CN111264390A - Method for regenerating plant by somatic cells of allium victorialis - Google Patents

Abstract

The invention relates to a method for regenerating a plant by somatic cells of a Allium victorialis, which comprises the following steps: s1 explant: bulb, S2 medium: adding different kinds and dosage of growth regulators into MS minimal medium to prepare medium for primary, secondary and rooting culture of Allium victorialis; the pH value of the culture medium is 5.5-6.0, the sucrose is 20 g.L < -1 >, and the culture medium is sterilized by a conventional method; s3 inoculation and culture: (1) inoculation, (2) primary culture, (3) subculture, (4) adventitious root induction, and (5) test-tube plantlet transplantation, compared with the prior art, the invention has the advantages that: solves the problem of low propagation rate of the Allium victorialis by the conventional method, improves the seedling rate of the Allium victorialis by tissue culture, and solves the key technology of industrialized seedling of the Allium victorialis.

Description

Method for regenerating plant by somatic cells of allium victorialis

Technical Field

The invention relates to the technical field of biology, in particular to a method for regenerating a plant from somatic cells of a Allium victorialis.

Background

Allium victorialis L is perennial herb of Allium of Liliaceae, has mild and pungent taste, no toxicity, enters lung meridian, is pungent and warm in flavor, is aromatic and can be used for treating common cold due to wind-cold and nausea, and is originated from mountain slope, forest, grassland ditch edge, and tender leaf for eating.

The Bulbus Allii Victorialis bulb is oblong, the bulb skin is in mesh shape, the length of the bulb is 8-20 cm, the width of the bulb is 3-9.5 cm, the base part of the bulb is wedge-shaped, slightly extends downwards along the stem of the leaf, the tip of the bulb is gradually sharp or short pointed, the leaf has a long stem, and the bulb is long oval or oblong, has whole edge, is soft and smooth, and is slightly pink; parallel veins, scape is cylindrical, the height is 25-80 cm, 1/4-1/2 is sheathed by leaves, the involucre 2 is split and lodged, the umbelliform inflorescence is spherical, a plurality of dense flowers are arranged, the small pedicles are approximately equal in length and cluster at the stem top to form umbelliform inflorescence arrangement; the flower stem is 30-60 cm long, the flower is small, green white or even light purple, the perianth 6, the stamen 6, the ovary upper position, the 3 rd chamber, the style of the flower pillar is filiform, and the stigma is small. Capsule, cracking of the back of the chamber, black seeds and 6-8 months of flowering phase. The bulbs grow singly or 2-3 bulbs grow together, the bulbs are nearly cylindrical, the outer skins of the bulbs are grey brown to black brown, and the bulbs are broken into fibers and are obviously netted.

Propagation of Allium victorialis is difficult. Like most allium plants, the propagation mode can be divided into sexual propagation and vegetative propagation, the sexual propagation is mainly a seed propagation method, but the allium victorialis grown for more than 3-4 years can blossom and fruit, and the seeds have short service life, deep dormancy, low seedling rate of seeding and seedling raising and low propagation rate of a plant division method.

Disclosure of Invention

The technical problem to be solved by the invention is to overcome the technical defects, and provide the method for regenerating the somatic cells of the allium victorialis, which effectively solves the problem of low reproduction rate of the allium victorialis in the conventional method and improves the seedling rate of the tissue culture allium victorialis.

The technical scheme provided by the invention is as follows: a method for regenerating a plant from somatic cells of Allium victorialis comprises the following steps:

s1 explant: a bulb. Collecting Bulbus Allii Victorialis, cleaning, and cutting into 1cm square;

s2 medium: adding different kinds and dosage of growth regulators into MS minimal medium to prepare medium for primary, secondary and rooting culture of Allium victorialis;

primary culture: MS +6-BA1.0mg/L + NAA0.2mg/L +2, 4-D0.2 mg/L;

subculturing: MS +6-BA1.0mg/L + NAA0.2mg/L +2, 4-D0.2 mg/L;

adventitious root induction: 1/2MS + IBA0.5mg/L;

the pH value of the culture medium is 5.5-6.0, the sucrose is 20 g.L < -1 >, and the culture medium is sterilized by a conventional method;

s3 inoculation and culture:

(1) inoculation: washing the bulbs with tap water, then adding a detergent into a tap water solution to scrub the surfaces of the bulbs, washing the detergent solution with the tap water, putting the washed cold onion bulbs into a sterilized beaker in a sterile room, pouring 0.1% mercuric chloride surface disinfectant to soak for 8min, finally washing the surfaces of the bulbs with the sterile water for 5 times, inoculating the washed cold onion bulbs on an MS +6-BA1.0mg/L + IBA0.1mg/L culture medium in a sterile state, culturing in a culture room after inoculation, wherein the temperature is 23-25 ℃, the illumination intensity is 3300-3500 lux, and the illumination time is 12 h;

(2) primary culture: cutting leaf from bulb sprout, inoculating on MS +6-BA1.0mg/L + NAA0.2mg/L +2, 4-D0.2 mg/L culture;

(3) subculturing: the culture medium MS +6-BA1.0mg/L + NAA0.2mg/L +2, 4-D0.2 mg/L with a subculture period of 40 days;

(4) adventitious root induction: inoculating strong individual seedling on 1/2MS + IBA0.5mg/L culture medium, culturing for 25-35 days to start rooting, wherein the root system is strong, has 3-6 pieces, has root system length of 0.8-1.2cm, and has leaf height of 7-10 cm.

(5) Transplanting test-tube seedlings: opening a bottle cap of an individual seedling with complete growth and rooting in a culture room for 24 hours, taking out a test-tube seedling, cleaning a culture medium adhered to the root, carrying out traditional Chinese medicine bath in 800 times of carbendazim aqueous solution, planting the test-tube seedling on a matrix of humus soil and loam soil in a ratio of 1: 1, and controlling the temperature to be 22 +/-2 ℃ during seedling hardening; the relative humidity of the seedlings is 80-90% at the initial stage of seedling exercising, and then the relative humidity of the air is gradually reduced; spraying the bactericide once every 5 days to prevent the seedlings from infecting mixed bacteria, domesticating and culturing in a greenhouse for 3-4 weeks to realize natural growth, and the survival rate is more than 90%.

Compared with the prior art, the invention has the advantages that:

(1) primary culture: cutting leaf from bulb sprout, and inoculating on MS +6-BA1.0mg/L + NAA0.2mg/L +2, 4-D0.2 mg/L culture.

(2) Subculturing: the culture medium MS +6-BA1.0mg/L + NAA0.2mg/L +2, 4-D0.2 mg/L, the subculture period is 40 days.

(3) Adventitious root induction: the strong individual seedlings are inoculated on 1/2MS culture medium, after about 30 days of culture, the seedlings start to root, the root system is strong, 3-6 seedlings exist, and the root system is about 1cm long. The height of the leaf is about 7-10 cm.

Solves the problem of low propagation rate of the Allium victorialis by the conventional method, improves the seedling rate of the Allium victorialis by tissue culture, and solves the key technology of industrialized seedling of the Allium victorialis.

Drawings

FIG. 1 is a schematic representation of the primary culture of explants of the invention.

Fig. 2 is a schematic diagram of the embryogenesis of Allium victorialis body according to the present invention.

Fig. 3 is a schematic view of subculture of Allium victorialis according to the present invention.

FIG. 4 is a schematic illustration of adventitious root induction according to the present invention.

FIG. 5 is a schematic diagram of the tube plantlet transplanting of the present invention.

Detailed Description

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

Referring to the accompanying drawings 1-5, a method for regenerating a plant from somatic cells of Allium victorialis comprises the following steps:

s1 explant: a bulb. In spring, in season with vigorous growth of Allium victorialis, digging Bulbus Allii Victorialis, cleaning, and cutting into 1cm cubes;

s2 medium: adding different kinds and dosage of growth regulators into MS minimal medium to prepare medium for primary, secondary and rooting culture of Allium victorialis;

primary culture: MS +6-BA1.0mg/L + NAA0.2mg/L +2, 4-D0.2 mg/L;

subculturing: MS +6-BA1.0mg/L + NAA0.2mg/L +2, 4-D0.2 mg/L;

adventitious root induction: 1/2MS + IBA0.5mg/L;

the pH value of the culture medium is 5.5-6.0, the sucrose is 20 g.L < -1 >, and the culture medium is sterilized by a conventional method;

s3 inoculation and culture:

(1) inoculation: washing the bulbs with tap water, then adding a detergent into a tap water solution to scrub the surfaces of the bulbs, washing the detergent solution with the tap water, putting the washed cold onion bulbs into a sterilized beaker in a sterile room, pouring 0.1% mercuric chloride surface disinfectant to soak for 8min, finally washing the surfaces of the bulbs with the sterile water for 5 times, inoculating the washed cold onion bulbs on an MS +6-BA1.0mg/L + IBA0.1mg/L culture medium in a sterile state, culturing in a culture room after inoculation, wherein the temperature is 23-25 ℃, the illumination intensity is 3300-3500 lux, and the illumination time is 12 h;

(2) primary culture: cutting leaf from bulb sprout, inoculating on MS +6-BA1.0mg/L + NAA0.2mg/L +2, 4-D0.2 mg/L culture;

(3) subculturing: the culture medium MS +6-BA1.0mg/L + NAA0.2mg/L +2, 4-D0.2 mg/L with a subculture period of 40 days;

(4) adventitious root induction: inoculating strong individual seedling on 1/2MS + IBA0.5mg/L culture medium, culturing for 25-35 days to start rooting, wherein the root system is strong, has 3-6 pieces, has root system length of 0.8-1.2cm, and has leaf height of 7-10 cm.

(5) Transplanting test-tube seedlings: opening a bottle cap of an individual seedling with complete growth and rooting in a culture room for 24 hours, taking out a test-tube seedling, cleaning a culture medium adhered to the root, carrying out traditional Chinese medicine bath in 800 times of carbendazim aqueous solution, planting the test-tube seedling on a matrix of humus soil and loam soil in a ratio of 1: 1, and controlling the temperature to be 22 +/-2 ℃ during seedling hardening; the relative humidity of the seedlings is 80-90% at the initial stage of seedling exercising, and then the relative humidity of the air is gradually reduced; spraying the bactericide once every 5 days to prevent the seedlings from infecting mixed bacteria, domesticating and culturing in a greenhouse for 3-4 weeks to realize natural growth, and the survival rate is more than 90%.

Claims (1)

1. A method for regenerating a plant from somatic cells of Allium victorialis is characterized by comprising the following steps:

s1 explant: a bulb. Collecting Bulbus Allii Victorialis, cleaning, and cutting into 1cm square;

s2 medium: adding different kinds and dosage of growth regulators into MS minimal medium to prepare medium for primary, secondary and rooting culture of Allium victorialis;

primary culture: MS +6-BA1.0mg/L + NAA0.2mg/L +2, 4-D0.2 mg/L;

subculturing: MS +6-BA1.0mg/L + NAA0.2mg/L +2, 4-D0.2 mg/L;

adventitious root induction: 1/2MS + IBA0.5mg/L;

the pH value of the culture medium is 5.5-6.0, the sucrose is 20 g.L < -1 >, and the culture medium is sterilized by a conventional method;

s3 inoculation and culture:

(1) inoculation: washing the bulbs with tap water, then adding a detergent into a tap water solution to scrub the surfaces of the bulbs, washing the detergent solution with the tap water, putting the washed cold onion bulbs into a sterilized beaker in a sterile room, pouring 0.1% mercuric chloride surface disinfectant to soak for 8min, finally washing the surfaces of the bulbs with the sterile water for 5 times, inoculating the washed cold onion bulbs on an MS +6-BA1.0mg/L + IBA0.1mg/L culture medium in a sterile state, culturing in a culture room after inoculation, wherein the temperature is 23-25 ℃, the illumination intensity is 3300-3500 lux, and the illumination time is 12 h;

(2) primary culture: cutting the leaf from the bulb and inoculating on MS +6-BA1.0mg/L + NAA0.2mg/L +2, 4-D0.2 mg/L culture;

(3) subculturing: the culture medium MS +6-BA1.0mg/L + NAA0.2mg/L +2, 4-D0.2 mg/L with a subculture period of 40 days;

(4) adventitious root induction: inoculating strong individual seedling on 1/2MS + IBA0.5mg/L culture medium, culturing for 25-35 days to start rooting, wherein the root system is strong, has 3-6 pieces, has root system length of 0.8-1.2cm, and has leaf height of 7-10 cm.

(5) Transplanting test-tube seedlings: opening a bottle cap of an individual seedling with complete growth and rooting in a culture room for 24 hours, taking out a test-tube seedling, cleaning a culture medium adhered to the root, carrying out traditional Chinese medicine bath in 800 times of carbendazim aqueous solution, planting the test-tube seedling on a matrix of humus soil and loam soil in a ratio of 1: 1, and controlling the temperature to be 22 +/-2 ℃ during seedling hardening; the relative humidity of the seedlings is 80-90% at the initial stage of seedling exercising, and then the relative humidity of the air is gradually reduced; spraying the bactericide once every 5 days to prevent the seedlings from infecting mixed bacteria, domesticating and culturing in a greenhouse for 3-4 weeks to realize natural growth, and the survival rate is more than 90%.

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