CN111328713B - A kind of method for constructing regeneration system of red cotyledon - Google Patents

Abstract

The invention discloses a method for constructing a cotyledon regeneration system, comprising the following steps: 1. pretreatment; 2. sterilization; 3. adventitious bud induction; 4. elongation and strong bud culture; plant. In the present invention, the cotyledons of the red flower are used as explants to complete the in vitro regeneration, and the in vitro regeneration of the red flower is carried out by using the technical scheme provided by the present invention, and the induced germination rate and the value-added coefficient are improved. With the advantages of robustness, healthy main root and high survival rate of seedling transplanting, the method for constructing the cotyledon regeneration system of the present invention provides a new technology and method for the genetic transformation and molecular breeding of the rose.

Description

A kind of method for constructing regeneration system of red cotyledon

technical field

The invention belongs to the technical field of plant tissue culture, and in particular relates to a method for constructing a cotyledon regeneration system.

Background technique

Malus asiatica, Rosaceae, Malus, small deciduous trees. Huahong, also known as "Shaguo" and "Ringo", is flat in nature, sweet and sour in taste, rich in various nutrients, and has the effects of quenching thirst and promoting body fluid, digestion and stagnation. In addition to fresh food, the fruit can also be processed into dried fruit, fruit dan skin or wine. Huahong is not only an ornamental tree species with beautiful flowers and fruits, but also an excellent rootstock for apples because of its good grafting compatibility with apples. However, the main method of grafting is to propagate the bonus. In addition, the severe deforestation leads to a serious shortage of germplasm resources for the bonus. This problem can be solved by establishing a regeneration system.

Plant regeneration technology in vitro is the basis for researches such as flower red germplasm conservation and genetic improvement. At present, the commonly used explants for establishing plant regeneration systems include organs such as leaves, cotyledons, hypocotyls, and stem segments. In vitro regeneration of stem segments, the optimal medium proliferation coefficient was 3.8, and no rooted seedlings were obtained; with immature embryos as explants, the optimal medium formed an average of 6 shoots and buds longer than 1 cm in 30 days, but, Individuals developed from embryos may have trait segregation, which will affect the quality of the variety and are not suitable for explants in regeneration systems. As the exophyte of the in vitro rapid propagation system, the stem segment with buds can effectively promote the proliferation of sterile seedlings of C. carinata on the MS medium supplemented with 2.0 mg/L 6-BA and 0.25 mg/LNAA, which is a perennial tree. In this plant, the stem segments are easy to lignify, while the cotyledon explants have the advantages of hypertrophy, rich nutrients, easy access to materials, and easy operation.

The establishment of the regeneration system is immature, the regeneration is difficult, the regeneration difference between different genotypes is large, there are many deformed seedlings, the regeneration speed is slow, and the rooting is difficult. Therefore, the establishment of the regeneration system of red rose needs to be solved urgently, and an efficient and stable regeneration system of red rose is established to provide technical support for genetic engineering and molecular breeding.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a method for using the red cotyledons as explants to induce differentiation into roots or shoots, and to construct a regeneration system for the red cotyledons, which overcomes the deficiencies of the original regeneration system and solves the problems of difficult regeneration and difficult rooting.

The technical scheme adopted in the present invention is:

A method for constructing a red cotyledon regeneration system, using the red cotyledon as an explant to complete in vitro regeneration, comprising the following steps:

(1) pretreatment: choose mature and plump red rose seeds, soak in sterile water for 10-12min;

(2) Sterilization: soak the pretreated seeds with alcohol for 30-35s, rinse with sterile water for 2-3 times, then soak with sodium hypochlorite solution for 8-10min, and finally rinse with sterile water for 3-4 times;

(3) Induction of adventitious buds: the sterilized seeds are stripped of the embryos, after cutting off part of the cotyledons at the adaxial and abaxial ends, inoculated into the adventitious bud induction medium, and cultivated in the dark for 7 days and then cultivated under light conversion;

(4) elongation and strong bud cultivation: transfer the culture material in step (3) to the elongation and strong bud culture medium, and cultivate under the light for 30-40 d;

(5) rooting culture: transfer the material with a stem height of 1-2 cm cultivated in step (4) to rooting medium, and cultivate under light for 30-40 d;

(6) Transplanting: when the root length induced in step (5) is 2-3 cm, rinse the root medium, transplant it into sterilized nutrient soil, and place it in a 25° C. indoor environment for natural growth.

Preferably, the alcohol concentration of the step (2) is 75%, and the mass fraction of the sodium hypochlorite is 10%.

Further, the components of the adventitious bud induction medium in the step (3) are: MS medium supplemented with TDZ at a concentration of 1 mg/L and NAA at a concentration of 0.05 mg/L.

Further, in the step (4), the components of the elongation and strong bud medium are: MS medium supplemented with 6-BA with a concentration of 1.5 mg/L and NAA with a concentration of 0.3 mg/L.

Further, the components of the rooting medium in the step (5) are: MS medium supplemented with IBA with a concentration of 1 mg/L and NAA with a concentration of 0.2 mg/L.

Preferably, the culture conditions under the light are as follows: the temperature is kept at 24-26° C., the illumination time is 16 h/d, the dark culture is 8 h/d, and the illumination intensity is 3000-3500 lux.

Further, the growth conditions of the step (6) are as follows: the illumination time is 16h/d, the dark culture is 8h/d, and the illumination intensity is 4000-6000lux.

The beneficial effects of the present invention are: compared with the existing method, the scheme of the present invention is adopted to construct a red regeneration system, and the red cotyledons are used as explants, and the cotyledon explants are hypertrophic, rich in nutrients, easy to obtain materials, easy to operate, etc. It has the advantages that it can efficiently complete in vitro regeneration and completely retain the genetic traits of red rose, which is the best choice for establishing a stable regeneration system of red rose. Using the induction medium MS+1mg/L TDZ+0.05mg/L NAA, the induction rate of cotyledon cluster buds reached 38.89%. After elongation and strong bud culture, the regenerated buds were numerous and robust, and the proliferation coefficient reached 5.13±0.96, which was higher than The stem segment is the proliferation coefficient of explants; the components of the rooting medium are MS+1mg/L IBA+0.2mg/LNAA, cultured for 30-40 days, the rooting rate is 60%, and the average rooting number is 9.67±3.04 roots. With the extension of subculture time, the rooting rate can reach 100%, and the root system is thicker, which is conducive to the survival of transplanting, which is obviously better than the rooting situation with immature embryos as explants.

The invention provides a stable and high-efficiency method, and solves the problems such as limited material selection of the red rose regeneration system, easy browning of the explants and the like.

Instruction drawings

Fig. 1 is a picture of the stripping of cotyledons;

Fig. 2 is that 20d Huahong cotyledon induces regenerated buds;

Fig. 3 is the bud growth condition after cultivating 50d;

Fig. 4 is the regeneration bud of elongation culture;

Figure 5 shows the culture of proliferating strong buds: Figure A is the proliferation of regenerated buds, and Figure B is the strong buds of regenerated buds;

Figure 6 is a rooting culture.

Detailed ways

The present invention will be further explained in detail below in conjunction with the specific embodiments, and the given examples are only for illustrating the present invention and do not limit the scope of the present invention.

The implementation methods in the following examples are conventional methods unless otherwise specified.

The materials, reagents, etc. used in the following examples are obtained from general commercial sources.

The MS medium used in the examples of the present invention was purchased from Hangzhou Best Biotechnology Co., Ltd.

TDZ: Thidiazuron, a cytokinin;

NAA: Naphthalene Acetic Acid, an auxin;

6-BA: 6-benzylaminopurine, a cytokinin;

IBA: indole butyric acid, an endogenous growth hormone.

The culture conditions under the light were as follows: the temperature was kept at 24-26°C, the light time was 16h/d, the dark culture was 8h/d, and the light intensity was 3000 lux.

Example 1

A method for constructing a regeneration system of red cotyledons, using the red cotyledons as explants to complete in vitro regeneration, comprising the following steps:

(1) pretreatment: select mature and plump red rose seeds soaked in sterile water for 10min;

(2) Sterilization: soak the above-mentioned seeds with alcohol with a concentration of 75% for 30s, rinse with sterile water for 2-3 times, then soak with sodium hypochlorite solution with a mass fraction of 10% for 10min, and rinse with sterile water for 3-4 times;

(3) adventitious bud induction: the sterilized seeds are stripped of the germ, and after cutting off part of the cotyledons at the abaxial end and abaxial end, inoculated on the adventitious bud induction medium, and cultivated in the dark for 7 days and then cultivated under light conversion;

The components of adventitious bud induction medium are: MS+1mg/LTDZ+0.05mg/LNAA;

(4) elongation and strong bud cultivation: transfer the culture material in step (3) to the elongation and strong bud culture medium, and cultivate under the light for 30-40d;

The components of elongation and strong bud medium are: MS+1.5mg/L 6-BA+0.3mg/LNAA;

(5) rooting culture: transfer the material with a stem height of 1-2 cm cultivated in step (4) to rooting medium, and cultivate under light for 30-40 d;

The components of rooting medium are: MS+1mg/L IBA+0.2mg/LNAA;

(6) Transplanting: when the root length induced in step (5) is 2-3 cm, rinse the root medium, transplant it into sterilized nutrient soil, and under the conditions of 25 ° C and 4000 lux of light, light for 16 hours per day, The cells were cultured in the dark for 8 hours, and after 14 days of culture, they were grown naturally under the condition of 6000 lux of light at 25°C.

Example 2

A method for constructing a red cotyledon regeneration system, using the red cotyledon as an explant to complete in vitro regeneration, specifically comprising the following steps:

(1) pretreatment: choose full rose red seeds and soak them in sterile water for 10min;

(2) Sterilization: wash with 75% alcohol for 30s, wash with sterile water for 2-3 times, then treat with 10% sodium hypochlorite solution for 10 minutes, and wash with sterile water for 3-4 times;

(3) adventitious bud induction: the sterilized seeds are stripped of the germ, and after cutting off the adaxial end and abaxial end part of the cotyledons, they are inoculated on the adventitious bud induction medium, and cultivated under the light;

The components of adventitious bud induction medium are: MS+1mg/LTDZ+0.05mg/LNAA;

(4) elongation and strong bud cultivation: the step (3) culture material is transferred to the elongation and strong bud culture medium, and cultivated under the light for 30-40 days;

The components of elongation and strong bud medium are: MS+1.5mg/L 6-BA+0.3mg/LNAA;

(5) rooting culture: transfer the material with the stem height of 1-2cm cultivated in step (4) to rooting medium, and cultivate under light for 30-40 days;

The components of rooting medium are: MS+1mg/L IBA+0.2mg/LNAA;

(6) Transplanting: when the root length induced in step (5) is 2-3 cm, rinse the root medium, transplant it into sterilized nutrient soil, and under the conditions of 25 ° C and 4000 lux of light, light for 16 hours per day, The cells were cultured in the dark for 8 hours, and after 14 days of culture, they were grown naturally under the condition of 6000 lux of light at 25°C.

Example 3

A method for constructing a red cotyledon regeneration system, using the red cotyledon as an explant to complete in vitro regeneration, specifically comprising the following steps:

(1) pretreatment: choose full rose red seeds and soak them in sterile water for 10min;

(2) Sterilization: wash with 75% alcohol for 30s, wash with sterile water for 2-3 times, then treat with 10% sodium hypochlorite solution for 10 minutes, and wash with sterile water for 3-4 times;

(3) adventitious bud induction: the sterilized seeds are stripped of the germ, and some cotyledons at the adaxial end and abaxial end are cut off, inoculated on the adventitious bud induction medium, and cultivated under the light after dark cultivation for 14 days;

The components of adventitious bud induction medium are: MS+1mg/LTDZ+0.05mg/LNAA;

(4) elongation and strong bud cultivation: the step (3) culture material is transferred to the elongation and strong bud culture medium, and cultivated under the light for 30-40 days;

The components of elongation and strong bud medium are: MS+1.5mg/L 6-BA+0.3mg/LNAA;

(5) rooting culture: transfer the material with the stem height of 1-2cm cultivated in step (4) to rooting medium, and cultivate under light for 30-40 days;

The components of rooting medium are: MS+1mg/L IBA+0.2mg/LNAA;

(6) Transplanting: when the root length induced in step (5) is 2-3 cm, rinse the root medium, transplant it into sterilized nutrient soil, and under the conditions of 25 ° C and 4000 lux of light, light for 16 hours per day, The cells were cultured in the dark for 8 hours, and after 14 days of culture, they were grown naturally under the condition of 6000 lux of light at 25°C.

For Example 1-3, a culture medium test group of different components was set up, and the adventitious bud regeneration of Example 1-3 was observed and recorded in real time:

Table 1 Effects of different media and different dark cultivation time on the induction of adventitious buds

According to the experimental results, during the induction of adventitious buds, using MS+1mg/L TDZ+0.05mg/LNAA medium, the cotyledons were cultivated in the dark for 7 days and then cultivated in the light for 2 weeks, that is, buds grew from the cotyledons. The time-averaged number of buds was the highest, the regeneration rate of adventitious buds was the highest, and the growth quality of buds was the best, which was conducive to the continued cultivation in later stages.

According to the culture method of Example 1 (cultivation in dark for 7d), the medium of different concentrations of TDZ, NAA and IBA combined was used for the induction culture of adventitious buds. At 25° C., 16 hours of light and 8 hours of dark culture were carried out every day for 40 days. Callus induction rate and cluster bud induction rate, the results are shown in Table 2:

Table 2 Effects of different concentrations of TDZ, NAA and IBA on the regeneration of cotyledons

The experiment showed that when the concentration of TDZ was 1.0 mg/L and the concentration of NAA was 0.05 mg/L, the induction rate of adventitious buds was the highest, which was 38.89%, and the average number of buds was 11.71.

The adventitious bud induced proliferation culture material was transferred to the elongation and strong bud medium for cultivation, and the elongation and strong bud medium was set with a combination of 6-BA and NAA at different concentrations. Cultivated for 8h and cultured for 40 days. The results are shown in Table 3.

Table 3 Effects of different concentrations of 6-BA and NAA on the proliferation of regenerated buds and strong buds

It can be seen from Table 3 that when the concentration of 6-BA is 1.5 mg/L and the concentration of NAA is 0.3 mg/L, the growth of regenerated buds is robust, and there are many regenerated buds, and the proliferation coefficient reaches 5.13±0.96.

The regenerated seedlings with a height of 1-3 cm cultivated by elongating and strong buds were transferred to the rooting medium to induce rooting, using different concentrations of IBA and NAA combined medium, under the conditions of 25 ° C and 3000 lux of light, light for 16 hours per day , cultured in dark for 8h, cultured for 40 days, and the rooting rate was counted. The results are shown in Table 5.

Table 4. Rooting situation of rhododendron seedlings in the medium with different concentrations of IBA and NAA combined

It can be seen from Table 4 that when the IBA concentration is 1 mg/L and the NAA concentration is 0.2 mg/L, the rooting rate of the regenerated seedlings is the highest, which is 60%.

Referring to Fig. 2-Fig. 6, it is the picture of cotyledon-induced regeneration bud and regeneration bud elongation and rooting of the embodiment of the present invention 1, it can be seen that this method can induce the regenerated bud of red flower more efficiently, and use this method to construct the regeneration system of red flower red It is more suitable and efficient than the original method.

The invention selects the cotyledons native to Guizhou as the explant to obtain the adventitious buds of the red rose, selects the formula of the adventitious bud induction medium, and constructs the red regeneration system.

The above-described embodiments are only some of the embodiments of the present invention, but not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

Claims (3)

1. a method for constructing a regeneration system of red cotyledon, is characterized in that: be explant with described red cotyledon, complete in vitro regeneration, comprise the following steps: (1) Pretreatment: Select mature and plump rosette seeds and soak them in sterile water for 10-12min; (2) Sterilization: soak the pretreated seeds in alcohol for 30-35s, rinse with sterile water for 2-3 times, then soak in sodium hypochlorite solution for 8-10min, and finally rinse with sterile water for 3-4 times; Described alcohol concentration is 75%, and the mass fraction of described sodium hypochlorite is 10%, the seed after sterilization is peeled off the germ, after cutting off the partial cotyledon of the abaxial end and the abaxial end for subsequent use; (3) Adventitious bud induction: Inoculate the spare material in the adventitious bud induction medium, cultivate in the dark for 7 days, and then cultivate under the light. The components of the adventitious bud induction medium are: MS medium supplemented with a concentration of 1 mg/L TDZ and NAA at a concentration of 0.05mg/L; (4) Elongation and strong bud culture: transfer the culture material in step (3) to the elongation and strong bud medium, and cultivate it under the light for 30-40 days. 1.5mg/L of 6-BA and 0.3mg/L of NAA; (5) Rooting culture: transfer the material with a stem height of 1-2 cm cultivated in step (4) to a rooting medium, and cultivate under light for 30-40 d. The rooting medium is composed of: MS medium with a concentration of 1 mg /L of IBA and 0.2mg/L of NAA; (6) Transplanting: When the root length induced in step (5) is 2-3 cm, rinse the root medium, transplant it into sterilized nutrient soil, and place it in a 25°C indoor environment to grow naturally. 2. a kind of method of constructing a regeneration system of cotyledon of rhododendron according to claim 1, is characterized in that, the culture condition under described light is: temperature keeps 24-26 ℃, illumination time 16h/d, dark culture 8h/d, Light intensity 3000-3500lux. 3. The method for constructing a regeneration system for cotyledons of floristry according to claim 1, wherein the growth conditions of the step (6) are: illumination time 16h/d, dark culture 8h/d, illumination intensity 4000- 6000lux.

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