CN112493129A

CN112493129A - Tissue culture seedling breeding method for Nandina domestica

Info

Publication number: CN112493129A
Application number: CN202011339282.XA
Authority: CN
Inventors: 季翔; 胡春宏; 常苹; 王婷婷
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 nandina seedling breeding, and particularly discloses a tissue culture seedling breeding method of nandina domestica. The breeding method comprises the following steps: selecting an explant; sterilizing explants; and (3) sprouting culture: inoculating the explant into an induction culture medium, wherein the induction culture medium comprises 1/2WPM culture medium, 6-BA, NAA, GA, agar and sucrose, and culturing for 7-8 weeks to obtain a germinated plant; and (3) proliferation culture: inoculating the germinated plant into a multiplication culture medium, wherein the multiplication culture medium comprises 1/2MS culture medium, BA, NAA, GA, Huabao No. 1, agar and sucrose, and culturing for 7-8 weeks to obtain a seedling plant; rooting culture: inoculating the seedling plant into a rooting culture medium, wherein the rooting culture medium comprises 1/2MS culture medium, NAA, adenine, activated carbon, agar and sucrose, and culturing for 7-8 weeks to obtain a rooting plant. The method can effectively promote the plants to generate cluster buds and root, the rooting rate can reach more than 95%, and the rooting rate of the plants is improved to a greater extent.

Description

Tissue culture seedling breeding method for Nandina domestica

Technical Field

The application relates to the field of nandina domestica breeding, in particular to a tissue culture seedling breeding method of nandina domestica.

Background

The Nandina domestica Thunb of berberidaceae, Nandina, plant is short and small, the plant shape is compact, the blade is oval or oval, the blade appears red in winter, and frost does not fall, cold resistance is stronger, often apply to the garden construction, have higher horticulture ornamental value.

The propagation mode of Nandina domestica fire-flaming mainly comprises cutting seedling and tissue culture seedling, the cutting seedling is generally carried out in 5-8 months, the semi-lignified branch of the current year is selected as the cutting shoot, and the cutting shoot is planted on the substrate after being soaked by auxin. The tissue culture seedling raising mainly adopts the current-year branch as an explant, after surface disinfection, the explant is transferred into a sterile environment for culture, and after the stages of germination, proliferation, seedling strengthening and rooting, the sterile rooted seedling is transferred into a greenhouse for transplantation life, thus completing the whole process.

Aiming at the related technologies, the inventor finds that the existing tissue culture seedling raising mode is adopted to breed the nandina domestica, the rooting rate of a single plant is about 85% generally based on 3 or more rooting rates of the single plant, and when the rooting rate is less than 3 rooting rates, the living rate of the plant is influenced to a certain extent, and the market demand for the rooting rate cannot be met.

Disclosure of Invention

In order to improve the rooting rate of Nandina domestica Thunb, the application provides a tissue culture seedling breeding method of Nandina domestica Thunb.

The tissue culture seedling breeding method for Nandina domestica Thunb provided by the application adopts the following technical scheme:

a tissue culture seedling breeding method of Nandina domestica Thunb comprises the following steps:

s1, selecting explants;

s2, disinfecting explants;

s3, sprouting culture: inoculating the explant in the S2 into an induction culture medium, wherein the induction culture medium comprises 1/2WPM culture medium, 1.3-1.7 mg/L6-BA, 0.08-0.13mg/L NAA, 0.8-1.2 mg/L GA, 5.8-6.3mg/L agar and 28-32g/L sucrose, the pH value of the induction culture medium is 5.80-5.90, and culturing for 7-8 weeks to obtain a sprouting plant;

s4, proliferation culture: inoculating the germinated plant into a multiplication culture medium, wherein the multiplication culture medium comprises 1/2MS culture medium, 1.7-2.3mg/L BA, 0.3-0.7mg/L NAA, 0.8-1.3mg/L GA, 0.8-1.2g/L Huabao No. 1, 5.9-6.2mg/L agar and 28-32g/L sucrose, the pH value of the multiplication culture medium is 5.80-5.90, and culturing for 7-8 weeks to obtain a seedling plant;

s5, rooting culture: inoculating the seedling plant into a rooting culture medium, wherein the rooting culture medium comprises 1/2MS culture medium, 2.7-3.3mg/L NAA, 0.8-1.2g/L adenine, 0.1-0.5g/L active carbon, 5.9-6.3mg/L agar and 28-32g/L sucrose, the pH value of the rooting culture medium is 5.80-5.90, and culturing for 7-8 weeks to obtain the rooting plant.

By adopting the technical scheme, the WPM culture medium can provide trace elements, major elements and vitamins for plant tissue culture; the MS culture medium has higher inorganic salt concentration, can provide mineral nutrition required by tissue growth, has reasonable quantity and proportion of inorganic nutrients, and can meet the nutritional and physiological needs of plant cells; 6-BA can promote cell division and bud differentiation, and is favorable for promoting plant germination; BA can promote the explant to sprout; NAA can promote the proliferation and growth of buds, and has stable performance and high temperature and high pressure resistance; GA can promote the elongation growth of the stem of the seedling and promote the development of an adventitious embryo into a plant, and meanwhile GA and NAA can cooperate to jointly promote the plant to generate cluster buds; huabao No. 1 provides nitrogen, phosphorus, potassium and other elements for plants, and has the effect of strengthening the plants; adenine can promote the proliferation of buds and the growth of seedlings; the activated carbon can adsorb harmful substances generated in the process of culturing the plants, so that the browning reaction of the plants is reduced; agar can solidify a culture medium, the operation is simple and convenient, and workers can observe and study the state of the explant conveniently; sucrose can provide necessary carbon source substances for tissue growth, and can also adjust osmotic pressure in a culture medium, so that the homeostasis of plant cells can be maintained.

According to the technical scheme, the induction culture medium, the multiplication culture medium and the rooting culture medium are prepared by selecting specific components and specific mixing amount, so that the generation of cluster buds and rooting of plants can be effectively promoted, the rooting rate can reach more than 95%, the rooting rate of the plants is improved to a large extent, meanwhile, the possibility of generating callus by the plants can be reduced to a certain extent, the phenomenon of character separation or variation of the plants is reduced, and the plants can keep the excellent characteristics of female parents.

Preferably, the induction medium comprises 1/2WPM medium, 1.5 mg/L6-BA, 0.1mg/L NAA, 1.0mg/L GA, 6.1mg/L agar and 30g/L sucrose.

By adopting the technical scheme, the specific components and the specific mixing amount are selected to prepare the induction culture medium, so that the induction culture medium can promote the axillary bud germination of the explant to a greater degree, and the generation of the callus can be reduced.

Preferably, the proliferation medium comprises 1/2MS medium, 2.0mg/L BA, 0.5mg/L NAA, 1.0mg/L GA, 1.0g/L Huabao No. 1, 6.1mg/L agar and 30g/L sucrose.

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 promote the axillary bud multiplication of the germinated plant to a greater extent, and the bud body is strong and has good repeatability.

Preferably, the rooting medium comprises 1/2MS medium, 3.0mg/L NAA, 1.0g/L adenine, 0.3g/L activated carbon, 6.1mg/L agar and 30g/L sucrose.

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 promote the seedling plants to take root to a greater extent, the connection of the roots generated by the plants and the plant body structure is promoted, the occurrence of callus can be reduced, the generated roots are short and strong, and the rooting rate can reach more than 95%.

Preferably, the culture conditions in steps S3, S4 and S5 are all: the culture temperature is 26-30 ℃, and the illumination intensity is 2000-2100 lux.

Through adopting above-mentioned technical scheme, specific temperature is favorable to the plant to sprout and take root, and specific illumination intensity can reduce each stage plant and appear stress response, effectively reduces brown stain intensity, is favorable to the plant to adapt to new business fast also, promotes the plant to grow fast.

Preferably, the culture conditions in steps S3, S4 and S5 are all: culturing in dark for 48-72 hr after inoculation, and then illuminating for 11-13 hr and darkness for 11-13 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 seedling plant to root.

Preferably, in step S2, the explant is wiped with 75% alcohol, and then the volume ratio of bleached water to water is 1: and (3) soaking the explants for 25-35min by using the mixed solution of (3-5), and washing with sterile water.

By adopting the technical scheme, the germs on the surface of the explant are removed, and the influence of the germs on the germination of the explant is reduced.

Preferably, in step S2, the explant is washed with sterile water once, then the petiole of the explant is cut off to expose axillary buds, and then the volume ratio of the bleached water to the water is 1: (3-5) soaking the explant for 15-25min in the mixed solution, and washing with sterile water for 3-4 times.

Through adopting above-mentioned technical scheme, carry out preliminary disinfection to the explant earlier, detach the germ on explant surface, expose the axillary bud after, disinfect once more, can detach the germ that remains in petiole department, further reduce the germ and produce the influence to the bud of explant.

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

1. the method utilizes the induction culture medium, the multiplication culture medium and the rooting culture medium to breed the nandina domestica, can effectively promote the plant to take root, has the rooting rate of over 95 percent, can promote the plant to survive to a certain degree, and can meet the requirement of the market on the number of the taken root.

2. The method for breeding the Nandina domestica fire-breeding seedlings by using the buds can reduce the possibility of producing callus by the plants in the culture process, reduce the possibility of character separation or convenience, and is favorable for maintaining the excellent characters of the female parent of the seedlings.

3. This application is disinfected earlier to the explant, disinfects once more after cutting off the petiole to reduce the germ and produce the influence to the bud of explant, be favorable to the explant bud.

Detailed Description

The following examples further illustrate the present application in detail.

The sources of the components in each example are shown in table 1:

TABLE 1 sources of the components

Example 1-composition of induction medium, multiplication medium and rooting medium in example 5 are shown in table 2:

TABLE 2 compositions of Induction Medium, multiplication Medium and rooting Medium in examples 1-5

The method for cultivating seedlings and breeding the nandina domestica fire-resistant plants in the embodiment 1-5 comprises the following steps:

s1, selecting explants: 2cm of current-year semi-lignified branches are cut and taken as explants;

s2, explant disinfection: removing redundant leaves on a sterile operating platform, reserving 3 petioles, wiping off dust on the surface of the explant by using a sterile towel dipped with 75% alcohol, soaking the explant for 30min by using mixed liquid of bleaching water and water in a volume ratio of 1:3, washing the explant by using sterile water once, cutting off the petiole part, and exposing axillary buds wrapped by the petioles; soaking the explant for 20min by using mixed liquor with the volume ratio of bleaching water to water being 1:4, and then washing the explant for 3 times by using sterile water;

s3, sprouting culture: cutting off 0.3cm of the end of the sterilized explant in S2, inoculating the explant into an induction culture medium, wherein the pH value of the induction culture medium is 5.85, culturing in a dark room for 48 hours after inoculation, then, culturing in the dark for 12 hours with the light intensity of 2060lux at the culture temperature of 28 ℃ every day and the light intensity of 2060lux for 7 weeks to obtain a germinated plant;

s4, proliferation culture: inoculating the germinated plant in S3 into a multiplication culture medium, wherein the pH value of the multiplication culture medium is 5.85, culturing in a dark room for 48 hours after inoculation, then illuminating for 12 hours every day and in darkness for 12 hours at the culture temperature of 28 ℃ and the illumination intensity of 2060lux, and culturing for 7 weeks to obtain a seedling plant;

s5, rooting culture: selecting seedling plants in S4 with the plant height of more than 2cm, inoculating the seedling plants into a rooting culture medium, wherein the pH value of the rooting culture medium is 5.85, culturing in a dark room for 48 hours after inoculation, then, culturing in light for 12 hours and dark for 12 hours every day at the culture temperature of 28 ℃ and the light intensity of 2060lux for 7 weeks to obtain the rooting plants.

Example 6

This example differs from example 3 in that: in the stages of germination culture, proliferation culture and rooting culture, after inoculation, the seeds are cultured in a dark room for 48 hours, then the seeds are irradiated by light for 11 hours and dark for 13 hours every day, the culture temperature is 26 ℃, and the illumination intensity is 2000 lux.

Example 7

This example differs from example 3 in that: in the stages of germination culture, proliferation culture and rooting culture, after inoculation, the seeds are cultured in a dark room for 48 hours, then the seeds are irradiated for 13 hours and 11 hours in the dark every day, the culture temperature is 27 ℃, and the illumination intensity is 2030 lux.

Example 8

This example differs from example 3 in that: in the stages of germination culture, proliferation culture and rooting culture, after inoculation, the seeds are cultured in a dark room for 48 hours, then the seeds are irradiated by light for 11 hours and dark for 13 hours every day, the culture temperature is 30 ℃, and the illumination intensity is 2100 lux.

Example 9

This example differs from example 3 in that: in step S2, soaking the explant for 25min by using a mixed solution of bleaching water and water in a volume ratio of 1: 4; after axillary buds are exposed, the explants are soaked for 18min by using mixed liquor of bleaching water and water in a volume ratio of 1: 3.

Example 10

This example differs from example 3 in that: in step S2, soaking the explant for 35min by using a mixed solution of bleaching water and water in a volume ratio of 1: 5; after axillary buds are exposed, the explants are soaked for 23min by using mixed liquor of bleaching water and water in a volume ratio of 1: 5.

Comparative example 1

Nandina domestica was bred by the breeding method described in example 1 of related art (CN 103314853B).

Comparative example 2

This comparative example differs from example 3 in that: the GA in the induction medium was replaced with equal concentrations of NAA.

Comparative example 3

This comparative example differs from example 3 in that: the NAA in the induction medium was replaced with equal concentrations of GA.

Comparative example 4

This comparative example differs from example 3 in that: the NAA in the induction medium was replaced with an equal concentration of IAA.

Comparative example 5

This comparative example differs from example 3 in that: 6-BA in the induction medium was replaced with KT at an equal concentration.

Comparative example 6

This comparative example differs from example 3 in that: the same breeding method is adopted to breed the yellow reed wood of berberis of berberidaceae.

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

testing one:

in each example and each proportion, 30 flame nandina seedlings are respectively bred, and the germination rate and the browning rate of each group of plants in the germination culture stage are respectively counted, wherein the germination rate = the number of the germinated plants/the total number of the plants × 100%, and the browning rate = the number of the plants generating the browning/the total number of the plants × 100%.

And (2) testing:

in each embodiment and each proportion, respectively selecting 30 germinated plants for continuous breeding, respectively counting the axillary bud multiplication times and the browning rates of each group of plants in the multiplication culture stage, wherein the axillary bud multiplication times are = the number of cluster buds/axillary buds generated by a single axillary bud, and averaging after the axillary bud multiplication rates of each group of plants are removed from a maximum value and a minimum value; browning rate = number of plants producing browning/total number of plants 100%.

And (3) testing:

in each example and each proportion, 30 seedling plants are respectively selected for continuous breeding, the rooting rate and the browning rate of each group of plants in the rooting culture stage are respectively counted, the rooting rate is determined based on that 3 or more than 3 roots are generated by a single plant, the rooting rate = the number of rooted plants/total plants × 100%, and the browning rate = the number of plants generating browning/total plants × 100%.

And (4) testing:

in each example and each proportion, 30 rooted plants were selected for transplanting, and after 3 weeks of transplanting, the transplanting survival rate of each group of plants was counted, wherein the transplanting survival rate = the number of surviving plants/total number of plants × 100%.

The test results are shown in table 3:

TABLE 3 test results of test one to test four

Referring to table 3, compared with comparative example 1, in the process of breeding nandina domestica thunb of examples 1 to 5, the germination rate, the axillary bud multiplication factor, the rooting rate and the transplanting survival rate are all greater than those of comparative example 1, which shows that the breeding method disclosed by the application can effectively improve the germination rate, the axillary bud multiplication factor, the rooting rate and the transplanting survival rate of nandina domestica thunb, and the rooting rate reaches more than 95%. The probability of browning of plants in each culture stage in examples 1 to 5 is lower than that in comparative example 1, which shows that the induction medium, the proliferation medium and the rooting medium disclosed by the application can effectively reduce the possibility of browning of plants and can ensure the normal growth of the plants to a certain extent.

Compared with the comparative examples 2 and 3, the germination rate in the example 3 is higher than that in the comparative examples 2 and 3, which shows that the added NAA and GA can cooperate with each other to effectively promote the germination of the explant, and reduce the possibility of browning of the plant. In addition, the axillary bud multiplication multiple, the rooting rate and the transplanting survival rate of the plant in the embodiment 3 are all larger than those of the comparative example 2 and the comparative example 3, and the NAA and the GA added in the application can promote the sprouting and the proliferation of the axillary buds of the explant and influence the axillary bud proliferation, the rooting and the transplanting survival of the plant in the subsequent breeding process.

Compared with the comparative example 4 and the comparative example 5, the explant germination rate in the example 3 is higher than that in the comparative example 4 and the comparative example 5, which shows that the NAA and the GA selected by the application are suitable for promoting the germination of the Nandina domestica fire explant, and simultaneously influence the axillary bud proliferation and rooting of the plant in the subsequent breeding process.

Compared with the comparative example 6, the germination rate, the axillary bud multiplication times, the rooting rate and the transplanting survival rate of the plant in the example 3 are all larger than those in the comparative example 6, which shows that the breeding method disclosed by the application is only suitable for breeding the Nandina domestica Thunb, and can effectively promote rooting and transplanting survival of the Nandina domestica Thunb.

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 of Nandina domestica Thunb is characterized by comprising the following steps:

s1, selecting explants;

s2, disinfecting explants;

2. The tissue culture seedling breeding method of Nandina domestica Thunb as claimed in claim 1, wherein: the induction medium comprises 1/2WPM medium, 1.5 mg/L6-BA, 0.1mg/L NAA, 1.0mg/L GA, 6.1mg/L agar and 30g/L sucrose.

3. The tissue culture seedling breeding method of Nandina domestica Thunb as claimed in claim 1, wherein: the proliferation culture medium comprises 1/2MS culture medium, 2.0mg/L BA, 0.5mg/L NAA, 1.0mg/L GA, 1.0g/L Huabao No. 1, 6.1mg/L agar and 30g/L sucrose.

4. The tissue culture seedling breeding method of Nandina domestica Thunb as claimed in claim 1, wherein: the rooting medium comprises 1/2MS medium, 3.0mg/L NAA, 1.0g/L adenine, 0.3g/L active carbon, 6.1mg/L agar and 30g/L sucrose.

5. The tissue culture seedling breeding method of Nandina domestica Thunb as claimed in claim 1, wherein: the culture conditions in the steps S3, S4 and S5 are all as follows: the culture temperature is 26-30 ℃, and the illumination intensity is 2000-2100 lux.

6. The tissue culture seedling breeding method of Nandina domestica Thunb as claimed in claim 1, wherein: the culture conditions in the steps S3, S4 and S5 are all as follows: culturing in dark for 48-72 hr after inoculation, and then illuminating for 11-13 hr and darkness for 11-13 hr every day.

7. The tissue culture seedling breeding method of Nandina domestica Thunb as claimed in claim 1, wherein: in step S2, the explant is wiped with 75% alcohol, and then the volume ratio of bleached water to water is 1: and (3) soaking the explants for 25-35min by using the mixed solution of (3-5), and washing with sterile water.

8. The tissue culture seedling breeding method of Nandina domestica Thunb as claimed in claim 7, wherein: in the step S2, the explant is washed with sterile water for one time, then the petiole of the explant is cut off to expose axillary buds, and then the volume ratio of bleaching water to water is 1: (3-5) soaking the explant for 15-25min in the mixed solution, and washing with sterile water for 3-4 times.