CN114711145B

CN114711145B - Tissue culture method of potentilla glandulifera

Info

Publication number: CN114711145B
Application number: CN202210505299.0A
Authority: CN
Inventors: 董丽; 张浩然; 范舒欣; 郝培尧; 赵琳; 郑亦卿; 张启翔; 程堂仁; 王佳
Original assignee: Beijing Forestry University
Current assignee: Beijing Forestry University
Priority date: 2022-05-10
Filing date: 2022-05-10
Publication date: 2022-11-18
Anticipated expiration: 2042-05-10
Also published as: CN114711145A

Abstract

The invention provides a tissue culture method of Potentilla glandulifera, which is characterized by comprising the following steps: young and tender leaves are taken as explants, the surface of the young and tender leaves is sterilized and then inoculated in a callus induction culture medium to induce callus, the induced callus is inoculated in an adventitious bud differentiation culture medium to differentiate adventitious buds, wherein the callus induction culture medium is MS + NAA 0.1-05 mg/L + TDZ 0.1-1.0 mg/L, and the adventitious bud differentiation culture medium is MS + NAA0.5mg/L + TDZ 0.1-1.0 mg/L. According to the invention, the hormone combination of NAA + TDZ is applied in the callus induction stage of tissue culture of the Potentilla plant for the first time, the induction method can enable the induction rate of the leaf callus of Potentilla adeniana to reach more than 90.00%, the callus growth state is good, and obvious bud points can be seen on the surface in the later culture stage. The callus differentiation culture medium can lead the differentiation rate to reach 46.67 percent, lead the adventitious bud to grow robustly and lay a good foundation for the successful tissue culture of the potentilla glandulifera.

Description

Tissue culture method of potentilla glandulifera

Technical Field

The invention belongs to the field of plant tissue culture, and particularly relates to a tissue culture method of potentilla adenanthera.

Background

Beijing is in North China plain, belongs to a typical northern temperate continental monsoon climate, is hot and rainy in summer and cold and dry in winter, so that alternative materials for the rural greening ground cover are limited, and potentilla plants are considered to be an ideal Beijing greening rural greening ground cover. Relevant researchers carry out field investigation on various garden green lands in counties and regions and urban areas in Beijing area to find that the Potentilla plants show good ecological benefit and landscape effect in garden application, but the applied Potentilla plants are single and mainly comprise Potentilla stolonifera, potentilla anserine and Potentilla fraxinifolia. Zhao Fanyu 2016, the research on Potentilla plant resources in Beijing and its surrounding areas began to be carried out in 2016, and it was ascertained that 20 species (varieties) of Potentilla plants in Beijing and its surrounding areas account for about 1/4 of the Potentilla plants in China, but most of the plant resources are in a wild state. The potentilla is used as a local cover plant and has great development potential, and the researches of predecessors find that the potentilla still has good ornamental value in the drought or insufficient illumination environment, but the systematic researches in the aspect of propagation technology limit the popularization and application of the potentilla in landscaping.

The penchant finds out through consulting the literature that the current research on the propagation technology of the potentilla plants shows 2 singleness: one is to study single species and the other is to study single mode. There are three or more existing documents on the research of Potentilla plant propagation techniques, of which only twelve species have been explored and show a high degree of coincidence in species selection, such as Potentilla sericea Mao Pufu, potentilla anserine, and Potentilla anserine stock appearing many times. Regarding the tissue culture technology, the prior literature only searches about 6 kinds of potentilla including potentilla stock, and the selection of explants mostly utilizes stem tips and stem segments; the disinfectant adopts mercuric chloride with different concentrations; in the induction mode, the stem tip is usually used for directly inducing to generate adventitious buds; in the callus induction stage, 6BA +2.4D is a common combination; in the callus differentiation stage, 6BA + NAA or ZT + NAA is adopted.

The green period of Potentilla adenanthera is more than 220, effective ground surface coverage can be formed in more than 60% of the whole year, the flowering period of Potentilla adenanthera is concentrated in 4-5 months in spring, the flowers are yellow, bright and delicate, and the ornamental value of the lower layer of a spring plant community can be effectively enriched; on the aspect of garden application potential, the results of previous researches show that potentilla adenantha is usually grown in a half-shade environment with trees and shrubs on the upper layer and can be used as an excellent plant material for roadside afforestation.

However, no relevant research aiming at the potentilla glandulifera tissue culture technology exists at present, and compared with the traditional propagation mode, the tissue culture method has the outstanding advantages of less required raw materials, short growth cycle, large propagation coefficient, artificially controllable culture conditions, isolation of natural condition interference and the like, can culture a large number of high-quality seedlings with consistent properties in a short time, and has great significance for the development of local cover plants. As a plant with wide application prospect in the Potentilla, the method is considered to provide theoretical basis and technical support for large-scale production of Potentilla adenantherae and lay a solid foundation for garden application.

Disclosure of Invention

In order to solve the above problems, the present application aims to provide a tissue culture method of Potentilla tormentosa.

The tissue culture method of Potentilla glandulifera provided by the invention comprises the following steps: young and tender leaves are taken as explants, the surface of the young and tender leaves is sterilized and then inoculated in a callus induction culture medium to induce callus, the induced callus is inoculated in an adventitious bud differentiation culture medium to differentiate adventitious buds, wherein the callus induction culture medium is MS + NAA 0.1-1.0 mg/L + TDZ1.0 mg/L, and the adventitious bud differentiation culture medium is MS + NAA0.5mg/L + TDZ 0.1-1.0 mg/L.

Preferably, the callus induction medium is one of: MS + NAA0.1mg/L + TDZ0.1 mg/L, MS + NAA0.5mg/L + TDZ0.1 mg/L, MS + NAA0.1mg/L + TDZ0.5mg/L, MS + NAA0.5mg/L + TDZ0.5mg/L, MS + NAA0.1mg/L + TDZ1.0 mg/L, MS + NAA0.5mg/L + TDZ1.0 mg/L.

More preferably, the callus induction medium is MS + NAA0.5mg/L + TDZ1.0 mg/L.

Preferably, the adventitious bud differentiation medium is: MS + NAA0.5mg/L + TDZ0.5mg/L, MS + NAA0.5mg/L + TDZ1.0 mg/L

More preferably, the adventitious bud differentiation medium is: MS + NAA0.5mg/L + TDZ1.0 mg/L.

In a specific embodiment of the invention, the explant is surface sterilized by the following surface sterilization method: cutting young and tender leaves, soaking in liquid detergent for 1-5min, washing under running water for 30-60min, placing on a clean bench, sterilizing with 75% alcohol solution for 35-120s, pouring off alcohol, washing with sterile water for 3-4 times, and soaking with 2% sodium hypochlorite solution for 5-10 min.

According to the invention, the potentilla glandulifera leaves are used as the explants, and the explants are sterilized by using alcohol and sodium hypochlorite, so that the method has the advantages of convenience in material acquisition, economy, practicability, environmental friendliness and high culture efficiency.

According to the method, the hormone combination of NAA and TDZ is applied to the callus induction stage of tissue culture of the Potentilla plant for the first time, the induction method can enable the callus induction rate of the Potentilla adenanthera leaves to reach more than 90.00%, the callus growth state is good, and obvious bud points can be seen on the surface at the later stage of culture.

The callus differentiation culture medium can lead the differentiation rate to reach 46.67 percent, lead the adventitious bud to grow robustly and lay a good foundation for the successful tissue culture of the potentilla glandulifera.

Drawings

FIG. 1 is a photograph showing the callus induced from the leaves of Potentilla tormentosa as in example 2. A: milky white callus produced by the induction of potentilla glandulifera leaves; b: green callus induced by potentilla glandulifera leaves; c: and (3) differentiation of callus of potentilla glandulifera leaves to obtain the bud points.

FIG. 2 is a photograph showing adventitious buds formed by callus differentiation in example 3. A. B: adventitious buds differentiated from the potentilla glandulifera callus; c: potentilla glandulifera callus which is not differentiated to form adventitious buds and begins to brown.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1 explant Disinfection

Taking healthy Potentilla glandulifera plants as test materials, shearing young leaves, soaking in detergent water for 1min, washing under running water for 30min, putting into a clean bench, disinfecting with 75% alcohol solution for 1min, pouring off alcohol, washing with sterile water for 3-4 times, soaking with 2% and 4% sodium hypochlorite solutions for several minutes (the specific time is shown in table 3.1), shaking continuously during the period to make explants fully contact with the solution, then washing with sterile water for 5-6 times, and sucking surface water with sterile filter paper. Cutting leaves into leaf disks with 0.5 x 0.5mm, inoculating the leaf disks on MS +6-BA0.5mg/L +2,4-D0.5mg/L culture media, inoculating 30 explants for each treatment, repeating the steps for 3 times, respectively counting the pollution rate, the death rate, the callus induction rate and the like of different disinfection schemes after 3 weeks, and screening out the optimal disinfection scheme of the explants.

TABLE 1 method for Disinfection of explants

Different disinfection modes have different sterilization effects on the leaves of the potentilla glandulifera, and certain influence is generated on the state of the callus. As can be seen from Table 2, different disinfection methods have significant effects on the contamination rate, mortality rate and callus induction rate of Potentilla glandulifera leaves. When the concentration of sodium hypochlorite is 2%, the death rate of leaves is gradually increased and the callus induction rate is gradually reduced along with the prolonging of the sterilization time. 2% sodium hypochlorite is used for 5min, the death rate of the leaves is the lowest and is only 13.33%, and compared with the death rates of 8min and 10min, the death rates are respectively reduced by 26.67% and 60.0%; the callus induction rate of the leaf is the highest and reaches 86.67%, and compared with the callus induction rates of 8min and 10min treatment, the callus induction rates of the leaf are respectively improved by 40.00% and 60.00%, and the differences are obvious (P is less than 0.05). The result shows that 2% and 4% sodium hypochlorite can achieve good sterilization effect when treated with sodium hypochlorite with different concentrations for the same time, the pollution rate is 0.00%, but the higher concentration sodium hypochlorite can kill microbes on the surface of the explant and cause serious damage to plant cells, so that the death rate of the explant is obviously improved to 60.00%, and adverse effect is generated on callus induction, the callus induction rate of the leaf treated with the concentration is 40.00%, and is reduced by 46.67% compared with the treatment with the 2% sodium hypochlorite, the significant difference (P < 0.05) is achieved, the induced callus is dark in color, small in volume and not strong in vitality, and the leaf can not be used for subsequent research. Therefore, for the leaves of Potentilla tormentosa, treatment with 75% alcohol for 1min +2% sodium hypochlorite for 5min is the best mode of disinfection.

TABLE 2 Effect of different sterilization regimes on explant sterilization

Example 2 callus induction

The sterilized explants were inoculated into MS minimal medium supplemented with different concentrations of 6BA, 2,4D, NAA and TDZ, 30 explants per treatment, 3 replicates. And after 30 days, counting the induction rate of the explant callus, observing and recording the growth state and color of the callus, wherein the culture medium for inducing the callus is shown in a table 3.

TABLE 3 callus induction Medium

As can be seen from Table 4, the ratio of hormones in the culture medium was different, and the callus induction rate and callus state of the corresponding Potentilla glandulifera leaves were significantly different. The results show that when a single cytokinin 6-BA is added to the culture medium, the leaf can not generate callus regardless of the concentration of the 6-BA.

The difference of the callus induction rate of the potentilla glandulifera leaves is obvious (P is less than 0.05) on various culture media with different 6-BA and 2.4D mass concentrations. The mass concentration of 6-BA is 0.5mg/L, when different concentrations of 2.4D are added, the leaf can be successfully induced to generate callus, and the callus induction rate of the leaf shows a trend of continuous reduction along with the continuous increase of the concentration of 2.4D, wherein the callus induction rate is the highest A4 and reaches 100.00 percent, and the callus induction rate is the lowest A8 and is only 30.00 percent. The mass concentration of 6-BA is increased to 1.0mg/L, when different concentrations of 2.4D are added, the callus induction effect of the leaf is poor, except A9 and A12, the callus does not occur under other treatments, the leaf is dead finally, the induction rate of the treated A9 callus is the highest in the same group, reaches 83.33 percent and is improved by 73.33 percent compared with A12, the difference between the induction rate and the induction rate is obvious (P is less than 0.05), but compared with the treated A4-A8, the induction rate of the callus is greatly reduced, and the volume of the callus is obviously reduced. The above indicates that higher concentrations of 6-BA may inhibit callus production from Potentilla glandulosa leaves.

6-BA and NAA with different mass concentrations are added into a culture medium, the potentilla glandulifera leaves can be induced to generate callus, but the induction rate of the callus is obviously different (P is less than 0.05), experiments show that the healing time under the matching of 6-BA and NAA is averagely delayed by about 5d compared with other treatments, 14d after inoculation, most leaves are almost unchanged except for deepening the color of a few leaves and curling the leaf margin, and the callus commonly occurs after 20d inoculation, but compared with A4-A8 treatment, the callus has small volume, is white green granules and has compact texture. When 6-BA (0.5 and 1.0 mg/L) with a certain mass concentration is added, the induction rate of the leaf tends to rise continuously along with the increase of the concentration of the NAA. In the treatments A14-A19, the induction rate of the leaf callus is highest by adding 0.5/1.0mg/L6-BA +1.0mg/L NAA (A16, A19), and is 43.33 percent, which is obviously higher than that of the callus under other treatments. When the NAA concentration is the same, the callus induction rate is lower as the 6-BA concentration is higher, and the callus induction rates of the A17 and A18 treated by the method are respectively reduced by 6.67 percent and 10.00 percent compared with those of the A14 and A15 treated by the method. The results show that NAA may have a certain promotion effect on the callus induction of Potentilla tormentosa leaves, and the higher the concentration is, the more obvious the promotion effect is; the other side also reflects that higher concentration of 6-BA adversely affects callus induction.

After the Potentilla adenanthera leaves are inoculated on NAA and TDZ culture media with different mass concentrations to be cultured for 7D, the leaves are deepened and curled, the callus at the wound of the 14D back leaves is the initial callus, the color is mainly green or white green, the surface is glossy, the volume of the callus is increased by about 20D, the callus is different from loose milky-white lumpy callus (shown in a figure 1-A) induced in the culture media with 6-BA and 2.4D, the callus induced in the culture media with NAA and TDZ is compact green callus (shown in a figure 1-B), and green bud points (shown in a figure 1-C) are visible on the surface of the callus after subculture in the later period, which shows that the callus induced by the hormone combination of NAA and TDZ has stronger organ regeneration capacity. The induction rate of the callus tissues treated by the A20-A25 is in the range of 60.00% -100.00%, wherein the highest induction rate is A21 and is 100.00%, and secondly, the A25 is treated, the induction rate of the callus tissues is 90.00%, and no significant difference exists among 2 treatments (P > 0.05). From the growth state of the callus tissues, the callus tissues in the A25 are not only uniformly distributed and compact in structure, but also have obvious surface bud points at the later stage of culture, so that the callus tissues in the culture medium have stronger differentiation capability.

In conclusion, the optimal culture medium for inducing the Potentilla tormentosa leaves to generate the callus is MS + NAA0.5mg/L + TDZ1.0 mg/L.

TABLE 4 influence of different hormone combinations on the induction of Potentilla adenlandi leaf callus

Note: data are mean ± sem; the lower case letters in the same column indicate significant differences (P < 0.05).

EXAMPLE 3 callus differentiation

The calli that grew vigorously were selected and transferred to a differentiation medium, the medium ratio is shown in table 5. Each treatment was inoculated with 10 calli, 3 replicates. And after 30d, counting the differentiation rate of the callus, observing and recording the growth state and color of the callus, and recording the growth state of the buds if the buds occur.

TABLE 5 callus differentiation Medium

The callus with good state induced by the leaves of Potentilla tormentilla is inoculated on a differentiation medium, the statistical result after 30 days is shown in table 3.6, the data in the table shows that different hormone ratios have important influence on the callus differentiation of Potentilla tormentilla, only the callus in 4 culture media in 18 differentiation culture media has adventitious buds differentiated, and the callus in the other 14 culture media has browning death (see figure 2-C).

As can be seen from Table 6, the combination of 6BA and NAA did not induce the differentiation of adventitious buds in Potentilla adeniana callus, and the differentiation rates of the F1-F8 callus were all 0.00%. The browning rate of the callus under each treatment is different greatly, and the browning rates of the calluses of F2, F7 and F8 are the highest and reach 100.00 percent, which is obviously higher than that of other treatments (P is less than 0.05) in the same group. It was observed that when the callus was inoculated on these 3 kinds of media, the volume of the callus did not change with the lapse of the culture time, the lower part of the callus in contact with the media gradually browned, the upper part of the callus turned yellow and dried, and finally the whole callus died. The browning rate of the callus of F5 is the lowest in 8 culture media, and is 0.000%, namely no callus is browned in the culture period of 30d, and the observation shows that the volume of the part of callus is continuously increased after inoculation, the newly-grown white callus presents a white cluster shape, and no bud point appears after multiple subcultures.

The culture medium is added with NAA and TDZ simultaneously, so that adventitious buds can be differentiated from Potentilla adenlandii callus (see figure 2-A/B), the callus differentiation rates of different treatments are obviously different, and the growth state of the adventitious buds is greatly influenced by hormone concentration. As can be seen from Table 6, the callus differentiation rate of Potentilla adenianae increased with the increase of NAA concentration when the TDZ concentration was constant, and the callus differentiation rates of F10, F12 and F14 were increased by 10.00%, 20.00% and 46.67% as compared with those of F9, F11 and F13, respectively, which all reached significant differences (P < 0.05). When the mass concentration of NAA in the culture medium is 0.5mg/L, the callus differentiation rate shows a trend of increasing along with the increase of the TDZ concentration, the callus differentiation rate of F14 is respectively improved by 26.67 percent and 36.67 percent compared with that of F12 and F10, and the differentiation rate of 3 has a significant difference (P is less than 0.05). F10, F12 and F14 were 2.67, 2.00, 14.14 respectively in terms of average adventitious bud number, with F14 having an average adventitious bud number significantly higher than the other two treatments (P < 0.05), and although F12 had an average adventitious bud number slightly lower than F10, there was no significant difference between the two (P > 0.05). It was observed that the adventitious bud growth state also shows a trend from weak to strong with the increase of the mass concentration of TDZ when the mass concentration of NAA is constant. Adventitious buds in the culture medium F10 are in a bubbling state, a few adventitious buds are differentiated into stems and leaves, the color of the leaves is light green, and the overall growth vigor is weak; the F12 and the F14 have more adventitious buds, and the complete plant is differentiated at the same time, so that the new buds grow well.

The callus of the leaf of Potentilla tormentosa is inoculated into a culture medium added with 2.4D and TDZ with different mass concentrations, the differentiation condition of the callus is extremely undesirable, in 4 treatments of F15-F18, only the callus in F18 is differentiated into adventitious buds, but the differentiation rate is only 6.67 percent, and the differentiation rate is not significantly different from that of other treatments in the same group (P is more than 0.05). The callus tissues in the F15-F17 are not differentiated successfully, and the browning rate is in the range of 43.33% -100.00%.

In conclusion, the optimal culture medium for inducing the callus differentiation of the potentilla glandularis leaves is MS + NAA0.5mg/L + TDZ1.0 mg/L.

TABLE 6 influence of different hormone combinations on Potentilla adenanthera callus differentiation

Note: data are mean ± sem; the difference in lower case letters in the same column indicates significant difference (P < 0.05).

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A tissue culture method of Potentilla glandulifera is characterized by comprising the following steps: taking tender leaves as explants, inoculating the tender leaves to a callus induction culture medium after surface sterilization to induce callus, inoculating the induced callus to an adventitious bud differentiation culture medium to differentiate adventitious buds, wherein the callus induction culture medium is MS + NAA 0.1-0.5 mg/L + TDZ 0.1-1.0 mg/L, the adventitious bud differentiation culture medium is MS + NAA0.5mg/L + TDZ 0.1-1.0 mg/L, and the explants are subjected to surface sterilization by adopting the following surface sterilization method: cutting tender leaves, soaking in detergent water for 1-5min, washing under running water for 30-60min, placing on a clean bench, sterilizing with 75% alcohol solution for 35-120s, pouring off alcohol, washing with sterile water for 3-4 times, and soaking with 2% sodium hypochlorite solution for 5-10 min.

2. The method for tissue culture of Potentilla tormentilla according to claim 1, wherein the callus induction medium is MS + NAA0.5mg/L + TDZ1.0 mg/L.

3. The tissue culture method of Potentilla tormentosa as claimed in claim 1, wherein the adventitious bud differentiation medium is MS + NAA0.5mg/L + TDZ1.0 mg/L.