CN110521596B

CN110521596B - Method for inducing exocarpium citri grandis callus to bud

Info

Publication number: CN110521596B
Application number: CN201910830033.1A
Authority: CN
Inventors: 黄新敏; 韩寒冰; 欧阳乐军; 李小双; 林珏; 彭召敏
Original assignee: Guangdong University of Petrochemical Technology
Current assignee: Guangdong University of Petrochemical Technology
Priority date: 2019-09-02
Filing date: 2019-09-02
Publication date: 2023-03-31
Anticipated expiration: 2039-09-02
Also published as: CN110521596A

Abstract

The invention belongs to the technical field of plant tissue culture, and particularly relates to a method for inducing germination of exocarpium citri grandis callus; the method comprises the following steps: explant selection and sterilization, callus formation, subculture, rooting induction after callus budding and bud formation, seedling hardening and transplanting; the method can induce calluses to form a large number of buds and develop into independent plants on the basis of the calluses by adopting trehalose to replace cane sugar and adding pummelo leaf extracting solution and acetosyringone on the basis of the original formula of a calluse forming culture medium on the basis of inducing the stem bud sites of pummelo peel to form the calluses.

Description

Method for inducing exocarpium citri grandis callus to bud

Technical Field

The invention belongs to the technical field of plant tissue culture, and particularly relates to a method for inducing germination of exocarpium citri grandis callus.

Background

Pummelo peel has the effects of dispelling cold, eliminating dampness, promoting qi, eliminating diseases, relieving cough, strengthening spleen, promoting digestion and the like, is named as 'southern ginseng', is one of famous south Chinese medicines and one of ten broad-spectrum medicines, and is listed as the national geographical sign product for protection in 2006. Genuine pummelo peel is prepared from exocarp of immature fruit of Citrus grandis of Rutaceae. The pummelo peel propagation technology mostly adopts modes of grafting, layering on high branches and the like, which greatly limits the popularization of good varieties of pummelo peel. Therefore, the tissue culture technology has important advantages for expanding propagation of the good varieties of the pummelo pee. At present, research on tissue culture technology of pummelo peel exists, but stem segments with buds in specific time need to be selected as explants to be directly subjected to bud induction, callus cannot be formed, the culture bud is less, a large amount of explants need to be consumed, and therefore mass production of tissue culture seedlings is difficult to realize. In addition, a large amount of phenolic substances can be formed in the process of culturing the exocarpium citri grandis explant, so that callus is browned, and callus differentiation and sprouting are inhibited.

Disclosure of Invention

The invention aims to produce a special culture medium for inducing callus to bud by adopting trehalose to replace cane sugar and adding pummelo leaf extracting solution and acetosyringone on the basis of inducing stem bud sites of pummelo peel to form callus on the basis of an original callus forming culture medium formula on the basis of inducing stem bud sites of the pummelo peel to form callus.

The technical scheme of the invention is as follows:

a method for inducing the germination of exocarpium Citri Grandis callus comprises the following steps: explant selection and sterilization, callus formation, subculture, rooting induction after callus budding and bud formation, seedling hardening and transplanting;

wherein the callus formation step adopts a callus formation culture medium;

preparing a callus formation culture medium: MS + benzyladenine (6-BA) 0.8-1.2mg/L + Naphthaleneacetic acid (NAA)

0.03-0.06mg/L, 100-120 mg/L of vitamin C, 50g/L of sucrose, 100g/L of potato and 7g/L of agar, and the pH value of the culture medium is 5.8; wherein the callus induction sprouting step adopts an induction culture medium;

preparation of an induction culture medium: MS, benzyladenine (6-BA) 0.8-1.2mg/L, naphthylacetic acid (NAA) 0.03-0.06mg/L, vitamin C100-120 mg/L, acetosyringone 0.196mg/L, trehalose 15g/L, pummelo leaf extract 0.02% (namely 0.2 mg/L), potato 100g/L and agar 7g/L, wherein the pH value of the culture medium is 5.8.

The preparation method of the pummelo peel extracting solution comprises the following steps:

fresh exocarpium Citri Grandis leaf is placed in a 110 deg.C oven for deactivating enzyme, and then placed in a 60 deg.C oven for drying to constant weight, and pulverized and sieved with 40 mesh sieve to obtain powder smaller than 40 mesh for extraction. Respectively taking 5g of leaf sample powder, placing the 5g of leaf sample powder in a 250mL extraction tube, adding 250mL of 60% ethanol solution, fully shaking up, and carrying out ultrasonic treatment for 20-30 min at 40-50 ℃ by using an ultrasonic instrument for 2 times. All extracts were combined and freeze dried, dissolved in ultrapure water and kept at 4 ℃ until use.

Wherein in the callus forming step, dark culture is carried out for 15-20 days at the temperature of 26 +/-2 ℃.

Wherein in the step of inducing callus to bud, the callus is cultured for 25-35 days under the condition that the illumination intensity is 2300-2400lx and 169h/8 h in light and dark (16-hour illumination culture, 8-hour dark culture and light and dark culture are alternately and circularly carried out) and the temperature is 26 +/-2 ℃.

Wherein the explant adopts the current-year semi-lignified stem segment of pummelo peel.

Further, the current-year semi-lignified stem segment of pummelo peel is obliquely cut with 1.5-2.0cm of axillary bud as explant by using a sterile scalpel.

As a further improvement of the invention, the preparation process of the callus formation culture medium comprises the steps of preparing a working solution from MS + benzyladenine (6-BA) 0.8-1.2mg/L, naphthylacetic acid (NAA) 0.03-0.06mg/L, vitamin C100-120 mg/L, sucrose 50g/L, potato 100g/L and agar 7g/L, subpackaging the working solution according to 50mL of each erlenmeyer flask, sucking air from each erlenmeyer flask through a dust collector after subpackaging, sealing the erlenmeyer flask and putting the erlenmeyer flask into a sterilization pot for high-pressure sterilization. Because the working solution is easy to generate bubbles after being subpackaged, and the bubbles in the culture medium are difficult to escape due to the high air pressure of the sealed conical bottle in the high-pressure disinfection process, the bubbles solidified on the surface cause poor contact between the explant and the surface of the culture medium (as shown in figure 2). Through simple vacuum pumping of the dust collector, the air pressure in the conical flask can be greatly reduced, small bubbles can escape favorably, and a smoother culture medium surface can be formed finally. The induction medium does not need to be pumped, because the callus needs to be cut into small pieces during induction, and the surface flatness of the medium is not high.

The invention has the beneficial effects that: the research of the tissue culture technology of pummelo peel exists, but stem segments with buds in specific time are required to be selected as explants to be directly subjected to bud induction, callus cannot be formed, the culture budding is less, a large amount of explants are required to be consumed, and therefore the mass production of tissue culture seedlings is difficult to realize. The method adopts the callus forming culture medium to effectively induce the pummelo peel stem segments to form a large amount of callus, and adopts trehalose to replace cane sugar and adds acetosyringone to prepare the special callus budding inducing culture medium on the basis of the original callus forming culture medium formula for inducing the callus to form buds and develop into independent plants, and in addition, the callus can be propagated more callus through subculture. The method has the advantages that the explant is not limited by seasons, more callus is generated, the callus can effectively bud to form plants, the obtained plants grow tidily and maintain the excellent characters of the original explant, the test result is stable, the repeatability is good, and the method can be applied to large-scale commercial production. Trehalose and acetosyringone can promote callus to form a large number of buds and develop into independent plants by inducing callus differentiation. The exocarpium Citri Grandis leaf extractive solution contains a large amount of antioxidant active substances such as rhoifolin and naringin, and can inhibit peroxidation process in callus culture process, reduce formation of phenols in callus, and further inhibit callus browning. In addition, rhoifolin, naringin and other substances in the pummelo peel leaf extracting solution also have the bacteriostatic action, and can inhibit the pollution of explants and callus tissues. In addition, the pummelo peel leaf extracting solution is from pummelo peel plants, and pummelo peel callus has no rejection to the substances, so that the absorption and utilization of the substances by the callus are facilitated.

Drawings

FIG. 1 is a schematic process flow diagram of the present invention;

FIG. 2 is a photograph of a bubble-carrying medium.

Detailed Description

The following further describes embodiments of the present invention in conjunction with the attached figures:

example 1

As shown in fig. 1, the main steps of the present invention are as follows:

(1) Selecting and sterilizing explants: selecting current-year semi-lignified stem segments of pummelo peel, ultrasonically cleaning for 10 minutes by using a washing powder solution with the mass concentration of 0.5%, and then washing by using tap water; soaking in 75% alcohol for 30s in an ultraclean workbench, washing with sterile water for 3-4 times, wiping water with sterile filter paper, sterilizing with 0.1% mercuric chloride for 15-17 min, washing with sterile water for 7-8 times, sucking surface water with sterile filter paper, and obliquely cutting 1.5-2.0cm axillary bud with a sterile scalpel to obtain explant.

(2) Callus formation: inoculating the explant to a callus formation culture medium which is sterilized at high temperature for culture, and performing dark culture at 26 +/-2 ℃ for 15-20 days. The callus forming culture medium comprises MS, benzyladenine (6-BA) 1mg/L, naphthylacetic acid (NAA) 0.05mg/L, vitamin C100-120 mg/L, 5% sucrose, 10% potato and agar 7g/L, and the pH value of the culture medium is 5.8.

(3) Subculturing: selecting sterile, light-colored and transparent viable callus, cutting into small pieces under sterile conditions, placing in callus formation culture medium for subculture at the culture temperature of 26 + -2 deg.C, and dark culturing.

(4) Inducing callus to bud: selecting sterile, light-colored and transparent viable callus, placing the callus in a callus budding culture medium, and culturing for 25-35 days under the conditions of light intensity of 2300-2400lx and 169h/8 h in light and dark at the temperature of 26 +/-2 ℃ to induce budding. The callus budding culture medium is a special culture medium for inducing callus budding by adopting trehalose to replace sucrose and adding acetosyringone on the basis of the original callus forming culture medium formula, and the specific formula comprises MS, benzyladenine (6-BA) 1mg/L, naphthylacetic acid (NAA) 0.05mg/L, vitamin C100-120 mg/L, acetosyringone 100 mu mol/L, 15% trehalose, 0.02% pummelo leaf extract, 10% potato and agar 7g/L, and the pH value of the culture medium is 5.8.

(5) After the buds are formed, rooting induction, seedling hardening and transplanting can be carried out.

Example 2

The preparation process of the callus formation culture medium comprises the steps of preparing working solution from MS, 0.8-1.2mg/L of benzyladenine (6-BA), 0.03-0.06mg/L of naphthylacetic acid (NAA), 100-120 mg/L of vitamin C, 50g/L of cane sugar, 100g/L of potato and 7g/L of agar, subpackaging the working solution according to 50mL of each conical flask, sucking air from each conical flask through a dust collector after subpackaging, sealing the conical flasks and placing the conical flasks into a sterilization pot for high-pressure sterilization. Because the working solution is easy to generate bubbles after being subpackaged, the bubbles in the culture medium are difficult to escape due to the high air pressure of the sealed conical bottle in the high-pressure disinfection process, and the contact between the explant and the surface of the culture medium is poor due to the bubbles solidified on the surface. Through simple vacuum pumping of the dust collector, the air pressure in the conical flask can be greatly reduced, small bubbles can escape favorably, and a smoother culture medium surface can be formed finally.

When the vacuum cleaner is used for extracting the conical flask, the air suction pipe of the vacuum cleaner is aligned to the bottle mouth of the conical flask, the bottle mouth and the air suction pipe are held by hands, the hands are equivalent to a sealing ring, and the hand of a general adult can be simply sealed because the requirement on the vacuum degree is low during air extraction and the air suction pipe is not strictly vacuumized.

Experimental data:

100 samples are obtained by the method, and the number of callus formation, callus induced budding success and seedling hardening survival are counted.

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In addition, the number of shoots induced (i.e., the number of shoots generated) per callus (which is referred to herein as the number of calli formed on the callus formation medium, not the number after division) was counted to be 12.3 on average, i.e., about 12.3 shoots were generated from one callus. Only this experiment could produce 98 × 12.3 × 90/98=1107 viable seedlings.

The data show that the invention has obvious effect on the pummelo peel which is difficult to induce to sprout originally, and is suitable for industrial production.

The foregoing embodiments and description have been provided to illustrate the principles and preferred embodiments of the invention, and various changes and modifications may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. A method for inducing the germination of exocarpium Citri Grandis callus comprises the following steps: explant selection and sterilization, callus formation, subculture, rooting induction after callus budding and bud formation, seedling hardening and transplanting;

wherein the callus formation step adopts a callus formation culture medium;

preparing a callus formation culture medium: MS + benzyladenine (6-BA) 0.8-1.2mg/L + naphthylacetic acid (NAA) 0.03-0.06mg/L + vitamin C100-120 mg/L + sucrose 50g/L + potato 100g/L + agar 7g/L, and the pH value of the culture medium is 5.8;

wherein the callus bud induction step adopts an induction culture medium;

preparation of an induction culture medium: MS, 0.8-1.2mg/L of benzyladenine (6-BA), 0.03-0.06mg/L of naphthylacetic acid (NAA), 100-120 mg/L of vitamin C, 0.196mg/L of acetosyringone, 15g/L of trehalose, 0.2mg/L of an extract of pummelo peel leaves, 100g/L of potatoes and 7g/L of agar, wherein the pH value of a culture medium is 5.8;

placing fresh exocarpium Citri Grandis leaf in 110 deg.C oven for deactivating enzyme, drying at 60 deg.C to constant weight, pulverizing, and sieving with 40 mesh sieve to obtain powder smaller than 40 mesh for extraction; respectively taking 5g of leaf sample powder, placing the leaf sample powder in a 250mL extraction tube, adding 250mL of 60% ethanol solution, fully shaking, and extracting for 2 times by adopting an ultrasonic instrument under the condition of ultrasonic waves at 40 to 50 ℃ for 20 to 30 min; mixing all extractive solutions, freeze drying, dissolving with ultrapure water, and standing at 4 deg.C;

in the callus forming step, culturing in the dark at the temperature of 26 +/-2 ℃ for 15 to 20 days; in the step of inducing callus to bud, culturing for 25-35 days under the conditions that the illumination intensity is 2300-2400lx and 169h/8 h in light and dark and the temperature is 26 +/-2 ℃; cutting 1.5-2.0cm axillary bud of exocarpium Citri Grandis in current year with sterile scalpel to obtain explant; the preparation process of the callus formation culture medium comprises the following steps: preparing working solution from 0.8-1.2mg/L of MS + benzyladenine (6-BA), 0.03-0.06mg/L of naphthylacetic acid (NAA), 100-120 mg/L of vitamin C, 50g/L of sucrose, 100g/L of potato and 7g/L of agar, subpackaging the working solution according to 50mL of each conical flask, sucking air from each conical flask by a dust collector after subpackaging, sealing the conical flasks, and putting the conical flasks into a sterilization pot for high-pressure sterilization.