CN114711143B

CN114711143B - Method for asexually and rapidly propagating Acer ginnala Maxim seedlings through tissue culture

Info

Publication number: CN114711143B
Application number: CN202210422027.4A
Authority: CN
Inventors: 由香玲; 种增斌; 赵曦阳; 陶雷; 曲冠证; 张世凯
Original assignee: Northeast Forestry University
Current assignee: Northeast Forestry University
Priority date: 2022-04-21
Filing date: 2022-04-21
Publication date: 2023-08-08
Anticipated expiration: 2042-04-21
Also published as: CN114711143A

Abstract

A method for asexually and rapidly propagating Acer ginnala Maxim seedlings through tissue culture belongs to the technical field of forestry biology. The main technical process is as follows: taking dormant buds of Acer ginnala Maxim in early spring as explants, germinating the dormant buds and differentiating adventitious buds in a DKW culture medium added with a plant growth regulator, and growing joints; the axillary buds of the joints are used as explants, the axillary buds are induced to germinate and grow, and the axillary buds growing the joints can be used as the explants again for germination and propagation. The terminal buds of the germinated axillary buds are induced to root in a 1/2DKM culture medium added with auxin plant growth regulator to form neat and uniform complete regenerated plants, and the regenerated plants are transplanted into soil for growth after seedling training and can be used for forestation. The regenerated Acer ginnala Maxim plant obtained by dormancy bud and axillary bud can inherit the characters of the female parent well, and the nursery stock produced in this way is more neat and stronger.

Description

Method for asexually and rapidly propagating Acer ginnala Maxim seedlings through tissue culture

Technical Field

The invention relates to a method for asexually and rapidly propagating Acer ginnala Maxim seedlings through tissue culture, and belongs to the technical field of forestry biology.

Background

Acer ginnala Maxim (Acer trifluorum Komarov.) is a genus Acer of Aceraceae, and is distributed in southeast of Heilongjiang, jilin, liaoning, etc., and also distributed in Korea; heilongjiang provinces are mainly distributed in Ningan, dongning, shangzhi, five-normal mountains (Zhou Yiliang, heilongjiang tree-like structures, 1992). The Acer ginnala Maxim has tidy crowns, beautiful leaf shapes and bright red leaves in late autumn, and is an excellent tree species for garden and scenic spot afforestation and pavement appreciation (Wang Yongjun and the like, acer ginnala Maxim seeding and cluster tree cultivation technology, 2017 (01): 69-70).

Along with the rapid development of modern landscape construction and economic tree species breeding in China, the demand for improved seedling of Acer ginnala Maxim is increasing. Under natural conditions, the acer ginnala Maxim has obvious irregular alternation phenomenon in abundant and defaults, the seed maturation rate is low, the seeds which are generally immature in year growth account for 30% -50% (Shan Chunlan and the like), and the acer ginnala Maxim sowing propagation technology is adopted for exploring, 2018 (08): 82-83). Moreover, because the pericarp has a certain thickness and hardness, the dormancy can be released only by low-temperature cellaring treatment for half a year, and the pericarp is used for artificial sowing (Shan Chunlan and the like, and the acer ginnala sowing propagation technology is adopted for exploring, 2018 (08): 82-83). These all result in limited production of Acer ginnala Maxim seedlings and longer seedling period. At present, the market demand cannot be completely met by means of sowing and seedling raising. The tissue culture technique is not affected by environmental, temperature and seasonal changes, and a large number of seedlings with excellent properties can be cultivated in a short time. Research on asexual rapid propagation of Acer ginnala Maxim tissue culture is not reported at home and abroad at present.

The invention mainly aims to provide a method for asexually and rapidly propagating Acer ginnala Maxim seedlings by a tissue culture mode, which not only can inherit the characters of female parents well, but also can realize rapid mass production of the seedlings, and provides technical support for rapid propagation and genetic improvement of good varieties.

Disclosure of Invention

In order to develop the color leaf tree species in northeast, the invention provides a method for asexually and rapidly propagating Acer ginnala Maxim seedlings by tissue culture. The specific contents are as follows: selecting branches of Acer ginnala Maxim without plant diseases and insect pests in late March, cutting off buds on the branches, placing the branches into a detergent solution for cleaning, washing for 2 hours with running water, placing the branches into an ultra-clean bench for conventional disinfection (5 min for disinfection by 75% alcohol and 12min for disinfection by 5% sodium hypochlorite), washing by sterile water for 5 times), removing outer scales and small leaves of the buds, cutting off small buds, inoculating the small buds into DKW culture medium containing 25g/L sucrose, 7.47g of Agar plant gel, different plant growth regulators (BA (0.1-1.0 mg/L), TDZ (0.01-0.1 mg/L), ZT (0.1-2.0 mg/L) and NAA (0.01-0.5 mg/L)) and inducing germination of the buds and proliferation of adventitious buds. After 4 weeks of culture, the buds germinate, and the best culture conditions for the proliferation of adventitious buds from the buds are: DKW medium+0.01 mg/LNAA+0.03mg/L TDZ+25g/L sucrose+7.47 g Agar plant gel, the culture conditions for better adventitious bud growth are: DKW medium+0.01 mg/LNAA+1.0mg/L ZT+25g/L sucrose+7.47 g Agar plant gel, and the adventitious bud grown has a node. The axillary buds of the adventitious bud are excised and used as explants, and inoculated with DKW culture medium containing 0.01mg/L NAA, 25g/L sucrose, 7.47g of Agar plant gel and ZT (0.1-2.0 mg/L) for culture, and the axillary buds are induced to germinate and grow. After 4 weeks the axillary buds germinate and grow, and suitable conditions are: DKW medium+0.01 mg/LNAA+1.0mg/L ZT+25g/L sucrose 7.47g Agar plant gel.

Cutting adventitious bud obtained by the induction and terminal bud of germinated axillary bud, with length of 2.0cm, inoculating into 1/2DKW containing IAA (0.5-1.5 mg/L), NAA (0.2 mg/L), IBA (0.5 mg/L), 20g/L sucrose and 7.47g/L Agar plant gel, and culturing for 4 weeks to induce bud rooting to form complete regenerated plant. The regenerated plants are subjected to soil transplanting after seedling training in a culture medium for 3 weeks, and soil matrixes must be subjected to high-pressure steam sterilization before transplanting. The seedling training is to open a rooting culture bottle cap, and put the bottle cap in a culture room to continue growing so as to lignify tender stems. The matrix components are humus soil, vermiculite and perlite in a ratio of 5:2:1.

The culture medium designed by the invention is conventionally treated: the pH value is adjusted to 5.8, and the mixture is sterilized under high pressure and then used; autoclaving means: sterilizing at 121 deg.C under 0.15MPa for 20min. Culture chamber conditions: the culture conditions are 25+ -1deg.C, 1000-1500Lx, 16h of light and 8h of dark.

Drawings

FIG. 1 germination of Acer ginnala Maxim buds

A is that Acer ginnala Maxim branches without plant diseases and insect pests are selected in a forest plant garden in Heilongjiang province in early spring;

b is the bud in A inoculated into culture conditions: DKW+0.03mg/L TDZ+0.01mg/L NAA+25 g/L sucrose+7.47 g Agar for 3 d;

c is the inoculation of the shoots in a to culture conditions: DKW medium+0.01 mg/LNAA+0.03mg/L TDZ+25g/L sucrose+7.47 g Agar plant gel for 4 weeks;

d is the inoculation of the shoots in A to culture conditions: DKW medium+0.01 mg/LNAA+1.0mg/L ZT+25g/L sucrose+7.47 g Agar plant gel for 4 weeks

FIG. 2 axillary bud germination and growth of joints

A is the axillary bud of the cut-off abutment bud in FIG. 1D;

b is the axillary bud in A under the following conditions: DKW culture medium+0.01 mg/LNAA+1.0mg/L ZT+25g/L sucrose+7.47 g Agar plant gel, germinating and growing in joints after 4 weeks culture

FIG. 3 rooting of shoots

A. B is the condition that young terminal buds are cultured for 4 weeks under the culture conditions of 1/2DKW+1.5mg/L IAA+20g/L sucrose+7.47 g/L Agar to root and form regenerated plants

FIG. 4 soil transplanting of regenerated plants

A. B, C the soil transplanting of the regenerated plants after 3 weeks of seedling training

Detailed Description

Example 1: germination of Acer ginnala Maxim buds

In early spring, selecting Acer ginnala Maxim branches without diseases and insect pests, placing the branches in a detergent solution for cleaning, washing for 2 hours with running water, and then placing in an ultra-clean bench for conventional disinfection as follows: 75% alcohol for 5min,5% sodium hypochlorite for 12min, and finally washing with sterilized water for 5 times, and finally sucking the surface moisture on sterile filter paper. The sterilized buds are stripped of external scales and leaflets, cut off small buds, inoculated into DKW medium containing BA (0.1-1.0 mg/L), TDZ (0.01-0.1 mg/L), ZT (0.1-2.0 mg/L), NAA (0.01-0.5 mg/L), 25g/L sucrose and 7.47g of Agar plant gel, and induced bud germination and adventitious bud proliferation. After 4 weeks of culture, the buds germinated, some had clumped adventitious buds produced, and some had joints grown. The statistical results are shown in table 1, from which it can be seen that: the germination and proliferation effects of ZT and TDZ on buds are better than those of BA, and the TDZ is more beneficial to the proliferation of adventitious buds, and the ZT is more beneficial to the growth of the proliferated adventitious buds. The best culture conditions for propagating adventitious buds from buds are: DKW medium+0.01 mg/LNAA+0.03mg/L TDZ+25g/L sucrose+7.47 g Agar plant gel, the culture conditions for better adventitious bud growth are: DKW medium+0.01 mg/LNAA+1.0mg/L ZT+25g/L sucrose+7.47 g Agar plant gel.

TABLE 1 Effect of plant growth regulator on germination of Acer ginnala Maxim bud in early spring and proliferation of adventitious bud

Note that the data were analyzed by multiple comparisons using SPSS software. "-" means absent; the same letter in the same column indicates that the difference is insignificant (P < 0.05).

The following is the same as

Example 2: acer ginnala Maxim axillary bud germination and growth and proliferation of extirpation

Axillary buds of adventitious bud with the node obtained in example 1 are excised as explants, inoculated into DKW medium containing ZT (0.1-2.0 mg/L), NAA (0.01 mg/L), sucrose (25 g/L) and Agar (7.47 g) at different concentrations, and cultured to induce axillary bud germination and growth. The germination and growth of axillary buds were counted after 4 weeks and the results are shown in Table 2. From the results, the DKW culture medium, 0.01mg/LNAA, 1.0mg/L ZT, 25g/L sucrose and 7.47g of Agar plant gel have better germination rate of the axillary buds, growth of the axillary buds and jointing condition. Each node can be used as an explant to continuously induce the germination and proliferation of the axillary buds. The method can obtain uniform and tidy strong buds.

TABLE 2 Acer ginnala Maxim axillary bud germination growth and proliferation

Example 3: rooting of shoots

The adventitious buds obtained in example 1 and the germinated axillary buds obtained in example 2 were excised, their terminal buds (2.0 cm in length), inoculated into 1/2DKW containing IAA (0.5-1.5), NAA (0.2), IBA (0.5), 20g/L sucrose and 7.47g/L Agar plant gel and cultured, and the buds were rooted after 4 weeks to form complete regenerated plants. The statistical results are shown in table 3, from which it can be seen: the culture conditions suitable for rooting are: 1/2DKW+1.5mg/L IAA+20g/L sucrose+7.47 g/L Agar plant gel.

TABLE 3 Effect of IAA, IBA, NAA on Acer ginnala Maxim bud rooting

Example 4: seedling hardening and soil transplanting of Acer ginnala Maxim regenerated plants

To increase the survival rate of the regenerated plants formed in example 3, the stems were lignified, and soil transplanting was performed after 3 weeks of seedling hardening in the flask. In order to reduce the germ infection, the soil must be sterilized at high temperature before transplanting. The soil components are humus soil, vermiculite and perlite in a ratio of 5:2:1. At the early stage of transplanting survival, the soil surface is sprayed with medicaments such as carbendazim and the like periodically every week, and the seedlings can be slowed down to once a month after growing.

Claims

1. A method for asexually and rapidly propagating Acer ginnala Maxim seedlings by tissue culture is characterized by comprising the following steps:

in early spring, selecting Acer ginnala Maxim branches without diseases and insect pests, placing the branches in a detergent solution for cleaning, washing for 2 hours with running water, and then placing in an ultra-clean bench for conventional disinfection as follows: 5min of 75% alcohol and 12min of 5% sodium hypochlorite, washing with sterilized water for 5 times, and finally sucking the surface water on sterile filter paper; removing external scales and lobules from the sterilized buds, cutting off small buds, inoculating the small buds into a culture medium consisting of DKW culture medium+0.01 mg/LNAA+0.03mg/L TDZ+25g/L sucrose+7.47 g agar plant gel, culturing for 4 weeks, germinating the buds, and generating clustered adventitious buds, wherein the adventitious buds grow with joints; the culture conditions for the adventitious bud growth joint are as follows: DKW medium+0.01 mg/L NAA+1.0mg/L ZT+25g/L sucrose+7.47 g agar plant gel;

taking the axillary buds of the adventitious bud for the jointing as an explant, inoculating the explant into a culture medium consisting of DKW culture medium+0.01 mg/LNAA+1.0mg/LZT+25g/L sucrose+7.47 g of agar plant gel, wherein the axillary buds can germinate and jointing to grow, and the jointed axillary buds can continue to be used as the explant for proliferation;

cutting the terminal buds of the obtained adventitious buds and terminal buds of axillary buds, inoculating the terminal buds and the terminal buds with the length of 2.0cm into a culture medium which is suitable for rooting of buds and consists of 1/2DKW culture medium+1.5 mg/L IAA+20g/L sucrose+7.47 g/L Agar plant gel, and culturing for four weeks to form complete regenerated plants;

performing soil transplanting after hardening seedlings in a culture medium for 3 weeks, and performing high-pressure steam sterilization on a soil matrix before transplanting;

the seedling hardening is to open a rooting culture bottle cap, and put in a culture room to continue growing so as to lignify tender stems;

the soil matrix comprises humus, vermiculite and perlite in a ratio of 5:2:1.