Method for promoting coconut callus induction by using puerarin

Abstract

The invention discloses a method for promoting coconut callus induction by using puerarin, and relates to the technical field of plant tissue culture. According to the method, tender inflorescences of coconut trees serve as tissue culture explants, a Y3 culture medium serves as a basic culture medium, 3 mg / L puerarin is added in the callus induction stage, it is found that the puerarin can partially replace 2, 4-D or dicamba, the use amount of the 2, 4-D or dicamba is reduced, the risk of subsequent regeneration malformation is reduced, the browning rate of the explants is reduced by 43%, the callus induction rate is increased by 16%, and the survival rate of the explants is increased. And the induction time is shortened by 30 days. Aiming at the problems of explant browning and low callus induction rate caused by high-concentration herbicide plant hormones (2, 4-D or dicamba), puerarin is added in the coconut tissue culture regeneration process, so that the dosage of 2, 4-D or dicamba is reduced, the explant browning rate is reduced, the callus induction rate is improved, the induction time is shortened, and the tissue culture regeneration method has the advantages that the tissue culture regeneration efficiency is improved, and the tissue culture regeneration cost is reduced. The method has good popularization and application prospects in the field of coconut tissue culture and rapid propagation.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, and in particular to a method for promoting coconut callus induction using puerarin. Background Technology

[0002] Coconut is Hainan's most distinctive tropical woody oilseed crop, playing a vital role in the province's socio-economic development. Fresh coconut meat contains 35% oil, with a yield of up to 2.26 tons per hectare, making it a significant source of edible oil in tropical regions. Currently, coconut cultivation primarily relies on seedling propagation, which suffers from problems such as poor variety uniformity and limited seedling quantity, failing to meet the high-standard development needs of the coconut planting and downstream processing industries. Establishing an efficient tissue culture and rapid propagation technology system for coconuts is one of the important ways to solve these problems.

[0003] In coconut tissue culture, young inflorescences are commonly used as explant material due to their high induction rate. In inflorescence culture, young inflorescences with undifferentiated male / female flowers are selected, and the florets are induced to dedifferentiate and redifferentiate to form somatic embryos. Compared to using zygotic embryos (heterozygotes) or microspores (haploids), the main advantage of using young inflorescences is that they originate from somatic cell tissue of the parent plant, thus preserving the parent's genetic characteristics intact and ensuring that the offspring's genotype is consistent with the mother plant, thereby achieving stable transmission of superior traits. Furthermore, the obtained tissue seedlings are diploid and can be directly used in actual production.

[0004] In coconut tissue culture using young inflorescences as material, 2,4-dichlorophenoxyacetic acid (2,4-D) is the commonly used inducing hormone in the callus induction stage. As the mainstream hormone for inducing callus, 2,4-D has a significant effect on callus induction in various explants such as young coconut inflorescences, zygotic embryos, and leaves. However, the concentration of 2,4-D used in existing callus induction media is mostly 100~200 mg / L, and the usage period is 6~9 months or even longer, which has many drawbacks: (1) Browning of explants: High concentrations of 2,4-D stimulate explant cells to produce excessive phenolic compounds, leading to browning of the culture medium and tissues. At the same time, it disrupts the balance of reactive oxygen species (ROS) in cells, leading to oxidative stress, damaging cell membrane structure, accelerating the browning process and affecting cell viability. (2) Callus malformation: High concentrations of 2,4-D easily lead to waterlogging of callus tissue, resulting in rapid growth, but difficulty in differentiation and easy browning and death; (3) Difficulty in differentiation: 2,4-D concentrations exceeding the appropriate range will remain in the tissue, are not easily metabolized, interfere with normal cell division and differentiation, and affect subsequent somatic embryo induction and regeneration. In general, although the traditional 2,4-D-based callus induction system is effective, it still has limitations in terms of explant browning and callus induction rate.

[0005] To address the aforementioned issues, a recent research trend in tissue culture has emerged towards using auxin-like substances derived from natural plants to replace synthetic growth regulators. Puerarin, a major active ingredient extracted from the traditional Chinese medicine kudzu root, belongs to the isoflavone class of compounds. Therefore, this invention adds puerarin and other natural plant active substances to the callus culture medium, which is of great significance for establishing efficient and rapid propagation of coconut tissue culture. Summary of the Invention

[0006] To address the aforementioned deficiencies in existing technologies, this invention proposes a method for promoting coconut callus induction using puerarin, thereby solving the problems mentioned in the background section. This invention uses puerarin (CAS No.: 3681-99-0) to partially replace 2,4-D or dicamba for rapid tissue culture propagation of coconut, effectively reducing the browning rate of young coconut inflorescences during callus induction, increasing the callus induction rate, and shortening the callus induction time.

[0007] To achieve the above objectives, the present invention provides the following technical solution: A method for promoting coconut callus induction using puerarin, the method comprising the following steps: (1) Explant pretreatment: Using young coconut inflorescences as explants, the explants were disinfected and cut into 0.5~1.5cm segments for later use; (2) Callus induction: Small explant segments were inoculated onto callus induction medium and induced and subcultured in sequence to obtain callus tissue; The callus induction medium is based on Y3 medium, and each liter of the basic medium contains 1-5 mg / L of puerarin, 1-3 g / L of activated carbon, 2-5 g / L of plant gel, 30-60 g / L of sucrose, and any one of 20-50 mg / L of dicamba or 60-120 mg / L of 2,4-D.

[0008] Preferably, the young coconut inflorescences in step (1) are from Hainan native cultivated coconut varieties that have been bearing fruit normally for 2-3 years.

[0009] Preferably, the length of the young coconut inflorescence in step (1) is 10-14 cm, and the two spathes are intact.

[0010] Preferably, the disinfection method in step (1) includes the following steps: S1. Rinse the outer layer of the young coconut inflorescence 2-4 times with 75% alcohol by volume. Then remove the first spathe and wipe the entire inflorescence with 75% alcohol by volume. Let it dry. S2. Remove the second spathe again, take out the spikelets inside, select the top 2 / 3 of each spikelet, soak it in sterile water, change the sterile water every 15 minutes, and obtain the sterilized explant.

[0011] Preferably, the soaking time in step S2 is 20-30 minutes.

[0012] Preferably, the pH value of the callus induction medium in step (2) is 5.60~5.90.

[0013] Preferably, the Y3 culture medium formula (per liter) is: calcium chloride dihydrate 29.4 g / L, potassium chloride 149.6 g / L, ammonium chloride 53.6 g / L, potassium nitrate 202.4 g / L, sodium dihydrogen phosphate 27.56 g / L, boric acid 0.3092 g / L, magnesium sulfate heptahydrate 24.8 g / L, manganese sulfate 0.848 g / L, potassium iodide 0.832 g / L, copper sulfate 0.0248 g / L, and zinc sulfate 0.7 g / L. 2 g / L, nickel chloride 0.0024 g / L, cobalt chloride 0.024 g / L, sodium molybdate dihydrate 0.024 g / L, disodium EDTA 3.724 g / L, ferrous sulfate heptahydrate 1.5 g / L, thiamine hydrochloride 0.1012 g / L, pyridoxine hydrochloride 0.1028 g / L, nicotinic acid 0.1 g / L, L-asparagine 8.8 g / L, L-arginine 10 g / L, L-glutamine 10 g / L.

[0014] Preferably, the callus induction medium in step (2) is based on Y3 medium, and each liter of basic medium contains 5 mg / L of puerarin, 2.5 g / L of activated carbon, 4 g / L of plant gel, 40 g / L of sucrose, and any one of 35 mg / L of dicamba or 90 mg / L of 2,4-D.

[0015] Preferably, the puerarin mentioned in step (2) is added after being dissolved in dimethyl sulfoxide. The specific steps are as follows: dissolve the puerarin in dimethyl sulfoxide (DMSO) to prepare a 10 g / L storage solution, filter it through a 0.22 μm filter membrane for sterilization, and store it at -4℃ in the dark for later use.

[0016] Preferably, the induction culture and subculture proliferation culture conditions in step (2) are: temperature 25℃~28℃, humidity 55%~65%, dark culture; subculture cycle is 25~30 days.

[0017] The present invention relates to the application of the method of promoting coconut callus induction using puerarin in reducing the browning rate of young coconut inflorescences during the callus induction process, increasing the callus induction rate, and shortening the callus induction time.

[0018] Compared with the prior art, the beneficial effects of the present invention are: This invention is the first to add puerarin during the callus induction stage of young coconut inflorescences, which can reduce the browning degree of explants, increase the callus induction rate, and accelerate the callus induction speed. It was also found that adding 3 mg / L puerarin during the callus induction stage can partially replace the efficacy of 2,4-D or dicamba, and the browning rate of explants was reduced by 43%, the callus induction rate was increased by 16%, and the induction time was shortened by 30 days. Attached Figure Description

[0019] Figure 1 The effect of adding puerarin on the browning of young coconut inflorescences was investigated. A represents the browning results after 30 days of inoculation with the commonly used coconut young inflorescence callus induction medium (120 mg / L 2,4-D + 0 mg / L puerarin, control group); B represents the browning results after 30 days of inoculation with the coconut young inflorescence callus induction medium used in this invention (90 mg / L 2,4-D + 3 mg / L puerarin, treatment group). Figure 2 The effect of adding puerarin on callus induction in young coconut inflorescences was investigated. A represents the effect of the commonly used coconut young inflorescence callus induction medium (120 mg / L 2,4-D + 0 mg / L puerarin, control group) after 120 days of inoculation; B represents the effect of the coconut young inflorescence callus induction medium used in this invention (90 mg / L 2,4-D + 3 mg / L puerarin, treatment group) after 120 days of inoculation. Detailed Implementation

[0020] To enable those skilled in the art to better understand the technical content of this invention, the technical solution of this invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the embodiments of this invention are conventional methods. In the following embodiments and comparative examples, unless otherwise specified, all raw materials can be obtained commercially or prepared by conventional methods.

[0021] Example 1 A method for promoting coconut callus induction using puerarin, the method comprising the following steps: (1) Explant pretreatment: Select young inflorescences of Hainan tall coconut trees that have been bearing fruit normally for 2 years, with a length of 10cm and two intact spathes, as explants. Disinfect the explants and cut them into 0.5cm segments for later use. (2) Callus induction: Small explant segments were inoculated onto callus induction medium at pH 5.60 for induction culture and subculture proliferation culture. The culture conditions were 25℃, 55% humidity, dark culture, and a subculture cycle of 25 days to obtain callus tissue. The callus induction medium is based on Y3 medium, and each liter of the basic medium contains 1 mg / L puerarin, 1 g / L activated carbon, 2 g / L plant gel, 30 g / L sucrose, and 20 mg / L dicamba.

[0022] The disinfection method includes the following steps: S1. Rinse the outer layer of the young coconut inflorescence twice with 75% alcohol by volume. Then remove the first spathe and wipe the entire inflorescence with 75% alcohol by volume. Let it air dry. S2. Remove the second spathe again, take out the spikelets inside, select the top 2 / 3 of each spikelet of the inflorescence, soak it in sterile water for 20 minutes, change the sterile water every 15 minutes, and obtain the sterilized explant.

[0023] Example 2 A method for promoting coconut callus induction using puerarin, the method comprising the following steps: (1) Explant pretreatment: Select young inflorescences of “Wenye No. 2” coconut trees that have been bearing fruit normally for 3 years, with a length of 14cm and two intact spathes, as explants. Disinfect the explants and cut them into 1.5cm segments for later use. (2) Callus induction: Small explant segments were inoculated onto callus induction medium at pH 5.90 for induction culture and subculture proliferation culture. The culture conditions were 28℃, 65% humidity, dark culture, and a subculture cycle of 30 days to obtain callus tissue. The callus induction medium is based on Y3 medium, and each liter of the basic medium contains 5 mg / L puerarin, 3 g / L activated carbon, 5 g / L plant gel, 60 g / L sucrose, and 50 mg / L dicamba.

[0024] The disinfection method includes the following steps: S1. Rinse the outer layer of the young coconut inflorescence four times with 75% alcohol by volume. Then remove the first spathe and wipe the entire inflorescence with 75% alcohol by volume. Let it air dry. S2. Remove the second spathe again, take out the spikelets inside, select the top 2 / 3 of each spikelet of the inflorescence, soak it in sterile water for 30 minutes, change the sterile water every 15 minutes, and obtain the sterilized explant.

[0025] Example 3 A method for promoting coconut callus induction using puerarin, the method comprising the following steps: (1) Explant pretreatment: Select young inflorescences of “Wenye No. 3” coconut trees that have been bearing fruit normally for 3 years, with a length of 12cm and two intact spathes as explants. Disinfect the explants and cut them into 1.0cm segments for later use. (2) Callus induction: Small explant segments were inoculated onto callus induction medium at pH 5.90 for induction culture and subculture proliferation culture. The culture conditions were 27℃, 60% humidity, dark culture, and a subculture cycle of 30 days to obtain callus tissue. The callus induction medium is based on Y3 medium, and each liter of the basic medium contains 2 mg / L puerarin, 2 g / L activated carbon, 3 g / L plant gel, 40 g / L sucrose, and 90 mg / L 2,4-D.

[0026] The disinfection method includes the following steps: S1. Rinse the outer layer of the young coconut inflorescence three times with 75% alcohol by volume. Then remove the first spathe and wipe the entire inflorescence with 75% alcohol by volume. Let it dry. S2. Remove the second spathe again, take out the spikelets inside, select the top 2 / 3 of each spikelet of the inflorescence, soak it in sterile water for 30 minutes, change the sterile water every 15 minutes, and obtain the sterilized explant.

[0027] Experimental Example 1: Browning Rate and Callus Induction Time of Young Coconut Inflorescences The experiment was set up according to the table below, where the browning rate (%) = number of browned explants / total number of explants × 100%. The test results are shown in Table 1 and... Figure 1 As shown, the commonly used callus induction medium supplemented with 2,4-D (120 mg / L) and dicamba (50 mg / L) was used as the control group. The hormone concentration was gradually reduced, and 0-5 mg / L puerarin was added at the same time. After 30 days, the browning rate of young inflorescences and the time of initial callus formation were counted.

[0028] The results showed that when the concentrations of 2,4-D or dicamba remained constant, the addition of puerarin increased the browning rate of explants. As the concentrations of 2,4-D or dicamba decreased, the browning rate of explants decreased, while the callus induction time was correspondingly prolonged. Adding puerarin to the callus induction medium while reducing the plant hormone concentration significantly reduced the browning rate and shortened the callus induction period. When the hormone concentration was below a specific threshold (60 mg / L), the effect of adding puerarin on shortening the induction time was not significant. Therefore, within a suitable range of plant hormone concentrations, adding puerarin to the callus induction medium can effectively inhibit browning and shorten the callus induction time.

[0029] Table 1. Effects of puerarin on browning rate and callus induction time of young coconut inflorescences.

[0030] Note: The browning rate of explants was statistically analyzed 30 days after inoculation.

[0031] Experimental Example 2: Coconut Callus Induction Rate The experiment was set up according to the table below, where the callus rate (%) = number of explants that induced callus / total number of explants × 100%. The test results are shown in Table 2 and... Figure 2 As shown, using commonly used culture media 2,4-D (120 mg / L) and dicamba (50 mg / L) as control groups, the concentration of plant hormones was gradually reduced, while 0-5 mg / L puerarin was added. The callus induction rate of young inflorescences was counted after 120 days.

[0032] The results showed that adding puerarin decreased callus induction rate when the concentrations of 2,4-D or dicamba remained constant. The callus induction rate gradually decreased with decreasing concentrations of 2,4-D or dicamba, but adding puerarin while decreasing the concentration significantly improved the induction rate. When the plant hormone concentration was below a specific threshold (60 mg / L), adding puerarin had no significant effect on improving the callus induction rate. Therefore, within a suitable range of plant hormone concentrations, adding puerarin to the callus induction medium can effectively improve the callus induction rate.

[0033] Table 2. Effects of puerarin on coconut callus induction rate

[0034] Note: The callus induction rate was statistically analyzed 120 days after inoculation.

[0035] In coconut tissue culture callus induction, the main factors affecting callus induction are the degree of explant browning and the ratio of plant hormones in the culture medium. Browning is mainly caused by the oxidation of large amounts of phenolic substances secreted by the explant after external stimulation and the imbalance of reactive oxygen species (ROS) caused by cell damage at the wound site. Puerarin, as a natural isoflavone compound, has strong antioxidant activity. First, it can scavenge reactive oxygen free radicals (ROS) generated by young inflorescences during culture, thereby reducing oxidative stress damage to explant cells and lowering the browning rate. Second, puerarin can inhibit the activity of polyphenol oxidase (PPO) and peroxidase (POD), blocking the oxidative transformation of phenolic compounds into quinones. Puerarin can reduce the formation of brown polymers by competitively binding to the active site of phenol oxidase. In addition, as an isoflavone compound, puerarin may also affect the distribution, activity and content of auxin carriers, or indirectly regulate cell membrane fluidity, protein kinase / phosphatase activity, related protein expression, cellular environment, etc., affecting the transport and distribution of plant hormones such as auxin, and ultimately playing a role in regulating callus induction.

[0036] In summary, adding puerarin to the callus induction medium can reduce the concentration of 2,4-D or dicamba, while also reducing the browning degree of young inflorescences and improving callus induction efficiency. Considering the browning rate, callus induction time, and callus induction rate, the optimal combination for callus induction is: 2,4-D 90 mg / L + puerarin 3 mg / L.

[0037] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for promoting coconut callus induction using puerarin, characterized in that, The method includes the following steps: (1) Explant pretreatment: Using young coconut inflorescences as explants, the explants were disinfected and cut into small sections for later use; (2) Callus induction: Small explant segments were inoculated onto callus induction medium and induced and subcultured in sequence to obtain callus tissue; The callus induction medium is based on Y3 medium, and each liter of the basic medium contains 1-5 mg / L of puerarin, 1-3 g / L of activated carbon, 2-5 g / L of plant gel, 30-60 g / L of sucrose, and any one of 20-50 mg / L of dicamba or 60-120 mg / L of 2,4-D.

2. The method according to claim 1, characterized in that, The young coconut inflorescences mentioned in step (1) are from local Hainan coconut varieties that have been bearing fruit normally for 2-3 years.

3. The method according to claim 1, characterized in that, The length of the young coconut inflorescence in step (1) is 10-14cm, and the two spathes are intact.

4. The method according to claim 1, characterized in that, The disinfection method described in step (1) includes the following steps: S1. Rinse the outer layer of the young coconut inflorescence 2-4 times with 75% alcohol by volume. Then remove the first spathe and wipe the entire inflorescence with 75% alcohol by volume. Let it air dry. S2. Remove the second spathe again, take out the spikelets inside, select the top 2 / 3 of each spikelet, soak it in sterile water, change the sterile water every 15 minutes, and obtain the sterilized explant.

5. The method according to claim 4, characterized in that, The soaking time in step S2 is 20-30 minutes.

6. The method according to claim 1, characterized in that, The length of the segment is 0.5~1.5cm.

7. The method according to claim 1, characterized in that, The pH value of the callus induction medium in step (2) is 5.60~5.

90.

8. The method according to claim 1, characterized in that, The callus induction medium in step (2) is based on Y3 medium, and each liter of basic medium contains 5 mg / L of puerarin, 2.5 g / L of activated carbon, 4 g / L of plant gel, 40 g / L of sucrose, and any one of 35 mg / L of dicamba or 90 mg / L of 2,4-D.

9. The method according to claim 1, characterized in that, The induction culture and subculture proliferation culture conditions in step (2) are: temperature 25℃~28℃, humidity 55%~65%, dark culture; subculture cycle is 25~30 days.

10. The application of the method according to any one of claims 1 to 9 in reducing the browning rate of young coconut inflorescences during callus induction, increasing the callus induction rate, and shortening the callus induction time.

Images