阅读说明：本技术 一种铁筷子组培繁殖方法 (Tissue culture propagation method for Chimonanthus praecox ) 是由 段青 王继华 王祥宁 杨晓 杜文文 马璐琳 田敏 于 2021-08-10 设计创作，主要内容包括：本发明提供一种铁筷子组培繁殖方法,包括以下步骤：(1)外植体的获取与处理；(2)不定芽诱导培养；(3)不定芽继代增殖培养；(4)生根培养；(5)炼苗和移栽,得铁筷子种苗。本发明通过对激素、培养环境的综合调节,使铁筷子不定芽增殖倍数达到3.5以上,生根率达到90%以上,解决了铁筷子繁殖系数低、生根难、生产周期长的难题。另外,通过将优良株系的优良性状保持下来,可以生产优良性状稳定的种苗,易于标准化、工厂化操作,为市场提供统一标准的优良种苗。(The invention provides a tissue culture propagation method of Chimonanthus praecox, which comprises the following steps: (1) obtaining and processing an explant; (2) adventitious bud induction culture; (3) subculturing and proliferating the adventitious bud; (4) rooting culture; (5) hardening and transplanting to obtain the chopsticks seedling. The method comprehensively adjusts hormone and culture environment, so that the multiplication factor of adventitious buds of the chopsticks reaches more than 3.5, the rooting rate reaches more than 90%, and the problems of low propagation coefficient, difficult rooting and long production period of the chopsticks are solved. In addition, by maintaining the excellent properties of the excellent strains, seedlings with stable excellent properties can be produced, the standardization and the industrial operation are easy, and the uniform and standard excellent seedlings are provided for the market.)

1. A tissue culture propagation method of Chimonanthus praecox is characterized by comprising the following steps:

(1) obtaining and processing explants: cutting small axillary buds with undeveloped leaves in 8 months, soaking in 15-30% washing powder water for 3-6min, washing with running water for more than 30min, soaking in 70% alcohol for 20-35s, soaking in 0.2% Fungicide for 15min, soaking in 0.15% mercury bichloride for 20-30min, washing with sterile water for 3 times, and filtering to remove water to obtain treated small axillary buds;

(2) adventitious bud induction culture: inoculating the axillary buds treated in the step (1) on the following adventitious bud induction culture medium: MS +2iP 2mg/L +6-BA 1-2 mg/L + NAA 0.1-0.3 mg/L, agar 6.5g/L, cane sugar 30g/L, and pH5.8;

performing induction culture for 40-60 days under the conditions that the temperature is 15 +/-2 ℃, the illumination intensity is 2000-3000 lx and the illumination time is 8-12h/d until the explant is differentiated into adventitious buds;

(3) subculture multiplication culture of adventitious buds: cutting the adventitious bud induced by the step (2), and transferring the adventitious bud into the following proliferation culture medium:

MS +2iP 2mg/L +6-BA 1mg/L + NAA 0.1-0.3 mg/L, agar 6.5g/L, cane sugar 30g/L, and pH5.8;

subculturing at the temperature of 15-23 ℃, the illumination intensity of 2000-3000 lx and the illumination time of 8-12h/d, and subculturing once every 30d until adventitious buds with the length of 1-2 cm are obtained;

(4) rooting culture: transferring the adventitious bud subjected to the subculture multiplication in the step (3) into the following rooting medium:

MS + IBA 0.5-1.0 mg/L, agar 6.50g/L, sucrose 30g/L, pH5.8;

performing induced rooting culture for 25-35 d under the conditions that the temperature is 15 +/-2 ℃, the illumination intensity is 2000-3000 lx and the illumination time is 8-12h/d to obtain sterile seedlings with 5-8 fibrous roots growing at plant base parts;

(5) hardening and transplanting seedlings: taking out the sterile seedlings subjected to rooting culture in the step (4), cleaning root agar, putting the sterile seedlings into a carbendazim solution with the mass concentration of 0.1-0.2%, disinfecting for 1-2 min, and transplanting the sterile seedlings into a substrate with the following mass ratio: the humus soil, the laterite and the perlite are = 1: 1, and the seedlings can be transplanted after acclimation and hardening in a greenhouse for 40-60 days according to the conventional method, wherein the transplanting survival rate is over 95 percent.

Technical Field

The invention relates to a tissue culture propagation method, in particular to a tissue culture propagation method of Chimonanthus praecox, belonging to the technical field of plant breeding.

Background

Iron chopsticks (Helleborus) Is a perennial plant of Ranunculaceae, about 22 species of the genus are mainly distributed in Europe and Western Asia, and China only has chopsticks: (Helleborus thibetanus) One kind of the medicine. The plant type of the Chimonanthus praecox is short, the leaf color is dark green, the flower and leaf are peculiar, the Chimonanthus praecox is used as a potted plant, and the Chimonanthus praecox blossoms in winter and spring or early spring and is called as 'Christmas rose', and the ornamental value is extremely high. The chopsticks are mainly based on seed propagation, but because the seeds have dormancy characteristics and are limited by environmental conditions, the fertilized seeds need 10-12 weeks to mature, and the time from sowing to germination of the mature seeds needs 26-34 weeks, so that the production period is too long, and the large-scale production and commercial application of the chopsticks are severely restricted. The existing tissue culture of the chopsticks has the problems of difficult in vitro culture starting, low proliferation rate, difficult rooting and domestication and the like caused by browning, endophyte and the like. There is therefore a need for improvements in the prior art.

Disclosure of Invention

The invention provides a tissue culture propagation method for chopsticks, aiming at solving the problems of overlong growth cycle, difficult in vitro culture starting, high pollution rate, low increment rate, difficult rooting and domestication and the like caused by the dormancy characteristic of seeds of the existing chopsticks.

The invention is completed by the following technical scheme: a tissue culture propagation method of Chimonanthus praecox is characterized by comprising the following steps:

(1) obtaining and processing explants: cutting small axillary buds with undeveloped leaves in 8 months, soaking in 15-30% washing powder water for 3-6min, washing with running water for more than 30min, soaking in 70% alcohol for 20-35s, soaking in 0.2% Fungicide for 15min, soaking in 0.15% mercury bichloride for 20-30min, washing with sterile water for 3 times, and filtering to remove water to obtain treated small axillary buds;

(2) adventitious bud induction culture: inoculating the axillary buds treated in the step (1) on the following adventitious bud induction culture medium: MS +2iP 2mg/L +6-BA 1-2 mg/L + NAA 0.1-0.3 mg/L, agar 6.5g/L, cane sugar 30g/L, and pH5.8;

performing induction culture for 40-60 days under the conditions that the temperature is 15 +/-2 ℃, the illumination intensity is 2000-3000 lx and the illumination time is 8-12h/d until the explant is differentiated into adventitious buds;

(3) subculture multiplication culture of adventitious buds: cutting the adventitious bud induced by the step (2), and transferring the adventitious bud into the following proliferation culture medium:

MS +2iP 2mg/L +6-BA 1mg/L + NAA 0.1-0.3 mg/L, agar 6.5g/L, cane sugar 30g/L, and pH5.8;

subculturing at the temperature of 15-23 ℃, the illumination intensity of 2000-3000 lx and the illumination time of 8-12h/d, and subculturing once every 30d until adventitious buds with the length of 1-2 cm are obtained;

(4) rooting culture: transferring the adventitious bud subjected to the subculture multiplication in the step (3) into the following rooting medium:

MS + IBA 0.5-1.0 mg/L, agar 6.50g/L, sucrose 30g/L, pH5.8;

performing induced rooting culture for 25-35 d under the conditions that the temperature is 15 +/-2 ℃, the illumination intensity is 2000-3000 lx and the illumination time is 8-12h/d to obtain sterile seedlings with 5-8 fibrous roots growing at plant base parts;

(5) hardening and transplanting seedlings: taking out the sterile seedlings subjected to rooting culture in the step (4), cleaning root agar, putting the sterile seedlings into a carbendazim solution with the mass concentration of 0.1-0.2%, disinfecting for 1-2 min, and transplanting the sterile seedlings into a substrate with the following mass ratio: the humus soil, the laterite and the perlite are = 1: 1, and the seedlings are domesticated and acclimated in a greenhouse for 40-60 days according to the conventional method and then can be transplanted to a field, wherein the transplanting survival rate is more than 95%.

The invention has the following advantages and effects: by adopting the scheme, the multiplication rate of the adventitious buds of the chopsticks can reach more than 3.5 and the rooting rate can reach more than 90 percent through the comprehensive regulation of hormones and culture environment, and the problems of low propagation coefficient, difficult rooting and long production period of the chopsticks are solved. In addition, by maintaining the excellent properties of the excellent strains, seedlings with stable excellent properties can be produced, the standardization and the industrial operation are easy, and the uniform and standard excellent seedlings are provided for the market.

Detailed Description

The present invention will be described in further detail with reference to specific examples.

Example 1

A method for tissue culture propagation of Chimonanthus praecox comprises the following steps:

(1) selection and sterilization of explants: in autumn 8 months, cutting small axillary buds with undeveloped leaves, soaking in 15% washing powder water for 6min, washing with running water for more than 30min, placing on a clean bench, soaking in 70% alcohol for 30s, soaking in 0.2% Fungicide for 15min, soaking in 0.15% mercury bichloride for 20min, washing with sterile water for 3 times, and filtering to remove excessive water to obtain treated small axillary buds;

(2) adventitious bud induction culture: inoculating the axillary buds treated in the step (1) on the following adventitious bud induction culture medium:

MS +2iP 2mg/L +6-BA 1mg/L + NAA 0.1mg/L, agar 6.5g/L, sucrose 30g/L, pH5.8;

performing induction culture for 60 days under the conditions that the temperature is 15 ℃, the illumination intensity is 2000lx and the illumination time is 10h/d until the explant differentiates into adventitious buds;

(3) subculture multiplication culture of adventitious buds: cutting the adventitious bud induced by the step (2), and transferring the adventitious bud into the following proliferation culture medium:

MS +2iP 2mg/L +6-BA 1mg/L + NAA 0.1mg/L, agar 6.5g/L, sucrose 30g/L, pH5.8;

subculturing at 15 deg.C under illumination intensity of 2000lx and illumination time of 10h/d, and subculturing once every 30d until adventitious bud of 2cm in length is obtained;

(4) rooting culture: transferring the adventitious bud subjected to the subculture multiplication in the step (3) into the following rooting medium:

MS + IBA 0.5mg/L, agar 6.50g/L, sucrose 30g/L, pH5.8;

culturing for 35d under the conditions that the temperature is 15 ℃, the illumination intensity is 2000lx and the illumination time is 10h/d to obtain sterile seedlings with 5 fibrous roots growing at the plant base;

(5) hardening and transplanting seedlings: taking out the sterile seedlings with 5 fibrous roots induced to root and cultured in the step (4), cleaning root agar, putting the sterile seedlings into a carbendazim solution with the mass concentration of 0.1 percent for disinfection for 2min, and transplanting the sterile seedlings to humus soil: laterite: perlite = 1: 1: 1, domesticating and hardening the seedlings in a greenhouse for 60 days according to a conventional mode, and transplanting the seedlings to a field.

The multiplication times of adventitious buds of the chopsticks in the embodiment reach 4.1, the rooting rate reaches 96%, and the transplanting survival rate is 98%.

Example 2

A method for tissue culture propagation of Chimonanthus praecox comprises the following steps:

(1) selection and sterilization of explants: in autumn 8 months, cutting small axillary buds with undeveloped leaves, soaking in 30% washing powder water for 3min, washing with running water for more than 30min, placing on a clean bench, soaking in 70% alcohol for 30s, soaking in 0.2% Fungicide for 15min, soaking in 0.15% mercury bichloride for 20min, washing with sterile water for 3 times, and filtering to remove excessive water to obtain treated small axillary buds;

(2) adventitious bud induction culture: inoculating the small axillary buds treated in the step (1) into an adventitious bud induction culture medium, and carrying out induction culture for 40d under the conditions of the temperature of 13 ℃, the illumination intensity of 3000lx and the illumination time of 8h/d until the explant is differentiated into adventitious buds;

the adventitious bud induction culture medium comprises: MS +2iP 2mg/L +6-BA 1.5mg/L + NAA 0.2mg/L, agar 6.5g/L, sucrose 30g/L, pH5.8;

(3) subculture multiplication culture of adventitious buds: transferring the adventitious bud induced in the step (2) into a proliferation culture medium, and carrying out subculture under the conditions that the temperature is 20 ℃, the illumination intensity is 3000lx and the illumination time is 8h/d, wherein subculture is carried out once every 30d until the adventitious bud with the length of 1cm is obtained;

the proliferation culture medium is as follows: MS +2iP 2mg/L +6-BA 1mg/L + NAA 0.2mg/L, agar 6.5g/L, sucrose 30g/L, pH5.8;

(4) rooting culture: transferring the adventitious bud cultured by the subculture multiplication in the step (3) to a rooting culture medium for inducing rooting, and culturing for 25d under the conditions that the temperature is 13 ℃, the illumination intensity is 3000lx and the illumination time is 8h/d to obtain a sterile seedling with 6 fibrous roots growing on the plant base;

the rooting culture medium comprises: MS + IBA 0.8mg/L, agar 6.50g/L, sucrose 30g/L, pH5.8;

(5) hardening and transplanting seedlings: taking out the sterile seedlings induced to root and cultured in the step (4), cleaning root agar, putting the sterile seedlings into a carbendazim solution with the mass concentration of 0.1-0.2%, disinfecting for 1-2 min, and transplanting the sterile seedlings to humus soil: laterite: perlite = 1: 1: 1, domesticating and hardening the seedlings in a greenhouse for 50 days according to a conventional mode, and transplanting the seedlings to a field.

The multiplication times of the adventitious buds of the chopsticks in the embodiment reach 3.6, the rooting rate reaches 92%, and the survival rate is 96%.

Example 3

A method for tissue culture propagation of Chimonanthus praecox comprises the following steps:

(1) selection and sterilization of explants: cutting small axillary buds with undeveloped leaves in autumn in 8 months, soaking the small axillary buds in 20% washing powder water for 5min, washing the small axillary buds with running water for more than 30min, placing the small axillary buds on a super-clean workbench, soaking the small axillary buds in 70% alcohol for 30s, then soaking the small axillary buds in 0.2% Fungicide for 15min, finally soaking the small axillary buds in 0.15% mercury bichloride for 20min, washing the small axillary buds with sterile water for 3 times, and filtering off redundant water to obtain treated small axillary buds;

(2) adventitious bud induction culture: inoculating the small axillary buds treated in the step (1) into an induction culture medium, and carrying out induction culture for 50d under the conditions that the temperature is 17 ℃, the illumination intensity is 2500lx and the illumination time is 12h/d until the explant is differentiated into adventitious buds;

the adventitious bud induction culture medium comprises: MS +2iP 2mg/L +6-BA 2mg/L + NAA 0.3mg/L, agar 6.5g/L, sucrose 30g/L, pH5.8;

(3) subculture multiplication culture of adventitious buds: transferring the adventitious bud induced in the step (2) into a proliferation culture medium, and carrying out subculture under the conditions that the temperature is 23 ℃, the illumination intensity is 2500lx and the illumination time is 12h/d, wherein subculture is carried out once every 30d until the adventitious bud with the length of 1.5cm is obtained;

the proliferation culture medium is as follows: MS +2iP 2mg/L +6-BA 1mg/L + NAA 0.3mg/L, agar 6.5g/L, sucrose 30g/L, pH5.8;

(4) rooting culture: transferring the adventitious bud cultured by the subculture multiplication in the step (3) to a rooting culture medium for inducing rooting, and culturing for 25d under the conditions that the temperature is 16 ℃, the illumination intensity is 2500lx and the illumination time is 12h/d to obtain a sterile seedling with 7 fibrous roots growing on the plant base;

the rooting culture medium comprises: MS + IBA 0.5-1.0 mg/L, agar 6.50g/L, sucrose 30g/L, pH5.8;

(5) hardening and transplanting seedlings: taking out the sterile seedlings induced to root and cultured in the step (4), cleaning root agar, putting the sterile seedlings into a carbendazim solution with the mass concentration of 0.1-0.2%, disinfecting for 1-2 min, and transplanting the sterile seedlings to humus soil: laterite: perlite = 1: 1: 1, domesticating and hardening the seedlings in a greenhouse for 40 days according to a conventional mode, and then transplanting the seedlings into a field.

The multiplication times of the adventitious buds of the chopsticks in the embodiment reach 3.5, the rooting rate reaches 90%, and the survival rate is 95%.

Comparative example 1

A method for tissue culture propagation of Chimonanthus praecox comprises the following steps:

(1) selection and sterilization of explants: cutting axillary buds with unfolded leaves in autumn in 4 months, soaking in washing powder water for 5min, washing with running water for more than 30min, soaking in 70% ethanol for 30s, soaking in 0.15% mercuric chloride for 20min, washing with sterile water for 3 times, and filtering to remove excessive water;

(2) adventitious bud induction culture: inoculating the axillary buds treated in the step (1) on the following adventitious bud induction culture medium: MS +6-BA 2mg/L + NAA 0.2mg/L, agar 6.5g/L, cane sugar 30g/L, pH5.8; and (3) dying the dead plants due to fungal contamination after culturing for 30 days under the conditions that the temperature is 22 ℃, the illumination intensity is 2000-3000 lx and the illumination time is 10 h/d.

Comparative example 2

A method for tissue culture propagation of Chimonanthus praecox comprises the following steps:

(1) selection and sterilization of explants: cutting axillary buds with unfolded leaves in autumn in 4 months, soaking in washing powder water for 5min, washing with running water for more than 30min, sequentially soaking in 70% alcohol for 30s, 0.2% Fungicide for 15min, 0.15% mercuric chloride for 20min, washing with sterile water for 3 times, and filtering to remove excessive water;

(2) adventitious bud induction culture: inoculating the axillary buds treated in the step (1) into an adventitious bud induction culture medium, and carrying out induction culture for 60 days under the conditions that the temperature is 22 ℃, the illumination intensity is 2000-3000 lx and the illumination time is 10h/d until the explant is differentiated into adventitious buds;

the adventitious bud induction culture medium comprises: MS +6-BA 2mg/L + NAA 0.2mg/L, agar 6.5g/L, sucrose 30g/L, pH5.8;

(3) subculture multiplication culture of adventitious buds: transferring the adventitious bud induced in the step (2) into a proliferation culture medium, and carrying out subculture once every 30d under the conditions that the temperature is 22 ℃, the illumination intensity is 2000-3000 lx and the illumination time is 10 h/d;

the proliferation culture medium is as follows: MS +6-BA 1mg/L + NAA 0.2mg/L, agar 6.5g/L, sucrose 30g/L, pH5.8.

(4) Rooting culture: transferring the adventitious buds with the length of 1-2 cm cultured by the subculture multiplication in the step (3) to a rooting culture medium for inducing rooting, and culturing for 30d under the conditions that the temperature is 22 ℃, the illumination intensity is 2000-3000 lx and the illumination time is 10h/d until the plant base grows to 2-3 fibrous roots;

the rooting culture medium comprises: MS + IBA 0.5mg/L, agar 6.50g/L, sucrose 30g/L, pH5.8;

(5) hardening and transplanting seedlings: taking out the sterile seedlings with 2-3 fibrous roots induced to root and cultured in the step (4), cleaning root agar, putting the sterile seedlings into a carbendazim solution with the mass concentration of 0.1-0.2%, disinfecting for 1-2 min, and transplanting the sterile seedlings to humus soil: laterite: perlite = 1: 1: 1, domesticating and hardening the seedlings in a greenhouse for 50 days according to a conventional mode, and transplanting the seedlings, wherein the survival rate is 85%.

The chopsticks of the comparative example have slow adventitious bud proliferation, the multiplication factor of value increase is 1.8, the rooting rate is as low as 65%, and the rooting number is small.

Comparative example 3

A method for tissue culture propagation of Chimonanthus praecox comprises the following steps:

(1) selection and sterilization of explants: cutting axillary buds with unfolded leaves in autumn in 4 months, soaking in washing powder water for 5min, washing with running water for more than 30min, sequentially soaking in 70% alcohol for 30s, 0.2% Fungicide for 15min, 0.15% mercuric chloride for 20min, washing with sterile water for 3 times, and filtering to remove excessive water;

(2) adventitious bud induction culture: inoculating the small axillary buds treated in the step (1) into an induction culture medium, and carrying out induction culture for 60 days under the conditions that the temperature is 22 ℃, the illumination intensity is 2000-3000 lx and the illumination time is 10h/d until the explant is differentiated into adventitious buds;

the adventitious bud induction culture medium comprises: MS +2iP 2mg/L +6-BA 2mg/L + NAA 0.3mg/L, agar 6.5g/L, sucrose 30g/L, pH5.8;

(3) subculture multiplication culture of adventitious buds: transferring the adventitious bud induced in the step (2) into a proliferation culture medium, and carrying out subculture once every 30d under the conditions that the temperature is 22 ℃, the illumination intensity is 2000-3000 lx and the illumination time is 10 h/d;

the proliferation culture medium is as follows: MS +2iP 2mg/L +6-BA 1mg/L + NAA 0.3mg/L, agar 6.5g/L, sucrose 30g/L, pH5.8;

(4) rooting culture: transferring the adventitious buds with the length of 1-2 cm cultured in the subculture multiplication in the step (3) to a rooting culture medium for inducing rooting, and culturing for 25d under the conditions that the temperature is 22 ℃, the illumination intensity is 2000-3000 lx and the illumination time is 10h/d until 3-5 fibrous roots grow out of the plant base;

the rooting culture medium comprises: MS + IBA 0.5mg/L, agar 6.50g/L, sucrose 30g/L, pH5.8;

(5) hardening and transplanting seedlings: taking out the sterile seedlings with 3-5 fibrous roots induced to root and cultured in the step (4), cleaning root agar, putting the sterile seedlings into a carbendazim solution with the mass concentration of 0.1-0.2%, disinfecting for 1-2 min, and transplanting the sterile seedlings to humus soil: laterite: perlite = 1: 1: 1, domesticating and hardening the seedlings in a greenhouse for 60 days according to a conventional mode, and transplanting the seedlings, wherein the survival rate is 90%.

The adventitious buds of the chopsticks in the comparative example have slower proliferation, the multiplication factor is 2.4, and the rooting rate is 80%.

Comparative analysis

Table 1 compares the differences between the conventional tissue culture method control example 2 and the method of using the present invention example 1 for the breeding of chopsticks, including: the contamination rate of the explant, the induction quantity of adventitious buds, the multiplication times, the rooting rate, the quantity of fibrous roots and the transplanting survival rate. The results show that the adventitious bud induction quantity, the multiplication times, the rooting rate and the fibrous root quantity are obviously higher than those of the conventional tissue culture method except that the explant pollution rate of the invention is lower than that of the conventional tissue culture method.

TABLE 1

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