CN109479714B - Tissue culture method for mature zygotic embryo of coconut - Google Patents
Tissue culture method for mature zygotic embryo of coconut Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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- A01H4/008—Methods for regeneration to complete plants
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Abstract
The invention discloses a tissue culture method of coconut mature zygotic embryo, which takes the coconut mature zygotic embryo as an explant, strictly controls the culture medium formula at each stage of tissue culture, and has the callus induction rate of 98.0 percent, the somatic embryo induction rate of 98.4 percent, the somatic embryo regeneration rate of 98.7 percent and the transplanting survival rate of 98.1 percent through tissue culture. The culture medium formula and the method are more broad-spectrum, are suitable for a plurality of varieties such as Hainan local high-variety, Wen coconut No. 2, Wen coconut No. 3 and the like, have good repeatability and short period, and are beneficial to industrial and commercial popularization and application.
Description
Technical Field
The invention relates to a tissue culture method of mature zygotic embryos of coconuts, belonging to the technical field of plant tissue culture.
Background
Tissue culture techniques are methods for rapid propagation of clones from explants of roots, stems, leaves, flowers, fruits, etc. of plants, and have been successful in a variety of plants. Tissue culture techniques are the most efficient method of asexual propagation of coconut for rapid propagation. In the tissue culture of coconut, researchers have succeeded in vitro culture of zygotic embryos, immature inflorescences, young ovaries, stem tips, young leaves and the like in sequence to different degrees. As early as 1978, Fisher and Tsai attempted tissue regeneration from coconut embryos, but only callus was obtained and plants were not successfully regenerated. Verdeil et al, 1994, succeeded for the first time in obtaining regenerated plants from immature inflorescences of coconut. Chan et al (1998) obtained regenerated plants from the embryos of coconut by means of somatic embryos. Fernando et al (2000 years) induced the immature zygotic embryo of coconut for 7-9 months to obtain spherical callus, and further induced somatic embryogenesis to finally obtain the regenerated plant. Prasanthi et al (2007) obtained regenerated plants using the unfertilized ovary of coconut as the explant. Perera et al (2009) use microspores from coconut anthers as explants to obtain regenerated plants. Sandoval-Cancino et al (2016) successfully obtained regenerated plants using mature zygotic embryos of coconut as explants.
The tissue culture technology of coconut has obtained certain research results, but the research related to tissue culture by adopting mature zygotic embryos is less, and some obvious problems still exist, which hinder the development of the rapid propagation technology of coconut.
1. Culture method without general use for different coconut genotypes
Coconut is a highly heterozygous plant, but none of the culture media and culture methods published so far are widely applicable and are only limited to a certain genotype, even in the same variety, some plants can successfully induce callus, and some cannot. The srilanca coconut institute has a specialized tissue culture center, and it has been difficult to overcome this problem for decades.
2. Poor repeatability of the test
The same medium, the same explant (from the same plant), is difficult to replicate to achieve the same or similar results. The first obtained regenerated plant of the Ruditapes philippinarum has been fruited already, but the subsequent experiments have not been successful.
3. The tissue culture period is long
Due to the growth characteristics of coconuts and the existing tissue culture technology, the production period of coconut tissue culture seedlings is generally longer. Taking the inflorescence explant as an example, the callus induction stage is about 3 months, the callus propagation stage is about 3 months, the somatic embryo formation stage is 1 month, the somatic embryo germinates and takes root for 6 months, the plant becomes seedling for 6 months, and the total period of the whole test process is 17 months. If a mature technical system which can be used on a large scale is to be established, the time is longer.
In addition, the problems of low propagation coefficient, difficult propagation and the like are also needed to be solved.
In summary, no mature technical solution for production is available at present.
Disclosure of Invention
In view of the above, the invention provides a culture medium formula and a method with wider spectrum and good repeatability, which can improve callus induction rate, somatic embryo regeneration rate and transplanting survival rate of coconut explants and further shorten the tissue culture period.
The technical scheme adopted by the invention is as follows:
a tissue culture method of coconut mature zygotic embryo comprises the following steps:
(1) selection of explants: maturing zygotic embryos;
(2) sequentially adopting a callus induction culture medium, a callus propagation culture medium, a somatic embryo induction culture medium, a somatic embryo maturation culture medium, a somatic embryo germination culture medium, a regenerated plant strong root culture medium and a tissue culture seedling transplanting culture medium for culturing;
wherein the content of the first and second substances,
the callus induction culture medium comprises the following components: adding 100-150 mu M of 2,4-D, 5-10 mu M of 6-BA, 2-8 g/L of plant gel, 1-8 g/L of activated carbon and 20-50 g/L of cane sugar into a Y3 culture medium;
the callus propagation culture medium is the same as the callus induction culture medium;
the somatic embryo induction culture medium comprises the following components: adding 10-20 mu M of 2,4-D, 2-8 mu M of ABA, 2-8 g/L of plant gel, 1-8 g/L of active carbon and 20-50 g/L of cane sugar into a WPM culture medium;
the somatic embryo maturation medium comprises the following components: adding 2-8 g/L of plant gel, 1-8 g/L of active carbon and 20-50 g/L of cane sugar into a WPM culture medium;
the somatic embryo germination culture medium comprises the following components: adding 8-16 mu M of NAA, 2-8 mu M of ABA, 2-8 g/L of plant gel, 1-8 g/L of active carbon and 20-50 g/L of cane sugar into a WPM culture medium;
the regenerated plant strong root culture medium comprises the following components: adding 0.20-0.60 mu M GA3, 1-8 g/L plant gel, 1-8 g/L active carbon and 20-60 g/L cane sugar into 1/2CRI72 culture medium or WPM culture medium; the 1/2CRI72 medium comprises the following components: 5 to 10mM ammonium nitrate, 5 to 10mM potassium nitrate, 0.5 to 1mM sodium dihydrogen phosphate, 0.5 to 1.5mM calcium chloride, 0.5 to 1.5mM magnesium sulfate, 100 to 150. mu.M boric acid, 80 to 100. mu.M manganese sulfate, 20 to 40. mu.M zinc sulfate, 1.0 to 1.5. mu.M copper sulfate, 0.5 to 1. mu.M sodium molybdate, 0.5 to 1. mu.M cobalt chloride, 2 to 5. mu.M potassium iodide, 80 to 100. mu.M ferrous sulfate, 80 to 100. mu.M Na2EDTA, 500-650 μ M sodium sulfate, 500-600 μ M inositol, 20-40 μ M nicotinic acid, 20-40 μ M thiamine hydrochloride, 0.5-1 μ M biotin, 2-5 μ M D-calcium pantothenate, 3-6 μ M pyridoxine hydrochloride, 5-10 μ M riboflavin, 5-10 μ M ascorbic acid, 100-120 μ M L-cysteine hydrochloride, and 30-50 μ M glycine;
the tissue culture seedling transplanting culture medium comprises the following components: vermiculite according to mass ratio: coconut husk: perlite is (2-6), (1-2) and (1-2).
Preferably, the callus induction medium comprises the following components: adding 110 mu M2,4-D, 8.8 mu M6-BA, 4g/L plant gel, 3g/L active carbon and 40g/L sucrose into Y3 culture medium;
preferably, the somatic embryo induction medium comprises the following components: adding 16 mu M2,4-D, 5 mu M ABA, 4g/L plant gel, 3g/L activated carbon and 40g/L sucrose into WPM culture medium;
preferably, the somatic embryo maturation medium comprises the following components: adding 4g/L of plant gel, 3g/L of activated carbon and 40g/L of cane sugar into the WPM culture medium;
preferably, the somatic embryo germination medium comprises the following components: adding 10 mu M NAA, 5 mu M ABA, 4g/L plant gel, 3g/L active carbon and 40g/L sucrose into WPM culture medium;
preferably, the regenerated plant strong root culture medium comprises the following components: adding 0.46 mu M GA3, 2g/L plant gel, 3g/L activated carbon and 45g/L sucrose into 1/2CRI72 culture medium or WPM culture medium;
preferably, the tissue culture seedling transplanting culture medium comprises the following components: vermiculite according to mass ratio: coconut husk: perlite is 2:1: 1.
Preferably, in the callus induction culture stage, the pH value of a culture medium is 5.75-5.80, the culture condition is 27-29 ℃ dark culture, and the culture time is 1-3 months.
Preferably, in the callus propagation culture stage, the somatic embryo induction culture stage and the somatic embryo maturation culture stage, the pH value of a culture medium is 5.75-5.80, the culture condition is 27-29 ℃ dark culture, subculture is carried out for 1-2 times every 1 month, and subculture is carried out for 2-3 times.
Preferably, in the somatic embryo germination culture stage, the pH value of a culture medium is 5.75-5.80, the culture condition is 27-29 ℃, the photoperiod is 12-18 h/d, and subculture is carried out for 1-2 times every month until the plant is regenerated.
Preferably, in the regeneration plant strong root culture stage, the pH value of a culture medium is 5.75-5.80, the culture condition is 27-29 ℃, the photoperiod is 12-18 h/d, and the subculture is performed for 1-2 times every month and 2-3 times in total.
Preferably, in the transplanting culture stage of the tissue culture seedlings, the culture conditions are 27-29 ℃, the photoperiod is 12-18 h/d, the moisture of the tissue culture seedlings is preserved by a moisture-preserving cover, the moisture-preserving cover is opened every day to expose the tissue culture seedlings to the external environment for 1-2 h, the exposure time is prolonged day by day until the tissue culture seedlings are exposed for 24h every day, and then the tissue culture seedlings can be transplanted to a nursery.
Preferably, the coconut variety is selected from Hippocampus locally high variety, Wen coconut No. 2, Wen coconut No. 3, Wen coconut No. 4, Brown dwarf or Malva.
Compared with the prior art, the invention has the beneficial effects that:
(1) according to the invention, the culture medium formula and the culture method are strictly controlled at each stage of tissue culture, so that the callus induction rate, the somatic embryo regeneration rate and the transplanting survival rate of explants are effectively improved; the callus inductivity can reach 98.0 percent, the somatic embryo inductivity can reach 98.4 percent, the somatic embryo regeneration rate can reach 98.7 percent, and the transplanting survival rate can reach 98.1 percent.
(2) As known in the art, because the genotypes of coconut tissue culture are greatly different, the same culture medium formula and the same culture method have response to some varieties and no response to some varieties. The culture medium and the culture method provided by the invention have better broad spectrum, and can be simultaneously suitable for tissue culture of a plurality of varieties such as Hainan local high-variety, Wenchau No. 2, Wenchau No. 3 and the like, in addition, as can be seen from the table 1, each group is repeatedly tested for 50 times, the test result of each time of the embodiment has no obvious deviation, the comparative example result has larger difference, and the method is excellent in repeatability and stable.
(3) From the test results of the embodiment 1 and the embodiments 4 to 8, the culture medium and the culture method provided by the invention have the advantages that the callus induction rate, the somatic embryo regeneration rate and the transplanting survival rate are all more than 90%, the best effect can be obtained when the culture medium is applied to the tissue culture of Wenchun No. 2, and the callus induction rate, the somatic embryo regeneration rate and the transplanting survival rate are all 98% higher than those of other varieties.
(4) The invention effectively shortens the tissue culture period, which is shortened by about 5 months compared with the conventional tissue culture method;
(5) the invention is suitable for coconut tissue culture taking mature zygotic embryos as explants;
(6) based on the technical scheme of the invention, the industrial and commercial production of the coconut tissue culture seedlings is facilitated.
Detailed Description
The present invention will be described in further detail with reference to the following embodiments.
The experimental methods used in the examples of the present invention are all conventional methods unless otherwise specified.
The materials, reagents and the like used in the examples of the present invention can be obtained commercially without specific description.
Some abbreviations in the present invention have the following meanings:
6-BA: 6-benzyladenine
2, 4-D: 2, 4-Dichlorophenoxyacetic acid
GA 3: gibberellins
ABA: abscisic acid
NAA: naphthylacetic acid
2 iP: dimethylpropiopurine
The Y3 culture medium is a culture medium containing the following components: 535mg/L of ammonium chloride, 2020mg/L of potassium nitrate, 247mg/L of magnesium sulfate, 294mg/L of calcium chloride, 1492mg/L of potassium chloride, 312mg/L of ammonium dihydrogen phosphate, 8.3mg/L of potassium iodide, 3.1mg/L of boric acid, 11.2mg/L of manganese sulfate, 7.2mg/L of zinc sulfate, 0.25mg/L of copper sulfate, 0.24mg/L of cobalt chloride, 0.24mg/L of sodium molybdate, 0.024mg/L of nickel chloride, 27.8mg/L of ferrous sulfate, 37.3mg/L of sodium chelate, 1mg/L of pyritinol hydrochloride, 1mg/L of thiamine hydrochloride, 102mg/L of inositol, 100mg/L of L-glutamine, 121mg/L of L-arginine and 88mg/L of L-asparagine.
Example 1
A tissue culture method of coconut mature zygotic embryo comprises the following steps:
taking a mature zygotic embryo of the Wen coconut No. 2 variety as an explant; sequentially carrying out callus induction stage, callus propagation stage, somatic embryo induction stage, somatic embryo maturation stage, somatic embryo germination stage, regenerated plant root strengthening stage and tissue culture seedling transplanting culture stage for culture;
1.1 callus induction phase:
the callus induction culture medium comprises the following components: adding 110 mu M of 2,4-D, 8.8 mu M of 6-BA, 4g/L of plant gel, 3g/L of activated carbon and 40g/L of cane sugar into a Y3 culture medium;
the pH value of the culture medium is 5.75, the culture condition is dark culture at 28 ℃, and the culture time is 1 month.
1.2 callus propagation stage:
the callus propagation culture medium is the same as the callus induction culture medium;
the pH value of the culture medium is 5.75, the culture condition is 28 ℃ dark culture, and the subculture is carried out for 1 time every 1 month and 2 times in total.
1.3 somatic embryo induction culture stage, somatic embryo maturation culture stage, and somatic embryo germination culture stage
The somatic embryo induction culture medium comprises the following components: adding 16 mu M2,4-D, 5 mu M ABA, 4g/L plant gel, 3g/L activated carbon and 40g/L sucrose into WPM culture medium;
the somatic embryo maturation medium comprises the following components: adding 4g/L of plant gel, 3g/L of activated carbon and 40g/L of cane sugar into the WPM culture medium;
the somatic embryo germination culture medium comprises the following components: adding 10 mu M NAA, 5 mu M ABA, 4g/L plant gel, 3g/L activated carbon and 40g/L sucrose into WPM culture medium;
in the induction culture stage and mature culture stage of somatic embryo, the pH value of culture medium is 5.75, the culture condition is dark culture at 28 ℃, and the subculture is carried out for 1 time every 1 month and 2 times in total.
In the germination culture stage of somatic embryos, the pH value of a culture medium is 5.75, the culture condition is 28 ℃, the photoperiod is 16h/d, and subculture is carried out for 1 time every month until plants are regenerated.
1.4 regeneration of plants at the root-strengthening stage
The regenerated plant strong root culture medium comprises the following components: adding 0.46 mu M GA3, 2g/L plant gel, 3g/L activated carbon and 45g/L sucrose into 1/2CRI72 culture medium; the 1/2CRI72 medium comprises the following components: 10mM ammonium nitrate, 10mM potassium nitrate, 1mM sodium dihydrogen phosphate, 1.5mM calcium chloride, 1.5mM magnesium sulfate, 150. mu.M boric acid, 100. mu.M manganese sulfate, 40. mu.M zinc sulfate, 1.5. mu.M copper sulfate, 1. mu.M sodium molybdate, 1. mu.M cobalt chloride, 5. mu.M potassium iodide, 100. mu.M ferrous sulfate, 100. mu.M Na2EDTA, 650 u M sodium sulfate, 600 u M inositol, 40 u M nicotinic acid, 40 u M thiamine hydrochloride, 1 u M biotin, 5 u M D-calcium pantothenate, 6 u M pyridine hydrochloride, 10 u M riboflavin, 10 u M ascorbic acid, 120 u M L-cysteine hydrochloride and 50 u M glycine; in the strong root culture stage of the regeneration plant, the pH value of a culture medium is 5.75, the culture condition is 28 ℃, the photoperiod is 16h/d, and the subculture is carried out for 1 time per month and 2 times in total.
1.5 transplanting and culturing stage of tissue culture seedlings
The tissue culture seedling transplanting culture medium comprises the following components: vermiculite according to mass ratio: coconut husk: perlite is 2:1: 1.
In the transplanting culture stage of the tissue culture seedlings, the culture condition is 28 ℃, the photoperiod is 16h/d, the moisture of the tissue culture seedlings is kept by a moisture-keeping cover, the moisture-keeping cover is opened every day to expose the tissue culture seedlings to the external environment for 1h, the exposure time is prolonged day by day until the tissue culture seedlings are exposed for 24h every day, and then the tissue culture seedlings are transplanted to a nursery.
Example 2
A tissue culture method of coconut mature zygotic embryo comprises the following steps:
taking a mature zygotic embryo of the Wen coconut No. 2 variety as an explant; sequentially carrying out callus induction stage, callus propagation stage, somatic embryo induction stage, somatic embryo maturation stage, somatic embryo germination stage, regenerated plant root strengthening stage and tissue culture seedling transplanting culture stage for culture;
1.1 callus induction phase:
the callus induction culture medium comprises the following components: adding 100 mu M of 2,4-D, 5 mu M of 6-BA, 2g/L of plant gel, 1g/L of activated carbon and 20g/L of cane sugar into a Y3 culture medium;
the pH value of the culture medium is 5.75, the culture condition is dark culture at 27 ℃, and the culture time is 1 month.
1.2 callus propagation stage:
the callus propagation culture medium is the same as the callus induction culture medium;
the pH value of the culture medium is 5.75, the culture condition is 27 ℃ dark culture, and the subculture is carried out for 1 time every 1 month and 2 times in total.
1.3 somatic embryo induction culture stage, somatic embryo maturation culture stage, and somatic embryo germination culture stage
The somatic embryo induction culture medium comprises the following components: adding 10 μ M2,4-D, 2 μ M ABA, 2g/L plant gel, 1g/L activated carbon and 20g/L sucrose into WPM culture medium;
the somatic embryo maturation medium comprises the following components: adding 2g/L of plant gel, 1g/L of activated carbon and 20g/L of cane sugar into the WPM culture medium;
the somatic embryo germination culture medium comprises the following components: adding 8 mu M NAA, 2 mu M ABA, 2g/L plant gel, 1g/L activated carbon and 20g/L sucrose into WPM culture medium;
in the induction culture stage and mature culture stage of somatic embryo, the pH value of culture medium is 5.75, the culture condition is 27 deg.C dark culture, and the subculture is carried out for 1 time every 1 month and 2 times in total.
In the somatic embryo germination culture stage, the pH value of the culture medium is 5.75, the culture condition is 27 ℃, the photoperiod is 12h/d, and subculture is carried out for 1 time every month until the plant is regenerated.
1.4 regeneration of plants at the root-strengthening stage
The regenerated plant strong root culture medium comprises the following components: adding 0.20 mu M GA3, 1g/L plant gel, 1g/L active carbon and 20g/L sucrose into 1/2CRI72 culture medium; the 1/2CRI72 medium comprises the following components: 5mM ammonium nitrate, 5mM potassium nitrate, 0.5mM sodium dihydrogen phosphate, 0.5mM calcium chloride, 0.5mM magnesium sulfate, 100. mu.M boric acid, 80. mu.M manganese sulfate, 20. mu.M zinc sulfate, 1.0. mu.M copper sulfate, 0.5. mu.M sodium molybdate, 0.5. mu.M cobalt chloride, 2. mu.M potassium iodide, 80. mu.M ferrous sulfate, 80. mu.M Na2EDTA, 500. mu.M sodium sulfate, 500. mu.M inositol, 20. mu.M nicotinic acid, 20. mu.M thiamine hydrochloride, 0.5. mu.M biotin, 2. mu. M D-calcium pantothenate, 3. mu.M pyridoxine hydrochloride, 5. mu.M riboflavin, 5. mu.M ascorbic acid, 100. mu. M L-cysteine hydrochloride, and 30. mu.M glycine;
in the strong root culture stage of the regeneration plant, the pH value of a culture medium is 5.75, the culture condition is 27 ℃, the photoperiod is 12h/d, and the subculture is carried out for 1 time per month and 2 times in total.
1.5 transplanting and culturing stage of tissue culture seedlings
The tissue culture seedling transplanting culture medium comprises the following components: vermiculite according to mass ratio: coconut husk: perlite is 1:1: 1.
In the transplanting culture stage of the tissue culture seedlings, the culture condition is 27 ℃, the photoperiod is 12h/d, the moisture of the tissue culture seedlings is kept by a moisture-keeping cover, the moisture-keeping cover is opened every day to expose the tissue culture seedlings to the external environment for 1h, the exposure time is prolonged day by day until the tissue culture seedlings are exposed for 24h every day, and then the tissue culture seedlings are transplanted to a nursery.
Example 3
A tissue culture method of coconut mature zygotic embryo comprises the following steps:
taking a mature zygotic embryo of the Wen coconut No. 2 variety as an explant; sequentially carrying out callus induction stage, callus propagation stage, somatic embryo induction stage, somatic embryo maturation stage, somatic embryo germination stage, regenerated plant root strengthening stage and tissue culture seedling transplanting culture stage for culture;
1.1 callus induction phase:
the callus induction culture medium comprises the following components: adding 150 mu M of 2,4-D, 10 mu M of 6-BA, 8g/L of plant gel, 8g/L of activated carbon and 50g/L of cane sugar into a Y3 culture medium;
the pH value of the culture medium is 5.80, the culture condition is 29 ℃ dark culture, and the culture time is 3 months.
1.2 callus propagation stage:
the callus propagation culture medium is the same as the callus induction culture medium;
the pH value of the culture medium is 5.80, the culture condition is 29 ℃ dark culture, and the subculture is carried out for 2 times every 1 month and 3 times in total.
1.3 somatic embryo induction culture stage, somatic embryo maturation culture stage, and somatic embryo germination culture stage
The somatic embryo induction culture medium comprises the following components: adding 20 mu M2,4-D, 8 mu M ABA, 8g/L plant gel, 8g/L activated carbon and 50g/L sucrose into WPM culture medium;
the somatic embryo maturation medium comprises the following components: adding 8g/L of plant gel, 8g/L of activated carbon and 50g/L of cane sugar into the WPM culture medium;
the somatic embryo germination culture medium comprises the following components: adding 16 mu M NAA, 8 mu M ABA, 8g/L plant gel, 8g/L activated carbon and 50g/L sucrose into WPM culture medium;
in the induction culture stage and mature culture stage of somatic embryo, the pH value of culture medium is 5.80, the culture condition is 29 deg.C dark culture, and the subculture is carried out for 2 times every 1 month and 3 times in total.
In the germination culture stage of somatic embryo, the pH value of the culture medium is 5.80, the culture condition is 29 ℃, the photoperiod is 18h/d, and the subculture is carried out for 2 times per month until the plant is regenerated.
1.4 regeneration of plants at the root-strengthening stage
The regenerated plant strong root culture medium comprises the following components: adding 0.60 mu M GA3, 8g/L plant gel, 8g/L active carbon and 60g/L sucrose into WPM culture medium;
in the strong root culture stage of the regeneration plant, the pH value of a culture medium is 5.80, the culture condition is 29 ℃, the photoperiod is 18h/d, and the subculture is carried out for 2 times per month and 3 times in total.
1.5 transplanting and culturing stage of tissue culture seedlings
The tissue culture seedling transplanting culture medium comprises the following components: vermiculite according to mass ratio: coconut husk: perlite is 6:1: 1.
In the transplanting culture stage of the tissue culture seedlings, the culture condition is 29 ℃, the photoperiod is 18h/d, the moisture of the tissue culture seedlings is preserved by a moisture-preserving cover, the moisture-preserving cover is opened every day to expose the tissue culture seedlings to the external environment for 2h, the exposure time is prolonged day by day until the tissue culture seedlings are exposed for 24h every day, and then the tissue culture seedlings are transplanted to a nursery.
Examples 4 to 8
The varieties selected in the embodiments 4-8 are Hainan local high variety, Wen coconut No. 3, Wen coconut No. 4, Brown dwarf and Mawa respectively. The other operations were the same as in example 1.
Comparative example 1
Comparative example 1 differs from example 1 in that:
the callus induction culture medium comprises the following components: adding 80 mu M of 2,4-D, 3 mu M of 6-BA, 2g/L of plant gel, 4g/L of activated carbon and 40g/L of cane sugar into a Y3 culture medium;
the somatic embryo induction culture medium comprises the following components: adding 8 mu M of 2,4-D, 1 mu M of ABA, 2g/L of plant gel, 4g/L of activated carbon and 40g/L of cane sugar into a WPM culture medium;
the somatic embryo germination culture medium comprises the following components: adding 6 mu M NAA, 1 mu M ABA, 2g/L plant gel, 4g/L active carbon and 40g/L sucrose into WPM culture medium;
the regenerated plant strong root culture medium comprises the following components: adding 0.15 mu M GA3, 1g/L plant gel, 0.5g/L active carbon and 20g/L sucrose into 1/2CRI72 culture medium; the 1/2CRI72 medium comprises the following components: 10mM ammonium nitrate, 10mM potassium nitrate, 1mM sodium dihydrogen phosphate, 1.5mM calcium chloride, 1.5mM magnesium sulfate, 150. mu.M boric acid, 100. mu.M manganese sulfate, 40. mu.M zinc sulfate, 1.5. mu.M copper sulfate, 1. mu.M sodium molybdate, 1. mu.M cobalt chloride, 5. mu.M potassium iodide, 100. mu.M ferrous sulfate, 100. mu.M Na2EDTA, 650 u M sodium sulfate, 600 u M inositol, 40 u M nicotinic acid, 40 u M thiamine hydrochloride, 1 u M biotin, 5 u M D-calcium pantothenate, 6 u M pyridine hydrochloride, 3 u M riboflavin, 3 u M ascorbic acid, 80 u M L-cysteine hydrochloride and 20 u M glycine;
the tissue culture seedling transplanting culture medium comprises the following components: vermiculite according to mass ratio: coconut husk: perlite is 1:2: 1.
Comparative example 2
Comparative example 2 differs from example 1 in that:
the callus induction culture medium comprises the following components: adding 180 mu M of 2,4-D, 12 mu M of 6-BA, 2g/L of plant gel, 4g/L of activated carbon and 40g/L of cane sugar into a Y3 culture medium;
the somatic embryo induction culture medium comprises the following components: adding 25 mu M of 2,4-D, 8 mu M of ABA, 2g/L of plant gel, 4g/L of activated carbon and 40g/L of cane sugar into the WPM culture medium;
the somatic embryo germination culture medium comprises the following components: adding 20 mu M NAA, 10 mu M ABA, 2g/L plant gel, 4g/L active carbon and 40g/L sucrose into WPM culture medium;
the regenerated plant strong root culture medium comprises the following components: adding 0.80 mu M GA3, 8g/L plant gel, 10g/L activated carbon and 50g/L sucrose into 1/2CRI72 culture medium; the 1/2CRI72 medium comprises the following components: 10mM ammonium nitrate, 10mM potassium nitrate, 1mM sodium dihydrogen phosphate, 1.5mM calcium chloride, 1.5mM magnesium sulfate, 150. mu.M boric acid, 100. mu.M manganese sulfate, 40. mu.M zinc sulfate, 1.5. mu.M copper sulfate, 1. mu.M sodium molybdate, 1. mu.M cobalt chloride, 5. mu.M potassium iodide, 100. mu.M ferrous sulfate, 100. mu.M Na2EDTA, 650 u M sodium sulfate, 600 u M inositol, 40 u M nicotinic acid, 40 u M thiamine hydrochloride, 1 u M biotin, 5 u M D-calcium pantothenate, 6 u M pyridine hydrochloride, 12 u M riboflavin, 12 u M ascorbic acid, 150 u M L-cysteine hydrochloride and 80 u M glycine;
the tissue culture seedling transplanting culture medium comprises the following components: vermiculite according to mass ratio: coconut husk: perlite is 7:1: 2.
Comparative example 3
Comparative example 3 differs from example 1 in that:
the callus induction medium also contains 5 mu M2 iP;
the somatic embryo induction culture medium and the somatic embryo maturation culture medium also contain 30 mu M pyrimidyl alcohol;
the somatic embryo germination culture medium does not contain NAA;
the tissue culture seedling transplanting culture medium comprises the following components: vermiculite according to mass ratio: and 2:1 of coconut coir.
Comparative example 4
Comparative example 4 differs from example 1 in that the WPM medium described in the somatic embryo induction medium, somatic embryo maturation medium, somatic embryo germination medium was changed to Y3 medium, and the 1/2CRI72 medium described in the regenerated plant strong root medium was changed to Y3 medium.
Test example:
coconut tissue culture was performed by the methods of examples and comparative examples, each group was repeatedly tested 50 times, and callus induction rate, somatic embryo regeneration rate, and transplant survival rate were counted. The results are shown in Table 1.
The callus induction rate is the number of explants inducing callus/the number of explants inoculated × 100%
The induction rate of somatic embryos is equal to the number of somatic embryos/the number of inoculated callus blocks multiplied by 100 percent
The regeneration rate of somatic embryos is the number of somatic embryos induced to produce whole plants/the number of inoculated somatic embryos x 100%
The survival rate of transplantation is equal to the survival rate of transplantation/total number of transplantation multiplied by 100 percent
TABLE 1
| Callus induction rate/% | Somatic embryo induction rate/%) | Somatic embryo regeneration rate/%) | Survival rate of transplantation/%) | |
| Example 1 | 98.0±0.47 | 98.4±0.51 | 98.7±0.40 | 98.1±0.24 |
| Example 2 | 92.7±0.14 | 91.8±0.43 | 90.7±0.47 | 92.4±0.14 |
| Example 3 | 93.8±0.47 | 92.4±0.74 | 93.1±0.01 | 93.0±0.47 |
| Example 4 | 91.6±0.05 | 92.1±0.45 | 93.5±0.22 | 90.5±0.25 |
| Example 5 | 93.0±0.45 | 92.4±0.42 | 91.7±0.40 | 90.7±0.70 |
| Example 6 | 91.9±0.27 | 90.1±0.17 | 92.6±0.60 | 90.5±0.58 |
| Example 7 | 92.7±0.23 | 91.0±0.14 | 93.8±0.27 | 92.8±0.12 |
| Example 8 | 94.4±0.24 | 93.8±0.20 | 91.2±0.28 | 92.9±0.61 |
| Comparative example 1 | 67.7±5.24 | 65.9±6.14 | 74.6±5.19 | 72.7±9.88 |
| Comparative example 2 | 74.0±4.40 | 70.4±4.01 | 70.1±6.61 | 67.4±8.06 |
| Comparative example 3 | 60.3±4.17 | 62.8±4.24 | 49.8±7.40 | 59.9±12.26 |
| Comparative example 4 | 81.8±7.12 | 80.0±5.19 | 85.6±6.00 | 82.5±10.03 |
The result shows that the callus induction rate, the somatic embryo regeneration rate and the transplanting survival rate of the invention can reach more than 90 percent by taking the mature zygotic embryo of coconut as an explant through tissue culture, the callus induction rate can reach 98.0 percent at most, the somatic embryo induction rate can reach 98.4 percent at most, the somatic embryo regeneration rate can reach 98.7 percent at most, and the transplanting survival rate can reach 98.1 percent at most.
The culture medium formula and the method are more broad-spectrum, are suitable for a plurality of varieties such as Hainan local high-variety, Wen coconut No. 2, Wen coconut No. 3 and the like, have good repeatability and short period, and are beneficial to industrial and commercial popularization and application.
The foregoing is a more detailed description of the present invention that is presented in conjunction with specific embodiments, and the practice of the invention is not to be considered limited to those descriptions. It will be apparent to those skilled in the art that a number of simple derivations or substitutions can be made without departing from the inventive concept.
Claims (8)
1. A tissue culture method of mature zygotic embryos of coconuts is characterized by comprising the following steps:
(1) selection of explants: maturing zygotic embryos;
(2) sequentially adopting a callus induction culture medium, a callus propagation culture medium, a somatic embryo induction culture medium, a somatic embryo maturation culture medium, a somatic embryo germination culture medium, a regenerated plant strong root culture medium and a tissue culture seedling transplanting culture medium for culturing;
wherein the content of the first and second substances,
the callus induction culture medium is: adding 100-150 mu M of 2,4-D, 5-10 mu M of 6-BA, 2-8 g/L of plant gel, 1-8 g/L of activated carbon and 20-50 g/L of cane sugar into a Y3 culture medium;
the callus propagation culture medium is the same as the callus induction culture medium;
the somatic embryo induction culture medium comprises: adding 10-20 mu M of 2,4-D, 2-8 mu M of ABA, 2-8 g/L of plant gel, 1-8 g/L of active carbon and 20-50 g/L of cane sugar into a WPM culture medium;
the somatic embryo maturation medium is as follows: adding 2-8 g/L of plant gel, 1-8 g/L of active carbon and 20-50 g/L of cane sugar into a WPM culture medium;
the somatic embryo germination culture medium comprises: adding 8-16 mu M of NAA, 2-8 mu M of ABA, 2-8 g/L of plant gel, 1-8 g/L of active carbon and 20-50 g/L of cane sugar into a WPM culture medium;
the regenerated plant strong root culture medium comprises: adding 0.20-0.60 mu M GA to 1/2CRI72 culture medium or WPM culture medium31-8 g/L of plant gel, 1-8 g/L of active carbon and 20-60 g/L of cane sugar; the 1/2CRI72 culture medium is: 5 to 10mM ammonium nitrate, 5 to 10mM potassium nitrate, 0.5 to 1mM sodium dihydrogen phosphate, 0.5 to 1.5mM calcium chloride, 0.5 to 1.5mM magnesium sulfate, 100 to 150. mu.M boric acid, 80 to 100. mu.M manganese sulfate, 20 to 40. mu.M zinc sulfate, 1.0 to 1.5. mu.M copper sulfate, 0.5 to 1. mu.M sodium molybdate, 0.5 to 1. mu.M cobalt chloride, 2 to 5. mu.M potassium iodide, 80 to 100. mu.M ferrous sulfate, 80 to 100. mu.M Na2EDTA, 500-650 μ M sodium sulfate, 500-600 μ M inositol, 20-40 μ M nicotinic acid, 20-40 μ M thiamine hydrochloride, 0.5-1 μ M biotin, 2-5 μ M D-calcium pantothenate, 3-6 μ M pyridoxine hydrochloride, 5-10 μ M riboflavin, 5-10 μ M ascorbic acid, 100-120 μ M L-cysteine hydrochloride, and 30-50 μ M glycine;
the tissue culture seedling transplanting culture medium comprises: vermiculite according to mass ratio: coconut husk: the perlite is 2-6: 1-2.
2. The tissue culture method of coconut mature zygotic embryo according to claim 1,
the callus induction culture medium is: adding 110 mu M of 2,4-D, 8.8 mu M of 6-BA, 4g/L of plant gel, 3g/L of activated carbon and 40g/L of cane sugar into a Y3 culture medium;
the somatic embryo induction culture medium comprises: adding 16 mu M of 2,4-D, 5 mu M of ABA, 4g/L of plant gel, 3g/L of activated carbon and 40g/L of cane sugar into a WPM culture medium;
the somatic embryo maturation medium is as follows: adding 4g/L of plant gel, 3g/L of activated carbon and 40g/L of cane sugar into the WPM culture medium;
the somatic embryo germination culture medium comprises: adding 10 mu M NAA, 5 mu M ABA, 4g/L plant gel, 3g/L active carbon and 40g/L sucrose into WPM culture medium;
root strengthening culture of regenerated plantThe base is as follows: 1/2CRI72 medium or WPM medium supplemented with 0.46 μ M GA32g/L plant gel, 3g/L active carbon and 45g/L cane sugar;
the tissue culture seedling transplanting culture medium comprises: vermiculite according to mass ratio: coconut husk: perlite is 2:1: 1.
3. The tissue culture method of coconut mature zygotic embryo according to claim 1, wherein in callus induction culture stage, the pH value of the culture medium is 5.75-5.80, the culture condition is 27-29 ℃ dark culture, and the culture time is 1-3 months.
4. The tissue culture method of coconut mature zygotic embryo according to claim 1, wherein in callus propagation culture stage, somatic embryo induction culture stage and somatic embryo maturation culture stage, pH value of culture medium is 5.75-5.80, culture condition is 27-29 ℃ dark culture, subculture 1-2 times every 1 month, and subculture 2-3 times.
5. The tissue culture method of coconut mature zygotic embryo according to claim 1, characterized in that in the somatic embryo germination culture stage, the pH value of the culture medium is 5.75-5.80, the culture conditions are 27-29 ℃, the photoperiod is 12-18 h/d, and subculture is performed 1-2 times per month until plant regeneration.
6. The tissue culture method of coconut mature zygotic embryo according to claim 1, characterized in that in the regeneration plant strong root culture stage, the pH value of the culture medium is 5.75-5.80, the culture conditions are 27-29 ℃, the photoperiod is 12-18 h/d, and the subculture is performed 1-2 times per month for 2-3 times.
7. The tissue culture method of coconut mature zygote embryo according to claim 1, characterized in that in the tissue culture seedling transplanting culture stage, the culture conditions are 27-29 ℃, the photoperiod is 12-18 h/d, the tissue culture seedling is moisturized by moisturizing cover, the moisturizing cover is opened every day to expose the tissue culture seedling to the external environment for 1-2 h, the exposure time is prolonged day by day until 24h is exposed every day, and then the tissue culture seedling is transplanted to the nursery.
8. The tissue culture method of mature zygotic embryo of coconut as claimed in claim 1, wherein the coconut variety used is selected from native high variety of Hainan, Wen coconut No. 2, Wen coconut No. 3, Wen coconut No. 4, Brown dwarf or Malva.
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