阅读说明：本技术 一种促进浙贝母鳞茎发生同步化的培养方法 (Culture method for promoting synchronization of fritillaria thunbergii bulbs ) 是由 丁楚蔚 陈志� 王忠华 于 2021-08-16 设计创作，主要内容包括：本公开提供了一种促进浙贝母鳞茎发生同步化的培养方法,旨在解决现有的培养方法同步化率低且生长速度慢的问题。促进浙贝母鳞茎发生同步化的培养方法,包括以下步骤：(1)不定芽诱导：将浙贝母组培苗叶柄切成0.5-1cm的小段,接种于培养基中,诱导不定芽；(2)成苗培养：不定芽诱导40d后,芽高1-2cm,将丛生芽从基部纵向切割成带芽小块,转移培养基,促进芽生长成苗；(3)生根培养：成苗培养后,将高为5cm的成苗切割成单株转移培养基进行生根培养；(4)移栽：组培苗基部长出3～8条长1～2cm的不定根后,将生根组培苗从培养瓶中取出,栽种到基质中。本发明的培养方法,生产浙贝母组培苗所用的时间为115天,在促进鳞茎同步化同时可大大缩短培养时间。(The invention provides a culture method for promoting synchronization of fritillaria thunbergii bulbs, and aims to solve the problems of low synchronization rate and low growth speed of the existing culture method. The culture method for promoting the synchronization of the bulbs of the thunberg fritillary bulb comprises the following steps: (1) adventitious bud induction: cutting the petiole of tissue culture seedling of Bulbus Fritillariae Thunbergii into 0.5-1cm small segments, inoculating in culture medium, and inducing adventitious bud; (2) seedling culture: after the adventitious bud is induced for 40 days, the height of the bud is 1-2cm, the cluster buds are longitudinally cut into small blocks with buds from the base part, and the culture medium is transferred to promote the buds to grow into seedlings; (3) rooting culture: after seedling culture, cutting the seedling with the height of 5cm into a single plant transfer culture medium for rooting culture; (4) transplanting: after 3-8 adventitious roots with the length of 1-2cm grow on the base of the tissue culture seedling, taking the rooted tissue culture seedling out of the culture bottle, and planting the rooted tissue culture seedling into a matrix. The culture method of the invention takes 115 days to produce the tissue culture seedling of the thunberg fritillary bulb, and can greatly shorten the culture time while promoting the synchronization of bulbs.)

1. A culture method for promoting synchronization of fritillaria thunbergii bulbs is characterized by comprising the following steps:

(1) adventitious bud induction: cutting the petiole of the tissue culture seedling of thunberg fritillary bulb into small sections with the length of 0.5-1cmd, inoculating the small sections into a culture medium of MS + 0.5-1.0 mg/L6-BA + 0.1-0.3 mg/L NAA +50g/L sucrose +7g/L agar, and inducing adventitious buds;

(2) seedling culture: after the adventitious bud is induced for 40 days, the bud is 1-2cm high, the cluster bud is longitudinally cut into small blocks with buds from the base part, and the small blocks are transferred to a culture medium of MS + 0.2-0.5 mg/L6-BA + 0.1-0.3 mg/L NAA +30g/L sucrose +7g/L agar to promote the bud to grow into seedlings;

(3) rooting culture: after seedling culture, cutting 5 cm-high seedlings into single plants, and transferring the single plants to a culture medium of MS + 0.1-0.5 mg/L NAA + 30-60 g/L sucrose +7g/L agar for rooting culture;

(4) transplanting: after 3-8 adventitious roots with the length of 1-2cm grow on the base of the tissue culture seedling, taking the rooted tissue culture seedling out of the culture bottle, washing off the culture medium on the surface, cutting off the residual leaves, and planting the rooted tissue culture seedling into a matrix.

2. The culture method for promoting synchronization of Bulbus Fritillariae Thunbergii bulb according to claim 1, wherein the pH of the culture medium is 5.8, and the culture medium is sterilized at 121 deg.C for 20 min.

3. The culture method for promoting synchronization of fritillaria thunbergii bulbs according to claim 1, wherein the culture method comprises the following steps: in the step (1), the culture temperature is 23 +/-1 ℃, the illumination intensity is 3000-4000 Lx, and the illumination time is 12 h/d.

4. The culture method for promoting synchronization of fritillaria thunbergii bulbs according to claim 1, wherein the culture method comprises the following steps: in the step (2), the culture temperature is 23 +/-1 ℃, the illumination intensity is 3000-4000 Lx, and the illumination time is 12 h/d.

5. The culture method for promoting synchronization of fritillaria thunbergii bulbs according to claim 1, wherein the culture method comprises the following steps: in the step (3), the culture temperature is 23 +/-1 ℃, the illumination intensity is 3000-4000 Lx, and the illumination time is 12 b/d.

6. The culture method for promoting synchronization of fritillaria thunbergii bulbs according to claim 1, wherein the culture method comprises the following steps: the culture temperature in the step (4) is 20-25 ℃, the relative humidity is more than 85%, and the illumination intensity is 2000-3000 Lx.

Technical Field

The invention belongs to the technical field of tissue culture of thunberg fritillary bulb, and particularly relates to a culture method for promoting synchronization of bulbs of thunberg fritillary bulb.

Background

Fritillaria thunbergii Miq is a perennial herb of Fritillaria of Liliaceae, and the bulb has effects of clearing heat, eliminating phlegm, relieving cough, removing toxic substance, resolving hard mass, and resolving carbuncle. The fritillary bulb is mainly propagated by bulbs, the propagation coefficient is lower than that of other medicinal materials, the growth period is long, the seed nature is degraded, and the production efficiency is greatly limited.

The plant tissue culture is not limited by season time, and a regeneration system can be established by adopting various organs or tissues, so that the aim of rapid propagation is fulfilled. The establishment of efficient tissue culture system by means of plant tissue culture technology is one main way of producing high quality seedling and obtaining great amount of seedling for production and application in short period. The thunberg fritillary bulb has high commodity value, can quickly obtain regenerated thunberg fritillary bulb seedlings through tissue culture, can overcome the defects of seed propagation and bulb propagation technology, and has high practical value. At present, aiming at a tissue culture and breeding system of thunberg fritillary bulb, more literature reports are that callus is induced by bulbs as explants and then is further differentiated, after multiple subculture proliferation, the bulb finally takes root and grows seedlings, and various nutrients and active substances are added into a culture medium at each stage. Further perfecting the optimized regeneration method, obtaining a regeneration system with high propagation coefficient, short period and low cost is a research direction in tissue culture industrial production of the thunberg fritillary bulb, and lays a solid foundation for further developing cultivation of detoxified seedlings and new variety breeding of the thunberg fritillary bulb.

Disclosure of Invention

The invention provides a culture method for promoting synchronization of fritillaria thunbergii bulbs, and aims to solve the problems of low synchronization rate and low growth speed of the existing culture method.

In order to solve the technical problem, the technical scheme adopted by the disclosure is as follows: a culture method for promoting synchronization of fritillaria thunbergii bulbs comprises the following steps:

(1) adventitious bud induction: cutting the petiole of the tissue culture seedling of the thunberg fritillary bulb into small sections of 0.5-1cm, inoculating the small sections into a culture medium of MS, 0.5-1.0 mg/L6-BA, 0.1-0.3 mg/L NAA, 50g/L sucrose and 7g/L agar, and inducing adventitious buds;

(2) seedling culture: after the adventitious bud is induced for 40 days, the bud is 1-2cm high, the cluster bud is longitudinally cut into small blocks with buds from the base part, and the small blocks are transferred to a culture medium of MS + 0.2-0.5 mg/L6-BA + 0.1-0.3 mg/L NAA +30g/L sucrose +7g/L agar to promote the bud to grow into seedlings;

(3) rooting culture: after seedling culture, cutting a seedling with the height of about 5cm into a single plant, and transferring the single plant to a culture medium of MS + 0.1-0.5 mg/L NAA + 30-60 g/L sucrose +7g/L agar for rooting culture;

(4) transplanting: after 3-8 adventitious roots with the length of 1-2cm grow on the base of the tissue culture seedling, taking the rooted tissue culture seedling out of the culture bottle, washing off the culture medium on the surface, cutting off the residual leaves, and planting the rooted tissue culture seedling into a matrix.

The further improved scheme is as follows: the medium had a pH of 5.8 and was sterilized at 121 ℃ for 20 min.

The further improved scheme is as follows: in the step (1), the culture temperature is 23 +/-1 ℃, the illumination intensity is 3000-4000 Lx, and the illumination time is 12 h/d.

The further improved scheme is as follows: in the step (2), the culture temperature is 23 +/-1 ℃, the illumination intensity is 3000-4000 Lx, and the illumination time is 12 h/d.

The further improved scheme is as follows: in the step (3), the culture temperature is 23 +/-1 ℃, the illumination intensity is 3000-4000 Lx, and the illumination time is 12 h/d.

The further improved scheme is as follows: the culture temperature in the step (4) is 20-25 ℃, the relative humidity is more than 85%, and the illumination intensity is 2000-3000 Lx.

The beneficial effect of this disclosure does:

the tissue culture method taking the petioles of the thunberg fritillary bulb as the inducing material establishes a tissue culture and rapid propagation system for promoting the bulb of the thunberg fritillary bulb to generate synchronization, the inductivity of the adventitious buds induced by 40 days of petiole culture is 89.18%, the inductivity of the adventitious buds is 5.21, the seedling culture is 50 days, the height of the seedling is 5.02cm, the rooting culture is 25 days, the rooting rate is 95%, the number of the rooting is 4.93, and the root length is 1.58 cm. The tissue culture method using the fritillaria thunbergii petioles as explants provided by the invention has the advantages that the time for producing the fritillaria thunbergii tissue culture seedlings is 115 days, and the bulb synchronization is promoted while the in-situ planting time can be greatly shortened, so that the tissue culture method can be widely applied to greenhouse intensive planting, the production period of fritillaria thunbergii medicinal materials is shortened, and the production cost is reduced.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the drawings that are required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present disclosure and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings may be obtained from the drawings without inventive effort.

FIG. 1 is a drawing of adventitious bud induction culture.

FIG. 2 is a diagram of multiple shoots.

FIG. 3 is a drawing of rooting culture.

Detailed Description

The technical solution in the embodiments of the present disclosure will be clearly and completely described below with reference to the drawings in the embodiments of the present disclosure. It should be understood that the specific embodiments described herein are merely illustrative of the disclosure and are not intended to limit the disclosure. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the disclosure without inventive step, are within the scope of the disclosure.

The embodiment provides a culture method for promoting synchronization of fritillaria thunbergii bulbs, which comprises the following steps:

(1) adventitious bud induction: cutting the petiole of the tissue culture seedling of the thunberg fritillary bulb into small sections of 0.5-1cm, inoculating the small sections into a culture medium of MS, 0.5-1.0 mg/L6-BA, 0.1-0.3 mg/L NAA, 50g/L sucrose and 7g/L agar, and inducing adventitious buds;

(2) seedling culture: after the adventitious bud is induced for 40 days, the bud is 1-2cm high, the cluster bud is longitudinally cut into small blocks with buds from the base part, and the small blocks are transferred to a culture medium of MS + 0.2-0.5 mg/L6-BA + 0.1-0.3 mg/L NAA +30g/L sucrose +7g/L agar to promote the bud to grow into seedlings;

(3) rooting culture: after seedling culture, cutting a seedling with the height of about 5cm into a single plant, and transferring the single plant to a culture medium of MS + 0.1-0.5 mg/L NAA + 30-60 g/L sucrose +7g/L agar for rooting culture;

(4) transplanting: after 3-8 adventitious roots with the length of 1-2cm grow on the base of the tissue culture seedling, taking the rooted tissue culture seedling out of the culture bottle, washing off the culture medium on the surface, cutting off the residual leaves, and planting the rooted tissue culture seedling into a matrix.

Wherein the pH of the culture medium is 5.8, and the culture medium is sterilized at 121 ℃ for 20 min.

In the step (1), the culture temperature is 23 +/-1 ℃, the illumination intensity is 3000-4000 Lx, and the illumination time is 12 h/d.

In the step (2), the culture temperature is 23 +/-1 ℃, the illumination intensity is 3000-4000 Lx, and the illumination time is 12 h/d.

In the step (3), the culture temperature is 23 +/-1 ℃, the illumination intensity is 3000-4000 Lx, and the illumination time is 12 h/d.

Wherein the culture temperature in the step (4) is 20-25 ℃, the relative humidity is more than 85%, and the illumination intensity is 2000-3000 Lx.

The present disclosure will be further described with reference to a more specific embodiment, and the cultivation method for promoting synchronization of fritillary bulb comprises:

(1) cutting materials:

cutting the leaves, petioles and bulbs of the tissue culture seedling of Fritillaria thunbergii into 0.5-1.0cm on a superclean bench.

(2) Adventitious bud induction culture:

preparing culture medium with different hormone concentrations, wherein the formula of the culture medium is MS +6-BA (0.5, 1.0, 1.5 and 2.0mg/L) + NAA (0.1, 0.2 and 0.3mg/L) +7.0g/L agar +50g/L sucrose, and the pH value is 5.8. Respectively inoculating the cut thunberg fritillary bulb leaves, petioles and bulb small sections into 8 culture media to induce adventitious buds, wherein the culture conditions are that the temperature is 23 +/-1 ℃, the illumination intensity is 3000-4000 Lx, and the illumination time is 12 h/d. After several days of culture, the leaves die gradually; after 20 days, adventitious buds begin to be induced from the petioles and the bulbs, and the leaves tend to be stable after 40 days; the result shows that the synchronization of the petioles for inducing the adventitious buds is higher, the petioles grow more vigorously, the petioles of the tissue culture seedlings are cut into 0.5-1cm sections and are connected to a flat plate for culture, the formula of a suitable culture medium is MS + 0.5-1.0 mg/L6-BA + 0.1-0.3 mg/LNAA +50g/L sucrose +7g/L agar, the adventitious bud induction rate measured after 40 days is 89.18%, and the adventitious bud induction coefficient is 5.21.

(1) Seedling culture:

the induced multiple shoots were cut longitudinally from the base into single shoots and transferred to a seedling medium using a multifactorial multilevel assay (6-BA: 0.1, 0.2, 0.5 mg/L; NAA: 0.05, 0.1, 0.2, 0.3; sucrose: 30, 40, 50g/L), agar 7g/L, pH 5.8. The culture conditions are that the temperature is 23 +/-1 ℃, the illumination intensity is 3000-4000 Lx, and the illumination time is 12 h/d. The formula of the screened proper culture medium is MS + 0.2-0.5 mg/L6-BA + 0.1-0.3 mg/L NAA +30g/L cane sugar +7g/L agar, and the measured seedling height after 50 days of culture is 5.02 cm.

(2) Rooting culture:

after seedling formation, the seedlings are transferred into 7 culture media which are based on MS, contain no hormone or NAA (0.05, 0.1, 0.2 and 0.5mg/L) or IBA (0.05, 0.1 and 0.2mg/L) with different concentrations, 30 to 60g/L of cane sugar and 7g/L of agar and have the pH value of 5.8, the culture conditions are 23 +/-1 ℃, the illumination intensity is 3000 to 4000Lx, and the illumination time is 12 h/d. After 25 days of culture, a proper rooting culture medium is obtained by screening, the formula is MS + 0.1-0.5 mg/LNAA + 30-60 g/L sucrose +7g/L agar, the rooting rate is 95%, the number of roots is 4.93, and the root length is 1.58 cm.

(3) Transplanting:

after 3-8 adventitious roots with the length of 1-2cm grow on the basal part of the tissue culture seedling, taking the rooted seedling out of a culture bottle, washing off the culture medium on the surface, cutting off the residual leaves, planting the rooted seedling in a mixed matrix, and culturing at the temperature of 20-25 ℃, the relative humidity of more than 85 percent and the illumination intensity of 2000-3000 Lx.

The present disclosure is not limited to the above alternative embodiments, and any other various forms of products may be obtained by anyone in the light of the present disclosure, but any changes in shape or structure thereof fall within the scope of the present disclosure, which is defined by the claims of the present disclosure.