CN108925430B

CN108925430B - Method for inducing adventitious buds by germinating bud bodies of Korean pine germinating seeds

Info

Publication number: CN108925430B
Application number: CN201810975891.0A
Authority: CN
Inventors: 梁艳; 高美玲; 陈阳; 李诗佳; 赵艳; 徐洪国; 梁晓雪; 孙悦; 罗枫新; 赵蕊阳; 付雨晴
Original assignee: Qiqihar University
Current assignee: Qiqihar University
Priority date: 2018-08-24
Filing date: 2018-08-24
Publication date: 2020-01-14
Anticipated expiration: 2038-08-24
Also published as: CN108925430A

Abstract

A method for inducing adventitious buds by germinating buds by using Korean pine germinating seeds relates to the field of forest seedling propagation. The invention aims to solve the problems of difficult occurrence of organs of the Korean pine and low adventitious bud differentiation rate at present. The method comprises the following steps: (1) taking materials of explants and sterilizing; (2) performing induced differentiation culture on the adventitious bud; (3) performing elongation culture on the adventitious bud; (4) and (4) rooting induction culture of the regenerated plant. Inoculating the Korean pine seed subjected to pregermination treatment on a differentiation culture medium as an explant, and culturing for 30 d; after obtaining the adventitious bud, transferring the adventitious bud to an elongation culture medium, continuing culturing for 60d (once intermediate subculture), cutting and separating the cluster seedling when the cluster seedling grows to 2-3cm, and inoculating the cluster seedling into a rooting culture medium for acclimatization. The adventitious bud inductivity of the invention reaches 92.2 percent, the average bud number of each explant reaches 10-12, the bud height of the explant reaches 3.5cm after the explant is subjected to elongation growth culture for 60 days, and the rooting rate reaches 52 percent.

Description

Method for inducing adventitious buds by germinating bud bodies of Korean pine germinating seeds

Technical Field

The invention relates to the field of forest seedling propagation, in particular to a method for inducing adventitious buds by germinating bud bodies through accelerating germination of Korean pine seeds.

Background

Korean pine (Pinus koraiensis) is a tall arbor tree species of Pinus of Pinaceae of gymnosperm, is a group-establishing tree species of temperate zone apical pole community-Korean pine broadleaf forest, is a high-quality tree species produced by using materials and nuts, and has important ecological and economic values. At present, the demand of Korean pine seedlings in the market is very large, but due to the problems of long production period of Korean pine, low seed setting rate, reduced excellent properties of offspring and the like, the shortage of good seed stocks of Korean pine becomes the bottleneck of development of Korean pine industry, the in-vitro rapid propagation technology based on the tissue culture means can effectively solve the problems, the research on the in-vitro culture adventitious bud induction technology of Korean pine is developed, the effective way of accelerating the propagation speed of Korean pine through the tissue culture technology is explored, the cost can be reduced to the maximum extent, the propagation coefficient is improved, and the technical basis is provided for promoting the industrialized seedling of Korean pine. In addition, the establishment of the Korean pine in-vitro rapid propagation system is also a precondition for successfully constructing a Korean pine genetic transformation system, and a foundation can be laid for further developing the transgenic breeding work of Korean pine. At present, related reports about the Korean pine organogenesis technology are few, in the existing research, a Korean pine mature zygote embryo is mostly adopted as an explant, and adventitious buds are directly induced by isolated culture or callus induction is firstly carried out and then adventitious bud induction is carried out, so that a certain effect is obtained, but the problems that the adventitious buds are easy to generate variation and excellent characters are difficult to maintain exist in the callus induction are solved. In the prior art, mature zygotic embryo of Korean pine is directly induced to obtain adventitious bud, but the induction rate of the adventitious bud is only 30 percent, and the induction rate is low (research on in vitro culture of mature embryo of Korean pine, Liuyuxi, Sunwhong year, Chinese Louissima, Qizhi, university of northeast forestry, 1991).

Disclosure of Invention

The invention aims to solve the problems of difficult organogenesis and low adventitious bud differentiation rate of the prior Korean pine. The invention provides a method for inducing adventitious buds by germinating buds of Korean pine germinating seeds, which has high adventitious bud induction rate and high multiplication coefficient. The method mainly comprises four stages of adventitious bud induction culture: the method comprises the steps of material selection and disinfection treatment of a pregermination seed explant, induction of an adventitious bud, an elongation growth stage of the adventitious bud and a rooting domestication stage of the adventitious bud.

The invention relates to a method for inducing adventitious buds by germinating buds by using Korean pine germinating seeds, which is realized by the following steps:

(1) taking materials of explants and sterilizing: taking mature Korean pine seeds harvested in the previous year and subjected to mixed sand low-temperature kiln germination acceleration treatment, firstly taking out buds extracted from Korean pine from seed shells, removing endosperm, washing with washing powder, washing with running water, removing the seed shells, soaking with 10% by mass of sodium hypochlorite for 15min, washing with sterile water for 5 times, soaking with 70% by volume of alcohol for 1 mm, washing with sterile water for 5 times, placing in 0.1% by mass of mercuric chloride solution for disinfection for 10min, and washing with sterile water for 5 times to obtain pretreated explant of the Korean pine seeds;

(2) induction of adventitious buds: cutting off a radicle part of the explant obtained by the disinfection treatment in the step (1) in a super clean bench, and vertically placing the explant in a DCR culture medium according to the morphological direction for carrying out the induction culture of adventitious buds; wherein the DCR culture medium contains 1-3 mg/L KT, 0.2mg/L TDZ, 500mg/L acid hydrolyzed casein, 500mg/L L-glutamine, 3 mass percent of cane sugar and 0.7 mass percent of agar, and the pH value of the DCR culture medium is 5.7-5.8;

or the DCR culture medium contains 1-3 mg/L KT, 0.5mg/L TDZ, 500mg/L acid hydrolyzed casein, 500 mg/LL-glutamine, 3 mass percent of cane sugar and 0.7 mass percent of agar, and the pH value of the DCR culture medium is 5.7-5.8;

induction culture conditions: the temperature is 23 +/-2 ℃, the humidity is 60-70%, the illumination time is 14h/d, and the illumination intensity is as follows: 2000-3000lux, and the culture time is 30 days;

(3) elongation growth of adventitious buds: transferring the adventitious bud obtained after the induction culture in the step (2) for 30 days into a DCR culture medium for elongation and enrichment culture of the adventitious bud for 60 days, and replacing a fresh DCR culture medium once when the culture reaches 30 days; wherein the DCR culture medium contains NAA with the concentration of 0.1mg/L, 6-BA with the concentration of 0.2-1.0 mg/L, acid hydrolyzed casein with the concentration of 500mg/L, 500mg/L L-glutamine, 1g/L activated carbon, 3% of cane sugar and 0.7% of agar, and the pH value of the DCR culture medium is 5.7-5.8; induction culture conditions: the temperature is 23 +/-2 ℃, the humidity is 60-70%, the illumination time is 14h/d, and the illumination intensity is as follows: 2000-3000lux, culture time 60d, wherein 30d replaced fresh culture medium;

(4) rooting induction of adventitious buds: separating and cutting the adventitious bud cluster elongated and subjected to the enrichment culture in the step (3) into single buds, and then placing the single buds into 1/2DCR culture medium for rooting and domestication to complete the process; wherein the 1/2DCR culture medium contains 0.5mg/L NAA, 500mg/L acid hydrolyzed casein, 1g/L active carbon, 2% sucrose and 0.7% agar, and has a pH value of 5.7-5.8.

The invention has the following beneficial effects:

1. the induction method adopts DCR culture medium and adds KT and TDZ with different concentrations, the induction rate of the adventitious bud of the Korean pine is up to 92.2 percent by selecting explants and adjusting culture conditions, the average bud number of each explant is up to 10-12, NAA and 6-BA are added in the stage of the elongation growth of the adventitious bud, the bud treated for 60 days is up to 3.5cm, and the rooting rate is up to 54 percent.

2. The invention has definite explanation for the explant state of the germination accelerating seed of the Korean pine, and has definite explanation for the steps of disinfection treatment, inoculation mode, adventitious bud induction, elongation culture and rooting treatment. The method for selecting the Korean pine pregermination seeds as the explants has the advantages of convenient material acquisition, simple operation, high adventitious bud induction rate and the like, so that the adoption of the Korean pine pregermination seeds as the explants is more favorable for developing a Korean pine efficient regeneration system and related researches.

According to the invention, different hormones are added into the DCR culture medium to obtain the Korean pine adventitious bud with high induction rate, realize the elongation and the strengthening of the Korean pine adventitious bud and finally obtain the Korean pine rooting test-tube plantlet. Through experimental analysis, although the use of hormones for improving the germination rate of adventitious buds is reported in pine tissue culture at present, the difficulties encountered in tissue culture of different conifer species are different, so the means adopted are different, and the final ideal effect can not be achieved only by adjusting the type and the dosage of the hormones, but rather, the integration of multiple factors is generated. Such as selection of explant material, placement of explants, screening of hormones to establishment of culture systems, etc., may have a decisive influence on the final culture results. For the invention, experiments show that the DCR culture medium is added with KT, TDZ, NAA and 6-BA with different concentrations; the effects of the different combinations are also very different. Therefore, the present application can produce unexpected technical effects in terms of adventitious bud induction rate, adventitious bud elongation, and rooting rate by selecting the above technical means.

Drawings

FIG. 1 shows Korean pine seeds undergoing pregermination treatment;

FIG. 2 is a diagram of explants for adventitious bud induction just after inoculation;

FIG. 3 shows adventitious buds of Korean pine induced for 20 days under a microscope;

FIG. 4 shows the induction of adventitious buds of Korean pine for 30 days;

FIG. 5 shows adventitious buds of Korean pine growing for 30 d;

FIG. 6 shows adventitious buds of Korean pine growing for 60 d;

FIG. 7 shows the rooted red pine tube plantlets obtained by induction.

Detailed Description

The technical solution of the present invention is not limited to the following specific embodiments, but includes any combination of the specific embodiments. It is to be noted that the experimental methods described in the following embodiments are all conventional methods unless otherwise specified, and the reagents and materials are commercially available unless otherwise specified.

The first embodiment is as follows: the method for inducing adventitious buds by germinating the sprouted seeds with the Korean pine is realized by the following steps:

The second embodiment is as follows: the first difference between the present embodiment and the specific embodiment is: the mature Korean pine seeds in the step (1) are collected in the previous 10 months of the year and subjected to low-temperature sand storage and germination acceleration. The rest is the same as the first embodiment.

The third concrete implementation mode: the first difference between the present embodiment and the specific embodiment is: the explant material taking and disinfection treatment method in the step (1) comprises the following steps: taking mature Korean pine seeds harvested in the previous year and subjected to low-temperature hierarchical sand storage germination acceleration treatment, taking the embryo root with the epicotyl extending 0.5-1cm out of the episperm as an explant material, firstly taking the bud body extracted from the Korean pine out of a seed shell, removing endosperm, washing with washing powder, washing cleanly with running water, soaking with 10% by mass of sodium hypochlorite for 20min, washing with sterile water for 5 times, soaking with 70% alcohol for 1 mm in volume percentage for 5 times, and soaking with 0.1% mercuric chloride for 15min, and washing with sterile water for 5 times to finish the disinfection treatment.

The rest is the same as the first embodiment.

The fourth concrete implementation mode: the first difference between the present embodiment and the specific embodiment is: and (2) vertically inserting the cut radicle part of the germinated bud subjected to the sterilization treatment in the step one into a DCR culture medium according to the morphological direction for adventitious bud induction culture for 30d, wherein the DCR culture medium contains 2mg/L KT, 0.5mg/L TDZ, 500mg/L acid casein hydrolysate, 500 mg/LL-glutamine, 3% sucrose and 0.7% agar.

The rest is the same as the first embodiment.

The fifth concrete implementation mode: the first difference between the present embodiment and the specific embodiment is: placing the adventitious bud obtained after the induction culture in the step (2) for 30d in a DCR culture medium for elongation and enrichment culture of the adventitious bud for 60d in the step (3), and replacing a fresh DCR culture medium once when the culture reaches 30 d; the DCR culture medium in the step (3) contains 0.1mg/L NAA, 0.2 mg/L6-BA, 1g/L activated carbon, 500mg/L acid hydrolyzed casein, 500mg/L L-glutamine, 3% sucrose and 0.7% agar.

The rest is the same as the first embodiment.

The sixth specific implementation mode: the first difference between the present embodiment and the specific embodiment is: the rooting domestication conditions in the step (4) are as follows: the temperature is 23 +/-2 ℃, the humidity is 60-70%, the illumination time is 14h/d, and the illumination intensity is 2000-3000 lux.

The rest is the same as the first embodiment.

The seventh embodiment: the first difference between the present embodiment and the specific embodiment is: in the fourth step, the height of the adventitious bud bundle is 2-3 cm. The rest is the same as the first embodiment.

Preferred embodiments of the present invention are described below with reference to the accompanying drawings:

example 1

The method for inducing adventitious buds by germinating the seed germination accelerating Korean pine comprises the following steps:

firstly, test materials: the germination accelerating seed test material of the Korean pine in the embodiment is healthy, full and uniform mature Korean pine seeds which are harvested in a Korean dragon river Korean pine seed orchard in the current year for 10 months and are subjected to low-temperature germination accelerating treatment by conventional sand storage for half a year, seed shells are cracked, and radicle parts are exposed for 0.5-1 cm. FIG. 1 shows the germination-promoted Korean pine seeds used in the experimental study.

Secondly, selection and disinfection treatment of explants: in the first step, firstly, the extracted germinating bud is taken out, the endosperm of the germinating bud is removed, and then the germinating bud is soaked in a 10% sodium hypochlorite solution for 20min, washed by sterile water for 5 times, treated by 70% alcohol for 1min, washed by sterile water for 5 times, soaked in a 0.1% mercury bichloride solution for 15min and washed by sterile water for 5 times.

Thirdly, induction of adventitious buds and screening of hormone concentration: in the second step, the radicle part is cut off by a sterile scalpel and tweezers in a clean bench after the disinfection treatment in the first step, and then the radicle part is vertically inserted into a culture medium according to the morphological direction for adventitious bud induction culture, and fig. 2 shows an explant which is inoculated into the culture medium and used for adventitious bud induction. In the test, DCR is used as a basic culture medium, TDZ and KT (the combination is shown in table 1) with different concentrations are added, and the test further contains 500mg/L acid hydrolyzed casein, 500 mg/LL-glutamine, 3% of sucrose and 0.7% of agar, and the pH value is 5.7-5.8. Induction culture conditions: the temperature is 23 +/-2 ℃, the humidity is 60-70%, the illumination time is 14h/d, and the illumination intensity is as follows: 2000-3000lux, for 30 days. Each group was replicated in 10 flasks, each receiving 4 explants.

The result of the adventitious bud induction rate test of Korean pine is shown in Table 1. Research results show that each treatment combination has adventitious bud formation, but the influence of the hormone type and concentration on the adventitious bud induction rate of the Korean pine is obviously different. On the whole, the change of the concentration of KT has larger influence on the induction of the adventitious bud, and the induction rate of the adventitious bud shows a change trend of increasing firstly and then decreasing with the increase of the treatment concentration of KT. When the concentration of KT is 1mg/L, the concentration of TDZ is 0.2 or 0.5mg/L, and the concentration of NAA is 0 or 0.1mg/L, the induction rate of the adventitious buds is the lowest, the induction rate is between 25.52 and 41.56 percent, and the number of the induced buds is only 3 to 6; when the concentration of KT is increased to 2mg/L, the induction rate of the adventitious buds is obviously increased, the number of the adventitious buds induced by explants is increased, wherein KT 2mg/L and 0.5mg/L TDZ are added, the induction rate of the adventitious buds is obviously superior to that of other treatment combinations when the concentration of NAA is 0 or 0.1mg/L, the induction rate of the adventitious buds is higher than 90% after 30d of culture, and the average induction rate of each explant is 10-12; when the KT treatment concentration is further increased, the adventitious bud induction rate is reduced and the number of induced buds is also reduced when the concentration is increased to 3 mg/L. In conclusion, the optimal hormone combination for adventitious bud induction of the Korean pine germinating seeds is obtained by adding 2mg/L KT and 0.5mg/L TDZ into a DCR basic culture medium. As shown in figure 3, adventitious bud induction is carried out for 20d, and observation by a stereomicroscope shows that the surface of the expanded cotyledon base of the aseptic seedling grows into slightly green protruding bud points; the bud points were observed to develop gradually into macroscopic shoots when cultured for 30 days, see FIG. 4.

TABLE 1 Experimental design of adventitious bud induction of Korean pine germinating seed by different hormone combinations

And fourthly, transferring and subculturing the adventitious bud obtained by the induction culture of the step three for 30d in a DCR culture medium for elongation and strengthening culture of the adventitious bud for 60d, wherein the culture medium is replaced by fresh culture medium once for 30 d. DCR is used as a basic culture medium, and 0.1mg/L NAA is added to be combined with 6-BA and active carbon with different concentrations (each combination is shown in a table 2).

The results of the Korean pine adventitious bud elongation study are shown in Table 2. The results show that the treatment concentration of 6-BA has a significant effect on the elongation of adventitious buds. On the whole, with the increase of the treatment concentration of 6-BA, the length of the adventitious bud shows a change trend of increasing firstly and then decreasing, when no 6-BA is added, no matter whether activated carbon is added, the adventitious bud slightly extends before the extension treatment, but is not obvious, and even the adventitious bud only maintains the state of a bud point just differentiated; when the concentration of 6-BA is 0.2mg/L and the concentration of activated carbon is 1g/L, the average bud length is the highest and reaches 3.5cm, and the test-tube plantlet grows robustly; when the 6-BA concentration increased to 0.5mg/L or 1mg/L, the average shoot length decreased again (below 2cm), and the leaves were fine and elongation growth was slow. In conclusion, the optimal hormone combination for adventitious bud elongation of the Korean pine pregermination seeds is obtained by adding 0.2mg/L of 6-BA, 0.1mg/L of NAA and 1g/L of activated carbon into a DCR culture medium. FIG. 5 is a diagram showing adventitious buds of Korean pine growing for 30d, wherein the buds are slightly elongated when the adventitious buds are cultured for 30 d; FIG. 6 is a graph showing adventitious buds of Korean pine grown for 60d by elongation, and the average bud length was found to be 3.5cm when cultured for 60d by elongation.

TABLE 2 Experimental design of adventitious bud elongation of seed for accelerating germination of Korean pine by using different hormone combinations

Fifthly, separating and cutting the adventitious bud cluster (2-3cm high) elongated in the step three into single buds, and then placing the single buds in a DCR culture medium for rooting induction. The culture medium is adopted as follows: 1/2DCR culture medium (halving macroelement) contains 0.5mg/L NAA, 500mg/L acid hydrolyzed casein, 1g/L active carbon, 2% sucrose and 0.7% agar, pH value is 5.7-5.8, and the culture medium is changed once after 60 days and 30 days of co-culture.

After 60 days of culture, the test-tube plantlet has white root system generation, the test-tube plantlet is about 1cm long, and the statistical rooting rate is 52% (figure 7).

In the induction method of the embodiment, the DCR culture medium is adopted and the TDZ and KT with different concentrations are added, so that the induction rate of the adventitious bud is high and can reach as high as 92.2%, the average bud number of each explant reaches 10-12, the bud length of the explant after 60d of elongation growth is 3.5cm, the rooting rate reaches 52%, and the rooting effect is good.

The embodiment has specific explanation on the explant state of the Korean pine pregermination seeds, and also has specific explanation on the disinfection treatment, the inoculation mode, the adventitious bud induction, the elongation growth and the rooting steps. The Korean pine explant selected in the embodiment has the advantages of convenience in material acquisition, simplicity in operation of an adventitious bud induction process, high adventitious bud induction rate and the like, so that the adventitious bud induction technology of the Korean pine pregermination seed is more beneficial to developing research related to a Korean pine efficient regeneration system and molecular breeding.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.

The method provided by the present invention further includes a washing step before performing sterilization, and the method of washing is not particularly limited as long as impurities can be removed to obtain clean seeds.

Claims

1. A method for inducing adventitious buds by germinating buds of Korean pine germinating seeds is characterized by comprising the following steps:

(2) inducing adventitious buds, namely cutting off radicle parts of explants obtained by the disinfection treatment in the step (1) in an ultraclean workbench, vertically placing the explants in a DCR culture medium according to morphological direction for inducing and culturing the adventitious buds, wherein the DCR culture medium contains 2mg/L KT, 0.5mg/L TDZ, 500mg/L acid hydrolyzed casein, 500mg/L L-glutamine, 3 mass percent of sucrose and 0.7 mass percent of agar, and the pH value of the DCR culture medium is 5.7 ~ 5.8.8;

the induction culture conditions comprise the temperature of 23 +/-2 ℃, the humidity of 60% of ~ 70%, the illumination time of 14h/d, the illumination intensity of 2000-3000lux and the culture time of 30 d;

(3) elongation growth of adventitious buds: transferring the adventitious bud obtained after the induction culture in the step (2) for 30 days into a DCR culture medium for elongation and enrichment culture of the adventitious bud for 60 days, and replacing a fresh DCR culture medium once when the culture reaches 30 days;

wherein the DCR culture medium contains NAA with the concentration of 0.1mg/L, 6-BA with the concentration of 0.2 ~ 0.5.5 mg/L, acid hydrolyzed casein with the concentration of 500mg/L, 500mg/L L-glutamine, 1g/L activated carbon, 3 percent of cane sugar and 0.7 percent of agar, and the pH value of the DCR culture medium is 5.7 ~ 5.8.8, and the induction culture conditions comprise the temperature of 23 +/-2 ℃, the humidity of 60 percent ~ 70 percent, the illumination time of 14h/d, the illumination intensity of 2000-3000lux and the culture time of 60d, wherein the fresh culture medium is replaced once for 30 d;

(4) and (3) rooting induction of the adventitious bud, namely separating and cutting the adventitious bud clump subjected to elongation and strengthening culture in the step (3) into single buds, and then placing the single buds into 1/2DCR culture medium for rooting and domestication to finish the rooting and domestication, wherein the 1/2DCR culture medium contains 0.5mg/L of NAA, 500mg/L of acid hydrolyzed casein, 1g/L of activated carbon, 2% of sucrose and 0.7% of agar, and the pH value is 5.7 ~ 5.8.8.

2. The method for inducing adventitious bud in the germinated bud body by using the germination accelerating seed of the Korean pine according to claim 1, wherein the mature Korean pine seed in the step (1) is the mature Korean pine seed which is harvested and subjected to germination accelerating treatment by low-temperature sand storage in the previous 10 months.

3. The method for inducing adventitious bud by germinating bud bodies on the Korean pine pregermination seeds as claimed in claim 1, wherein the explant in the step (1) is obtained by the following steps: taking mature Korean pine seeds harvested in the previous year and subjected to low-temperature hierarchical sand storage germination acceleration treatment, taking the embryo root with the epicotyl extending 0.5-1cm out of the episperm as an explant material, firstly taking the bud body extracted from the Korean pine out of a seed shell, removing endosperm, washing with washing powder, washing cleanly with running water, soaking with 10% by mass of sodium hypochlorite for 20min, washing with sterile water for 5 times, soaking with 70% alcohol for 1 mm in volume percentage for 5 times, and soaking with 0.1% mercuric chloride for 15min, and washing with sterile water for 5 times to finish the disinfection treatment.

4. The method for inducing adventitious buds through germination accelerating seed germination of Korean pine and bud body induction according to claim 1, wherein in the step (3), the adventitious buds obtained after 30d of the induction culture in the step (2) are placed in a DCR culture medium for 60d of adventitious bud elongation and enrichment culture, and the DCR culture medium is replaced once after the culture is carried out for 30 d; the DCR culture medium in the step (3) contains 0.1mg/L NAA, 0.2 mg/L6-BA, 1g/L activated carbon, 500mg/L acid hydrolyzed casein, 500mg/L L-glutamine, 3% sucrose and 0.7% agar.

5. The method for inducing adventitious bud in the bud germination by using the Korean pine pregermination seed according to claim 1, wherein the rooting acclimation conditions in the step (4) are 23 +/-2 ℃, 60% humidity ~ 70%, 14h/d illumination time and 3000lux illumination intensity of 2000-.

6. The method for inducing adventitious bud in the germinated bud body of the seed germinated by using the Korean pine according to claim 1, wherein the height of the adventitious bud bundle in the fourth step is 2 ~ 3 cm.