 28-33g/L sucrose and 6.6-7.0g/L agar, the pH value of the multiplication culture medium is 5.75-5.90, and culturing for 4-6 weeks to obtain a germinated plant;

s5, rooting culture: and (3) inoculating the bud-growing plant in the S4 into a rooting culture medium, wherein the rooting culture medium comprises 1/2WPM culture medium, 1.2-1.8mg/L IBA, 13-17g/L sucrose and 6.5-7.0g/L agar, the pH value of the rooting culture medium is 5.75-5.90, and culturing for 3-4 weeks to obtain the rooting plant.

By adopting the technical scheme, the MS culture medium has higher inorganic salt concentration, can provide mineral nutrition required by tissue growth, has reasonable quantity and proportion of the inorganic nutrients, and can meet the nutritional and physiological needs of plant cells; BA can promote the explant to sprout; KT can induce the differentiation of axillary buds and inhibit the differentiation of roots at the same time, thereby being beneficial to the sprouting plants to generate cluster buds; IBA can promote the generation of adventitious buds and is beneficial to the generation of cluster buds of the germinated plants; the WPM culture medium can provide trace elements, major elements and vitamins for plant tissue culture, sucrose can provide necessary carbon source substances for tissue growth, and meanwhile osmotic pressure in the culture medium can be adjusted, so that the steady state in plant cells can be maintained; the agar can solidify the culture medium, the operation is simple and convenient, and the workers can observe and study the state of the explant conveniently.

According to the technical scheme, the starting culture medium, the propagation culture medium and the rooting culture medium are prepared by selecting specific components and specific mixing amount, so that the possibility of producing callus by the plant can be effectively reduced, the possibility of character separation of the plant due to regeneration of the plant by the callus is reduced, the bred seedling can keep excellent characters of a female parent, and meanwhile, the character consistency of the seedling can be ensured to a certain extent.

Preferably, in the step S3, the start-up medium includes MS medium, 0.5mg/L BA, 0.1mg/L IBA, 30g/L sucrose and 6.8g/L agar.

By adopting the technical scheme, the starting culture medium is prepared by selecting specific components and specific mixing amount, so that the starting culture medium can better promote the germination of the explant, and the possibility of the explant to generate callus is better reduced.

Preferably, in the step S4, the proliferation medium comprises MS medium, 0.3mg/L BA, 0.2mg/L KT, 0.1mg/L IBA, 30g/L sucrose and 6.8g/L agar.

By adopting the technical scheme, the specific components and the specific mixing amount are selected to prepare the multiplication culture medium, so that the multiplication culture medium can better promote the axillary buds of the plants to generate the cluster buds, and the buds are robust and have good repeatability.

Preferably, in the step S5, the rooting medium comprises 1/2WPM medium, 1.5mg/L IBA, 15g/L sucrose and 6.8g/L agar.

By adopting the technical scheme, the specific components and the specific mixing amount are selected to prepare the rooting culture medium, so that the rooting culture medium can better promote the plant to take root, the root generated by the plant is promoted to be connected with a plant body structure, the transplanting survival rate is improved, the generated root is short and thick, the damage can be reduced during transplanting, and the possibility of generating callus by the plant can be reduced.

Preferably, the culture conditions of step S3, step S4 and step S5 are all: culturing in dark for 48-72 hr after inoculation, and then illuminating for 15-17 hr and dark for 7-9 hr every day.

By adopting the technical scheme, the plant can be induced to sprout by utilizing illumination, the dedifferentiation of the plant can be induced by utilizing dark culture, and the cooperation of the illumination condition and the dark condition at a specific time every day is favorable for inducing the explant to sprout, inducing the sprouting plant to generate cluster buds and inducing the sprouting plant to root.

Preferably, the culture conditions of step S3, step S4 and step S5 are all: the illumination intensity is 1800-.

By adopting the technical scheme, the specific temperature is favorable for the plant to sprout and take root, the specific illumination intensity can reduce the possibility of stress reaction of the plant in each culture stage, the browning intensity is effectively reduced, the plant is favorable for being quickly adapted to new nutrient solution, and the plant is promoted to grow quickly.

Preferably, in step S2, alcohol with a concentration of 72-78% is selected to wipe the explant, and then the explant is immersed in a bleaching water to water volume ratio of 1: (3-5) adding the mixture into the mixed solution for 35-40 minutes, and shaking continuously during the soaking period; then washing with sterile water for 3-4 times, each time for 2-5 minutes.

Through adopting above-mentioned technical scheme, disinfect to explant and can reduce the germ on explant surface and produce the influence to the bud of explant to this kind of disinfection mode is simple easily to be operated, and the staff of being convenient for disinfects the explant.

In summary, the present application has the following beneficial effects:

1. the method has the advantages that the starting culture medium is utilized to promote the axillary buds of the explant to germinate, the value-added culture medium is utilized to promote the axillary buds to generate cluster buds, the rooting culture medium is utilized to promote the plant to take root, the possibility that the plant generates callus can be effectively reduced, the possibility that the plant generates character separation due to the regeneration of the callus is reduced, the bred seedling can keep the excellent characters of the female parent, and meanwhile, the character consistency of the seedling can be ensured to a certain extent.

2. This application chooses specific culture condition to breed the padauk, can effectively reduce each possibility that cultivates stage plant stress reaction and brown stain phenomenon appear, is favorable to the plant to adapt to new nutrient solution fast, impels the plant to grow fast, the bud is healthy and strong.

3. According to the method, only a small amount of explant materials are needed, a large amount of branches do not need to be collected according to a cutting mode, the branch taking damage to finished nursery stocks is reduced, plant seeds do not need to be relied on, and batch production can be carried out according to order requirements.

Detailed Description

The following examples further illustrate the present application in detail.

The sources of the raw material components in the present application are shown in table 1:

TABLE 1 sources of the raw material components

Examples 1-5 the compositions of the start medium, the multiplication medium and the rooting medium are shown in tables 2 and 3:

TABLE 2 composition of the priming medium, the multiplication medium and the rooting medium in examples 1 to 5

The method for breeding the daphne giraldii nitsche in the embodiments 1 to 5 comprises the following steps:

s1, selecting explants: in spring 4 months, cutting tender tips of the red raspberries, cutting branches with 3 axillary bud stem sections from top buds downwards, removing leaves, cutting single-node axillary bud stem sections as explants, wherein each stem section contains two axillary buds and is 2cm long;

s2, explant disinfection: wiping the explant with 75% alcohol on a sterile operating table, and immersing the explant in a volume ratio of bleaching water to water of 1: 5 for 40min, and shaking continuously during the soaking; then washing with sterile water for 3 times, each time for 3 min;

s3, start culture: respectively cutting off 0.4cm of both ends of the explant in S2, and inoculating the explant into a start culture medium, wherein the pH of the start culture medium is 5.80; after inoculation, dark culture is carried out in a culture room for 48 hours, then illumination is carried out for 16 hours every day and the dark culture is carried out for 8 hours, the culture temperature in the culture room is 25 ℃, the illumination intensity is 1950 lux, the culture is carried out for 3 weeks, axillary buds germinate to obtain germinated plants, the plants are robust, and the height of the plants reaches 3 cm;

s4, proliferation culture: inoculating the germinated plant in the S3 to a multiplication culture medium, wherein the pH of the multiplication culture medium is 5.80; culturing in dark for 48 hours in a culture room after inoculation, then illuminating for 16 hours and 8 hours in dark every day, wherein the culture temperature in the culture room is 25 ℃, the illumination intensity is 1950 lux, culturing for 6 weeks, and generating cluster buds by axillary buds to obtain a bud plant, wherein the height of the cluster buds reaches 2 cm;

s5, rooting culture: inoculating the budding plants in the S4 to a rooting culture medium, wherein the pH of the rooting culture medium is 5.80; after inoculation, the seedlings are cultured in a dark room for 48 hours, then the seedlings are irradiated by light for 16 hours and 8 hours in the dark every day, the culture temperature of the culture room is 25 ℃, the illumination intensity is 1950 lux, the seedlings are cultured for 3 weeks, the sprouting plants root, and the length of roots reaches 4 cm.

Example 6

This example differs from example 3 only in that: the culture conditions of the stages of starting culture, multiplication culture and rooting culture are as follows: after inoculation, the seeds are cultured in a dark room for 65 hours, then the seeds are irradiated by light for 15 hours and 9 hours in the dark every day, the culture temperature in the culture room is 23 ℃, and the illumination intensity is 1800 lux.

Example 7

This example differs from example 3 only in that: the culture conditions of the stages of starting culture, multiplication culture and rooting culture are as follows: after inoculation, the seeds are cultured in a dark culture room for 72 hours, then the seeds are irradiated for 17 hours and 7 hours in the dark every day, the culture temperature in the culture room is 26 ℃, and the illumination intensity is 2000 lux.

Comparative example 1

The method for breeding the red daphne wood by adopting a cuttage mode comprises the following steps:

s1, in the current 7 months, cutting semi-lignified young shoots with the thickness of 2.5mm from the Daphne giraldii Nitsche plants into cutting slips with the length of 7cm, reserving 2 leaves at the upper end, and removing the leaves and petioles at the lower end;

s2, dipping the cutting slips into indoleacetic acid solution with the concentration of 40mg/L for 12 hours, and then inserting the cutting slips into the ground, wherein the cutting depth is 4 cm; carrying out sun-shading treatment on the cutting shoots by using a sun-shading cover, wherein the transmittance of the sun-shading cover is 40%;

and S3, after cutting for one month, checking the rooting condition of the base part of the cutting shoot, and removing the sunshade net when 3 roots grow to ensure that the seedling grows.

Comparative example 2

This comparative example differs from example 3 in that: the BA in the start-up medium was changed to KT at an equal concentration.

Comparative example 3

This comparative example differs from example 3 in that: IBA in the start medium was changed to an equal concentration of IAA.

Comparative example 4

This comparative example differs from example 3 in that: the BA in the growth medium was changed to an equal concentration of PBA.

Comparative example 5

This comparative example differs from example 3 in that: KT in the multiplication medium was changed to PBA of equal concentration.

Comparative example 6

This comparative example differs from example 3 in that: IBA in the growth medium was replaced with NAA at equal concentration.

Comparative example 7

The breeding method in each embodiment and each proportion is tested, and the specific test process is as follows:

testing one:

each example, comparative example 2 to comparative example 7 bred 30 seedlings of daphne giraldii, respectively, and counted the germination rate = number of plants germinated/total number of plants 100% and the probability of callus generation = number of plants generating callus/total number of plants 100% for each group of plants in the initiation culture phase.

And (2) testing:

in each embodiment, comparative example 2 to comparative example 7, 30 germinated plants are respectively selected for continuous breeding, the multiplication multiple of axillary buds of each group of plants in the multiplication culture stage and the probability of generating callus are respectively counted, the multiplication multiple of the axillary buds = the number of cluster buds generated by a single axillary bud/the number of the axillary buds, and the average value is taken after the multiplication rate of the axillary buds of each group of plants is removed from a maximum value and a minimum value; probability of callus production = number of plants producing callus/total number of plants 100%.

And (3) testing:

in each example and comparative example 2-comparative example 7, 30 budding plants are respectively selected for continuous breeding, the rooting rate and the callus generation probability of each group of plants in the rooting culture stage are respectively counted, in comparative example 1, 30 cutting shoots are selected for cuttage treatment, the rooting rate and the callus generation probability of the base parts of the cutting shoots are counted after 3 weeks of cuttage, the rooting judgment is based on that 3 or more than 3 roots are generated from a single plant, the rooting rate = the number of rooted plants/total plants 100%, and the callus generation probability = the number of plants generating callus/total plants 100%.

And (4) testing:

in each example, comparative example 2 to comparative example 7, 30 rooted plants are respectively selected for transplanting, after 3 weeks of transplanting, the transplanting survival rate of each group of plants is respectively counted, in comparative example 1, 30 cuttings are selected for cuttage, and after 6 weeks of cuttage, the transplanting survival rate is counted, wherein the transplanting survival rate = the number of surviving plants/the total number of plants is 100%.

The test results of test one to test four are shown in table 3:

table 3 test results of tests one to four

Referring to table 3, compared with comparative example 1, in examples 1 to 5, in the process of breeding the red raspberries, the probability of generating the callus in the rooting culture stage is less than that in comparative example 1, and the probability of generating the callus in the initiation culture stage and the probability of generating the callus in the propagation culture stage are up to 10.0%, which shows that the method for breeding the red raspberries disclosed by the application can reduce the possibility of generating the callus in the tissue culture process of the plants and reduce the possibility of character separation of seedlings caused by regeneration of the plants by the callus. In addition, the transplanting survival rate of the daphne giraldii nitsche in the comparative example 1 is less than that in the examples 1 to 5, which shows that the breeding method disclosed by the application not only can reduce the possibility of generating callus of the plant, but also can promote the survival of the seedling.

Compared with the comparative examples 2 and 3, the germination rates of the plants in the starting culture stage in the example 3 are both higher than those in the comparative examples 2 and 3, and the callus generation probabilities are both lower than those in the comparative examples 2 and 3, which shows that the starting culture medium prepared by using BA and IBA can effectively promote the germination of the explants and reduce the possibility of the explants generating callus. In addition, the axillary bud multiplication multiple of the plant in the multiplication culture stage and the rooting rate and transplanting survival rate of the plant in the rooting culture stage are both greater than those in the comparative example 2 and the comparative example 3, which shows that the BA and IBA selected in the starting culture stage can influence the axillary bud multiplication and rooting of the subsequent plant to a certain extent, and the axillary bud multiplication multiple and rooting rate of the plant can be improved to a certain extent.

Compared with comparative examples 4 to 6, the multiplication multiples of the axillary buds of the plants in the multiplication culture stage in example 3 are all larger than those in comparative examples 4 to 6, and the probability of generating the callus is all smaller than those in comparative examples 4 to 6, which shows that the multiplication culture medium prepared by using BA, KT and IBA can effectively increase the number of the axillary buds generating the cluster buds, and simultaneously can reduce the possibility of generating the callus by the plants, thereby being beneficial to the axillary buds generating the cluster buds. In addition, the rooting rate and the transplanting survival rate of the plants in the rooting culture stage in the embodiment 3 are both greater than those in the comparative example 4 and the comparative example 6, which shows that the BA, KT and IBA selected by the application also have influence on the subsequent rooting and transplanting of the plants, and the rooting and transplanting survival of the plants can be effectively promoted to a certain extent.

Compared with the comparative example 7, the rooting rate of the plant in the rooting culture stage in the example 3 is greater than that in the comparative example 7, and the probability of generating the callus is less than that in the comparative example 7, which shows that the rooting culture medium prepared by selecting IBA can effectively promote the plant to root, and simultaneously can reduce the possibility of generating the callus by the plant and the possibility of character separation of the seedling caused by the regeneration of the plant by the callus.

The present embodiment is only for explaining the present application, and it is not limited to the present application, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present application.

Claims

1. A tissue culture seedling breeding method for commercially producing Rhus red seedlings is characterized by comprising the following steps:

s1, selecting explants;

s2, disinfecting explants;

2. The method for breeding tissue culture seedlings of commercially produced red raspberries according to claim 1, wherein: in the step S3, the start-up medium comprises MS medium, 0.5mg/L BA, 0.1mg/L IBA, 30g/L sucrose and 6.8g/L agar.

3. The method for breeding tissue culture seedlings of commercially produced red raspberries according to claim 1, wherein: in the step S4, the proliferation culture medium comprises MS culture medium, 0.3mg/L BA, 0.2mg/L KT, 0.1mg/L IBA, 30g/L sucrose and 6.8g/L agar.

4. The method for breeding tissue culture seedlings of commercially produced red raspberries according to claim 1, wherein: in the step S5, the rooting medium comprises 1/2WPM medium, 1.5mg/L IBA, 15g/L sucrose and 6.8g/L agar.

5. The method for breeding tissue culture seedlings of commercially produced red raspberries according to claim 1, wherein: the culture conditions of step S3, step S4 and step S5 are all as follows: culturing in dark for 48-72 hr after inoculation, and then illuminating for 15-17 hr and dark for 7-9 hr every day.

6. The method for breeding tissue culture seedlings of commercially produced red raspberries according to claim 1, wherein: the culture conditions of step S3, step S4 and step S5 are all as follows: the illumination intensity is 1800-.

7. The method for breeding tissue culture seedlings of commercially produced red raspberries according to claim 1, wherein: in step S2, alcohol with a concentration of 72-78% is selected to wipe the explant, and then the explant is immersed in bleaching water and water in a volume ratio of 1: (3-5) adding the mixture into the mixed solution for 35-40 minutes, and shaking continuously during the soaking period; then washing with sterile water for 3-4 times, each time for 2-5 minutes.