CN112385540B - Tissue culture rapid propagation method taking areca inflorescence as explant - Google Patents
Tissue culture rapid propagation method taking areca inflorescence as explant Download PDFInfo
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Abstract
The invention discloses a tissue culture rapid propagation method taking areca inflorescence as an explant, which comprises the steps of explant selection, induction culture, proliferation culture, germination culture and transplantation. The invention provides a method for successfully obtaining a plant regeneration rapid propagation system by taking an areca-nut inflorescence as an explant and a direct somatic embryo generation mode, wherein the induction rate of the somatic embryo of the areca-nut is 25.5%, the increment coefficient of the somatic embryo is up to 11 at most, the plant regeneration rate is 59.26%, and only 8 months are needed from the culture of an explant material to the final obtainment of a regeneration plant.
Description
Technical Field
The invention belongs to the technical field of tissue culture, and particularly relates to a tissue culture rapid propagation method taking areca inflorescence as an explant.
Background
Betel nut (Areca catechu L.) is a tropical and subtropical economic crop of Areca in Palmae, China is the second major producing area of Areca in the world, and the planting of Areca nut in China has been in history for more than 1500 years, wherein Hainan and Taiwan in China are major producing areas of Areca nut in China, and a small amount of Areca nut is cultivated in Guangdong, Guangxi, Yunnan, Fujian and other provinces. According to statistics, the planting area of the betel nuts in China in 2008 reaches 6.28 ten thousand hm2. The areca in China is mainly concentrated in Hainan, and the planting area of the areca in Hainan in 2008 is 6.27 ten thousand hm2Accounts for 99.97 percent of Chinese continent and has a harvest area of about 3.13 ten thousand hm2The yield reaches 11.65 ten thousand t, and the yield per unit is 16.54kg/hm2The total yield of areca nuts in the whole year is 23.30 hundred million yuan. At present, areca catechu has been developed as the second largest tropical economic crop in Hainan province, second only to natural rubber, and is one of the economic shores in Hainan.
At present, the betel nut seedlings are all in a spontaneous state, the breeding system of betel nut fine variety seedlings is not perfect, uniform market supervision and referential production standards are lacked, a large number of inferior seedlings with diseases, impure germplasm and unclear sources are caused to flow into the market, great threat is caused to the healthy development of the betel nut industry, and the cultivation of betel nut fine variety is a fundamental way for solving the problems.
The areca has only one growing point, so the areca cannot be bred by a conventional asexual propagation method, and the areca is high heterozygous genetically, so that the offspring can generate character segregation, and the application of sexual hybridization in the genetic improvement of the areca is limited. The method for establishing areca plant regeneration through plant somatic cell generation is a rapid, efficient and reliable areca fine variety breeding technology and is also an optimal way for realizing the rapid propagation of areca fine clone.
The research on the areca tissue culture technology in China is less, and Huang Liyun et al take 6-8 months areca nuts and preliminarily realize the in vitro culture of areca zygote embryos. Through retrieval, no relevant research report for establishing areca regeneration plants through a somatic embryogenesis way exists at present.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a tissue culture and rapid propagation method taking areca inflorescence as an explant.
The purpose of the invention is realized by the following technical scheme: a tissue culture rapid propagation method taking areca inflorescence as explant comprises the following steps:
s1, selection of explants: collecting young and tender inflorescences of areca nuts, and cleaning and disinfecting the young and tender inflorescences of the areca nuts to be used as explants for later use;
s2, induced culture: inoculating the explant sterilized in the step S1 to a somatic embryo induction culture medium for dark culture to obtain a somatic embryo; the somatic embryo induction culture medium is any one of Y3 minimal medium + 1-60 mg/L picloram +60g/L sucrose +2g/L plant gel, Y3 minimal medium + 1-60 mg/L2, 4-D +60g/L sucrose +2g/L plant gel or Y3 minimal medium + 1-60 mg/L dicamba +60g/L sucrose +2g/L plant gel, and the pH value of the culture medium is 5-6;
s3, propagation culture: transferring the somatic embryos to a proliferation medium for dark culture, wherein the proliferation medium comprises: y3 basic culture medium, 1-10 mg/L2, 4-D, 1-10 mg/L6-BA, 30g/L sucrose and 2g/L plant gel, wherein the pH value of the culture medium is 5-6;
s4, germination culture: transferring the somatic embryos subjected to proliferation culture to a germination culture medium for germination culture under the condition of illumination to obtain complete regeneration plants; wherein, the germination culture medium is as follows: MS basic culture medium, 0.5-5 mg/L TDZ, 2g/L active carbon, 30g/L cane sugar and 2g/L plant gel, wherein the pH value of the culture medium is 5-6;
s5, transplanting: and (3) transplanting 2-4 leaves of the completely rooted regeneration plant obtained by germination culture into a culture medium for culture under a spraying system, wherein the culture medium is a mixture of sand, soil and an organic fertilizer.
Further, the cleaning and disinfecting specific operations described in step S1 are: washing young and tender inflorescence of Arecae semen with flowing water for 30min, transferring to a clean bench, and rinsing with sterile water; soaking the mixture in 75% alcohol for 5min, then soaking the mixture in 8% sodium hypochlorite for 10-15 min, finally washing the mixture with sterile water for 5-6 times, and placing the mixture into the sterile water for later use.
Further, the cultivation time of the dark cultivation in the step S2 is 90-160 days, and the cultivation temperature is 26 +/-1 ℃.
Further, the somatic embryo induction medium in step S2 is: y3 minimal medium +50mg/L picloram +60g/L sucrose +2g/L plant gel.
Further, the cultivation time of the dark cultivation in the step S3 is 25 to 30 days, and the cultivation temperature is 26 +/-1 ℃.
Further, in step S3, the proliferation medium is: y3 minimal medium +2 mg/L2, 4-D +2mg/L6-BA +30g/L sucrose +2g/L plant gel.
Further, the lighting conditions in step S4 are: the illumination intensity is 1000-3000Lx, and the illumination time is 16-18 h/d.
Further, the culture time of the germination culture in the step S4 is 60-90 days, the culture temperature is 26 +/-1 ℃, and the subculture is carried out once every 30 days.
Further, the germination medium in step S4 is: MS minimal medium +2mg/L TDZ +2g/L active carbon +30g/L sucrose +2g/L plant gel.
Further, the weight ratio of the sand, the soil and the organic fertilizer in the step S5 is 10:2: 1.
The invention has the following advantages: the invention provides a method for successfully obtaining a plant regeneration rapid propagation system by taking an areca-nut inflorescence as an explant and a direct somatic embryo generation mode, wherein the induction rate of the somatic embryo of the areca-nut is 25.5%, the increment coefficient of the somatic embryo is up to 11 at most, the plant regeneration rate is 59.26%, and only 8 months are needed from the culture of an explant material to the final obtainment of a regeneration plant.
Drawings
FIG. 1 is a schematic diagram of somatic embryos obtained by direct induction of areca inflorescences;
FIG. 2 is a schematic diagram showing the proliferation of somatic embryos of betel nuts;
FIG. 3 is a schematic diagram of the regeneration plant of Areca catechu L.
Detailed Description
The invention is further described with reference to the following figures and examples, without limiting the scope of the invention to the following:
the experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
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Example 1: a tissue culture rapid propagation method taking areca inflorescence as explant comprises the following steps:
s1, selection of explants: collecting young inflorescences of Arecae semen, washing with running water for 30min, transferring to a clean bench, and rinsing with sterile water; soaking in 75% ethanol for 5min, soaking in 8% sodium hypochlorite for 10min, washing with sterile water for 5 times, placing in sterile water, and cleaning and sterilizing to obtain explant;
s2, induced culture: inoculating the explants sterilized in the step S1 to a somatic embryo induction culture medium, and performing dark culture to obtain somatic embryos, wherein the culture time is 90 days, and the culture temperature is 26 +/-1 ℃ as shown in figure 1; wherein the somatic embryo induction culture medium is Y3 minimal medium +1mg/L picloram +60g/L sucrose +2g/L plant gel, and the pH value of the culture medium is 5;
s3, propagation culture: the somatic embryos obtained were transferred to a multiplication medium for dark culture, as shown in FIG. 2, for 25 days at a temperature of 26. + -. 1 ℃ in the culture medium: y3 minimal medium +1 mg/L2, 4-D +1mg/L6-BA +30g/L sucrose +2g/L plant gel, and the pH value of the medium is 5;
s4, germination culture: transferring the somatic embryos subjected to proliferation culture to a germination culture medium for germination culture under the illumination condition, wherein the culture time is 60 days, the culture temperature is 26 +/-1 ℃, and subculturing once every 30 days to obtain complete regeneration plants, as shown in figure 3, wherein the illumination condition is as follows: the illumination intensity is 1000Lx, and the illumination time is 16 h/d; the germination culture medium comprises: MS basic culture medium, 0.5mg/L TDZ, 2g/L active carbon, 30g/L cane sugar and 2g/L plant gel, wherein the pH value of the culture medium is 5;
s5, transplanting: and (3) growing 2-4 leaves of the completely rooted regeneration plant obtained by germination culture, transplanting the regenerated plant into a culture medium, and culturing the regenerated plant under a spraying system, wherein the culture medium is a mixture of sand, soil and an organic fertilizer, and the weight ratio of the sand, the soil and the organic fertilizer is 10:2: 1.
Example 2: a tissue culture rapid propagation method taking areca inflorescence as explant comprises the following steps:
s1, selection of explants: collecting young inflorescences of Arecae semen, washing with running water for 30min, transferring to a clean bench, and rinsing with sterile water; soaking in 75% ethanol for 5min, soaking in 8% sodium hypochlorite for 15min, washing with sterile water for 6 times, placing in sterile water, and cleaning and sterilizing to obtain explant;
s2, induced culture: inoculating the explants sterilized in the step S1 to a somatic embryo induction culture medium, and performing dark culture to obtain somatic embryos, wherein the culture time is 160 days, and the culture temperature is 26 +/-1 ℃ as shown in figure 1; wherein the somatic embryo induction culture medium is Y3 minimal medium +60mg/L picloram +60g/L sucrose +2g/L plant gel, and the pH value of the culture medium is 6;
s3, propagation culture: the somatic embryos obtained were transferred to a multiplication medium for dark culture, as shown in FIG. 2, for 30 days at a temperature of 26. + -. 1 ℃ in the culture medium: y3 minimal medium, 10 mg/L2, 4-D, 1-10 mg/L6-BA, 30g/L sucrose and 2g/L plant gel, wherein the pH value of the minimal medium is 6;
s4, germination culture: transferring the somatic embryos subjected to proliferation culture to a germination culture medium for germination culture under the illumination condition, wherein the culture time is 90 days, the culture temperature is 26 +/-1 ℃, and subculturing once every 30 days to obtain complete regeneration plants, as shown in figure 3, wherein the illumination condition is as follows: the illumination intensity is 3000Lx, and the illumination time is 18 h/d; the germination culture medium comprises: MS basic culture medium, 5mg/L TDZ, 2g/L active carbon, 30g/L cane sugar and 2g/L plant gel, wherein the pH value of the culture medium is 6;
s5, transplanting: and (3) growing 2-4 leaves of the completely rooted regeneration plant obtained by germination culture, transplanting the regenerated plant into a culture medium, and culturing the regenerated plant under a spraying system, wherein the culture medium is a mixture of sand, soil and an organic fertilizer, and the weight ratio of the sand, the soil and the organic fertilizer is 10:2: 1.
Example 3: a tissue culture rapid propagation method taking areca inflorescence as explant comprises the following steps:
s1, selection of explants: collecting young inflorescences of Arecae semen, washing with running water for 30min, transferring to a clean bench, and rinsing with sterile water; soaking in 75% ethanol for 5min, soaking in 8% sodium hypochlorite for 13min, washing with sterile water for 5 times, placing in sterile water, and cleaning and sterilizing to obtain explant;
s2, induced culture: inoculating the explants sterilized in the step S1 to a somatic embryo induction culture medium, and performing dark culture to obtain somatic embryos, wherein the culture time is 100 days, and the culture temperature is 26 +/-1 ℃ as shown in figure 1; wherein the somatic embryo induction culture medium is Y3 minimal medium, 50mg/L picloram, 60g/L sucrose and 2g/L plant gel, and the pH value of the culture medium is 5;
s3, propagation culture: the somatic embryos obtained were transferred to a multiplication medium for dark culture, as shown in FIG. 2, for 27 days at a temperature of 26. + -. 1 ℃ in the culture medium: y3 minimal medium +2 mg/L2, 4-D +5mg/L6-BA +30g/L sucrose +2g/L plant gel, and the pH value of the medium is 6;
s4, germination culture: transferring the somatic embryos subjected to proliferation culture to a germination culture medium for germination culture under the illumination condition, wherein the culture time is 70 days, the culture temperature is 26 +/-1 ℃, and subculturing once every 30 days to obtain complete regeneration plants, as shown in figure 3, wherein the illumination condition is as follows: the illumination intensity is 1500Lx, and the illumination time is 17 h/d; the germination culture medium comprises: MS basic culture medium, 1mg/L TDZ, 2g/L active carbon, 30g/L cane sugar and 2g/L plant gel, wherein the pH value of the culture medium is 6;
s5, transplanting: and (3) growing 2-4 leaves of the completely rooted regeneration plant obtained by germination culture, transplanting the regenerated plant into a culture medium, and culturing the regenerated plant under a spraying system, wherein the culture medium is a mixture of sand, soil and an organic fertilizer, and the weight ratio of the sand, the soil and the organic fertilizer is 10:2: 1.
Example 4: a tissue culture rapid propagation method taking areca inflorescence as explant comprises the following steps:
s1, selection of explants: collecting young inflorescences of Arecae semen, washing with running water for 30min, transferring to a clean bench, and rinsing with sterile water; soaking in 75% ethanol for 5min, soaking in 8% sodium hypochlorite for 14min, washing with sterile water for 5 times, placing in sterile water, and cleaning and sterilizing to obtain explant;
s2, induced culture: inoculating the explants sterilized in the step S1 to a somatic embryo induction culture medium, and performing dark culture to obtain somatic embryos, wherein the culture time is 120 days, and the culture temperature is 26 +/-1 ℃ as shown in figure 1; wherein the somatic embryo induction culture medium is Y3 minimal medium +1 mg/L2, 4-D +60g/L sucrose +2g/L plant gel, and the pH value of the culture medium is 5;
s3, propagation culture: the somatic embryos obtained were transferred to a multiplication medium for dark culture, as shown in FIG. 2, for 25 days at a temperature of 26. + -. 1 ℃ in the culture medium: y3 minimal medium +2 mg/L2, 4-D +8mg/L6-BA +30g/L sucrose +2g/L plant gel, and the pH value of the medium is 5;
s4, germination culture: transferring the somatic embryos subjected to proliferation culture to a germination culture medium for germination culture under the illumination condition, wherein the culture time is 80 days, the culture temperature is 26 +/-1 ℃, and subculturing once every 30 days to obtain complete regeneration plants, as shown in figure 3, wherein the illumination condition is as follows: the illumination intensity is 2000Lx, and the illumination time is 18 h/d; the germination culture medium comprises: MS basic culture medium, 3mg/L TDZ, 2g/L active carbon, 30g/L cane sugar and 2g/L plant gel, wherein the pH value of the culture medium is 6;
s5, transplanting: and (3) growing 2-4 leaves of the completely rooted regeneration plant obtained by germination culture, transplanting the regenerated plant into a culture medium, and culturing the regenerated plant under a spraying system, wherein the culture medium is a mixture of sand, soil and an organic fertilizer, and the weight ratio of the sand, the soil and the organic fertilizer is 10:2: 1.
Example 5: a tissue culture rapid propagation method taking areca inflorescence as explant comprises the following steps:
s1, selection of explants: collecting young inflorescences of Arecae semen, washing with running water for 30min, transferring to a clean bench, and rinsing with sterile water; soaking in 75% ethanol for 5min, soaking in 8% sodium hypochlorite for 10min, washing with sterile water for 6 times, placing in sterile water, and cleaning and sterilizing to obtain explant;
s2, induced culture: inoculating the explants sterilized in the step S1 to a somatic embryo induction culture medium, and performing dark culture to obtain somatic embryos, wherein the culture time is 130 days, and the culture temperature is 26 +/-1 ℃ as shown in figure 1; wherein the somatic embryo induction culture medium is Y3 minimal medium +60 mg/L2, 4-D +60g/L sucrose +2g/L plant gel, and the pH value of the culture medium is 5;
s3, propagation culture: the somatic embryos obtained were transferred to a multiplication medium for dark culture, as shown in FIG. 2, for 30 days at a temperature of 26. + -. 1 ℃ in the culture medium: y3 minimal medium +3 mg/L2, 4-D +5mg/L6-BA +30g/L sucrose +2g/L plant gel, and the pH value of the medium is 6;
s4, germination culture: transferring the somatic embryos subjected to proliferation culture to a germination culture medium for germination culture under the illumination condition, wherein the culture time is 90 days, the culture temperature is 26 +/-1 ℃, and subculturing once every 30 days to obtain complete regeneration plants, as shown in figure 3, wherein the illumination condition is as follows: the illumination intensity is 2700Lx, and the illumination time is 16 h/d; the germination culture medium comprises: MS basic culture medium, 3mg/L TDZ, 2g/L active carbon, 30g/L cane sugar and 2g/L plant gel, wherein the pH value of the culture medium is 6;
s5, transplanting: and (3) growing 2-4 leaves of the completely rooted regeneration plant obtained by germination culture, transplanting the regenerated plant into a culture medium, and culturing the regenerated plant under a spraying system, wherein the culture medium is a mixture of sand, soil and an organic fertilizer, and the weight ratio of the sand, the soil and the organic fertilizer is 10:2: 1.
Example 6: a tissue culture rapid propagation method taking areca inflorescence as explant comprises the following steps:
s1, selection of explants: collecting young inflorescences of Arecae semen, washing with running water for 30min, transferring to a clean bench, and rinsing with sterile water; soaking in 75% ethanol for 5min, soaking in 8% sodium hypochlorite for 11min, washing with sterile water for 5 times, placing in sterile water, and cleaning and sterilizing to obtain explant;
s2, induced culture: inoculating the explants sterilized in the step S1 to a somatic embryo induction culture medium, and performing dark culture to obtain somatic embryos, wherein the culture time is 90 days, and the culture temperature is 26 +/-1 ℃ as shown in figure 1; wherein the somatic embryo induction culture medium is Y3 minimal medium +10 mg/L2, 4-D +60g/L sucrose +2g/L plant gel, and the pH value of the culture medium is 6;
s3, propagation culture: the somatic embryos obtained were transferred to a multiplication medium for dark culture, as shown in FIG. 2, for 27 days at a temperature of 26. + -. 1 ℃ in the culture medium: y3 minimal medium +8 mg/L2, 4-D +2mg/L6-BA +30g/L sucrose +2g/L plant gel, and the pH value of the medium is 6;
s4, germination culture: transferring the somatic embryos subjected to proliferation culture to a germination culture medium for germination culture under the illumination condition, wherein the culture time is 78 days, the culture temperature is 26 +/-1 ℃, and subculturing once every 30 days to obtain complete regeneration plants, as shown in figure 3, wherein the illumination condition is as follows: the illumination intensity is 3000Lx, and the illumination time is 16 h/d; the germination culture medium comprises: MS basic culture medium, 5mg/L TDZ, 2g/L active carbon, 30g/L cane sugar and 2g/L plant gel, wherein the pH value of the culture medium is 5;
s5, transplanting: and (3) growing 2-4 leaves of the completely rooted regeneration plant obtained by germination culture, transplanting the regenerated plant into a culture medium, and culturing the regenerated plant under a spraying system, wherein the culture medium is a mixture of sand, soil and an organic fertilizer, and the weight ratio of the sand, the soil and the organic fertilizer is 10:2: 1.
Example 7: a tissue culture rapid propagation method taking areca inflorescence as explant comprises the following steps:
s1, selection of explants: collecting young inflorescences of Arecae semen, washing with running water for 30min, transferring to a clean bench, and rinsing with sterile water; soaking in 75% ethanol for 5min, soaking in 8% sodium hypochlorite for 15min, washing with sterile water for 5 times, placing in sterile water, and cleaning and sterilizing to obtain explant;
s2, induced culture: inoculating the explants sterilized in the step S1 to a somatic embryo induction culture medium, and performing dark culture to obtain somatic embryos, wherein the culture time is 155 days, and the culture temperature is 26 +/-1 ℃ as shown in figure 1; wherein the somatic embryo induction culture medium is Y3 minimal medium, 1mg/L dicamba, 60g/L sucrose and 2g/L plant gel, and the pH value of the culture medium is 5;
s3, propagation culture: the somatic embryos obtained were transferred to a multiplication medium for dark culture, as shown in FIG. 2, for 25 days at a temperature of 26. + -. 1 ℃ in the culture medium: y3 minimal medium +10 mg/L2, 4-D +1mg/L6-BA +30g/L sucrose +2g/L plant gel, and the pH value of the medium is 6;
s4, germination culture: transferring the somatic embryos subjected to proliferation culture to a germination culture medium for germination culture under the illumination condition, wherein the culture time is 75 days, the culture temperature is 26 +/-1 ℃, and subculturing once every 30 days to obtain complete regeneration plants, as shown in figure 3, wherein the illumination condition is as follows: the illumination intensity is 3000Lx, and the illumination time is 16 h/d; the germination culture medium comprises: MS basic culture medium, 5mg/L TDZ, 2g/L active carbon, 30g/L cane sugar and 2g/L plant gel, wherein the pH value of the culture medium is 5;
s5, transplanting: and (3) growing 2-4 leaves of the completely rooted regeneration plant obtained by germination culture, transplanting the regenerated plant into a culture medium, and culturing the regenerated plant under a spraying system, wherein the culture medium is a mixture of sand, soil and an organic fertilizer, and the weight ratio of the sand, the soil and the organic fertilizer is 10:2: 1.
Example 8: a tissue culture rapid propagation method taking areca inflorescence as explant comprises the following steps:
s1, selection of explants: collecting young inflorescences of Arecae semen, washing with running water for 30min, transferring to a clean bench, and rinsing with sterile water; soaking in 75% ethanol for 5min, soaking in 8% sodium hypochlorite for 10min, washing with sterile water for 6 times, placing in sterile water, and cleaning and sterilizing to obtain explant;
s2, induced culture: inoculating the explants sterilized in the step S1 to a somatic embryo induction culture medium, and performing dark culture to obtain somatic embryos, wherein the culture time is 160 days, and the culture temperature is 26 +/-1 ℃ as shown in figure 1; wherein the somatic embryo induction culture medium is Y3 minimal medium, 60mg/L dicamba, 60g/L sucrose and 2g/L plant gel, and the pH value of the culture medium is 6;
s3, propagation culture: the somatic embryos obtained were transferred to a multiplication medium for dark culture, as shown in FIG. 2, for 30 days at a temperature of 26. + -. 1 ℃ in the culture medium: y3 minimal medium +10 mg/L2, 4-D +1mg/L6-BA +30g/L sucrose +2g/L plant gel, and the pH value of the medium is 5;
s4, germination culture: transferring the somatic embryos subjected to proliferation culture to a germination culture medium for germination culture under the illumination condition, wherein the culture time is 60 days, the culture temperature is 26 +/-1 ℃, and subculturing once every 30 days to obtain complete regeneration plants, as shown in figure 3, wherein the illumination condition is as follows: the illumination intensity is 3000Lx, and the illumination time is 18 h/d; the germination culture medium comprises: MS basic culture medium, 0.5mg/L TDZ, 2g/L active carbon, 30g/L cane sugar and 2g/L plant gel, wherein the pH value of the culture medium is 5;
s5, transplanting: and (3) growing 2-4 leaves of the completely rooted regeneration plant obtained by germination culture, transplanting the regenerated plant into a culture medium, and culturing the regenerated plant under a spraying system, wherein the culture medium is a mixture of sand, soil and an organic fertilizer, and the weight ratio of the sand, the soil and the organic fertilizer is 10:2: 1.
Example 9: a tissue culture rapid propagation method taking areca inflorescence as explant comprises the following steps:
s1, selection of explants: collecting young inflorescences of Arecae semen, washing with running water for 30min, transferring to a clean bench, and rinsing with sterile water; soaking in 75% ethanol for 5min, soaking in 8% sodium hypochlorite for 12min, washing with sterile water for 5 times, placing in sterile water, and cleaning and sterilizing to obtain explant;
s2, induced culture: inoculating the explants sterilized in the step S1 to a somatic embryo induction culture medium, and performing dark culture to obtain somatic embryos, wherein the culture time is 160 days, and the culture temperature is 26 +/-1 ℃ as shown in figure 1; wherein the somatic embryo induction culture medium is Y3 minimal medium, 35mg/L dicamba, 60g/L sucrose and 2g/L plant gel, and the pH value of the culture medium is 6;
s3, propagation culture: the somatic embryos obtained were transferred to a multiplication medium for dark culture, as shown in FIG. 2, for 30 days at a temperature of 26. + -. 1 ℃ in the culture medium: y3 minimal medium +8 mg/L2, 4-D +4mg/L6-BA +30g/L sucrose +2g/L plant gel, and the pH value of the medium is 5;
s4, germination culture: transferring the somatic embryos subjected to proliferation culture to a germination culture medium for germination culture under the illumination condition, wherein the culture time is 75 days, the culture temperature is 26 +/-1 ℃, and subculturing once every 30 days to obtain complete regeneration plants, as shown in figure 3, wherein the illumination condition is as follows: the illumination intensity is 1200Lx, and the illumination time is 18 h/d; the germination culture medium comprises: MS basic culture medium, 5mg/L TDZ, 2g/L active carbon, 30g/L cane sugar and 2g/L plant gel, wherein the pH value of the culture medium is 6;
s5, transplanting: and (3) growing 2-4 leaves of the completely rooted regeneration plant obtained by germination culture, transplanting the regenerated plant into a culture medium, and culturing the regenerated plant under a spraying system, wherein the culture medium is a mixture of sand, soil and an organic fertilizer, and the weight ratio of the sand, the soil and the organic fertilizer is 10:2: 1.
The following experiments illustrate the beneficial effects of the present invention:
1. effect of different concentrations and different types of auxin on induction of betel nut somatic embryogenesis
S1, washing collected young and tender inflorescences of betel nuts for 20min by using running water, removing surface dirt, transferring to a super-clean workbench, putting into a sterile beaker, and rinsing for 1 time by using sterile water; sterilizing the surface of inflorescence by soaking in 75% alcohol for 1min, soaking in 8% sodium hypochlorite for 10min, washing with sterile water for 5-6 times, and placing in sterile water;
s2, peeling off spathes on the outer side, taking out inflorescences, inoculating the inflorescences to different somatic embryo induction culture media, wherein the culture media comprise Y3 basic culture media, auxin, sucrose and plant gel, the factors and levels are different, the auxin, the sucrose and the plant gel are different in level, the pH value is 5.8, the processing factors and levels are shown in table 1, the culture temperature is 26 +/-1 ℃, subculture is carried out once every 30 days, and after dark culture is carried out for 120 days, somatic embryos are obtained;
s3, transferring the obtained somatic embryos to a multiplication culture medium for multiplication culture, wherein the multiplication culture medium comprises Y3 minimal medium, 2 mg/L2, 4-D, 2mg/L6-BA, 30g/L sucrose and 2g/L plant gel, the pH value is 5.8, the culture temperature is 26 +/-1 ℃, and the multiplication culture is carried out for 1 time every 30 days;
s4, transferring the obtained somatic embryos to a germination culture medium for culture, wherein the germination culture medium comprises: MS basic culture medium, 2mg TDZ, 2g/L active carbon, 30g/L sucrose and 2g/L plant gel, wherein the pH value is 5.8, the culture temperature is 26 +/-1 ℃, the illumination intensity is 1000-3000Lx, the illumination time is 16-18h/d, and a complete regeneration plant is obtained after 3 months of culture;
s5, transplanting the obtained rooted complete plant to a culture medium for culture under a spraying system after 2-4 leaves grow, wherein the culture medium is sand, soil and organic fertilizer in a ratio of 10:2:1, the transplanting survival rate is more than 90%, and the plant can be transplanted to a nursery for growth after 2-3 months of culture.
TABLE 1 Effect of different concentrations and different types of auxin on induction of Areca catechu somatic embryogenesis
As can be seen from Table 1, three auxins, picloram, 2,4-D and dicamba, all induced areca somatic embryos in the range of 1-60mg, wherein picloram was superior to the other 2 auxins, the induction rate was highest at the concentration of 50mg/L, and decreased after the concentration of more than 50mg/L, and the culture was strongly browned.
2. Induction rate of betel nut somatic embryogenesis under different basic culture medium conditions
S1, washing collected young and tender inflorescences of betel nuts with running water for 20min, removing surface dirt, transferring to a super-clean workbench, putting into a sterile beaker, rinsing with sterile water for 1 time, sterilizing the surfaces of the inflorescences, soaking with 75% alcohol for 1min, then soaking with 8% sodium hypochlorite for 10min, finally washing with sterile water for 5-6 times, and putting into sterile water for later use;
s2, peeling off spathes on the outer side, taking out inflorescences, inoculating the inflorescences to different somatic embryo induction culture media, treating the culture media by using different basic culture media, adding 50mg/L picloram, 60g/L sucrose and 2g/L plant gel, enabling the pH to be 5.8, specifically referring to table 2 for the different basic culture media, carrying out subculture once every 30 days at the culture temperature of 26 +/-1 ℃, and carrying out dark culture for 120 days to obtain somatic embryos;
s3, transferring the obtained somatic embryos to a multiplication culture medium for multiplication culture, wherein the multiplication culture medium comprises: y3 minimal medium +2 mg/L2, 4-D +2mg/L6-BA +30g/L sucrose +2g/L plant gel, pH 5.8, culture temperature 26 + -1 deg.C, subculture for 1 time every 30 days;
s4, transferring the obtained somatic embryos to a germination culture medium for culture, wherein the germination culture medium comprises: MS basic culture medium, 2mg TDZ, 2g/L active carbon, 30g/L sucrose and 2g/L plant gel, wherein the pH value is 5.8, the culture temperature is 26 +/-1 ℃, the illumination intensity is 1000-3000Lx, the illumination time is 16-18h/d, and a complete regeneration plant is obtained after 3 months of culture;
s5, transplanting the obtained rooted complete plant to a culture medium for cultivation under a spraying system after 2-4 leaves grow, wherein the culture medium is sand, soil and organic fertilizer in a ratio of 10:2:1, and the plant can be transplanted to a nursery for growth after 2-3 months of cultivation.
TABLE 2 induction rate of betel nut somatic embryogenesis under different minimal medium conditions
As shown in Table 2, the induction rates of the somatic embryos of the betel nuts are remarkably different by three different basic culture media, the overall induction rate of the Y3 culture medium is the highest, and the WPM is the lowest, so that the induction effect of the Y3 culture medium and picloram is the best at 50 mg/L.
3. Influence of different concentrations of 2,4-D and 6-BA ratio on betel nut somatic embryo proliferation
S1, washing collected young and tender inflorescences of betel nuts with running water for 20min, removing surface dirt, transferring to a super-clean workbench, putting into a sterile beaker, rinsing with sterile water for 1 time, sterilizing the surfaces of the inflorescences, soaking with 75% alcohol for 1min, then soaking with 8% sodium hypochlorite for 10min, finally washing with sterile water for 5-6 times, and putting into sterile water for later use;
s2, peeling off spathes on the outer side, taking out inflorescences, inoculating the inflorescences to a somatic embryo induction culture medium, wherein the culture medium comprises a Y3 basic culture medium, 50mg/L picloram, 60g/L sucrose and 2g/L plant gel, the pH value is 5.8, the culture temperature is 26 +/-1 ℃, subculture is carried out once every 30 days, and after dark culture is carried out for 120 days, a somatic embryo is obtained;
(3) transferring the obtained somatic embryos to a proliferation culture medium for proliferation culture, wherein the proliferation culture medium comprises Y3 minimal medium, 2,4-D with different concentrations, 6-BA with different concentrations, 30g/L sucrose and 2g/L plant gel, the pH value is 5.8, the culture temperature is 26 +/-1 ℃, subculture is carried out for 1 time every 30 days, and the specific concentrations of 2,4-D and 6-BA are shown in Table 3, wherein A1, A2, A3 and A4 respectively represent that the concentrations of 2,4-D are 1mg/L, 2mg/L, 5mg/L and 10mg/L, and B1, B2, B3 and B4 respectively represent that the concentrations of 6-BA are 1mg/L, 2mg/L, 5mg/L and 10 mg/L;
s4, transferring the obtained somatic embryos to a germination culture medium for culture, wherein the germination culture medium comprises: MS basic culture medium, 2mg TDZ, 2g/L active carbon, 30g/L sucrose and 2g/L plant gel, wherein the pH value is 5.8, the culture temperature is 26 +/-1 ℃, the illumination intensity is 1000-3000Lx, the illumination time is 16-18h/d, and a complete regeneration plant is obtained after 3 months of culture;
s5, transplanting the obtained rooted complete plant to a culture medium after 2-4 leaves grow, and culturing under a spraying system, wherein the culture medium is sand: soil: the organic fertilizer is 10:2:1, and can be transplanted to a nursery garden to grow after being cultured for 2-3 months.
TABLE 3 Effect of different concentrations of 2,4-D and 6-BA on Areca catechu somatic embryo proliferation
| Treatment of | Fold of proliferation | Treatment of | Fold of proliferation | Treatment of | Fold of proliferation | Treatment of | Fold of proliferation |
| A1B1 | 3.33 | A2B1 | 7.00 | A3B1 | 2.67 | A4B1 | 2.00 |
| A1B2 | 5.67 | A2B2 | 11.00 | A3B2 | 3.00 | A4B2 | 1.33 |
| A1B3 | 2.33 | A2B3 | 3.67 | A3B3 | 2.67 | A4B3 | 1.33 |
| A1B4 | 1.33 | A2B4 | 2.33 | A3B4 | 2.00 | A4B4 | 1.33 |
As can be seen from Table 3, the treatment of A2B2, namely the combination of 2 mg/L2, 4D and 2mg/L6-BA, has the best proliferation effect, and the proliferation fold can reach 11.00 at most.
4. Effect of different TDZ concentrations on somatic embryo Germination
S1, washing collected young and tender inflorescences of the betel nuts for 20min by using running water to remove surface dirt. Transferring to an ultra-clean workbench, putting into a sterile beaker, and rinsing with sterile water for 1 time; sterilizing the surface of inflorescence by soaking in 75% alcohol for 1min, soaking in 8% sodium hypochlorite for 10min, washing with sterile water for 5-6 times, and placing in sterile water;
s2, peeling off spathes on the outer side, taking out inflorescences, inoculating the inflorescences to a somatic embryo induction culture medium, wherein the culture medium comprises a Y3 basic culture medium, 50mg/L picloram, 60g/L sucrose and 2g/L plant gel, the pH value is 5.8, the culture temperature is 26 +/-1 ℃, subculture is carried out once every 30 days, and after dark culture is carried out for 120 days, a somatic embryo is obtained;
s3, transferring the obtained somatic embryos to a multiplication culture medium for multiplication culture, wherein the multiplication culture medium comprises Y3 minimal medium, 2 mg/L2, 4-D, 2mg/L6-BA, 30g/L sucrose and 2g/L plant gel, the pH value is 5.8, the culture temperature is 26 +/-1 ℃, and the multiplication culture is carried out for 1 time every 30 days;
s4, transferring the obtained somatic embryos to a germination culture medium for culture, wherein the germination culture medium comprises: MS basic culture medium, TDZ with different concentrations, 2g/L active carbon, 30g/L sucrose and 2g/L plant gel, the pH value is 5.8, the specific concentration of the TDZ is shown in a table 4, the culture temperature is 26 +/-1 ℃, the illumination intensity is 1000-3000Lx, the illumination time is 16-18h/d, and a complete regeneration plant is obtained after 3 months of culture;
s5, transplanting the obtained rooted complete plant to a culture medium after 2-4 leaves grow, and culturing under a spraying system, wherein the culture medium is sand: soil: the organic fertilizer is 10:2:1, and can be transplanted to a nursery garden to grow after being cultured for 2-3 months.
TABLE 4 Effect of different TDZ concentrations on somatic embryo Germination
| Numbering | TDZ concentration mg/L | Somatic embryo germination rate |
| 1 | 0.5 | 18.52％ |
| 2 | 1 | 48.15％ |
| 3 | 2 | 59.26％ |
| 4 | 3 | 40.74％ |
| 5 | 4 | 14.81％ |
| 6 | 5 | 7.41％ |
As can be seen from Table 4, TDZ has a significant effect on the germination of areca somatic cells, and the germination rate is the highest when the TDZ concentration is 2 mg/L.
5. Effect of different basic Medium on somatic embryo Germination of Areca catechu
S1, washing collected young and tender inflorescences of betel nuts with running water for 20min, removing surface dirt, transferring to a super-clean workbench, putting into a sterile beaker, rinsing with sterile water for 1 time, sterilizing the surfaces of the inflorescences, soaking with 75% alcohol for 1min, then soaking with 8% sodium hypochlorite for 10min, finally washing with sterile water for 5-6 times, and putting into sterile water for later use;
s2, peeling off spathes on the outer side, taking out inflorescences, inoculating the inflorescences to a somatic embryo induction culture medium, wherein the culture medium comprises a Y3 basic culture medium, 50mg/L picloram, 60g/L sucrose and 2g/L plant gel, the pH value is 5.8, the culture temperature is 26 +/-1 ℃, subculture is carried out once every 30 days, and after dark culture is carried out for 120 days, a somatic embryo is obtained;
s3, transferring the obtained somatic embryos to a multiplication culture medium for multiplication culture, wherein the multiplication culture medium comprises Y3 minimal medium, 2 mg/L2, 4-D, 2mg/L6-BA, 30g/L sucrose and 2g/L plant gel, the pH value is 5.8, the culture temperature is 26 +/-1 ℃, and the multiplication culture is carried out for 1 time every 30 days;
s4, transferring the obtained somatic embryos to a germination culture medium for culture, wherein the germination culture medium comprises: different basic culture media, 2mg TDZ, 2g/L active carbon, 30g/L sucrose and 2g/L plant gel, the pH value is 5.8, the different basic culture media are specifically shown in the table 5, the culture temperature is 26 +/-1 ℃, the illumination intensity is 1000 + 3000Lx, the illumination time is 16-18h/d, and a complete regeneration plant is obtained after 3 months of culture;
s5, transplanting the obtained rooted complete plant to a culture medium for cultivation under a spraying system after 2-4 leaves grow, wherein the culture medium is sand, soil and organic fertilizer in a ratio of 10:2:1, and the plant can be transplanted to a nursery for growth after 2-3 months of cultivation.
TABLE 5 Effect of different basic media on Areca catechu somatic embryo Germination
| Minimal medium | Number of inoculations per dish | Number of inoculation dishes | Number of sprouts | Germination rate |
| MS | 9 | 10 | 50 | 55.56％ |
| Y3 | 9 | 10 | 39 | 43.33％ |
| WPM | 9 | 10 | 32 | 35.56％ |
As can be seen from Table 5, there was a significant difference in the germination rates of somatic embryos of areca catechu among the different basal media, with the highest germination rate of MS medium.
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 person skilled in the art can substitute or change the technical solution of the present invention and the inventive concept within the technical scope of the present invention.
Claims (9)
1. A tissue culture rapid propagation method taking areca inflorescence as explant is characterized by comprising the following steps:
s1, selection of explants: collecting young and tender inflorescences of areca nuts, and cleaning and disinfecting the young and tender inflorescences of the areca nuts to be used as explants for later use; the specific operations of cleaning and sterilizing are as follows: washing young and tender inflorescence of Arecae semen with flowing water for 30min, transferring to a clean bench, and rinsing with sterile water; soaking the raw materials in 75% alcohol for 5min, then soaking the raw materials in 8% sodium hypochlorite for 10-15 min, finally washing the raw materials with sterile water for 5-6 times, and placing the raw materials in the sterile water for later use;
s2, induced culture: inoculating the explant sterilized in the step S1 to a somatic embryo induction culture medium for dark culture to obtain a somatic embryo; the somatic embryo induction culture medium is any one of Y3 minimal medium + 1-60 mg/L picloram +60g/L sucrose +2g/L plant gel, Y3 minimal medium + 1-60 mg/L2, 4-D +60g/L sucrose +2g/L plant gel or Y3 minimal medium + 1-60 mg/L dicamba +60g/L sucrose +2g/L plant gel, and the pH value of the culture medium is 5-6;
s3, propagation culture: transferring the somatic embryos to a proliferation medium for dark culture, wherein the proliferation medium comprises: y3 basic culture medium, 1-10 mg/L2, 4-D, 1-10 mg/L6-BA, 30g/L sucrose and 2g/L plant gel, wherein the pH value of the culture medium is 5-6;
s4, germination culture: transferring the somatic embryos subjected to proliferation culture to a germination culture medium for germination culture under the condition of illumination to obtain complete regeneration plants; wherein, the germination culture medium is as follows: MS basic culture medium, 0.5-5 mg/L TDZ, 2g/L active carbon, 30g/L cane sugar and 2g/L plant gel, wherein the pH value of the culture medium is 5-6;
s5, transplanting: and (3) transplanting 2-4 leaves of the completely rooted regeneration plant obtained by germination culture into a culture medium for culture under a spraying system, wherein the culture medium is a mixture of sand, soil and an organic fertilizer.
2. The tissue culture and rapid propagation method for areca catechu inflorescence explant according to claim 1, wherein the culture time of the dark culture in step S2 is 90-160 days, and the culture temperature is 26 ± 1 ℃.
3. The method for tissue culture and rapid propagation of areca catechu by using an inflorescence as an explant according to claim 1, wherein the somatic embryo induction medium in step S2 is: y3 minimal medium +50mg/L picloram +60g/L sucrose +2g/L plant gel.
4. The tissue culture and rapid propagation method for areca catechu inflorescence explant according to claim 1, wherein the culture time of the dark culture in step S3 is 25-30 days, and the culture temperature is 26 ± 1 ℃.
5. The tissue culture rapid propagation method taking areca inflorescence as explant according to claim 1, wherein the propagation medium in step S3 is: y3 minimal medium +2 mg/L2, 4-D +2mg/L6-BA +30g/L sucrose +2g/L plant gel.
6. The tissue culture rapid propagation method using betel palm inflorescence as explant according to claim 1, wherein the illumination conditions in step S4 are as follows: the illumination intensity is 1000-3000Lx, and the illumination time is 16-18 h/d.
7. The tissue culture rapid propagation method taking areca inflorescence as explant according to claim 1, wherein the culture time of germination culture in step S4 is 60-90 days, the culture temperature is 26 ± 1 ℃, and subculture is performed every 30 days.
8. The tissue culture rapid propagation method taking areca inflorescence as explant according to claim 1, wherein the germination medium in step S4 is: MS minimal medium +2mg/L TDZ +2g/L active carbon +30g/L sucrose +2g/L plant gel.
9. The tissue culture rapid propagation method taking areca inflorescence as explant according to claim 1, wherein the weight ratio of the sand, the soil and the organic fertilizer in step S5 is 10:2: 1.
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