CN107960326B - Tissue culture method of mesona chinensis benth - Google Patents

Abstract

the invention belongs to the technical field of biology, and particularly relates to a tissue culture method of a crystal grass, which comprises the following steps: s1, detoxification; s2, dedifferentiation; s3, propagation culture; s4, redifferentiation; s5, hardening seedlings. The invention solves the problem of browning phenomenon in the tissue culture process and improves the rooting rate and the rooting number of tissue culture seedlings.

Description

Tissue culture method of mesona chinensis benth

Technical Field

the invention belongs to the technical field of biology, and particularly relates to a tissue culture method of a mesona chinensis benth.

background

the herba Gelsemii Elegantis is a plant of Plumbum in Plumbago of Plumbago, has small flower, still remains flowers after death, and is similar to forget-me-not. The flowers and leaves of the grass are syngeneic, the stem height is about 10-50 cm, and the grass likes sunlight. The plants grow on hillsides, cliffs, stone seams and roadside areas. With the improvement of living standard of people, people like to send fresh flowers or dry flowers to friends and relatives or decorate own rooms with the fresh flowers or the dry flowers. The consumption of the crystal grass is greatly increased, so that the crystal grass is in short supply. The existing propagation of the mesona chinensis adopts seed propagation, and the propagation is slow.

disclosure of Invention

the invention aims to provide a tissue culture method of the crystal grass, which is used for improving the rooting rate and the rooting number of the crystal grass tissue culture seedlings.

in order to solve the technical problems, the technical scheme of the invention is as follows:

a tissue culture method of the crystal grass comprises the following steps:

S1, detoxification: placing the in-vitro tissue of the Mesona chinensis Benth in 0.05% -0.2% mercuric chloride solution for disinfection for 3-30 minutes, and then cleaning with sterile water for 0-5 times;

S2, dedifferentiation: placing the detoxified isolated tissue on a dedifferentiation culture medium, and culturing under the conditions that the temperature is 15-28 ℃, the illumination intensity is 3000-8000 Lx, and the illumination time is 8-16 hours/day to obtain a callus;

s3, proliferation culture: transferring the callus into a proliferation culture medium, and culturing under the conditions that the temperature is 15-28 ℃, the illumination intensity is 3000-8000 Lx, and the illumination time is 8-16 h/day;

S4, redifferentiation: transferring the proliferated callus to a redifferentiation culture medium, and culturing for 10-20 days under the conditions that the temperature is 15-28 ℃, the illumination intensity is 3000-8000 Lx, and the illumination time is 8-20 hours/day to obtain a rooting seedling;

S5, hardening seedlings: taking the rooted seedlings out of the culture bottle, washing the culture medium with clear water, and culturing in nutrient soil with the pH value of 5-8 and the EC value of 500-25000 mu S/cm, wherein the illumination intensity is 3000-20000 Lx, and the transplanting crystal grass seedlings are obtained after 15-25 days.

in the tissue culture method of the crystal grass provided by the invention, preferably, the in vitro tissue is a leaf, a petiole, a root, a stem, a flower or a flower spike.

In the tissue culture method of the mesona crystallina provided by the invention, preferably, the dedifferentiation culture medium comprises the following components in percentage by weight: MS +6-BA 0.2-1.0 mg/L + NAA 0.1-0.6 mg/L + forsythia suspense extract 0.8-1.6 mg/L;

Or N6+6-BA 0.2-1.0 mg/L + NAA 0.1-0.6 mg/L + forsythia suspensa extract 0.5-1.3 mg/L;

or B5+6-BA 0.2-1.0 mg/L + NAA 0.1-0.6 mg/L + forsythia suspensa extract 0.3-1.5 mg/L.

in the tissue culture method of the mesona crystallina provided by the invention, preferably, the components and contents of the proliferation culture medium are as follows: 0.1-1.0 mg/L of MS +6-BA, 0.05-0.8 mg/L of NAA and 1.0-2.0 mg/L of acanthopanax extract;

Or N6+6-BA 0.1-1.0 mg/L + NAA 0.05-0.8 mg/L + Acanthopanax senticosus extract 0.6-1.5 mg/L;

or B5+6-BA 0.1-1.0 mg/L + NAA 0.05-0.8 mg/L + Acanthopanax senticosus extract 1.0-2.0 mg/L.

In the tissue culture method of the mesona crystallina provided by the invention, preferably, the redifferentiation culture medium comprises the following components in percentage by weight: 1/2MS + NAA 0.2-2.0 mg/L + IBA 0-2.0 mg/L + IAA 0-10 mg/L + forsythia suspense extract 0.5-2.6 mg/L;

Or N6+ NAA 0.2-2.0 mg/L + IBA 0-2.0 mg/L + IAA 0-10 mg/L + forsythia suspensa extract 0.4-1.5 mg/L;

or B5+ NAA 0.2-2.0 mg/L + IBA 0-2.0 mg/L + IAA 0-10 mg/L + forsythia suspensa extract 0.8-1.8 mg/L.

In the tissue culture method of the Mesona chinensis Benth, the pH value is 6-7, the EC value is 1500-15000 mu S/cm, and the illumination intensity is 8000-15000 Lx in the step S5.

By adopting the technical scheme, the acanthopanax extract is added into the enrichment medium, so that the browning phenomenon is prevented; the forsythia suspense extract is added into the redifferentiation culture medium, so that the rooting rate and the rooting number of the tissue culture seedlings of the mesona chinensis benth are improved.

Drawings

FIG. 1 is a flow chart of the tissue culture method of the invention.

Detailed Description

The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

example 1

The embodiment provides a tissue culture method of a crystal grass, which comprises the following steps:

S1, detoxification: placing the leafstalks of the herba mesonae chinensis in 0.2% mercuric chloride solution for disinfection for 3 minutes, and then cleaning with sterile water for 5 times;

s2, dedifferentiation: placing the detoxified stem of the leaves of the gelsemium elegans on a dedifferentiation culture medium, and culturing under the conditions that the temperature is 28 ℃, the illumination intensity is 3000Lx and the illumination time is 8 hours/day to obtain callus; the components and contents of the dedifferentiation culture medium are as follows: MS +6-BA0.2 mg/L + NAA0.6mg/L + forsythia suspense extract 1.6 mg/L;

s3, proliferation culture: transferring the callus to a proliferation culture medium, and culturing at 28 deg.C under illumination intensity of 3000Lx for 16 hr/day; the proliferation culture medium comprises the following components in percentage by weight: MS +6-BA1.0mg/L + NAA0.05 mg/L + acanthopanax extract 1.0 mg/L;

s4, redifferentiation: transferring the proliferated callus to a redifferentiation culture medium, and culturing for 10 days at 15 deg.C under illumination intensity of 3000Lx for 20 hr/day to obtain rooted seedling; the redifferentiation culture medium comprises the following components in percentage by weight: 1/2MS + NAA 2.0mg/L + IBA 2.0mg/L + IAA 10mg/L + forsythia suspense extract 0.5 mg/L;

S5, hardening seedlings: taking out the rooted seedling from the culture bottle, washing the culture medium with clear water, and culturing in nutrient soil with pH of 8 and EC value of 25000 mu S/cm under illumination intensity of 20000Lx for 15 days to obtain the transplanting seedling of Mesona chinensis.

Example 2

the embodiment provides a tissue culture method of a crystal grass, which comprises the following steps:

S1, detoxification: placing the leaves of the herba mesonae chinensis in 0.05% mercuric chloride solution for disinfection for 30 minutes, and then cleaning with sterile water for 1 time;

S2, dedifferentiation: placing the detoxified herba Gei Piloselloidis leaf on dedifferentiation culture medium, culturing at 15 deg.C under illumination intensity of 8000Lx for 16 hr/day to obtain callus; the components and contents of the dedifferentiation culture medium are as follows: n6+6-BA1.0mg/L + NAA0.1mg/L + forsythia suspensa extract 1.3 mg/L;

s3, proliferation culture: transferring the callus to a proliferation culture medium, and culturing at 28 deg.C under illumination intensity of 3000Lx for 16 hr/day; the proliferation culture medium comprises the following components in percentage by weight: b5+6-BA0.1mg/L + NAA 0.8mg/L + Acanthopanax senticosus extract 1.0 mg/L;

s4, redifferentiation: transferring the proliferated callus to a redifferentiation culture medium, and culturing for 10 days at 15 deg.C under illumination intensity of 3000Lx for 20 hr/day to obtain rooted seedling; the redifferentiation culture medium comprises the following components in percentage by weight: b5+ NAA 2.0mg/L + IBA 1.0mg/L + IAA 2mg/L + forsythia suspense extract 1.8 mg/L;

s5, hardening seedlings: taking out the rooted seedling from the culture bottle, washing the culture medium with clear water, and culturing in nutrient soil with pH value of 5 and EC value of 500 μ S/cm under illumination intensity of 3000Lx for 25 days to obtain the transplanting seedling of herba Gerberae Piloselloidis.

example 3

the embodiment provides a tissue culture method of a crystal grass, which comprises the following steps:

s1, detoxification: sterilizing the roots of the Mesona chinensis Benth in 0.10% mercuric chloride solution for 20 min, and cleaning with sterile water for 3 times;

S2, dedifferentiation: placing the detoxified radix Cymbopogonis Citrari on dedifferentiation culture medium, and culturing at 20 deg.C under the condition of illumination intensity of 6000Lx and illumination time of 12 hr/day to obtain callus; the components and contents of the dedifferentiation culture medium are as follows: b5+6-BA 1.0mg/L + NAA0.1mg/L + forsythia suspense extract 1.5 mg/L;

S3, proliferation culture: transferring the callus to a proliferation culture medium, and culturing at 15 deg.C under illumination intensity of 8000Lx for 8 hr/day; the proliferation culture medium comprises the following components in percentage by weight: n6+6-BA1.0mg/L + NAA0.05 mg/L + acanthopanax extract 0.6 mg/L;

S4, redifferentiation: transferring the proliferated callus to a redifferentiation culture medium, and culturing for 10 days at 15 deg.C under illumination intensity of 3000Lx for 20 hr/day to obtain rooted seedling; the redifferentiation culture medium comprises the following components in percentage by weight: n6+ NAA 2.0mg/L + IBA 0.2mg/L + IAA 1mg/L + forsythia suspense extract 1.5 mg/L;

s5, hardening seedlings: taking out the rooted seedling from the culture bottle, washing the culture medium with clear water, and culturing in nutrient soil with pH of 7 and EC value of 15000 μ S/cm under illumination intensity of 15000Lx for 20 days to obtain transplanting seedling of herba Gerberae Piloselloidis.

Example 4

the embodiment provides a tissue culture method of a crystal grass, which comprises the following steps:

S1, detoxification: placing flowers of the Mesona chinensis Benth in 0.15% mercuric chloride solution for sterilizing for 15 minutes, and cleaning with sterile water for 2 times;

s2, dedifferentiation: placing detoxified herba Gerberae Piloselloidis flower on dedifferentiation culture medium, culturing at 16 deg.C under illumination intensity of 5000Lx for 10 hr/day to obtain callus; the components and contents of the dedifferentiation culture medium are as follows: b5+6-BA0.5mg/L + NAA 0.4mg/L + forsythia suspense extract 1.0 mg/L;

s3, proliferation culture: transferring the callus to a proliferation culture medium, and culturing at 20 deg.C under illumination intensity of 5000Lx for 13 hr/day; the proliferation culture medium comprises the following components in percentage by weight: b5+6-BA0.5mg/L + NAA 0.3mg/L + acanthopanax extract 1.2 mg/L;

S4, redifferentiation: transferring the proliferated callus to a redifferentiation culture medium, and culturing for 15 days under the conditions that the temperature is 25 ℃, the illumination intensity is 4000Lx and the illumination time is 10 hours/day to obtain a rooting seedling; the redifferentiation culture medium comprises the following components in percentage by weight: n6+ NAA 1.0mg/L + IBA 0.5mg/L + IAA 5mg/L + forsythia suspense extract 0.4 mg/L;

s5, hardening seedlings: taking out the rooted seedling from the culture bottle, washing the culture medium with clear water, and culturing in nutrient soil with pH value of 7 and EC value of 10000 μ S/cm under illumination intensity of 10000Lx for 18 days to obtain the transplanting seedling of herba Gerberae Piloselloidis.

Example 5

the embodiment provides a tissue culture method of a crystal grass, which comprises the following steps:

S1, detoxification: placing the leafstalks of the herba mesonae chinensis in 0.2% mercuric chloride solution for disinfection for 3 minutes, and then cleaning with sterile water for 5 times;

S2, dedifferentiation: placing the detoxified stem of the leaves of the gelsemium elegans on a dedifferentiation culture medium, and culturing under the conditions that the temperature is 28 ℃, the illumination intensity is 3000Lx and the illumination time is 8 hours/day to obtain callus; the components and contents of the dedifferentiation culture medium are as follows: MS +6-BA0.2 mg/L + NAA0.6mg/L;

S3, proliferation culture: transferring the callus to a proliferation culture medium, and culturing at 28 deg.C under illumination intensity of 3000Lx for 16 hr/day; the proliferation culture medium comprises the following components in percentage by weight: MS +6-BA1.0mg/L + NAA0.05 mg/L + acanthopanax extract 1.0 mg/L;

S4, redifferentiation: transferring the proliferated callus to a redifferentiation culture medium, and culturing for 30 days at 15 deg.C under illumination intensity of 3000Lx for 20 hr/day to obtain rooted seedling; the redifferentiation culture medium comprises the following components in percentage by weight: 1/2MS + NAA 2.0mg/L + IBA 2.0mg/L + IAA 10mg/L + forsythia suspense extract 0.5 mg/L;

s5, hardening seedlings: taking out the rooted seedling from the culture bottle, washing the culture medium with clear water, and culturing in nutrient soil with pH of 8 and EC value of 25000 mu S/cm under illumination intensity of 20000Lx for 15 days to obtain the transplanting seedling of Mesona chinensis.

example 6

the embodiment provides a tissue culture method of a crystal grass, which comprises the following steps:

S1, detoxification: placing the leafstalks of the herba mesonae chinensis in 0.2% mercuric chloride solution for disinfection for 3 minutes, and then cleaning with sterile water for 5 times;

S2, dedifferentiation: placing the detoxified stem of the leaves of the gelsemium elegans on a dedifferentiation culture medium, and culturing under the conditions that the temperature is 28 ℃, the illumination intensity is 3000Lx and the illumination time is 8 hours/day to obtain callus; the components and contents of the dedifferentiation culture medium are as follows: MS +6-BA0.2 mg/L + NAA0.6mg/L + forsythia suspense extract 1.6 mg/L;

s3, proliferation culture: transferring the callus to a proliferation culture medium, and culturing at 28 deg.C under illumination intensity of 3000Lx for 16 hr/day; the proliferation culture medium comprises the following components in percentage by weight: MS +6-BA1.0mg/L + NAA0.05 mg/L;

s4, redifferentiation: transferring the proliferated callus to a redifferentiation culture medium, and culturing for 32 days under the conditions that the temperature is 15 ℃, the illumination intensity is 3000Lx and the illumination time is 20 hours/day to obtain a rooting seedling; the redifferentiation culture medium comprises the following components in percentage by weight: 1/2MS + NAA 2.0mg/L + IBA 2.0mg/L + IAA 10mg/L + forsythia suspense extract 0.5 mg/L;

S5, hardening seedlings: taking out the rooted seedling from the culture bottle, washing the culture medium with clear water, and culturing in nutrient soil with pH of 8 and EC value of 25000 mu S/cm under illumination intensity of 20000Lx for 15 days to obtain the transplanting seedling of Mesona chinensis.

Example 7

The embodiment provides a tissue culture method of a crystal grass, which comprises the following steps:

S1, detoxification: placing the leafstalks of the herba mesonae chinensis in 0.2% mercuric chloride solution for disinfection for 3 minutes, and then cleaning with sterile water for 5 times;

S2, dedifferentiation: placing the detoxified stem of the leaves of the gelsemium elegans on a dedifferentiation culture medium, and culturing under the conditions that the temperature is 28 ℃, the illumination intensity is 3000Lx and the illumination time is 8 hours/day to obtain callus; the components and contents of the dedifferentiation culture medium are as follows: MS +6-BA0.2 mg/L + NAA0.6mg/L + forsythia suspense extract 1.6 mg/L;

S3, proliferation culture: transferring the callus to a proliferation culture medium, and culturing at 28 deg.C under illumination intensity of 3000Lx for 16 hr/day; the proliferation culture medium comprises the following components in percentage by weight: MS +6-BA1.0mg/L + NAA0.05 mg/L + acanthopanax extract 1.0 mg/L;

S4, redifferentiation: transferring the proliferated callus to a redifferentiation culture medium, and culturing for 40 days at 15 deg.C under illumination intensity of 3000Lx for 20 hr/day to obtain rooted seedling; the redifferentiation culture medium comprises the following components in percentage by weight: 1/2MS + NAA 2.0mg/L + IBA 2.0mg/L + IAA 10 mg/L;

s5, hardening seedlings: taking out the rooted seedling from the culture bottle, washing the culture medium with clear water, and culturing in nutrient soil with pH of 8 and EC value of 25000 mu S/cm under illumination intensity of 20000Lx for 15 days to obtain the transplanting seedling of Mesona chinensis.

In order to verify the beneficial effects of the invention, the tissue culture seedling of the crystal grass is carried out according to the method of each embodiment, and each index is examined, and the results are shown in table 1.

TABLE 1 results of each item of tissue-cultured seedlings according to the method of each example

group of

Height/cm of seedling

Blade/sheet

Rooting percentage/%)

Root number/strip

Root system status

Others

Example 1

>6

>9

96

6

Complete, well-developed and fresh

No disease

example 2

>6

>9

95

8

complete, well-developed and fresh

no disease

example 3

>7

>9

98

7

complete, well-developed and fresh

No disease

example 4

>6

>8

96

7

Complete, well-developed and fresh

no disease

Example 5

>4

<5

86

5

intact and weak

No disease

Example 6

<4

<6

85

5

intact and weak

browning

Example 7

<4

<6

73

3

Intact and weak

No disease

as can be seen from the above table, each index of the tissue culture seedlings obtained in examples 1 to 4 is superior to that of the tissue culture seedlings obtained in examples 5 to 6. In example 6, since acanthopanax extract was not added to the enrichment medium, browning occurred, and thus, it was found that adding acanthopanax extract to the enrichment medium could prevent browning. In example 7, since no forsythia suspense extract was added to the redifferentiation medium, the tissue culture seedling obtained in example 7 had a low rooting rate and a small number of roots, and thus, the addition of forsythia suspense extract to the redifferentiation medium had an effect of increasing the rooting rate and the number of roots of the tissue culture seedling.

the embodiments of the present invention have been described in detail, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.

Claims (3)

1. a tissue culture method of the crystal grass is characterized by comprising the following steps:

s1, detoxification: placing the in-vitro tissue of the Mesona chinensis Benth in 0.05% -0.2% mercuric chloride solution for disinfection for 3-30 minutes, and then cleaning with sterile water for 0-5 times;

s2, dedifferentiation: placing the detoxified isolated tissue on a dedifferentiation culture medium, and culturing under the conditions that the temperature is 15-28 ℃, the illumination intensity is 3000-8000 Lx, and the illumination time is 8-16 hours/day to obtain a callus;

s3, proliferation culture: transferring the callus into a proliferation culture medium, and culturing under the conditions that the temperature is 15-28 ℃, the illumination intensity is 3000-8000 Lx, and the illumination time is 8-16 h/day;

S4, redifferentiation: transferring the proliferated callus to a redifferentiation culture medium, and culturing for 10-20 days under the conditions that the temperature is 15-28 ℃, the illumination intensity is 3000-8000 Lx, and the illumination time is 8-20 hours/day to obtain a rooting seedling;

s5, hardening seedlings: taking out the rooted seedlings from the culture bottle, washing the culture medium with clear water, and cultivating the seedlings at a pH value of 5-8 and an EC value of 500 [ ] -E

In 25000 mu S/cm of nutrient soil, the illumination intensity is 3000-20000 Lx, and the transplanting crystal grass seedlings are obtained after 15-25 days;

The dedifferentiation culture medium comprises the following components in percentage by weight: MS +6-BA 0.2-1.0 mg/L + NAA 0.1-0.6 mg/L + forsythia suspense extract 0.8-1.6 mg/L;

or N6+6-BA 0.2-1.0 mg/L + NAA 0.1-0.6 mg/L + forsythia suspensa extract 0.5-1.3 mg/L;

or B5+6-BA 0.2-1.0 mg/L + NAA 0.1-0.6 mg/L + forsythia suspensa extract 0.3-1.5 mg/L;

The proliferation culture medium comprises the following components in percentage by weight: 0.1-1.0 mg/L of MS +6-BA, 0.05-0.8 mg/L of NAA and 1.0-2.0 mg/L of acanthopanax extract;

or N6+6-BA 0.1-1.0 mg/L + NAA 0.05-0.8 mg/L + Acanthopanax senticosus extract 0.6-1.5 mg/L;

Or B5+6-BA 0.1-1.0 mg/L + NAA 0.05-0.8 mg/L + Acanthopanax senticosus extract 1.0-2.0 mg/L;

The redifferentiation culture medium comprises the following components in percentage by weight: 1/2MS + NAA 0.2-2.0 mg/L + IBA 0-2.0 mg/L + IAA 0-10 mg/L + forsythia suspensa extract 0.5E

2.6mg/L；

Or N6+ NAA 0.2-2.0 mg/L + IBA 0-2.0 mg/L + IAA 0-10 mg/L + forsythia suspensa extract 0.4-1.5 mg/L;

Or B5+ NAA 0.2-2.0 mg/L + IBA 0-2.0 mg/L + IAA 0-10 mg/L + forsythia suspensa extract 0.8-1.8 mg/L.

2. the tissue culture method of the agropyron cristatum as claimed in claim 1, wherein the isolated tissue is a stem.

3. The tissue culture method of Mesona chinensis Benth according to claim 1, wherein in step S5, pH is 6-7, EC is 1500-15000 μ S/cm, and illumination intensity is 8000-15000 Lx.