CN112293260B - Method for obtaining tissue culture seedlings of reed in Africa - Google Patents

Abstract

The invention relates to a method for inducing differentiation and rooting of African reed into seedlings in one step, which comprises the following steps: exposing small buds of outdoor Phragmites australis branches, removing outer leaves around the buds, cleaning branch epidermis with 75% ethanol cotton ball, and removing the stripCutting the bud stem into 2cm, placing in a sterilizing bottle, and placing on an ultra-clean workbench; soaking in 75% ethanol for 30s, rinsing with sterile water for 3 times, and adding 0.1% HgCl ₂ Sterilizing the water solution for 10-15min, and washing with sterile water for 4-5 times; inoculating the sterilized reed stem segments to a culture medium for inducing differentiation and rooting; after each stem section with buds is cultured for 10 days, the first generation roots are then germinated, and after 20-30 days of culture, the transplanted tissue culture seedlings can be obtained. The method can achieve the purposes of induced differentiation and rooting in one step and rapid proliferation. After the seedlings are transplanted and hardened, the seedlings can be quickly adapted to soil cultivation and have strong activity. The invention is suitable for the reed variety.

Description

Method for obtaining tissue culture seedlings of reed in Africa

Technical Field

The invention belongs to the technical field of plant tissue culture, and through the optimization of a traditional rapid propagation tissue culture system, the method omits the conversion of culture media for a plurality of times, such as induction, differentiation, rooting and the like, saves the cost and time, can rapidly provide a batch of seedlings with uniform growth vigor, and provides a plurality of possibilities and guarantees for the application of the fields of ecology, medicine, gardens, animal husbandry and the like of the reed.

Background

The reed is one of emergent aquatic plants with the highest productivity in the plant community, is used as a main planting species of a wetland emergent aquatic plant community, widely grows in fresh water wetland, even coastal wetland and inland saline-alkali wetland with high salinity, and becomes a tool species for restoring wetland ecology by virtue of the characteristics of strong stress resistance, good adaptability, rapid propagation and the like. The reed root exudates are beneficial to removing nitrogen in a denitrification system and increasing the quantity of denitrifying bacteria, has a promoting effect on nitrification strength and an inhibiting effect on denitrification strength, and the reed has a protection effect on the diversity of soil fungi, and the variety of the rhizosphere soil fungi is 1.2 times of that of non-vegetation soil. The prior people research that the breeding of the bulrush usually consumes longer time and can not obtain a batch of plants with more uniform growth vigor at the same time, some bulrush varieties have seeds which can germinate and grow seedlings, while the root system of the special (superior) wild bulrush plant is developed, and the stem is hard like bamboo, and the biological characteristics of the bamboo are different from those of common bulrush; seedling basal growth, short blade, sharp blade tip, plant height of 3-4m, no heading and seed forming phenomenon in China and slow natural propagation. If the plant is widely planted on the banks of rivers, lakes and north and south China, the plant can block flood and silt, has the functions of storing flood, reducing flow rate and the like, and has the reputation of 'the kidney of the earth'. The energy is saved, the consumption is reduced, and the impact resistance is strong when the plant is planted in the wetland for treatment.

At present, some reports about reed tissue culture exist, the traditional reed seedling preparation includes stem cuttage, seed germination and the like, the photinia serrulata and the like and the royal sea and the like all utilize the stem cuttage, the Jordan and the like utilize the seed germination, and although an example that some wettable powder or disinfectant is used for increasing the germination rate of the seeds is provided, the practical operation still has certain limitations. The patent of Anshuqing et al, a culture method of non-callus of fresh water reed, also teaches the rapid propagation system of fresh water reed, but needs to be transformed by a plurality of culture media such as induction, differentiation, rooting, etc.

Therefore, it is very important to obtain the reed seedlings efficiently and quickly. Aiming at the defects, the method is invented by improving the prior art, starts with the reduction of explant browning, and has the advantages of rapidness, simplicity, convenience, high efficiency, economy and the like.

Disclosure of Invention

The invention summarizes a method for quickly, simply and efficiently obtaining the tissue culture seedling of the African reed by optimizing the experimental process and the formula of the culture medium for multiple times. The method utilizes the differentiation rooting one-step seedling culture medium to achieve the purposes of saving economic cost and quickly growing seedlings. The method is safe, rapid and simple to operate, and the seedling process can be completed in more than 30 days, so that the defects of long time, low induction rate, easy browning pollution and the like of the common technology are overcome, and the requirements of biological experiments are met.

In order to realize the purpose, the invention adopts the following technical scheme:

a method for rapidly obtaining tissue culture seedlings of African reeds is characterized in that stems of reeds are used for culturing, and adult African reeds are rapidly propagated, and the method comprises the following steps:

in sunny days, the outer leaves of each section of an outdoor African reed branch are stripped off to expose buds, the buds are wiped with 75% ethanol, the stem sections are cut into small sections, each section is 2-3cm long, the small sections are placed in a bottle, and then the small sections are placed on a clean bench. Soaking in 75% ethanol for 30s, rinsing with sterile water for 3 times, and adding 0.1% HgCl ₂ Sterilizing the water solution for 10-15min, and washing with sterile water for 4-5 times;

inoculating the sterilized reed stem segments to a culture medium for inducing differentiation and rooting; the culture medium is 1/2MS culture medium, IBA1.5mg/L, NAA1.5mg/L, 3% white sugar and 4g/L plant gel; the temperature of the culture room is 25 soil and 2 ℃, the light intensity is 2000lx, and the illumination is 12h every day, so that the tissue culture seedling which can be transplanted is obtained.

The pH of the medium was 5.8, and the medium was sterilized in an autoclave at 121 ℃ for 20 min.

The specific operation is that when the root of the test-tube seedling is about 2cm long, the cap of the bottle is opened and peat soil is added to harden the seedling for one week when the sprout is more than 3cm long, and then the seedling can be transplanted and raised. If the plant is used as mother bottle, one bud can be cut and one section can be used as proliferation when the plant grows to more than 5-6 sections.

The invention has the advantages and beneficial effects that:

(1) the induced differentiation rooting culture medium is used, so that the conversion of different culture media in multiple steps and different processes is omitted.

(2) The use of the culture medium is simple and can reduce the cost of the basic culture medium by half.

(3) Can achieve the effect of proliferation without cytokinin.

The invention has the beneficial effects that: the method is simple and quick to operate, and the method for quickly, simply and efficiently obtaining the tissue culture seedlings of the African reed is summarized, the aim of saving the economic cost and quickly growing the seedlings is achieved by utilizing the differentiation rooting one-step seedling culture medium, the seedling growing process can be completed within more than 30 days, the defects of long time, low induction rate, easy pollution and the like in the prior art are overcome, and the needs of biological experiments on the African reed can be better met.

Drawings

FIG. 1 shows a budded stem section of one week or so of cultivation of African reed;

FIG. 2 shows a bottle of Versa Veitchii to be transplanted obtained by the method;

FIG. 3 is a diagram of a large bottle of African reed seedlings which can be used for proliferation and obtained by the method;

FIG. 4 shows the status of the seedlings of the African reed bottles transplanted into peat soil;

FIG. 5 cultivation of 8 months pot seedlings.

Detailed Description

The invention is further described with reference to the following figures and examples, which are provided for the purpose of illustrating the invention in a clear manner and are not to be construed as limiting the invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And it is expressly intended that all such modifications or alterations to the methods, procedures, or conditions of the present invention, as expressed herein, are contemplated as falling within the scope of the present invention.

The embodiment is as follows: the tissue culture seedling of the African reed is obtained by the method.

In sunny days, leaf skins are wrapped outside clean buds of reed branches collected outdoors, buds are exposed, the twigs are wiped by 75% ethanol, the branches are cut into small sections with bud lengths of 2-3cm, the small sections are placed in bottles, and then the bottles are placed on an ultraclean workbench. Soaking in 75% ethanol for 30s, rinsing with sterile water for 3 times, sterilizing with 0.1% HgCl2 water solution for 10-15min, and washing with sterile water for 4-5 times;

inoculating the sterilized reed stem segments to a culture medium for inducing differentiation and rooting; the induction culture medium is 1/2MS culture medium, IBA1.5mg/L, NAA1.5mg/L, 3% white sugar and 4g/L plant gel; culturing at 25-2 deg.C under light intensity of 2000lx for 12-16h daily to obtain tissue culture seedling (FIG. 1).

When the root of the test-tube plantlet is about 2cm long, the cap of the bottle is opened when the sprout is drawn to be more than 3cm long, and peat soil is added to the sprout for hardening the sprout for one week (figure 2), and then the sprout can be transplanted for seedling (figure 3). If the plant is used as mother bottle, one bud can be cut and one section can be used as proliferation when the plant grows to more than 5-6 sections.

The stem with the bud is cultured for 5 days in an induction way, the stem with the bud is inoculated into the culture medium, about one week, the stem with the bud shoots, the stem grows and roots, and a good growth situation is formed (figure 1). After thirty days of culture, the root system grows vigorously, and the seedlings grow to 7-10cm and grow well (figure 2). When the seedlings grow to be higher than 10cm, the seedlings do not move outwards; allowing it to grow upwards continuously, cutting and propagating (one section and one bud) (figure 3) bottle seedling and transplanting when the extraction length is up to 15cm in an effective time, adopting peat soil as matrix, and the survival rate can reach more than 98% (figure 4). After transplanting, the seedlings can be cultivated for 8 months, and can be planted in the ground for colonization (figure 5).

The variety is propagated in different modes under the condition of tissue culture compared with the conventional tissue culture method of the reed. The reed stalks of the variety are hard and take the node number as the multiplication coefficient; and the tissue culture propagation mode of other reed varieties adopts bud multiplication, so that the multiplication coefficient is lower. Meanwhile, frequent culture medium replacement is omitted in the propagation step, differentiation and rooting can be achieved in the same culture medium, browning can be well inhibited, adult tissue culture seedlings can be grown successively after 30 days, the rooting rate can reach 100%, and the transplanting survival rate can reach more than 98%.

The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.

Claims (1)

1. A method for rapidly obtaining tissue culture seedlings of African reeds is characterized in that induced differentiation and rooting are carried out to form seedlings in one step, stem nodes of reeds are used for culture, and adult-plant African reed tissue culture seedlings are rapidly propagated, and the method comprises the following steps:

exposing small buds of an outdoor reed branch collected from Africa on sunny days, removing outer leaves around the buds, wiping the surface of the branch by using a cotton ball stained with 75% ethanol, then cutting the stem section with the buds into 2cm, placing the stem section with the buds in a sterilizing bottle, and placing the stem section on a clean bench; soaking in 75% ethanol for 30s, rinsing with sterile water for 3 times, and adding 0.1% HgCl ₂ Sterilizing the water solution for 10-15min, and washing with sterile water for 4-5 times;

inoculating the sterilized reed stem segments to a culture medium for inducing differentiation and rooting; the culture temperature is 25 +/-2 ℃, the light intensity is 2000lx, and the daily illumination is 12 h; after each stem section with buds is cultured for 10 days, the first generation roots then bud, and the transplanted tissue culture seedlings can be obtained after the culture for 20-30 days;

the culture medium for inducing differentiation and rooting comprises the following formula:

1/2MS culture medium + IBA1.5mg/L + NAA1.5mg/L +3% white sugar +4g/L plant gel;

the pH of the culture medium is 5.8, and the culture medium is sterilized in an autoclave at 121 ℃ for 20 min;

when the root of the bottle seedling is 2cm long, opening the bottle cover when the bud seedling is more than 3cm long, adding peat soil to harden the seedling for one week, and transplanting and growing seedlings;

when the plant grows to 5-6 days, one bud is cut and one section is used as proliferation.

Images