A tissue culture propagation method of Terminalia ferdinandiana

Abstract

The application belongs to the technical field of plant tissue culture and particularly relates to a tissue culture propagation method of Canarium ovatum. In order to protect the wild plant resources of Canarium ovatum, the application takes the seeds of Canarium ovatum as explants, screens the sterilization mode, primary culture, proliferation and rooting culture medium, and discusses the culture mode. Through screening the sterilization mode of the explants, the aseptic seedlings are successfully obtained, the seed germination rate is improved, and the basic material is provided for further tissue culture propagation. The proliferation and rooting are simultaneously realized by adopting the 1 / 2MS culture medium through the segment method, the production cost is saved, technical support is provided for further in vitro resource preservation and industrialized propagation of Canarium ovatum, and the protection of the germplasm resources of Canarium ovatum is facilitated.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of plant tissue culture, and particularly relates to a tissue culture propagation method of Terminalia myriocarpa. BACKGROUND

[0002] Terminalia myriocarpa is a tree plant of Combretaceae, is a national second-class protected wild plant, and is an endangered species. It is known to be distributed in Yunnan, Guangdong, Guangxi and Tibet, China, and is one of the common species in the upper layer of tropical evergreen rainforests. The plants are generally sporadically distributed, and there are also small patches of distribution, mainly distributed at an altitude of 600m to 1500m. It is known that the patch distribution range is within the altitude range of 600m to 1500m near Jiarexi to Xirang. Terminalia myriocarpa is a rare and endangered species, and with human felling and implementation of national strategic projects, the project occupies the wild resources and constitutes a threat. In order to protect the wild plant resources of Terminalia myriocarpa, it is urgent to carry out related researches such as artificial propagation technology to provide technical support for the protection of Terminalia myriocarpa.

[0003] At present, there are few researches on the propagation technology of Terminalia myriocarpa, and only a small amount of seed sowing and cutting propagation technology research reports. The tissue culture method refers to the separation of organs, tissues or cells required from the plant body, and the culture under artificial control conditions through aseptic operation to obtain regenerated whole plants or produce other products with economic value. The tissue culture propagation technology of Terminalia myriocarpa is not limited by external environmental conditions, and a large number of propagation offspring can be obtained in a short period of time, which has important significance for the protection of Terminalia myriocarpa resources, and there is no research on the tissue culture technology of Terminalia myriocarpa.

[0004] Although according to the theory of cell totipotency, any tissue or organ has the potential to develop into a complete plant, but from the actual operation process, it can be found that different types of plants and different types of organs or tissues of the same type have great differences in response to induction conditions. Therefore, it is not easy to successfully obtain a large number of Terminalia myriocarpa regenerated plants by tissue culture propagation method, and the successful tissue culture propagation method for obtaining a large number of Terminalia myriocarpa regenerated plants has important significance for the protection of wild plant resources of Terminalia myriocarpa and the promotion of varieties. SUMMARY

[0005] In order to protect the wild plant resources of Terminalia myriocarpa, the present application takes the Terminalia myriocarpa seeds without seed coat as explants, and establishes a Terminalia myriocarpa tissue culture regeneration system through tissue culture and screening of each link factor, fills the research gap of Terminalia myriocarpa tissue culture propagation technology, and provides technical support for the resource protection and further industrial application of Terminalia myriocarpa.

[0006] The application provides a tissue culture propagation method of Terminalia ferdinandiana, which comprises the following steps: taking Terminalia ferdinandiana seeds with removed pericarp as explants, disinfecting the explants, and sequentially performing start culture, proliferation culture and rooting culture on the disinfected explants.

[0007] Preferably, the alcohol is 75v / v% ethanol, and the volume concentration of sodium hypochlorite is 30%.

[0008] Preferably, the alcohol is 75v / v% ethanol, and the volume concentration of sodium hypochlorite is 30%.

[0009] Further preferably, the sterilization time of the 75% ethanol and 30% sodium hypochlorite is 5-15s respectively.

[0010] Preferably, the start culture medium is MS medium without added hormones.

[0011] Preferably, the start culture time is 30-32d.

[0012] Preferably, the proliferation culture medium is 1 / 2MS medium without added hormones.

[0013] Preferably, the proliferation culture time is 40-42d.

[0014] Preferably, the proliferation culture is to select Terminalia ferdinandiana aseptic seedlings with a height of more than 5cm in the start culture, cut the aseptic seedlings into 1.5-2.5cm stem segments with buds, and vertically inoculate the stem segments with a depth of 0.4-0.6cm.

[0015] Preferably, the rooting culture medium is 1 / 2MS medium without added hormones.

[0016] Preferably, the rooting culture time is 10-30d.

[0017] Preferably, the rooting culture is to select robust seedlings in the proliferation culture, cut the seedlings into stem segments with a length of more than 3cm, and inoculate the stem segments to root.

[0018] Preferably, the start culture, proliferation culture and rooting culture have the same conditions, including: environmental light / dark is 8-14 / 10-16h, light intensity is 2000Lux, and / or temperature is 24℃±2℃, and humidity is 60%±10%.

[0019] The application further provides a regenerated plant of Terminalia ferdinandiana prepared by the above tissue culture propagation method.

[0020] Beneficial effects: the application takes the seeds of Elaeocarpus acmonophyllus as explants, and screens the sterilization method, primary culture, proliferation culture and rooting culture, and discusses the culture method. The experimental results show that: the seed coat is completely removed, the explants are sterilized by 75% ethanol for 10s and 30% sodium hypochlorite for 10s, and the sterilization effect is best. The most suitable culture medium for each stage of the regeneration system is: (1) primary culture: MS; (2) proliferation culture: 1 / 2MS; (3) rooting culture: 1 / 2MS. The segment method is used to realize proliferation and rooting simultaneously in 1 / 2MS, and the rooting rate reaches 100%.

[0021] Elaeocarpus acmonophyllus is a national rare and endangered plant, and the tissue culture and propagation method of Elaeocarpus acmonophyllus provided by the application is not affected by the external environment, a large number of propagation offspring can be obtained in a short period in the tissue culture room, technical support is provided for the in vitro resource preservation of Elaeocarpus acmonophyllus, and the protection of Elaeocarpus acmonophyllus germplasm resources is beneficial.

[0022] Elaeocarpus acmonophyllus is also a representative and indicator species of tropical rainforest and monsoon forest in China, and the establishment of the tissue culture and propagation technical system of Elaeocarpus acmonophyllus can further industrialize to form a large number of high-quality seedlings, provide plant material support for the ecological restoration and further engineering application of tropical monsoon forest, and provide technical support for the resource preservation of the rare and endangered plant Elaeocarpus acmonophyllus. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 For the uncontaminated seeds of Elaeocarpus acmonophyllus in Example 2;

[0024] Figure 2 For the aseptic seedlings of Elaeocarpus acmonophyllus obtained by treatment number F in Example 3;

[0025] Figure 3 For the comparison chart of Elaeocarpus acmonophyllus proliferation culture medium screening in Example 4; wherein the upper four charts are the upper growth state, and the culture medium from left to right is G1, G2, MSH and E; the lower four charts are the root system growth state, and the culture medium from left to right is G1, G2, MSH and E. DETAILED DESCRIPTION

[0026] The application obtains regenerated Elaeocarpus acmonophyllus plants by seed tissue culture pretreatment, explant disinfection, starting culture, proliferation culture and rooting culture of collected Elaeocarpus acmonophyllus seeds. By screening the sterilization method of the explant, the aseptic seedlings are successfully obtained, the seed germination rate is improved, and the basic material for further tissue culture and propagation is provided. By using 1 / 2MS medium, proliferation and rooting can be realized simultaneously by the segment method, the production cost is saved, technical support is provided for further in vitro resource preservation and industrialized propagation of Elaeocarpus acmonophyllus, and the protection of Elaeocarpus acmonophyllus germplasm resources is beneficial.

[0027] Applicant found through a large number of research and screening experiments that, in the tissue culture propagation method of Terminalia ferdinandiana, after removing the seed and all the pericarp, the primary sterile seedlings are induced on MS to obtain the sterile seedlings, and the sterile seedlings are cut into stem segments with buds, and then the stem segments are cultured on 1 / 2MS medium to realize high-efficiency and synchronous rooting and propagation.

[0028] In a preferred embodiment of the present application, the sterilization method of 75% ethanol for 10s and 30% sodium hypochlorite for 10s can better improve the germination rate.

[0029] The scheme of the present application will be explained below in conjunction with examples. Those skilled in the art will understand that the following examples are only used to illustrate the present application and should not be regarded as limiting the scope of the present application. If the specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in the art or according to the product instructions are used. If the reagents or instruments are not specified by the manufacturer, they are all conventional products that can be obtained by purchase.

[0030] In the examples of the present application, the collection and treatment of Terminalia ferdinandiana seeds include: in mid-December, in Motuo, Tibet, using high pruning shears to cut off the pink fresh fruit clusters on the branches of Terminalia ferdinandiana, and using non-woven cloth bags to collect the fresh fruit clusters. The seeds in the non-woven cloth bags are placed in a sieve, and 1-2 layers of newspapers are placed underneath for indoor drying. The drying time is about 30d, the Terminalia ferdinandiana seeds are separated from the fruit clusters, the peduncle is removed, and the seeds are packed in plastic self-sealing bags and stored in a 4℃ refrigerator. The treated Terminalia ferdinandiana seeds are used for the following experimental research and screening.

[0031] The following experimental data are analyzed by using SPSS25. The contamination rate, germination rate, propagation coefficient, and rooting rate are tested by single factor ANOVA.

[0032] Example 1: Screening of seed tissue culture pretreatment methods of Terminalia ferdinandiana

[0033] After the Terminalia ferdinandiana seeds are harvested, the seeds are pretreated in three ways: with the complete pericarp, without the pericarp, and without the complete pericarp. Then the pretreated seeds are sterilized by 75% alcohol for 1min and 30% sodium hypochlorite for 5min, and then inoculated on MS medium. The contamination and germination conditions are observed and counted.

[0034] Contamination rate = number of contaminated seeds / total number of inoculated seeds x 100%;

[0035] Germination rate = number of germinated seeds / total number of inoculated seeds x 100%.

[0036] The results showed that the direct use of seed with skin inoculation, whether or not to remove the wing, can not obtain sterile seedlings, all fungal contamination in about 7d, can not germinate to obtain sterile seedlings. After removing all the fruit skin can significantly reduce the contamination rate of seed culture of Canarium ovatum seed, seed contamination rate is 3.33%, the germination rate is 16.7%, therefore, the explant for tissue culture of Canarium ovatum seed is the seed after removing the fruit skin, which can obtain sterile seedlings. The pollution rate of different pretreatment methods is shown in Table 1.

[0037] Table 1 Influence of different pretreatments on obtaining sterile seedlings of Canarium ovatum

[0038] Treatment No. Treatment Method Contamination Rate (%) Germination Rate (%) 1 With Intact Pericarp 100 a ]] 0 a ]] 2 Without Pericarp Wing 100 a ]] 0 a ]] 3 Without All Pericarp 3.33 b ]]> 16.7 b ]]>

[0039] Note: The numbers in the table with different letters represent significant differences at the 0.05 level. The same below.

[0040] Example 2 Screening of disinfection methods of explants in tissue culture propagation of Canarium ovatum

[0041] In order to further reduce the contamination rate and improve the germination rate, 75% alcohol and 30% sodium hypochlorite were used to sterilize the pretreated seeds, and the treatment time was set at 5s, 10s and 15s. Specifically, the pretreated seeds were first sterilized with 75% alcohol, washed with sterile water for 2 times, then sterilized with 30% sodium hypochlorite, and washed with sterile water for 6 times. After sterilization, inoculate on MS medium. After 7d, the contamination rate was counted (Table 2).

[0042] Through the screening of the above sterilization methods, it was found that the contamination rate of 75% alcohol 5s+30% sodium hypochlorite 5s was 5%, and the contamination rates of the other two sterilization methods were both 0%, and there was no significant difference among the three treatment methods. It is proved that the seed after removing the fruit skin has less bacteria and less pollution.

[0043] The germination rate of treatment 1 and treatment 2 was 45%, and the germination rate of treatment 3 was 22%. The seed after removing the fruit skin is small and tender, and too long sterilization time will cause certain damage to the seed and inhibit the seed germination. In combination with the contamination rate and the germination rate, the best sterilization method for tissue culture of Canarium ovatum seed after removing the fruit skin is 75% alcohol 10s+30% sodium hypochlorite 10s. The uncontaminated Canarium ovatum seed is shown in Figure 1 .

[0044] Table 2 Influence of different sterilization treatments on obtaining sterile seedlings of Canarium ovatum

[0045] Treatment No. Treatment Method Contamination Rate (%) Germination Rate (%) 1 75% Alcohol 5s + 30% Sodium Hypochlorite 5s 5 a ]] 45 a ]] 2 75% Alcohol 10s + 30% Sodium Hypochlorite 10s 0 a ]] 45 a ]] 3 75% Alcohol 15s + 30% Sodium Hypochlorite 15s 0 a ]] 22 b ]]

[0046] The existing technology on the germination rate of Euphoria jolkini seeds all shows that the germination rate of the seeds is low. In the research on the influence of different treatments on the germination of endangered plant Euphoria jolkini seeds by Yu Xiao et al. (Yu Xiao, Zhao Zhenning, Deng Lilan. Influence of Different Treatments on the Germination of Endangered Plant Euphoria jolkini Seeds [J]. Journal of Hubei University of Nationalities: Natural Science Edition, 2023, 44(3): 73-79.), the highest seed germination rate is 40%. In the test of the seed germination rate of Euphoria jolkini by Yang Lisha et al. (Yang Lisha, Li Mujie, Lang Wang, et al. Analysis of the Ecological Characteristics and Seedling Technique of Euphoria jolkini [J]. Green Technology, 2017(13): 2.), even the mature and full seeds have a germination rate of only 30% in the room, and the seed life is short. The seeds will completely lose the germination ability after being stored in the room with ventilation for more than 4 months, and the germination rate is about 20% after being stored in the desiccator and low-temperature state for 2 years. In the present application, the seed is used as an explant, the whole pericarp is peeled off and sterilized, and the germination rate of the aseptic seedling is up to 45%, which is obviously better than the seed germination rate in the prior art.

[0047] Example 3 Screening of the starting medium in the tissue culture propagation of Euphoria jolkini

[0048] The seed explants subjected to the optimal sterilization treatment screened in Example 2 are inoculated into MS and 1 / 2NN69 media containing different concentrations of hormone combinations, and the environmental light / dark is 14 / 10 h, the light intensity is 2000 Lux, the temperature is 24℃±2℃, and the humidity is 60%±10% during the culture. After inoculation for 30 days, the seed germination condition is observed, the primary induction rate is counted, and the optimal starting medium is screened, as shown in Table 3. Figure 2 The aseptic seedlings obtained for treatment No. F.

[0049] The primary induction rate = the number of germinated seeds / the total number of inoculated seeds x 100%.

[0050] Among them, the medium formula of treatment No. A is 1 / 2NN69 medium added with 0.1 mg / L IBA, and the medium formula of the rest is similar. The 1 / 2NN69 medium and the 1 / 2MS medium refer to that the macroelements are halved, and the medium components and ratios are known technologies.

[0051] Table 3 Influence of different media on the starting culture of Euphoria jolkini

[0052] Treatment No. Culture Medium Formula (mg / L) State Description Germination Rate (%) A 1 / 2 NN69 + 0.1 IBA No Germination 0 a ]] B 1 / 2 NN69 + 0.3 NAA + 0.05 IAA No Germination 0 a ]] C MS + 0.3 KT + 0.05 NAA No Germination 0 a ]] D MS + 0.3 GA + 0.05 NAA No Germination 0 a ]] E MS + 0.3 NAA + 0.05 IAA Only Long Root 0 a ]] F MS Normal Growth 16.7 b ]]

[0053] Example 4 Screening of the proliferation medium in the tissue culture propagation of Euphoria jolkini

[0054] The aseptic seedlings of E. ulmoides with the height of 5 cm or more in Example 3 were selected, and the seedlings were cut into stem segments with buds of about 2 cm in length by scissors or blades, and then the stem segments were vertically inoculated into 1 / 2MS and MS media with different hormone ratios at a depth of about 0.5 cm. After inoculation, the stem segments were placed in a culture room for culture. The environment of the culture room was the same as that of the start-up culture. After 40 days, the proliferation rate and the proliferation coefficient were counted to screen the best proliferation medium.

[0055] Proliferation rate = elongation growth or number of cluster buds produced / initial inoculation number x 100%;

[0056] Proliferation coefficient = number of stem segments or single number of cluster buds inoculated after 40 days of culture / initial inoculation number x 100%.

[0057] The aseptic seedlings of E. ulmoides with the height of 5 cm or more in Example 3 were selected, and the seedlings were cut into stem segments with buds of about 2 cm in length by scissors or blades, and then the stem segments were vertically inoculated into 1 / 2MS and MS media with different hormone ratios at a depth of about 0.5 cm. After inoculation, the stem segments were placed in a culture room for culture. The environment of the culture room was the same as that of the start-up culture. After 40 days, the proliferation rate and the proliferation coefficient were counted to screen the best proliferation medium. Figure 3 .

[0058] Table 4 Influence of different media on the proliferation of E. ulmoides

[0059]

[0060] Example 5 Screening of rooting medium in tissue culture propagation of E. ulmoides

[0061] The healthy seedlings of the proliferation culture in Example 4 were selected, and the seedlings were cut into stem segments of more than 3 cm in length, and then the stem segments were inoculated into 1 / 2MS media with different hormone ratios, and the environment of the culture room was the same as that of the proliferation culture. After 30 days, the rooting rate was counted.

[0062] Rooting rate = number of rooted stem segments / number of inoculated stem segments x 100%.

[0063] Three rooting media were used to induce rooting of the explants. The results showed that E medium could induce rooting, the rooting started at about 10 days, the root system and the upper growth were in good condition after 30 days, but the root system was too much to affect the upper growth. G2 medium could induce rooting, the rooting started at about 10 days, but the root system was still too much, and the upper leaves were yellow after 30 days. G1 medium could induce rooting and had no effect on the upper growth, the rooting started at about 10 days, and it was a good rooting medium. The rooting status of the explants in different media was shown in Table 5, and it was known that G1 medium could realize the rooting and multiplication simultaneously.

[0064] Table 5 The effect of different media on the rooting of Canarium ovatum

[0065]

[0066]

[0067] The above experiments showed that the explants with all the pericarps removed were sterilized with 75% ethanol for 10 s and 30% sodium hypochlorite for 10 s, and then the primary sterile seedlings were obtained on MS medium. The sterile seedlings were cut into stem segments with buds, and then the rooting and multiplication were realized simultaneously on 1 / 2MS medium.

Claims

1. A tissue culture propagation method of Elaeocarpus acuminatus, characterized by: The method comprises the following steps: The fruit of the seed of Anacardium occidentale is used as explants, the explants are sterilized, and the sterilized explants are sequentially subjected to initiation culture, proliferation culture and rooting culture; the initiation culture medium is MS medium without hormones; the proliferation culture medium is 1 / 2 MS medium without hormones; and the rooting culture medium is 1 / 2 MS medium without hormones.

2. The tissue culture propagation method of Terminalia ferdinandiana according to claim 1, characterised in that: The sterilization of the explants comprises 5-15 s of alcohol and 5-15 s of sodium hypochlorite.

3. The tissue culture propagation method of Terminalia ferdinandiana according to claim 2, characterised in that: The alcohol is 75 v / v % ethanol, and the volume concentration of sodium hypochlorite is 30 %.

4. The tissue culture propagation method of Terminalia ferdinandiana according to claim 3, characterised in that: The sterilization time of the alcohol and sodium hypochlorite is 10 s respectively.

5. The tissue culture propagation method of Terminalia ferdinandiana according to claim 1, characterised in that: The proliferation culture is to select the aseptic seedlings of Anacardium occidentale with a height of more than 5 cm after initiation culture, cut the seedlings into 1.5-2.5 cm stem segments with buds, vertically inoculate the stem segments, and the inoculation depth is 0.4-0.6 cm.

6. The tissue culture propagation method of Terminalia ferdinandiana according to claim 1, characterised in that: The rooting culture is to select the healthy seedlings of the proliferation culture, cut the seedlings into stem segments with a length of more than 3 cm, and inoculate the stem segments for rooting.

7. The tissue culture propagation method of Terminalia ferdinandiana according to claim 1, characterised in that: The initiation culture, proliferation culture and rooting culture have the same conditions, which comprise: 8-14 h of light and 10-16 h of darkness, a light intensity of 2000 Lux, and / or a temperature of 24 ℃±2 ℃ and a humidity of 60 %±10 %.

8. The tissue culture propagation method of Terminalia ferdinandiana according to claim 1, characterised in that: The initiation culture time is 30-32 d, the proliferation culture time is 40-42 d, and / or the rooting culture time is 10-30 d.

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