CN108633741A - The method for tissue culture of four seasons begonia - Google Patents

Abstract

The invention discloses a method for tissue culture of begonia four seasons, which comprises the following steps: (1) Sterilization: select the explant of begonia four seasons, wash it with tap water first, then soak it with alcohol, then clean it with sterile water, and then wash it with Disinfect with sodium hypochlorite, then rinse with sterile water for a second time, cut and inoculate the explants of Begonia four seasons; (2) Soak in nutrient solution: configure 1/2MS+9.0mg/L 6-BA nutrient solution, and inoculate the explants at 25°C. Put the plant into it and soak for 1 hour; (3) Leaf tissue culture: put the explant soaked in the nutrient solution in step (2) into the culture medium with different hormones and ratios, and proceed in sequence: induce the explant to form a callus tissue, induce callus to generate shoots, induce proliferation and differentiation of shoots, and induce shoots to take root to obtain seedlings; (4) seedling hardening and transplanting: put the seedlings of step (3) into a culture bottle, open and cultivate The bottle caps were used to harden the seedlings indoors for 2 days, and the basal medium of the seedlings was taken out and washed, and planted in a matrix of perlite: humus = 1:2.

Description

The Tissue Culture Method of Begonia Four Seasons

technical field

The invention relates to a propagation method of begonias, in particular to a tissue culture method of begonias in four seasons.

Background technique

Four Seasons Begonia, also known as Begonia and Melon Seed Begonia, is a perennial fleshy evergreen herbaceous flower belonging to Begoniaceae and Begonia. Stems erect, 15-30cm high; The leaves are mostly ovate, about 5-8cm in length and 1-3cm in width. The surface of the fleshy leaves is smooth and the lower part is slightly inclined. The edge of the leaves has coarse serrations, and can be divided into green-leaf strains and red-leaf strains according to leaf color. Four Seasons Begonia is a green-leaf strain, with shiny green leaves, reddish edges, and red flowers. They are clustered on the peduncle and appear luxuriant, and have a long flowering period. Under suitable temperature, humidity and light, they can bloom almost all year round. . Therefore, it is an excellent choice for ground covers, flower beds, flower mirrors, flower belts and indoor viewing. Begonias in four seasons like humid air environment and moist soil, and are afraid of drying and stagnant water. The optimum temperature for its growth is 20°C, so it needs to be placed in a ventilated, cool and shaded place in summer, and the plant needs to be placed in a sunny place in winter.

The seeds of perennial begonias are very small, so it is not easy to collect and store the seeds. In addition, the seed germination condition requires 100% humidity, the germination time is longer (about 20 days), and it takes about 3 months for the seedlings to emerge from the nursery. Therefore, cuttings are commonly used in production to carry out the asexual reproduction of begonias in four seasons. However, because the stems of begonias are fleshy and are susceptible to contamination by fungi and viruses, the stems are extremely easy to rot during cutting propagation.

In summary, it is necessary to improve the prior art to solve the above technical problems.

Contents of the invention

In view of this, the object of the present invention is to provide a tissue culture method of begonia perennials, which can more stably realize the reproduction of begonias four seasons, with fast reproduction speed and high survival rate. Specifically, it is realized through the following technical solutions:

The tissue culture method of Begonia four seasons of the present invention, comprises the following steps:

(1) Sterilization: select the explants of Begonia sibifolia, first rinse them with tap water, then soak them in alcohol, then wash them with sterile water, then sterilize them with low-concentration sodium hypochlorite, and then rinse them twice with sterile water. Explant cutting inoculation of begonia;

(2) Soaking in nutrient solution: prepare 1/2MS+9.0mg/L 6-BA nutrient solution, put the explants into it and soak for 1 hour at 25°C;

(3) Leaf tissue culture: put the explants soaked in the nutrient solution in step (2) into the MS basic medium containing different concentrations of hormones and proportions, and proceed sequentially: induce the explants to form callus, induce callus Wounded tissue generates young shoots, induces the proliferation and differentiation of young shoots, and induces young shoots to take root to obtain seedlings;

(4) Seedling hardening and transplanting: put the seedlings of step (3) into a culture bottle, open the lid of the culture bottle and harden the seedlings indoors for 2 days, take out the seedlings and wash the base medium, and plant perlite: humus =1:2 matrix.

Further, in the step (1), select medium-mature begonia leaves as explants.

Further, the medium for inducing explant differentiation in the step (3) is: MS medium + 0.2 mg/L TDZ.

Further, the medium for inducing the proliferation of sprouts in the step (3) is: MS+0.5mg/L 6-BA+0.12mg/LNAA.

Further, the medium for inducing shoots to take root in the step (3): 1/2MS+0.1mg/L NAA.

Further, in the step (3), the conditions in the process of inducing proliferation and differentiation of young shoots and inducing buds to take root to obtain seedlings are all temperature 23-27° C., light 14h/d, and light intensity stable at 2000 Lux in the cultivation indoor environment.

Further, the cultivation condition after transplanting in the step (4) is a greenhouse environment with a stable light intensity of 1200-2000 Lux, a temperature of 18-20° C. and a humidity of 69.5%.

Further, when sterilizing in the step (1), first rinse with tap water for 30 seconds, then soak for 5 seconds with alcohol with a volume fraction of 75%, then fully wash with sterile water for 5 times, and then use a mass fraction of 0.25% Disinfect with sodium hypochlorite for 15 minutes, and finally rinse with sterile water 5 times.

Further, in the step (1), the explant is cut into tissues with a size of about 0.8 cm×0.8 cm.

Beneficial effects of the present invention: in the tissue culture method of begonia four seasons of the present invention, explants are soaked in a nutrient solution containing a high concentration of 6-BA with the ability to promote tissue differentiation for 1 hour, which can enhance the differentiation ability of the explants, Increase the possibility and orderliness of its callus formation, and overcome the problems of difficult callus formation and uneven bud differentiation in the tissue culture process; other beneficial effects of the present invention will be further described in conjunction with the following specific examples .

Description of drawings

The present invention will be further described below in conjunction with accompanying drawing and embodiment:

Fig. 1 is the four seasons begonia picture of the strong plant of explant drawing source in the embodiment of the present invention;

Fig. 2 is the comparative figure of tender leaf, middle mature leaf and old leaf in the embodiment of the present invention one;

Fig. 3 is the explant figure after the sterilized section in the embodiment of the present invention is finished;

Fig. 4 is a stage diagram of callus formation in Example 1 of the present invention;

Fig. 5 is budding and multiplication stage figure in the embodiment of the present invention one;

Fig. 6 is the rooting stage figure in the embodiment of the present invention one;

Fig. 7 is the seedling hardening figure in the embodiment of the present invention one;

Fig. 8 is a transplanting figure in the embodiment of the present invention one;

Fig. 9 is a stage diagram of the whole experimental process in Embodiment 1 of the present invention

Detailed ways

The tissue culture method of Begonia four seasons of the present invention, comprises the following steps:

(1) Sterilization: select the explants of Begonia four seasons, rinse them with tap water first, then soak them in alcohol, then wash them with sterile water, then disinfect them with sodium hypochlorite, rinse them twice with sterile water, and wash the explants of Begonia four seasons. explant cutting inoculation;

(2) Soaking in nutrient solution: configure 1/2MS+9.0mg/L 6-BA nutrient solution, soak the explants in it for 1 hour at 25°C; 1/2MS refers to the MS medium according to Murashige and Skoog, It is an international common culture medium, in which a large number of elements are reduced to 1/2 of the original, and the rest of the elements remain unchanged. 6-BA refers to 6-benzylaminoadenine. 6-BA can promote tissue differentiation. Soaking explants in nutrient solution can enhance the differentiation ability of explants, increase the possibility of forming callus, and overcome Solved the problems of difficult callus formation and irregular differentiation of buds in the process of tissue culture;

(3) Leaf tissue culture: put the explants soaked in the nutrient solution in step (2) into the MS basic medium, and proceed sequentially: inducing the explants to form callus, inducing the callus to generate shoots, and inducing the Bud proliferation and differentiation, inducing young shoots to take root to obtain seedlings;

(4) Seedling hardening and transplanting: Open the lid of the culture bottle and harden the seedlings indoors for 2 days, take out the grown seedlings, wash the base medium, and plant them in the matrix of perlite: humus = 1:2.

In the present embodiment, in the step (1), medium-mature leaves of begonia four seasons are selected as explants. The specific standard of medium maturity refers to the 3rd to 5th leaf that is fully expanded from top to bottom. If the leaf is older, the degree of differentiation of plant cells will increase and the totipotency will decrease, so it is not suitable as an explant for tissue culture; if the leaf If the degree of maturity is low, the resistance to disinfectants is weak, and it is easy to vitrify after disinfection treatment. Therefore, moderately mature leaves should be selected as explants.

In this embodiment, the basic medium in the step (3) is MS medium, and different hormones TDZ, 6-BA and NAA are added according to different purposes of differentiation, proliferation and rooting. TDZ refers to phenylurea derivatives, which can significantly enhance the ability of cell division, and NAA refers to naphthaleneacetic acid, which plays a leading role in inducing rooting.

In this example, the conditions in the process of inducing the proliferation and differentiation of young shoots in the step (3) and inducing the buds to take root to obtain seedlings are all in a cultivation room with a temperature of 23-27° C., a light of 14 h/d, and a stable light intensity of 2000 Lux. surroundings. The survival rate of tissue culture under this condition is higher.

In the present embodiment, the cultivation condition after transplanting in the step (4) is a greenhouse environment where the light intensity is stable at 1200-2000Lux, the temperature is 18-20°C, and the humidity is 69.5%. The survival rate of tissue culture under this condition is higher.

In this embodiment, when sterilizing in the step (1), first rinse with tap water for 30 seconds, then soak for 5 seconds with alcohol with a volume fraction of 75%, then fully wash with sterile water for 5 times, and then use the mass fraction Disinfect with 0.25% sodium hypochlorite for 15 minutes, and finally rinse with sterile water for 5 times. Plant materials should be placed on an ultra-clean workbench during sterilization.

In this embodiment, in the step (1), the explant is cut into tissues with a size of about 0.8 cm×0.8 cm.

Table 1. The condition of the selected test medium for explant callus induction 30 days

Table 2. Situation after 30 days of bud induction by selection test medium

Table 3. The situation after 15 days of rooting induction of adventitious shoots by the selected test medium

The present invention will be further described below in conjunction with specific embodiment:

Embodiment one

Get a strong plant of Begonia four seasons, the begonia four seasons as shown in Figure 1, take its medium mature leaves as explants, as shown in Figure 2, the left is a young leaf, the middle is a medium mature leaf, and the right is an old leaf. Rinse with tap water for 30 seconds, then soak in alcohol with a volume fraction of 75% for 5 seconds, then fully wash with sterile water for 5 times, then disinfect with 0.25% sodium hypochlorite for 15 minutes, and finally rinse with sterile water for 5 minutes The second time, the explants were cut into tissues with a size of about 0.8cm×0.8cm. Configure 1/2MS+9.0mg/L 6-BA nutrient solution, put the explants into it and soak for 1h at 25℃. The explants are put into medium containing different concentrations of hormones and proportioning, and the explants are induced to form callus successively, as shown in Figure 4; the callus is induced to generate shoots, and the shoots are induced to proliferate The process is shown in Figure 5; the process of inducing young shoots to take root to obtain seedlings is shown in Figure 6; wherein, the medium for inducing explant differentiation, inducing bud proliferation, and inducing shoot rooting is respectively: MS+0.2 mg/L TDZ, MS+0.5mg/L 6-BA+0.12 mg/L NAA, 1/2MS+0.1mg/L NAA. The conditions in this process are the cultivation indoor environment with a temperature of 23-27°C, a light of 14 h/d, and a stable light intensity of 2000 Lux. The seedlings were put into the culture bottle, and the lid of the culture bottle was opened to harden the seedlings indoors for 2 days, as shown in Figure 7. Take out the seedlings and wash the basal medium, and plant them in the matrix of perlite: humus = 1:2. The culture conditions after transplanting are that the light intensity is stable at 1200-2000Lux, the temperature is 18-20°C, and the humidity is 69.5%. Greenhouse environment, the process is shown in Figure 8.

Finally, it is noted that the above embodiments are only used to illustrate the technical solutions of the present invention without limitation. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be carried out Modifications or equivalent replacements without departing from the spirit and scope of the technical solution of the present invention shall be covered by the claims of the present invention.

Claims (9)

1. a method for tissue culture of begonia four seasons, is characterized in that comprising the following steps: (1) Sterilization: choose the fully expanded leaves of Begonia four seasons as explants, rinse with tap water first, then soak with alcohol, then clean with sterile water, then disinfect with sodium hypochlorite, and then rinse with sterile water for the second time. Explant cutting and inoculation of Begonia four seasons; (2) Soaking in nutrient solution: prepare 1/2MS+9.0mg/L 6-BA nutrient solution, put the explants into it and soak for 1 hour at 25°C; (3) Leaf tissue culture: put the explants soaked in the nutrient solution in step (2) into the MS basic medium containing different hormones and proportions, and proceed sequentially: induce the explants to form callus, and induce callus The tissue generates sprouts, induces the proliferation of sprouts, and induces sprouts to take root to obtain seedlings; (4) Seedling hardening and transplanting: put the seedlings of step (3) into the culture bottle, open the lid of the culture bottle to harden the seedlings indoors for 2 days, take out the seedlings and clean the base culture medium, and plant perlite: humus= 1:2 matrix. 2. The tissue culture method of Begonia sibiris according to claim 1, characterized in that: in the step (1), medium-mature leaves of Begonia sibiran are selected as explants. 3. The method for tissue culture of Begonia perennials according to claim 2, characterized in that: the basic medium in the step (2) is MS medium+0.2mg/L TDZ+0.05mg/L NAA, soaked for 1h. 4. the tissue culture method of begonia four seasons according to claim 3, is characterized in that: in the described step (3), the substratum and culture condition of inducing explant differentiation are: MS+0.2mg/L TDZ; Culture time 30 days; culture conditions: temperature 23-27°C, light 14h/d, light intensity 2000Lux. 5. The tissue culture method of Begonia four seasons according to claim 4, characterized in that: the culture medium and the culture conditions for inducing the proliferation of sprouts in the step (3) are: MS+0.5mg/L 6-BA+0.12 mg/L NAA; the culture time is 30 days; the culture conditions are: temperature 23-27 ℃, light 14h/d, light intensity 2000Lux. 6. the tissue culture method of begonia four seasons according to claim 5, is characterized in that: in the described step (3), inducing bud to carry out the substratum and culture condition of rooting are: 1/2MS+0.1mg/L NAA; The time is 15 days; the culture conditions are temperature 23-27°C, light 14h/d, light intensity 2000Lux. 7. The tissue culture method of Begonia four seasons according to claim 6, characterized in that: the culture conditions after transplanting in the step (4) are that the light intensity is stable at 1200-2000Lux, the temperature is 18-20°C, and the humidity 69.5% of the greenhouse environment. 8. The tissue culture method of Begonia four seasons according to claim 7, characterized in that: when sterilizing in the step (1), first rinse with tap water for 30 seconds, then soak for 5 seconds with alcohol with a volume fraction of 75% , and then fully washed 5 times with sterile water, then disinfected with 0.25% sodium hypochlorite for 15 minutes, and finally fully rinsed 5 times with sterile water. 9 . The method for tissue culture of Begonia perennials according to claim 8 , characterized in that: in the step (1), the explants are cut into tissues with a size of about 0.8 cm×0.8 cm.