阅读说明：本技术 观赏向日葵快繁与组织培养植株再生方法 (Method for rapid propagation and tissue culture plant regeneration of ornamental sunflower ) 是由 陈珊宇 丁林云 阮关海 张慧 厉宝仙 怀燕 田燕 孟思洁 于 2021-07-26 设计创作，主要内容包括：本发明公开了观赏向日葵快繁与组织培养植株再生方法,挑选成熟的比较饱满的种子；温室,播种前催芽,种子尖的一头朝下,插进土壤中,再覆一层薄土,用喷壶喷水,让土壤保持湿润的状态,温室光照下出芽。本发明以观赏向日葵腋芽为外植体,研究了不同种类和浓度的植物生长调节剂对向日葵组织培养过程的影响,建立了其离体快速繁殖的技术体系；在向日葵快繁体系建立的基础上,建立向日葵愈伤胚性体细胞的诱导,以建立稳定、高效组织培养再生体系；在向日葵组织再生体系建立的基础上,不断优化再生体系条件,以建立稳定、高效再生体系为目标,进行基因重组载体构建,并建立向日葵的遗传转化体系,为深入基因功能研究提供服务。(The invention discloses a rapid propagation and tissue culture plant regeneration method for ornamental sunflower, which comprises the steps of selecting mature and plump seeds; and in the greenhouse, pregermination is carried out before sowing, one end of a seed tip faces downwards, the seed tip is inserted into soil, a layer of thin soil is covered, a watering can is used for spraying water to keep the soil in a moist state, and germination is carried out under the illumination of the greenhouse. The invention takes the axillary buds of the ornamental sunflower as the explant, researches the influence of plant growth regulators with different types and concentrations on the tissue culture process of the sunflower, and establishes a technical system for the in vitro rapid propagation of the plant growth regulators; on the basis of the establishment of a sunflower rapid propagation system, establishing the induction of somatic cells of a sunflower callus embryo so as to establish a stable and efficient tissue culture regeneration system; on the basis of establishing a sunflower tissue regeneration system, the conditions of the regeneration system are continuously optimized, a stable and efficient regeneration system is established as a target, the construction of a gene recombination vector is carried out, a genetic transformation system of the sunflower is established, and the service is provided for the deep gene function research.)

1. The rapid propagation and tissue culture plant regeneration method for ornamental sunflowers is characterized by comprising the following steps:

A. planting sunflowers:

a. seed selection: selecting mature and plump seeds;

b. sowing: a greenhouse, wherein the germination is accelerated before sowing, one end of the seed tip faces downwards, the seed tip is inserted into soil, a layer of thin soil is covered, a watering can is used for spraying water to keep the soil in a wet state, and the seeds germinate under the illumination of the greenhouse;

c. transplanting: transplanting the sunflowers after three pairs of true leaves are grown;

d. sampling: carrying out rapid propagation and callus induction experiments;

B. taking axillary buds as explants to induce adventitious buds:

a. sample processing

(1) Selecting sunflower plants with top flowers to be opened or already opened in the field;

(2) dividing the whole plant into several segments after harvest, and reserving stem segments with axillary buds;

(3) cleaning with liquid detergent, and washing with clear water for 0.5 hr;

b. explant preparation

(1) Cutting all axillary buds, washing with sterile water for one time, soaking in 75% ethanol for 30-60s, sterilizing with 10% hydrogen peroxide or 3% sodium hypochlorite solution for 15-20min or HgCl2 for 10-15min, and washing with sterile water for 4-5 times; then absorbing redundant moisture on a superclean workbench;

(2) cutting the top of axillary bud with the size of 5mm as an explant, and inoculating the explant onto a proper culture medium;

C. influence of different hormone concentrations on adventitious bud induction of axillary buds of ornamental sunflowers of different genotypes:

taking axillary buds as explants, adding 6-BA with different concentrations into an MS culture medium added with 0.03mg/L NAA with the same amount, carrying out concentration treatment on 6 concentration gradients including 0.3, 0.6, 0.9, 1.2, 1.5 and 1.8mg/L, taking the axillary buds with different genotypes to be tested 4-6d as explant materials, and researching the induction effect of adventitious buds;

D. elongation induction of adventitious buds:

3-4 combinations are set, and the optimal conditions suitable for adventitious bud elongation induction, namely MS +0.1mg/L BA +0.08mg/L NAA, are searched.

E. Regeneration induction:

MS+0.5mg/L 6-BA+0.2mg/L IAA+1.0gMgCl2

MS+1.0mg/L 6-BA+0.1mg/L IAA+1.0g MgCl2

F. rooting induction:

1/2MS+0.5mg/L IBA

G. training and culturing the regeneration strain: obtaining a sunflower regenerated plant with a developed root system, uncovering and hardening off the seedling for 2-3 days, spraying water to keep the leaf surface moist, cleaning a root system culture medium, and transplanting after the water culture survival;

H. the experimental results are as follows: obtaining callus and obtaining regenerated plants.

2. The method for rapid propagation and tissue culture plant regeneration of ornamental sunflower according to claim 1, wherein: in the step B, different hormone ratios and concentrations are set, 3-4 combined culture media are used, 1/2MS solid culture medium contains 1.8g/L agar, and the pH value is adjusted to 5.8-6.0 before sterilization.

3. The method for rapid propagation and tissue culture plant regeneration of ornamental sunflower according to claim 1, wherein: and B, inoculating the sunflower explant into culture medium jars containing different hormones, differentiating the explant into primary adventitious buds after culturing for 25-30 days, continuously culturing for 10-15 days, and then subculturing for 1 time at the temperature of 25-28 ℃, the sunlight/black night illumination time of 12h/12h and the illumination intensity of 1600 lux.

4. The method for rapid propagation and tissue culture plant regeneration of ornamental sunflower according to claim 1, wherein: in the step C, under the condition of different proportions of cytokinin and auxin, the difficulty of inducing adventitious buds by different materials is inconsistent with the bud emergence time.

Technical Field

The invention relates to the field of ornamental sunflowers, in particular to a rapid propagation and tissue culture plant regeneration method for ornamental sunflowers.

Background

Ornamental sunflowers, also known as colored sunflowers, include the genus helianthus, sunflower species, and some allied species. The flower disc is like the sun, the flower is bright, the color is bright, the strength is good, the flower disc is simple and natural, and the ornamental value is high. In foreign countries, enjoying sunflowers is deeply popular with people and widely used in fields such as cut flowers, pot flowers, dyeing flowers, yard beautification, and flower environment construction. The ornamental sunflower market in China just starts, and has certain development potential. The ornamental sunflower is an annual flower of Helianthus of Compositae, with plant height of 60-300 cm, flowering period of 25-35 days, and yellow, orange, red, pink and compound colors. Generally, the flower is planted in a piece-by-piece manner, the flower color is bright during blooming, and the flower is interesting and spectacular.

The method for obtaining high-quality sunflower varieties by utilizing biotechnology means is always the target of sunflower cultivation and breeding research, one of the important means of the biotechnology is isolated tissue culture, the number of the ornamental sunflower tissue college regeneration systems is small at present, and an efficient regeneration system is not established for the tissues. Therefore, a rapid propagation and tissue culture plant regeneration method for ornamental sunflowers is provided.

Disclosure of Invention

Based on the technical problems in the background technology, the invention provides a rapid propagation and tissue culture plant regeneration method for ornamental sunflowers, which aims to solve the problems in the background technology.

The invention provides the following technical scheme:

the rapid propagation and tissue culture plant regeneration method for ornamental sunflowers comprises the following steps:

A. planting sunflowers:

a. seed selection: selecting mature and plump seeds;

b. sowing: a greenhouse, wherein the germination is accelerated before sowing, one end of the seed tip faces downwards, the seed tip is inserted into soil, a layer of thin soil is covered, a watering can is used for spraying water to keep the soil in a wet state, and the seeds germinate under the illumination of the greenhouse;

c. transplanting: transplanting the sunflowers after three pairs of true leaves are grown;

d. sampling: carrying out rapid propagation and callus induction experiments;

B. taking axillary buds as explants to induce adventitious buds:

a. sample processing

(1) Selecting sunflower plants with top flowers to be opened or already opened in the field;

(2) dividing the whole plant into several segments after harvest, and reserving stem segments with axillary buds;

(3) cleaning with liquid detergent, and washing with clear water for 0.5 hr;

b. explant preparation

(1) Cutting all axillary buds, washing with sterile water for one time, soaking in 75% ethanol for 30-60s, sterilizing with 10% hydrogen peroxide or 3% sodium hypochlorite solution for 15-20min or HgCl2 for 10-15min, and washing with sterile water for 4-5 times; then absorbing redundant moisture on a superclean workbench;

(2) cutting the top of axillary bud with the size of 5mm as an explant, and inoculating the explant onto a proper culture medium;

C. influence of different hormone concentrations on adventitious bud induction of axillary buds of ornamental sunflowers of different genotypes:

taking axillary buds as explants, adding 6-BA with different concentrations into an MS culture medium added with 0.03mg/L NAA with the same amount, carrying out concentration treatment on 6 concentration gradients including 0.3, 0.6, 0.9, 1.2, 1.5 and 1.8mg/L, taking the axillary buds with different genotypes to be tested 4-6d as explant materials, and researching the induction effect of adventitious buds;

D. elongation induction of adventitious buds:

3-4 combinations are set, and the optimal conditions suitable for adventitious bud elongation induction, namely MS +0.1mg/L BA +0.08mg/LNAA, are searched.

E. Regeneration induction:

MS+0.5mg/L 6-BA+0.2mg/L IAA+1.0gMgCl2

MS+1.0mg/L 6-BA+0.1mg/L IAA+1.0g MgCl2

F. rooting induction:

1/2MS+0.5mg/L IBA

G. training and culturing the regeneration strain: obtaining a sunflower regenerated plant with a developed root system, uncovering and hardening off the seedling for 2-3 days, spraying water to keep the leaf surface moist, cleaning a root system culture medium, and transplanting after the water culture survival;

H. the experimental results are as follows: obtaining callus and obtaining regenerated plants.

Preferably, different hormone ratios and concentrations are set in the step B, 3-4 combined culture media are used, 1/2MS solid culture medium contains 1.8g/L agar, and the pH value is adjusted to 5.8-6.0 before sterilization.

Preferably, the sunflower explants are inoculated into culture medium jars containing different hormones in the step B, primary adventitious buds of the sunflower explants are differentiated after the sunflower explants are cultured for 25-30 days, subculture is carried out for 1 time after the sunflower explants are cultured for 10-15 days, the temperature is 25-28 ℃, the sunlight/night illumination time is 12h/12h, and the illumination intensity is 1600 lux.

Preferably, in the step C, the difficulty of inducing adventitious buds and the bud time of different materials are not consistent under different ratios of cytokinin and auxin.

The invention provides a method for rapid propagation and tissue culture plant regeneration of ornamental sunflower, which takes the ornamental sunflower axillary buds as explants, researches the influence of plant growth regulators of different types and concentrations on the tissue culture process of the sunflower, and establishes a technical system for in vitro rapid propagation; on the basis of the establishment of a sunflower rapid propagation system, establishing the induction of somatic cells of a sunflower callus embryo so as to establish a stable and efficient tissue culture regeneration system; on the basis of establishing a sunflower tissue regeneration system, the conditions of the regeneration system are continuously optimized, a stable and efficient regeneration system is established as a target, the construction of a gene recombination vector is carried out, a genetic transformation system of the sunflower is established, and the service is provided for the deep gene function research.

Drawings

FIG. 1 is a schematic diagram of the steps of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Referring to fig. 1, the present invention provides a technical solution:

the rapid propagation and tissue culture plant regeneration method for ornamental sunflowers comprises the following steps:

A. planting sunflowers:

a. seed selection: selecting mature and plump seeds;

b. sowing: a greenhouse, wherein the germination is accelerated before sowing, one end of the seed tip faces downwards, the seed tip is inserted into soil, a layer of thin soil is covered, a watering can is used for spraying water to keep the soil in a wet state, and the seeds germinate under the illumination of the greenhouse;

c. transplanting: transplanting the sunflowers after three pairs of true leaves are grown;

d. sampling: carrying out rapid propagation and callus induction experiments;

B. taking axillary buds as explants to induce adventitious buds:

a. sample processing

(1) Selecting sunflower plants with top flowers to be opened or already opened in the field;

(2) dividing the whole plant into several segments after harvest, and reserving stem segments with axillary buds;

(3) cleaning with liquid detergent, and washing with clear water for 0.5 hr;

b. explant preparation

(1) Cutting all axillary buds, washing with sterile water for one time, soaking in 75% ethanol for 30-60s, sterilizing with 10% hydrogen peroxide or 3% sodium hypochlorite solution for 15-20min or HgCl2 for 10-15min, and washing with sterile water for 4-5 times; then absorbing redundant moisture on a superclean workbench;

(2) cutting the top of axillary bud with the size of 5mm as an explant, and inoculating the explant onto a proper culture medium;

C. influence of different hormone concentrations on adventitious bud induction of axillary buds of ornamental sunflowers of different genotypes:

taking axillary buds as explants, adding 6-BA with different concentrations into an MS culture medium added with 0.03mg/L NAA with the same amount, carrying out concentration treatment on 6 concentration gradients including 0.3, 0.6, 0.9, 1.2, 1.5 and 1.8mg/L, taking the axillary buds with different genotypes to be tested 4-6d as explant materials, and researching the induction effect of adventitious buds;

D. elongation induction of adventitious buds:

setting 3-4 combinations, and searching for the optimal conditions suitable for adventitious bud elongation induction, namely MS +0.1mg/L BA +0.08 mg/LNAA;

adventitious bud proliferation, reducing 6-BA concentration is beneficial to bud proliferation, and adventitious bud cutting and subculture are carried out according to actual state.

MS+0.05mg/L BA+0.03mg/LNAA

MS+0.1mg/L BA+0.08mg/LNAA

MS+0.2mg/L BA+0.06mg/LNAA

Root induction

On the basis of the above experiments, rooting induction culture is set

1/2MS+0.5mg/L IAA

1/2MS+0.5mg/L IBA

E. Regeneration induction:

MS+0.5mg/L 6-BA+0.2mg/L IAA+1.0gMgCl2

MS+1.0mg/L 6-BA+0.1mg/L IAA+1.0g MgCl2

F. rooting induction:

1/2MS+0.5mg/L IBA

G. training and culturing the regeneration strain: obtaining a sunflower regenerated plant with a developed root system, uncovering and hardening off the seedling for 2-3 days, spraying water to keep the leaf surface moist, cleaning a root system culture medium, and transplanting after the water culture survival;

H. the experimental results are as follows: obtaining callus and obtaining regenerated plants.

Preferably, different hormone ratios and concentrations are set in the step B, 3-4 combined culture media are used, 1/2MS solid culture medium contains 1.8g/L agar, and the pH value is adjusted to 5.8-6.0 before sterilization.

Preferably, the sunflower explants are inoculated into culture medium jars containing different hormones in the step B, primary adventitious buds of the sunflower explants are differentiated after the sunflower explants are cultured for 25-30 days, subculture is carried out for 1 time after the sunflower explants are cultured for 10-15 days, the temperature is 25-28 ℃, the sunlight/night illumination time is 12h/12h, and the illumination intensity is 1600 lux.

Preferably, in the step C, the difficulty of inducing adventitious buds and the bud time of different materials are not consistent under different ratios of cytokinin and auxin.

MS+0.8mg/L6-BA+0.05mg/LNAA

MS+1.0mg/L6-BA+0.03mg/LNAA

MS+1.2mg/L6-BA+0.02mg/LNAA

Adventitious bud suitable induction culture medium MS +1.0-1.2mg/L6-BA +0.03 mg/LNAA. Through the screening of experimental conditions, under the condition that the concentration of cytokinin is about 1.0, the adventitious bud induction ratio is 50-60%.

Ornamental sunflower tissue culture and plant regeneration-screening target genotype

The explants were cut in a medium containing 1/2MS +0.001g/L CuSO4+20g/L sucrose +0.1g/L activated carbon. Pre-culturing at 26 + -1 deg.C for 7-10D; the relative humidity is 70-80%; the illumination time is 14 h/d; the illumination intensity was 2000 lx.

1. The cotyledon, cotyledonary node, hypocotyl, true leaf, etc. are subjected to callus induction

MS+0.5mg/L 6-BA+0.1mg/L NAA+0.5mg/L KT+0.2mg/L GA3

MS+0.5mg/L6-BA+0.5mg/LNAA+0.5mg/LIAA

MS+1.2mg/L 6-BA+0.03mg/L IAA

2. Callus proliferation induction-somatic cell induction-regeneration induction

MSB+1.5mg/L6-BA+0.1mg/LNAA

MSB*+0.1mg/LIAA+0.5mg/L KT

MSB2+0.5mg/L KT +0.1 mg/L2, 4-D +1.0g/L Glutamine +0.5g/L asparagine +1.9g/L KNO3+3.0g/L Phytagel

Condition exploration of different tissue cotyledon, cotyledonary node, hypocotyl and true leaf induced callus

Different genotypes of ornamental sunflower explants are inoculated on culture media with different concentrations, and all tissues are inoculated on three culture media to form callus or the callus is small, but the callus is obviously increased with the passage of time.

The sunflower material is researched to successfully generate adventitious buds under certain hormone induction, and different materials have certain difference in condition; callus induction conditions: the primary callus is obvious, and the difference between materials is not stable; the secondary metabolite exists, the condition needs to be searched for and removed, and the callus activity is improved under the regulation of certain hormone.

Is treated again

1. Planting potted sunflower seedlings on seeds, sampling, sterilizing, healing and inducing adventitious buds;

2. determining hormone combination and proportion induced by adventitious buds, and counting differences among different genotype materials;

3. solving secondary metabolites, and carrying out pretreatment culture;

4. preparing an explant: cutting an explant with a proper size, and trying to strip a stem tip for culture; leaf young leaves and immature flower buds are transversely cut to perform induced callus regeneration;

5. after explant callus induction and multiplication culture, somatic cell induction.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.