CN115039696A

CN115039696A - Method for inducing calluses and regenerating seedlings of myrtle

Info

Publication number: CN115039696A
Application number: CN202210501091.1A
Authority: CN
Inventors: 邓书林; 杨玲; 吕善武; 张艺; 杨恒
Original assignee: South China Botanical Garden of CAS
Current assignee: South China Botanical Garden of CAS
Priority date: 2022-05-09
Filing date: 2022-05-09
Publication date: 2022-09-13
Anticipated expiration: 2042-05-09
Also published as: CN115039696B

Abstract

The invention discloses a method for inducing calluses and regenerating seedlings of myrtle. The method comprises the following steps: A. inoculating sterile leaves or stem segments of the myrtle test-tube plantlet serving as an explant to a callus induction culture medium for induction culture to obtain callus; B. inoculating the callus onto a proliferation differentiation culture medium for proliferation differentiation culture to obtain compact-particle callus, and gradually forming cluster buds; C. cutting the cluster buds and transferring the cluster buds onto a rooting culture medium for rooting culture to obtain regenerated plantlets; D. removing a bottle cap of a rooting test-tube seedling with the height of 6-8 cm, putting the rooting test-tube seedling under natural illumination, hardening the seedling for 3-5 days, taking the test-tube seedling out of a culture bottle, washing off a root culture medium, and transplanting the test-tube seedling into a matrix mixed by humus soil and yellow sand mud. The method is simple to operate and easy to master, and is favorable for carrying out the subsequent work of propagation of the rhodomyrtus tomentosa seedlings.

Description

Method for inducing calluses and regenerating seedlings of myrtle

Technical Field

The invention relates to the technical field of modern Chinese herbal medicine cultivation, in particular to a method for inducing calluses and regenerating seedlings of myrtle.

Background

Myrtle (Rhodomyrtus tomentosa (air.) Hask) is evergreen shrub of Myrtaceae Myrtle genus, namely Rhodomyrtus tomentosa, Amygur montana, Myrtus communis, and the like, and is native to southeast, south and southwest parts of China and tropical and subtropical regions of Asia. As a traditional Chinese medicinal material, the traditional Chinese medicinal material mainly uses roots, leaves and fruits as the raw materials, and is a potential resource for exploring novel functional medicines. Many studies are currently focused on the functional properties of extracts of myrtle, which are traditionally used to combat various diseases and have pharmacological activity to protect plants against biotic or abiotic stress. In addition, myrtle can be used for making foods such as wine, jam and tea. Myrtle contains a large amount of volatile terpenoids, and the volatile oil plays an important role in life and health of people, such as respiratory drugs, perfume and the like.

At present, the myrtle is in a wild resource stage, and the habitat of the myrtle is often damaged along with frequent activities of human beings and livestock. Meanwhile, the excessive mining of the gene is caused to sharply reduce the number of individuals, thereby reducing the genetic diversity. Although the myrtle has high medical and health care values and great economic benefits, the large-area artificial domestication and cultivation are difficult due to seed fructification, short seed vigor, low natural germination rate and high heterogeneity of seedling populations, so the myrtle is still in a wild state, and the cultivation popularization and deep research of the myrtle are influenced.

Therefore, how to obtain high-quality embryogenic callus through a proper explant, then, proliferating and differentiating the embryogenic callus into tissues such as new buds or new roots and the like, establishing a myrtle callus regeneration seedling system, being beneficial to realizing the synchronization of seedling propagation, quickly and efficiently obtaining a large amount of regeneration seedlings to promote the realization of artificial cultivation and planting, and further, the perfect combination of reasonably utilizing the resources of the myrtle and protecting the ecological geographic diversity needs to be solved urgently. In addition, callus induction and plant regeneration are the basis for gene transformation research on huge characteristics of the myrtle, and theoretical practical basis is laid for further research work of the myrtle in future.

Disclosure of Invention

The invention aims to provide a method for inducing callus to regenerate seedlings of myrtle, which is simple to operate, easy to master and beneficial to developing propagation of myrtle seedlings.

The method for inducing the callus of the myrtle to regenerate the seedling comprises the following steps:

A. inoculating sterile leaves or stem segments of the myrtle test-tube plantlet serving as an explant to a callus induction culture medium for induction culture to obtain callus;

the callus induction culture medium is MS + 0.5-1 mg/L6-BA + 0.05-0.1 mg/L NAA + 0.005-0.01 mg/L TDZ + sucrose + agar;

B. inoculating the callus onto a proliferation differentiation culture medium for proliferation differentiation culture to obtain compact-particle callus at first and then gradually form cluster buds;

the proliferation and differentiation culture medium is 1/2MS + 1-1.5 mg/L6-BA + 0.1-0.5 mg/LNAA + sucrose + agar;

C. cutting the cluster buds and transferring the cluster buds onto a rooting culture medium for rooting culture to obtain regenerated plantlets;

the rooting culture medium is 1/2MS + sucrose + agar;

D. removing a bottle cap of a rooting test-tube seedling with the height of 6-8 cm, hardening the seedling under natural illumination for 3-5 days, taking the test-tube seedling out of a culture bottle, washing off a root culture medium, and transplanting the test-tube seedling into a matrix mixed by humus soil and yellow sand mud;

the conditions of the induction culture are that the illumination intensity is 1500-3000 lx, the light/dark condition is 16/8h, and the temperature is 26-32 ℃;

the conditions of proliferation and differentiation culture are that the illumination intensity is 1500-3000 lx, the light/dark condition of 16/8h and the temperature is 26-32 ℃;

the rooting culture conditions comprise the illumination intensity of 3000-5000 lx, the light/dark condition of 16/8h and the temperature of 26-32 ℃.

Preferably, the explant is obtained by performing scratch treatment on sterile leaves of the myrtle test-tube plantlet along the main vein direction of the leaves, wherein the number of the scratch treatment on each leaf is 2-3, and the length of each scratch is 1-2 mm, and then the treated leaves are used as the explant or the stem section of the sterile myrtle test-tube plantlet and cut into 1-2 cm to be used as the explant.

The callus induction culture medium is preferably MS +0.5 mg/L6-BA +0.05mg/L NAA +0.005mg/L TDZ +30g/L sucrose + agar 5 g/L.

The proliferation and differentiation culture medium is preferably 1/2MS +1 mg/L6-BA +0.1mg/L NAA +30g/L sucrose + agar 5 g/L;

the rooting medium is preferably 1/2MS +30g/L sucrose + 5g/L agar.

The matrix is preferably prepared by mixing humus soil and yellow sand mud according to the mass ratio of 3: 1.

Preferably, the conditions for inducing culture are illumination intensity of 3000lx, light/dark condition of 16/8h and temperature of 26 ℃;

the conditions of proliferation and differentiation culture are that the illumination intensity is 3000lx, the light/dark condition is 16/8h, and the temperature is 26 ℃;

the rooting culture conditions are that the illumination intensity is 5000lx, the light/dark condition is 16/8h, and the temperature is 26 ℃.

The MS culture medium is a conventional culture medium in the tissue culture field and can be purchased from commercial companies, and 1/2MS culture medium is prepared by halving macroelements in the MS culture medium and keeping other macroelements unchanged.

Compared with the prior art, the invention has the following beneficial effects:

in order to obtain high quality embryogenic callus, callus was induced from young leaves and stems on MS medium containing 0.5 mg/L6-BA, 0.05mg/L LNAA, 0.005mg/L TDZ, 30g/L sucrose, agar 5g/L, and was differentiated into adventitious buds in 60 days on 1/2MS medium containing 1 mg/L6-BA, 0.1mg/L LNAA, 30g/L sucrose, agar 5g/L, and adventitious buds on 1/2MS rooting medium consisting of 30g/L sucrose, agar 5g/L were rooted, and more than 90% of the plantlets were viable after the rooted regeneration was transplanted. Tender leaves and tender stems of test-tube seedlings of myrtle can be used as explants, the regeneration effect of tender leaves of seedlings is the best, the effect is the best when the temperature for callus proliferation and differentiation is 26 ℃ and the illumination intensity is 3000lx and the rooting culture temperature is 26 ℃ and the illumination intensity is 5000lx, the vitrification or browning phenomenon of the callus can be reduced, in addition, 1/2MS culture medium without plant exogenous hormone is more suitable for rooting, and the survival rate of the regenerated seedlings can reach 90%.

In addition, the method is simple to operate and easy to master, and is favorable for carrying out the subsequent work of propagation of the rhodomyrtus tomentosa seedlings.

Drawings

FIG. 1 is callus formation of explants of Myrtle of example 1, example 2, comparative example 1, comparative example 2 and comparative example 3;

FIG. 2 shows the callus of Myrtus communis proliferated and differentiated to form adventitious buds according to examples 1 and 2.

FIG. 3 is the rooting of the regenerated plants of Myrtus communis of examples 1 and 2.

FIG. 4 shows the survival regenerated seedlings of the transplantation of the myrtle in example 1.

Detailed Description

Experimental procedures for the invention not specifically indicated in the following examples are generally carried out under conventional conditions, or as recommended by the manufacturer. The various chemicals used in the examples are commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, article, or device that comprises a list of steps is not limited to only those steps or modules listed, but may alternatively include other steps not listed or inherent to such process, method, article, or device.

The "plurality" referred to in the present invention means two or more. "and/or" describes the association relationship of the associated objects, meaning that there may be three relationships, e.g., a and/or B, which may mean: a exists alone, A and B exist simultaneously, and B exists alone. The character "/" generally indicates that the former and latter associated objects are in an "or" relationship.

The embodiment provides a method for inducing callus to regenerate seedlings of myrtle, which comprises the following steps:

(1) inoculating a sterile myrtle test-tube plantlet explant to a callus induction culture medium for induction culture to obtain a callus;

(2) inoculating the callus onto a proliferation differentiation culture medium for proliferation differentiation culture to obtain compact-particle callus, and gradually forming cluster buds;

(3) cutting the cluster buds and transferring the cluster buds onto a rooting culture medium for rooting culture to obtain regenerated plantlets;

(4) removing a bottle cap of a rooting test-tube seedling with the height of about 6-8 cm, hardening the seedling under natural illumination for 3-5 days, taking the test-tube seedling out of a culture bottle, washing off a root culture medium, and transplanting the test-tube seedling into a matrix formed by mixing humus soil and yellow sand mud.

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

The following examples refer to sterile myrtle test-tube plantlet sources: the sterile downy rosemyrtle test-tube plantlet is a plantlet obtained by sterile propagation of downy rosemyrtle seeds, the downy rosemyrtle seeds are collected from a southern China plant garden in Guangzhou, and the seed collection time is 10 months in 2019.

Example 1

(1) Carrying out scratch treatment on sterile myrtle test-tube plantlet leaves along the main vein direction of the leaves, wherein the number of the scratch treatments of each leaf is 2-3, and the length of each scratch is 1-2 mm, then inoculating the treated leaves to a callus induction culture medium (MS, 0.5 mg/L6-BA, 0.05mg/LNAA, 0.005mg/L TDZ, 30g/L sucrose and 5g/L agar), adding sucrose and agar into the MS culture medium, sterilizing, and then adding 6-BA, NAA and TDZ solutions for filtration sterilization when the temperature is reduced to about 45 ℃, and carrying out induction culture (the light intensity is 3000lx, the light/dark condition is 16/8h, the temperature is 26 ℃) to obtain callus;

(2) inoculating the callus to a proliferation and differentiation culture medium (1/2MS, 1 mg/L6-BA, 0.1mg/LNAA, 30g/L sucrose and 5g/L agar), adding sucrose and agar into a 1/2MS culture medium, sterilizing, adding 6-BA and NAA solution for filtration sterilization when the temperature is reduced to about 45 ℃, performing proliferation and differentiation culture (illumination intensity is 3000lx, light/dark conditions of 16/8h and temperature is 26 ℃), obtaining compact-particle callus at first, and then gradually forming cluster buds;

(3) cutting the cluster buds, transferring the cluster buds into a rooting culture medium (1/2MS, 30g/L of sucrose and 5g/L of agar, and performing rooting culture (illumination intensity 5000lx, light/dark condition of 16/8h and temperature 26 ℃) after adding the sucrose and the agar into a 1/2MS culture medium and sterilizing to obtain regenerated plantlets;

The preparation of the culture medium needs high-temperature sterilization and disinfection, and the added exogenous hormone needs to be added after filtration sterilization.

The multiple buds are cut to be 3-5 cm away from a single bud.

And (3) operating in a sterile environment.

The matrix for transplanting is formed by mixing humus soil and yellow sand mud according to the mass ratio of 3: 1.

Example 2

This example differs from example 1 in that: in the step (1), the explant is a sterile stem section of a myrtle test-tube plantlet, is cut into 1-2 cm and is inoculated to a callus induction culture medium, and other steps are the same.

Comparative example 1

The difference between the comparative example and the example 1 is that in the step (1), the explant is the tender leaf at the top end of the disinfected wild myrtle which is harvested in three to five years, and other steps are the same.

Comparative example 2

The difference between the comparative example and the example 1 is that the explant is a tender stem of a three-five-year-old myrtle picked in a disinfected field, and other steps are the same.

Comparative example 3

The comparative example is different from example 1 in that the explant is a disinfected wild myrtle petal picked in three to five years, and other steps are the same.

Comparative example 4

The difference between this comparative example and example 1 is that the explant was a disinfected field picked three to five year old stamen of myrtle flowers, with the other steps being the same.

Comparative example 5

The comparative example differs from example 1 in that the light intensity of callus induction culture and proliferation differentiation culture was 1500 lx.

Comparative example 6

The comparative example differs from example 1 in that the light intensity for callus induction culture and proliferation differentiation culture is 1500lx and the temperature is 20 ℃.

Comparative example 7

This comparative example differs from example 1 in that the temperature for callus induction culture and proliferation differentiation culture was 20 ℃.

Comparative example 8

The comparative example is different from example 1 in that the light intensity for callus induction culture and proliferation differentiation culture is 1500lx and the temperature is 32 ℃.

Comparative example 9

This comparative example differs from example 1 in that the temperature for callus induction culture and proliferation differentiation culture was 32 ℃.

Comparative example 10

This comparative example is different from example 1 in that 0.5mg/L IBA was added to the rooting medium, and the other steps were the same.

Comparative example 11

This comparative example differs from example 1 in that 0.05mg/LNAA was added to the rooting medium and the other steps were the same.

Comparative example 12

This comparative example differs from example 1 in that 0.5mg/LNAA was added to the rooting medium, and the other steps were the same.

Comparative example 13

This comparative example differs from example 1 in that 0.5mg/L IBA and 0.05mg/L LNAA were added to the rooting medium, and the other steps were the same.

And (3) performance testing:

callus induction rate (%) ═ number of callus/number of inoculated explants × 100

Adventitious bud formation rate (%). adventitious bud number/number of inoculated callus × 100

Plant regeneration frequency (%). The number of regenerated shoots of callus/number of inoculated callus X100

Rooting percentage (%). regenerated seedling number/inoculated adventitious bud number × 100

Survival rate (%). survival regenerated seedling/number of transplanted regenerated plant) × 100

As shown in FIGS. 1 to 4 and tables 1 to 3, callus was induced from young leaves and stems on an MS medium containing 0.5 mg/L6-BA, 0.05mg/L LNAA, 0.005mg/L TDZ, 30g/L sucrose and agar 5g/L, 98.48% of young leaves and 96.93% of stem segments induced pale yellow-green, granular callus, wherein 77.07% of young leaf-formed callus was transformed into adventitious shoots within 60 days on a 1/2MS medium containing 1 mg/L6-BA, 0.1mg/L LNAA, 30g/L sucrose and agar 5g/L, and the callus was transformed into adventitious shoots at a temperature suitable for callus proliferation and differentiation of 26 to 32 ℃ and a light intensity of 3000lx, and further, adventitious shoots were rooted on a 1/2MS medium consisting of 30g/L sucrose and agar 5g/L, more than 90% of the plantlets survived after transplanting the rooted regenerated plants.

TABLE 1

TABLE 2

TABLE 3

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent should be subject to the appended claims.

Claims

1. A method for inducing callus of myrtle to regenerate seedlings is characterized by comprising the following steps:

A. inoculating sterile leaves or stem segments of the myrtle test-tube plantlet as an explant to a callus induction culture medium for induction culture to obtain callus;

B. inoculating the callus onto a proliferation differentiation culture medium for proliferation differentiation culture to obtain compact-particle callus, and gradually forming cluster buds;

the proliferation and differentiation culture medium is 1/2MS + 1-1.5 mg/L6-BA + 0.1-0.5 mg/L NAA + sucrose + agar;

the rooting culture medium is 1/2MS + sucrose + agar;

the rooting culture conditions comprise the light intensity of 3000-5000 lx and the light/dark condition of 16/8h at the temperature of 26-32 ℃.

2. The method as claimed in claim 1, wherein the explant is prepared by incising leaves of sterile myrtle test-tube plantlets along the main vein direction of the leaves, wherein the number of incised leaves is 2-3, and the length of each incised leaf is 1-2 mm, and then cutting the leaves as the explant or the stem of the sterile myrtle test-tube plantlets into 1-2 cm.

3. The method of claim 1 or 2, wherein the callus induction medium is MS +0.5 mg/L6-BA +0.05mg/L NAA +0.005mg/L TDZ +30g/L sucrose + agar 5 g/L.

4. The method according to claim 1 or 2, wherein the proliferation and differentiation medium is 1/2MS +1 mg/L6-BA +0.1mg/L NAA +30g/L sucrose + 5g/L agar.

5. The method according to claim 1 or 2, wherein the rooting medium is 1/2MS +30g/L sucrose + 5g/L agar.

6. The method according to claim 1 or 2, wherein the matrix is formed by mixing humus soil and yellow sand mud according to a mass ratio of 3: 1.

7. The method according to claim 1 or 2, wherein the conditions for inducing culture are illumination intensity 3000lx, light/dark 16/8h, temperature 26 ℃; the conditions of proliferation and differentiation culture are that the illumination intensity is 3000lx, the light/dark condition is 16/8h, and the temperature is 26 ℃; the conditions of rooting culture are that the illumination intensity is 5000lx, the light/dark condition is 16/8h, and the temperature is 26 ℃.