CN111280060A

CN111280060A - Efficient stem section propagation method of glorious bead

Info

Publication number: CN111280060A
Application number: CN202010231058.2A
Authority: CN
Inventors: 彭绿春; 李世峰; 瞿素萍; 宋杰; 解玮佳; 蔡艳飞; 张露
Original assignee: Flower Research Institute of YAAS
Current assignee: Flower Research Institute of YAAS
Priority date: 2020-03-27
Filing date: 2020-03-27
Publication date: 2020-06-16

Abstract

The invention relates to a stem section efficient propagation method of gloriopsis labrata' pearl, belonging to the field of plant tissue culture. According to the efficient propagation method of the stem section of the Diraphanus orientalis Levl, the shoots growing on the inflorescence of the Diraphanus orientalis Levl are used as the explants, seedlings produced by the tissue culture method are guaranteed, the excellent properties of the mother plant can be well maintained, meanwhile, the high disinfection success rate and the lateral bud germination rate of the explants are obtained, and the stem section of the Diraphanus orientalis Levl is efficiently propagated by matching with a special culture medium, so that provenance is provided for industrial development, and the predicament that the seedlings depend on import is eliminated.

Description

Efficient stem section propagation method of glorious bead

Technical Field

The invention belongs to the field of plant tissue culture, and relates to an in vitro efficient propagation method of stem segments of gloriopsis labrata 'bright pearl'.

Background

The 'Callicarpa kwangsiensis' is a perennial rare woody ornamental plant of the genus Callicarpa of the class of the Campylobacter eye (Telopoea), has unique flower shape, purplish red flower color and long flowering period, can be used as a cut flower and can also be used for landscaping. At present, seedlings and cut flower products of 'bright pearl' mainly depend on import, the price is high, the main reason is that the propagation efficiency is low, and the seedlings are not produced independently yet.

Because the seed shell of the bright pearl is hard and has dormancy characteristic, and the characters of the seed reproduction offspring are easy to separate; the cuttage rooting rate is low, the needed time is long, and the conventional seeds and cuttage propagation method are difficult to meet the requirement of the industry on seed sources. Tissue culture is a way for efficiently producing seedlings by plants, and although researches related to tissue culture of bright pearl exist at present, the conventional stem section is difficult to disinfect and low in sprouting rate, so that a regeneration system is difficult to establish and low in efficiency, most seeds are used as explants for tissue culture, and the condition of character separation of later generations cannot be avoided. Aiming at an excellent single plant, the stable inheritance of the excellent character of the single plant is difficult to guarantee by seed propagation, so that an efficient propagation method taking a nutritive organ as an explant is needed to be invented so as to realize the commercial application of tissue culture seedlings.

Disclosure of Invention

The invention aims to solve the technical problem that seeds are used as explants in the production of tissue culture seedlings of Diuo flower bright pearl, so that the stability of excellent properties of female parents of the produced seedlings is difficult to ensure, and provides an in vitro stem section efficient propagation method of Diuo flower bright pearl and the in vitro stem section efficient propagation method.

In order to solve the technical problems, the invention adopts the following technical scheme:

a stem section efficient propagation method of gloriosa aurantiaca' specifically comprises the following steps:

s1, selecting and disinfecting explants, cutting twigs growing on the open inflorescence of the bright pearl in 3-5 months in spring every year, removing leaves, keeping petioles, cleaning dirt on the surface, cutting the twigs into stem sections with the length of about 3cm, wherein each stem section has 2-3 petioles, washing with running water, disinfecting on a superclean bench, cleaning with sterile water, and drying the surface moisture in the air;

s2, performing lateral bud induction culture, after the stem segments are disinfected and fully aired to dry the surface moisture, inoculating the stem segments to a lateral bud induction culture medium MS +6-BA1mg/L + NAA0.1mg/L;

s3, performing enrichment culture, cutting and inoculating the lateral buds to an enrichment culture medium after the lateral buds grow to be 2-4 cm long, and performing enrichment culture;

s4, rooting culture, wherein after the propagation culture is carried out for 40-50 days, the propagation seedlings with the plant height of about 3cm are cut in a branch manner and inoculated to a rooting culture medium.

Further, the step S1 of cleaning the surface dirt is specifically cleaning the surface dirt with the laundry powder water.

Further, the running water flushing in step S1 takes 20 min.

Further, in step S1, the solution is soaked in 0.1% mercuric chloride solution for 12min, and then washed with sterile water for 5 times.

Further, the proliferation medium in step S3 is MS +6-BA0.4mg/L + NAA0.05mg/L.

Further, the rooting medium in the step S4 is MS + IBA0.75mg/L + NAA1 mg/L.

Compared with the prior art, the invention has the beneficial effects that: according to the efficient propagation method of the stem section of the Diraphanus orientalis Levl, the shoots growing on the inflorescence of the Diraphanus orientalis Levl are used as the explants, seedlings produced by the tissue culture method are guaranteed, the excellent properties of the mother plant can be well maintained, meanwhile, the high disinfection success rate and the lateral bud germination rate of the explants are obtained, and the stem section of the Diraphanus orientalis Levl is efficiently propagated by matching with a special culture medium, so that provenance is provided for industrial development, and the predicament that the seedlings depend on import is eliminated.

Detailed Description

The technical scheme of the invention is explained in detail by combining the specific embodiments as follows:

example 1

A high-efficiency propagation method of stem segments of glorious putamen comprises the following steps:

s1 selection and disinfection of explants

Cutting twigs growing on the open inflorescence of the bright pearl in spring for 3-5 months every year, removing leaves, keeping petioles, cleaning dirt on the surface with washing powder water, cutting into stem sections with the length of about 3cm, wherein each stem section has 2-3 petioles, washing for 20min with running water, and disinfecting on a clean bench. The disinfectant is soaked in 0.1% mercuric chloride solution for 12min, and then washed with sterile water for 5 times. And (4) airing the surface moisture of the disinfected stem segments on a superclean bench, and then carrying out lateral bud induction culture.

S2 lateral bud induction culture

After the stem sections are disinfected and the surface moisture is fully dried in the air, the stem sections are inoculated to a lateral bud induction culture medium MS +6-BA (6 benzyl adenine) 1mg/L + NAA (naphthalene acetic acid) 0.1mg/L, the explant contamination rate is 21.5%, the lateral bud germination rate is 73%, and the table 1 shows.

S3 proliferation culture

Cutting off and inoculating the lateral buds to a proliferation culture medium when the lateral buds grow to be 2-4 cm long, and performing proliferation culture;

wherein the multiplication culture medium contains 0.4mg/L of MS +6-BA (6-benzyladenine) and 0.05mg/L of NAA (naphthylacetic acid), the multiplication rate reaches 663 percent, the average plant height is 4.32cm, and the color of the multiplication seedlings and the growth of branches and leaves are normal.

S4 rooting culture

After the propagation culture is carried out for 40-50 days, the propagation seedlings with the plant height of about 3cm are cut separately and inoculated to a rooting culture medium. Wherein, the rooting culture medium is MS + IBA (indolebutyric acid) 0.75mg/L + NAA (naphthylacetic acid) 1mg/L, and the rooting rate is 70%.

Example 2

Explant sterilization validation test

Different explant disinfection time is tested by adopting the explant disinfection method in S1 and the lateral bud induction culture method in S2, and the explant disinfection method in the embodiment 1 and the control groups 1-3 are set to be completely the same as the disinfectant concentration and the culture medium in the embodiment 1, except that the disinfection time is respectively set to be 10min, 14min and 16min, so that the explant disinfection method adopted by the invention has the best effect.

The effect of different explant sterilization methods is shown in table 1:

TABLE 1

Example 3

Minimal medium validation test

The minimal medium used in the present invention was verified as the optimal medium by using the enrichment culture method in S3, selecting different minimal media, the minimal medium used in example 1, which was MS +6-BA0.4, and controls 1-2

mg/L + NAA0.05mg/L, the culture medium of the control group 1 is 1/2MS +6-BA0.4mg/L + NAA0.05mg/L, and the culture medium of the control group 2 is WPM +6-BA0.4mg/L + NAA0.05mg/L.

The growth effect of 'bright pearl' in different minimal media is shown in table 2:

TABLE 2

The SPAD value is an index reflecting the chlorophyll content of the plant, the SPAD value is obviously and positively correlated with the chlorophyll content in unit leaf area, the relative content of the chlorophyll can reflect the physiological condition of the plant leaves, and the larger the SPAD value is, the better the physiological condition of the plant is.

Example 4

Proliferation Medium validation test

The enrichment medium used in the present invention was verified as the optimum medium by using the enrichment culture method in S3, by selecting culture media of different concentrations of phytohormones, by using the enrichment medium of example 1, wherein the culture medium used in example 1 was MS +6-BA, and the control groups 1 to 3

0.4mg/L + NAA0.05mg/L, the culture medium used in the control group 1 is MS +6-BA0.8mg/L + NAA0.05mg/L, the culture medium used in the control group 2 is MS +6-BA1.2mg/L + NAA0.05mg/L, and the culture medium used in the control group 3 is MS +6-BA1.6mg/L + NAA0.05mg/L. The proliferation rate and plant height of the bright pearls in different media are shown in table 3:

TABLE 3

Example 5

Root growth medium validation test

The rooting culture method in S4 is adopted, culture media with different concentrations of plant hormones are selected, the rooting culture medium in the embodiment 1 and the control groups 1-4 are used for verifying that the rooting culture medium adopted by the invention is the optimal culture medium, wherein the rooting culture medium in the embodiment 1 is MS + IBA

0.75mg/L + NAA1mg/L, the rooting medium of the control group 1 is MS + IBA0.4mg/L + NAA0.5mg/L, the rooting medium of the control group 2 is MS + IBA0.8mg/L, the rooting medium of the control group 3 is MS + IBA0.2mg/L + NAA0.2mg/L, and the rooting medium of the control group 3 is MS + NAA0.8mg/L. The rooting effect of 'bright pearl' in different rooting media is shown in table 4:

TABLE 4

And (4) experimental conclusion: the method for sterilizing young branch and bud of bright pearl comprises soaking in 0.1% mercuric chloride solution for 12min with a contamination rate of 21.5%, and culturing explant in MS +6-BA1mg ∙ L^-1+NAA0.1mg∙L^-1On the culture medium, the lateral bud germination rate is 73 percent at most, the minimal medium suitable for the growth of the proliferation bud is MS, and the optimal culture medium for proliferation is MS +6-BA0.4mg ∙ L^-1+

NAA0.05mg∙L^-1The proliferation rate is 663%; the suitable culture medium for rooting is MS + IBA0.75mg ∙ L^-1+NAA1mg∙L^-1The rooting rate is 70%.

The above description is only an embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that are not thought of through the inventive work should be included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope defined by the claims.

Claims

1. A stem section efficient propagation method of gloriosa aurantiaca' is characterized by comprising the following steps:

s1, selecting and disinfecting explants, cutting twigs growing on the open inflorescence of the bright pearl in 3-5 months in spring every year, removing leaves, keeping petioles, cleaning surface dirt, cutting into stem sections with the length of about 3cm, wherein each stem section has 2-3 petioles, washing with running water, disinfecting on a superclean workbench, washing with sterile water again, and then drying the surface moisture in the air;

s2, performing lateral bud induction culture, after the stem segments are disinfected and fully dried, inoculating the stem segments to a lateral bud induction culture medium MS +6-BA1mg/L + NAA0.1 mg/L;

2. The method of claim 1, wherein the step S1 of cleaning the surface soil is specifically cleaning the surface soil with laundry powder water.

3. The method of claim 1, wherein the running water flush in step S1 takes 20 min.

4. The method of claim 1, wherein the sterilizing in step S1 is performed by soaking in 0.1% mercuric chloride solution for 12min and washing with sterile water for 5 times.

5. The method according to claim 1, wherein the proliferation medium in step S3 is MS +6-ba0.4mg/L + NAA0.05 mg/L.

6. The method of claim 1, wherein the rooting medium in step S4 is MS + iba0.75mg/L + NAA1 mg/L.