CN111149703B - Simple, convenient, efficient and high-quality papaya tissue culture seedling rooting method - Google Patents

Abstract

The invention belongs to the technical field of tissue culture seedling culture, and particularly relates to a rooting method for quickly propagating and culturing papaya fine seedlings based on a tissue culture technology. The papaya tissue culture seedling rooting method provided by the invention utilizes side buds of a plant as an explant, obtains a large number of differentiated buds after primary culture and multiplication culture respectively, can effectively promote root differentiation of the plant by a method of soaking cut base ends of the differentiated buds in rooting induction liquid containing indolebutyric acid, lanthanum chloride and phlorizin for rooting induction, and can be implemented by culturing the induced differentiated buds through a rooting matrix. The rooting method of the invention has the advantages that the rooting rate of the tissue culture seedlings can reach 100 percent, the rooting quantity is improved by 3-10 times, the average length of the root system is improved by 1-2 times, the seedling culture period is shortened by 10-20 days, the fibrous root is more, the root hair is developed, the root system can not be puffed and callized, the survival rate of the temporary planting reaches more than 98 percent, and the rooting method is suitable for culturing papaya seedlings.

Description

Simple, convenient, efficient and high-quality papaya tissue culture seedling rooting method

Technical Field

The invention belongs to the technical field of tissue culture seedling culture, and particularly relates to a rooting method for quickly propagating and culturing papaya fine seedlings based on a tissue culture technology.

Background

Papaya is also known as papaya, milk melon and papaya, belongs to evergreen soft woody large perennial herbaceous plants, is one of tropical famous fruits widely cultivated in tropical and subtropical regions, and is listed as the first ten fruits with the highest nutritional value by the world health organization. At present, the commercial planting of the papaya is mainly to carry out seedling propagation by using seeds, but the seedling performance of the papaya seeds is complex and the agronomic characters are seriously separated, so that the papaya seeds are not beneficial to modern standardized cultivation production.

Plant tissue culture, also called isolated culture, refers to a technique of separating desired tissues, organs or cells, protoplasts, etc. from a plant body, and culturing under an artificial control condition by aseptic manipulation to obtain regenerated whole plants or produce other products having economic value. The plant tissue culture technology is widely applied to the aspects of plant rapid propagation, genetic engineering improvement, excellent germplasm resource preservation and the like. A large amount of high-quality papaya seedlings can be efficiently obtained through a tissue culture technology, and popularization and utilization of papaya varieties with stable excellent agronomic characters and genetic characters are facilitated. However, the problems of difficult rooting, poor root system quality, complex operation, low efficiency, low transplanting survival rate and the like of the current papaya tissue culture seedlings seriously limit the production and popularization of papaya tissue culture seedlings in China.

The method for promoting the rooting of papaya tissue culture seedlings disclosed in papaya biotechnology comprises the following steps: adopting a culture medium of 1/2MS + KT0.2mg/L + IBA1.0mg/L + sucrose 2% + AC2g/L +5g/L agar for rooting culture to obtain a complete plant with roots. But the rooting effect in the actual production is not ideal, the deformed spongy roots with expanded bases can be generated, and the transplanting survival rate is not ideal. The papaya rooting method reported by Wangfang et al in papaya tissue culture seedling rooting medium and transplanting matrix screening is to inoculate the subculture seedling on 1/2MS + IBA0.4mg/L + carrageenan/agar traditional rooting medium to obtain complete plant. The rooting method greatly improves the rooting rate, but has the condition that partial roots are deformed and expanded and normal roots with fine and dense root hairs are few, thereby influencing the transplanting survival rate. A 'one-step rooting method', 'two-step rooting method' and a cutting rooting method are proposed in a 'Wanglou Master thesis' research on papaya tissue culture and rapid propagation technology system, wherein the 'one-step rooting' and the 'two-step rooting' are based on a traditional tissue culture medium formula MS + carrageenan/agar + hormone to root, and the generated roots all have an expanded abnormal sponge-shaped root system; the 'cutting rooting method' adopts the method that rootless seedlings are soaked in mixed liquid prepared by a root inducing agent and a bactericide and then are inserted into a matrix, the method is time-saving and labor-saving, but the death rate of the cutting method is extremely high, the reported death rate of the best scheme is over 50 percent, and the requirement of future large-scale production cannot be met. The rooting method provided by Zhangxiu spring published 'papaya' Tainong hybrid No. 2 'tissue culture technology' obtains normal rooting plants through two times of switching, and vermiculite is added into a culture medium for the second time of switching, which is also an improvement on the basis of the traditional culture medium in recent years, the obtained plants have less expanded and malformed roots, the normal root plants are increased, but the problems that the frequent switching of the plants can not only cause damage, increase the pollution rate but also increase the cost, and the method is very unfavorable for the subsequent large-scale production are solved.

Therefore, in modern large-scale papaya seedling production, the root promoting efficiency and the transplanting survival rate of the tissue culture seedlings are improved on the basis of ensuring the root system quality of the tissue culture seedlings, the seedling raising time is shortened, the production cost is reduced, and the method has important significance for promoting the saving and large-scale industrial production of papaya tissue culture seedlings in China.

Disclosure of Invention

Therefore, the technical problem to be solved by the invention is to provide a rooting method for quickly propagating and cultivating papaya excellent seedlings based on a tissue culture technology, so as to solve the problems of difficult rooting of tissue culture seedlings, complex technical operation, long seedling growing period and low efficiency in the papaya tissue culture quick propagation process in the prior art.

In order to solve the technical problems, the simple, convenient, efficient and high-quality papaya tissue culture seedling rooting method comprises the following steps:

(1) selecting proper papaya lateral buds as explants, and performing disinfection and sterilization treatment on the selected papaya lateral buds for later use;

(2) carrying out primary culture and multiplication culture on the treated explants to obtain a large number of papaya differentiated buds;

(3) soaking the papaya differentiated bud in rooting inducing liquid for adventitious root induction, and transplanting the papaya differentiated bud into a rooting substrate for rooting cultivation;

the rooting inducing liquid is a mixed solution containing indolebutyric acid with the mass concentration of 0.2-5g/L, lanthanum chloride with the mass concentration of 0.1-1mg/L and phlorizin with the mass concentration of 10-80 mg/L.

Specifically, in the step (3), the adventitious root induction step is to soak the stem base part of the mature tissue culture seedling differentiated bud with 5 to 8 leaves in a rooting induction liquid for adventitious root induction.

Specifically, in the step (3), the rooting matrix comprises a mixed solution of a solid matrix and a nutrient solution, and the mass ratio of the solid matrix to the volume ratio of the nutrient solution is 1-3: 1 g/mL.

Specifically, the solid matrix comprises the following components in a mass ratio of 1-2: 1-2: 1-2 peat, perlite and zeolite.

Specifically, the nutrient solution comprises the following components: 1/2MS +40-60mg/L choline chloride +25-35g/L sucrose, pH 5.5-6.0.

Specifically, in the step (3), the rooting cultivation step is to insert the transplanted leaves of the differentiated buds in the rooting matrix in an inverted manner, so that the cut at the base of the stem segment of the differentiated bud is suspended for cultivation;

the rooting culture step is carried out under the conditions of 28-30 ℃ and daily illumination culture for 12-14h, wherein the illumination intensity is 12000-15000 lx.

Specifically, in the step (1), the explant is obtained by selecting a side bud which is germinated after a single plant with good field age is cut and dried, and cutting 2-3cm from the top of the single plant after leaves and petioles are removed.

Specifically, in the step (1), the sterilization and disinfection step includes: soaking in 300mg/L rifampicin for sterilizing for 1 hr, sterilizing with 75% alcohol for 30s, sterilizing with 0.1% mercuric chloride solution for 7-8min, and washing with sterile water for 3 times.

Specifically, in the step (2), the primary culture step includes a step of inoculating a top tender part with 2-3 bud sites into a pre-culture medium for culture;

the pre-culture medium comprises the following components: MS +0.1-0.2 mg/L6-BA +0.2-0.3mg/LKT +0.4-0.5mg/L IAA +25-35g/L sucrose +5-10g/L agar, pH 5.5-6.0;

the primary culture step is carried out under the condition of 25-30 ℃ for 10-14h each day under illumination, the illumination intensity is controlled to be 4000-.

Specifically, in the step (2), the proliferation culture step comprises the steps of intercepting explant stems which germinate in the primary culture, and placing the explant stems in a proliferation culture medium for proliferation culture;

the components of the proliferation culture substrate comprise: MS +0.1-0.3 mg/L6-BA +0.2-0.3mg/L KT +0.1-0.2mg/L NAA +0.1-0.2mg/L IBA +25-35g/L sucrose +5-10g/L agar, pH 5.5-6.0;

the biological conditions of the proliferation culture step are that the daily illumination culture is 10-14h, the illumination intensity is 7000-9000lx, the subculture period is 25-30d, and a large amount of clustered adventitious buds can be generated after 8-10 subculture periods.

The invention also discloses a papaya tissue culture seedling method, which specifically comprises the steps of rooting and cultivating according to the method, and transplanting the rooted tissue culture seedling and carrying out conventional cultivation.

The simple, convenient, efficient and high-quality papaya tissue culture seedling rooting method provided by the invention is characterized in that papaya seedlings with excellent agronomic characters are cultured by a tissue culture and rapid propagation technology, side buds of a plant are used as explants, a large number of differentiated buds are obtained after primary culture and multiplication culture, the cut base ends of the differentiated buds are soaked in a rooting induction solution containing indolebutyric acid, lanthanum chloride and phlorizin for rooting induction, so that the root differentiation of the plant can be effectively promoted, and the induced differentiated buds are cultured by a rooting matrix and can be transplanted and cultured to grow into normal plantlets. Compared with the traditional rooting technology, the rooting method can improve the rooting rate of the tissue culture seedlings from 70-95% to 100%, improve the rooting number by 3-10 times, improve the average length of root systems by 1-2 times, shorten the seedling culture period by 10-20 days, have more fibrous roots and developed root hairs, avoid the expansion and callus phenomena of the root systems, effectively solve the problems of difficult rooting of the tissue culture seedlings, complicated technical operation, long seedling culture period and low efficiency in the existing tissue culture rapid propagation process of the papaya, and is suitable for the cultivation of the papaya seedlings.

The simple, convenient, efficient and high-quality papaya tissue culture seedling rooting method is characterized in that the rooting substrate is preferably improved solid substrate comprising turf, perlite and zeolite, and is formed by mixing MS culture medium added with choline chloride, 7-50 normal roots with root hairs can grow from the healing part of the cut base end of the stem section of the differentiated bud after the differentiated bud is cultured for 7-8 days by illumination, most of the roots can grow to 2-7cm after the differentiated bud is cultured for 21 days conventionally, the rooting rate, the number, the length and the quality of explants can be effectively improved, the expansion and callus of the roots can be avoided, the rooting time and the seedling period can be shortened, and the seedling raising efficiency can be greatly improved.

Detailed Description

Example 1

The rooting method of the papaya tissue culture seedling in the embodiment specifically comprises the following steps:

(1) selecting a variety of 'tassel yellow' papaya, selecting adult bearing trees of the papaya with excellent comprehensive agronomic characters according to the fruiting characters, disease resistance, stress resistance and strain performance, selecting individual plants with excellent field age in sunny or cloudy weather, cutting off 80-100cm of plant stems, culturing for 20-30 days to obtain strong lateral buds after germination and growth of latent buds, removing leaves and leaf stalks, and cutting off 2-3cm of top ends to serve as explants; sterilizing selected explants by 300mg/L rifampicin, then sterilizing the explants for 30s by 75% alcohol, then sterilizing the explants for 7min by 0.1% mercuric chloride solution, and washing the explants for at least 3 times by sterile water for later use;

(2) inoculating the top tender part with 2-3 bud positions of the sterilized explant into a pre-culture medium for primary culture, performing illumination culture for 12h every day at 28-30 ℃, wherein the illumination intensity is 5000lx, performing culture for 25-28d, and observing the pollution condition and the budding condition of the explant culture;

the pre-culture medium comprises the following components: MS +6-BA 0.2mg/L + KT 0.3mg/L + IAA0.5mg/L + sucrose 30g/L + agar 7g/L, pH5.8;

intercepting buds of explants after primary culture, inoculating the buds of the explants to a proliferation culture medium for proliferation culture of differentiation buds, performing illumination culture for 12h every day at the temperature of 28 ℃, controlling the illumination intensity to be 8000lx, continuously culturing for 25-30d as a subculture period, and culturing for 8-10 subculture periods to generate a large amount of cluster adventitious buds;

the components of the proliferation culture substrate comprise: MS +6-BA 0.2mg/L + KT 0.3mg/L + NAA0.1mg/L + IBA 0.1mg/L + sucrose 30g/L + agar 7g/L, pH5.8;

(3) after proliferation culture, selecting a single adventitious bud with 5-8 leaves and relatively mature, soaking the cut of the stem end of the single adventitious bud in a rooting induction liquid (containing 1g/L of indolebutyric acid, 0.1mg/L of lanthanum chloride and 10mg/L of phlorizin) for 2min for adventitious root induction so as to promote the differentiation of a root system;

after induction by the induction liquid, inserting the stem base of the tissue culture seedling into a rooting substrate which is sterilized at high temperature, and performing illumination culture for 12 hours at 28-30 ℃ every day with the illumination intensity of 12000-; the rooting medium comprises 100g of solid matrix and 50mL of nutrient solution; wherein the solid matrix comprises a mixture of a solid matrix and a solid matrix, and the solid matrix comprises the following components in a mass ratio of 1: 1: 1 peat, perlite and zeolite; the nutrient solution comprises the following components: 1/2MS + choline chloride 50mg/L + sucrose 30g/L, pH5.8.

After rooting culture is carried out for 7-8 days according to the method, the root tip can be seen to grow out from the healing position of the stem cut of the tissue culture seedling, most of root systems grow for 2-7cm after 21 days of culture, the number of the root systems is 7-35, and the root systems can be transplanted to a substrate culture medium and grow into normal plantlets.

Example 2

The rooting method of the present embodiment is the same as that of embodiment 1, and the difference is that the rooting inducing solution comprises the following components: 2g/L indolebutyric acid, 0.25mg/L lanthanum chloride and 20mg/L phlorizin.

Example 3

The rooting method of the present embodiment is the same as that of embodiment 1, and the difference is that the rooting inducing solution comprises the following components: 3g/L indolebutyric acid, 0.5mg/L lanthanum chloride and 40mg/L phlorizin.

Example 4

The rooting method of the embodiment is the same as that of embodiment 1, and is only different in that a papaya variety of 'one square melon' is selected, and the rooting inducing liquid comprises the following components: 3g/L indolebutyric acid, 0.5mg/L lanthanum chloride and 40mg/L phlorizin.

Example 5

The rooting method of the embodiment is the same as that of embodiment 1, and is only different in that a papaya variety of 'daily liter' is selected, and the rooting inducing solution comprises the following components: 3g/L indolebutyric acid, 0.5mg/L lanthanum chloride and 40mg/L phlorizin.

Comparative example 1

The rooting method in the comparative example is the same as that in example 1, and the difference is that the rooting matrix consists of: 1/2MS + IBA 2mg/L + sucrose 30g/L + agar 7g/L, pH5.8, the transplanting method is to insert the stem segment of the tissue culture seedling into the rooting culture medium for rooting.

Comparative example 2

The rooting method in the comparative example is the same as that in example 4, and the difference is that the rooting matrix consists of: 1/2MS + IBA 2mg/L + sucrose 30g/L + agar 7g/L, pH5.8, the transplanting method is to insert the stem segment of the tissue culture seedling into the rooting culture medium for rooting.

Comparative example 3

The rooting method in the comparative example is the same as that in example 5, and the difference is that the rooting matrix consists of: 1/2MS + IBA 2mg/L + sucrose 30g/L + agar 7g/L, pH5.8, the transplanting method is to insert the suspended leaves of the differentiated bud stem into the rooting culture medium for rooting.

Comparative example 4

The rooting method in the comparative example is the same as that in comparative example 1, and is different in that the base of the stem segment of the tissue culture seedling is inserted into the rooting culture medium for rooting.

Comparative example 5

The rooting method in the comparative example is the same as that in the comparative example 2, and is different in that the base of the stem segment of the tissue culture seedling is inserted into the rooting culture medium for rooting.

Comparative example 6

The rooting method in the comparative example is the same as that in the comparative example 3, and is different in that the base of the stem segment of the tissue culture seedling is inserted into the rooting culture medium for rooting.

Comparative example 7

The rooting method in the comparative example is the same as that in example 1, and is different in that sterile water is used to replace rooting inducing liquid to soak the stem cut of the tissue culture seedling.

Comparative example 8

The rooting method of the comparative example is the same as that of example 1, and is only different in that sterile water is used for replacing rooting inducing liquid to soak the stem section cut of the tissue culture seedling, and the rooting matrix comprises the following components: 1/2MS + IBA 2mg/L + sucrose 30g/L + agar 7g/L, pH5.8.

Comparative example 9

The rooting method of the comparative example is the same as that of example 1, and is different from the rooting inducing solution only in that the rooting inducing solution does not contain lanthanum chloride.

Comparative example 10

The rooting method of the comparative example is the same as that of example 1, and is different only in that the rooting inducing solution does not contain phlorizin.

Comparative example 11

The rooting method of this example is the same as example 1 except that the rooting matrix does not contain choline chloride.

Comparative example 12

The rooting method in the comparative example is the same as that in example 1, and is only different in that the rooting matrix comprises the following components: 1/2MS + choline chloride 50mg/L + sucrose 30g/L + agar 7g/L, pH5.8.

Examples of the experiments

The rooting was measured and recorded for the methods of examples 1-5 and comparative examples 1-9, respectively, and the results are reported in table 1 below.

TABLE 1 rooting of papaya tissue culture seedlings in different treatments

As can be seen from the data in the table above, compared with the known rooting technology (comparative examples 1-6) in the prior art, the rooting method disclosed by the invention can improve the rooting rate of the tissue culture seedling from about 70% to 100%, improve the rooting quantity by 3-10 times, improve the average length of the root system by 1-2 times, shorten the seedling culture period by 10-20d, have more fibrous roots and developed root hairs, avoid the swelling and callus phenomena of the root system, achieve the temporary planting survival rate of more than 98%, and effectively solve the problems of difficult rooting of the tissue culture seedling, complex technical operation, long seedling culture period and low efficiency in the conventional tissue culture and rapid propagation process of papaya.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. 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 obvious variations or modifications therefrom are within the scope of the invention.

Claims (5)

1. A simple, convenient, efficient and high-quality papaya tissue culture seedling rooting method is characterized by comprising the following steps:

(1) selecting proper papaya lateral buds as explants, and performing disinfection and sterilization treatment on the selected papaya lateral buds for later use;

(2) carrying out primary culture and multiplication culture on the treated explants to obtain a large number of papaya differentiated buds;

(3) soaking the papaya differentiated bud in rooting inducing liquid for adventitious root induction, and transplanting the papaya differentiated bud into a rooting substrate for rooting cultivation;

the rooting inducing liquid consists of a mixed solution of indolebutyric acid with the mass concentration of 0.2-5g/L, lanthanum chloride with the mass concentration of 0.1-1mg/L and phlorizin with the mass concentration of 10-80 mg/L;

the adventitious root induction step is to select the stem base part of an aged tissue culture seedling differentiation bud with 5 to 8 leaves to be soaked in a rooting induction liquid for adventitious root induction;

the rooting matrix is a mixed solution of a solid matrix and a nutrient solution, and the mass ratio of the solid matrix to the volume ratio of the nutrient solution is (1-3): 1 g/mL; the solid matrix comprises the following components in a mass ratio of 1-2: 1-2: 1-2 peat, perlite and zeolite; the nutrient solution comprises the following components: 1/2MS +40-60mg/L choline chloride +25-35g/L sucrose, pH5.5-6.0;

the rooting cultivation step is to insert the base part of the stem section of the tissue culture seedling into a rooting matrix which is sterilized at high temperature;

the rooting culture step is carried out under the conditions of 28-30 ℃ and daily illumination culture for 12-14h, wherein the illumination intensity is 12000-15000 lx.

2. The simple, efficient and high-quality rooting method for papaya tissue culture seedlings according to claim 1 is characterized in that in the step (1), the explant is a lateral bud which is germinated after a single plant with good field age is selected and cut, and 2-3cm of the top end of the single plant is cut to be used as the explant after leaves and petioles are removed.

3. The simple, efficient and high-quality rooting method for papaya tissue culture seedlings according to claim 1, wherein in the step (2), the primary culture step comprises the step of inoculating a top tender part with 2-3 bud sites into a pre-culture medium for culture;

the pre-culture medium comprises the following components: MS +0.1-0.2 mg/L6-BA +0.2-0.3mg/L KT +0.4-0.5mg/L IAA +25-35g/L sucrose +5-10g/L agar, pH 5.5-6.0;

the primary culture step is carried out under the condition of 25-30 ℃ for 10-14h each day under illumination, the illumination intensity is controlled to be 4000-.

4. The simple, efficient and high-quality rooting method for the papaya tissue culture seedlings as claimed in claim 1, wherein in the step (2), the proliferation culture step comprises the steps of intercepting explant stems sprouting in primary culture, and placing the explant stems in a proliferation culture medium for proliferation culture;

the proliferation culture medium comprises the following components: MS +0.1-0.3 mg/L6-BA +0.2-0.3mg/L KT +0.1-0.2mg/L NAA +0.1-0.2mg/L IBA +25-35g/L sucrose +5-10g/L agar, pH 5.5-6.0;

the biological conditions of the proliferation culture step are that the daily illumination culture is 10-14h, the illumination intensity is 7000-9000lx, the subculture period is 25-30d, and a large amount of clustered adventitious buds can be generated after 8-10 subculture periods.

5. A method for tissue culture of papaya seedlings, characterized in that it comprises a step of rooting culture according to any one of claims 1 to 4, and a step of transplanting the rooted tissue culture seedlings and performing conventional culture.