CN109479714A - A kind of coconut mature zygotic embryos method for tissue culture - Google Patents

Abstract

The invention discloses a tissue culture method for coconut mature zygotic embryos. The coconut mature zygotic embryos are used as explants, and the medium formula is strictly controlled in each stage of tissue culture. After tissue culture, the callus induction rate can reach 98.0%, and the somatic embryos The highest induction rate can reach 98.4%, the highest somatic embryo regeneration rate can reach 98.7%, and the highest transplant survival rate can reach 98.1%. The culture medium formula and method of the invention has a broader spectrum, is suitable for Hainan local high species, Wenye No. 2, Wenye No. 3 and other varieties, and has good repeatability and short cycle, which is conducive to industrialization and commercialization. .

Description

A kind of coconut mature zygotic embryo tissue culture method

technical field

The invention relates to a method for culturing coconut mature zygotic embryo tissue, belonging to the technical field of plant tissue culture.

Background technique

Tissue culture technology is a method of rapidly multiplying clones using explants such as roots, stems, leaves, flowers, and fruits of plants, and has been successfully used in many plants. Tissue culture technology is the most effective asexual reproduction method for rapid propagation of coconut. In terms of coconut tissue culture, researchers have successively used zygotic embryos, immature inflorescences, young ovaries, shoot tips, and young leaves to culture in vitro, all of which have achieved success to varying degrees. As early as 1978, Fisher and Tsai tried to use coconut embryos for tissue regeneration, but only obtained callus and failed to regenerate plants. In 1994, Verdeil et al. were the first to successfully obtain regenerated plants from immature coconut inflorescences. Chan et al. (1998) obtained regenerated plants from coconut germs by somatic embryo approach. Fernando et al. (2000) used the immature zygotic embryos of coconut 7-9 months to obtain spherical callus, and further induced somatic embryogenesis to finally obtain regenerated plants. Prasanthi et al. (2007) obtained regenerated plants using the unfertilized cells of coconut as explants. Perera et al. (2009) used microspores in coconut anther as explants to obtain regenerated plants. G. Sandoval-Cancino et al. (2016) used coconut mature zygotic embryos as explants to successfully obtain regenerated plants.

The tissue culture technology of coconut has obtained certain research results, but there are few related researches on using mature zygotic embryos for tissue culture, and there are still some obvious problems, which hinder the development of coconut rapid propagation technology.

1. There is no common cultivation method for different coconut genotypes

Coconut is a highly heterozygous plant, and none of the media and cultivation methods released so far is widely applicable, and is only limited to a certain type of genotype, even in the same variety, some Plants can successfully induce callus, while others cannot. Sri Lanka's Coconut Research Institute has a special tissue culture center, and they have been facing this problem for decades, which is difficult to overcome.

2. The repeatability of the test is poor

The same medium, the same explants (from the same plant), it is difficult to repeat the same or similar results. The regenerated plants obtained by the Philippine Coconut Agency for the first time already bear fruit, but subsequent experiments have not been successful.

3. Long tissue culture cycle

Due to the growth characteristics of coconut itself and the existing tissue culture technology, the production cycle of coconut tissue culture seedlings is generally longer. Taking the inflorescence explants 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 for 6 months, and the plants grow into 6 seedlings. month, the entire trial process totaled 17 months. It will take longer to establish a mature technology system that can be used on a large scale.

In addition, problems such as low reproduction coefficient and difficulty in proliferation also need to be solved urgently.

In summary, there is currently no mature technical solution for production.

SUMMARY OF THE INVENTION

In view of this, the present invention proposes a more broad-spectrum, repeatable medium formulation and method to improve callus induction rate, somatic embryo induction rate, somatic embryo regeneration rate and transplant survival rate of coconut explants, and simultaneously Further shorten the tissue culture cycle.

The technical scheme adopted by the present invention is as follows:

A method for culturing coconut mature zygotic embryo tissue, comprising the following steps:

(1) Selection of explants: mature zygotic embryos;

(2) Use callus induction medium, callus propagation medium, somatic embryo induction medium, somatic embryo maturation medium, somatic embryo germination medium, regenerated plant strong root medium and tissue culture seedling transplanting medium in sequence to cultivate;

in,

The callus induction medium includes the following components: add 100-150 μM 2,4-D, 5-10 μM 6-BA, 2-8 g/L phytogel, 1-8 g/L activated carbon and 20-50 g/L in Y3 medium. L sucrose;

The callus propagation medium is the same as the callus induction medium;

The somatic embryo induction medium includes the following components: add 10-20 μM 2,4-D, 2-8 μM ABA, 2-8 g/L phytogel, 1-8 g/L activated carbon and 20-50 g/L sucrose in WPM medium ;

The somatic embryo maturation medium includes the following components: adding 2-8 g/L plant gel, 1-8 g/L activated carbon and 20-50 g/L sucrose to the WPM medium;

The somatic embryo germination medium includes the following components: adding 8-16 μM NAA, 2-8 μM ABA, 2-8 g/L phytogel, 1-8 g/L activated carbon and 20-50 g/L sucrose in WPM medium;

The root-strengthening medium for regenerated plants includes the following components: add 0.20-0.60 μM GA3, 1-8 g/L phytogel, 1-8 g/L activated carbon and 20-60 g/L sucrose to 1/2 CRI72 medium or WPM medium The 1/2 CRI72 medium comprises the following components: 5-10mM ammonium nitrate, 5-10mM potassium nitrate, 0.5-1mM sodium dihydrogen phosphate, 0.5-1.5mM calcium chloride, 0.5-1.5mM magnesium sulfate, 100-150μM Boric acid, 80～100μM manganese sulfate, 20～40μM zinc sulfate, 1.0～1.5μM copper sulfate, 0.5～1μM sodium molybdate, 0.5～1μM cobalt chloride, 2～5μM potassium iodide, 80～100μM ferrous sulfate, 80～100μM Na ₂ EDTA, 500～650μM sodium sulfate, 500～600μM inositol, 20～40μM niacin, 20～40μM thiamine hydrochloride, 0.5～1μM biotin, 2～5μM D-calcium pantothenate, 3～6μM pyridoxine hydrochloride, 5-10 μM riboflavin, 5-10 μM ascorbic acid, 100-120 μM L-cysteine hydrochloride and 30-50 μM glycine;

The tissue culture seedling transplanting medium includes the following components: in terms of mass ratio, vermiculite: coconut bran: perlite=(2-6):(1-2):(1-2).

Preferably, the callus induction medium comprises the following components: 110 μM 2,4-D, 8.8 μM 6-BA, 4 g/L phytogel, 3 g/L activated carbon and 40 g/L sucrose are added to the Y3 medium;

Preferably, the somatic embryo induction medium includes the following components: 16 μM 2,4-D, 5 μM ABA, 4 g/L phytogel, 3 g/L activated carbon and 40 g/L sucrose are added to the WPM medium;

Preferably, the somatic embryo maturation medium comprises the following components: adding 4g/L phytogel, 3g/L activated carbon and 40g/L sucrose to the WPM medium;

Preferably, the somatic embryo germination medium comprises the following components: 10 μM NAA, 5 μM ABA, 4 g/L phytogel, 3 g/L activated carbon and 40 g/L sucrose are added to the WPM medium;

Preferably, the root-strengthening medium for regenerated plants includes the following components: adding 0.46 μM GA3, 2 g/L phytogel, 3 g/L activated carbon and 45 g/L sucrose to 1/2 CRI72 medium or WPM medium;

Preferably, the tissue culture seedling transplanting medium includes the following components: vermiculite: coconut bran: perlite=2:1:1 in mass ratio.

Preferably, in the callus induction culture stage, the pH value of the medium is 5.75-5.80, the culture condition is dark culture at 27-29°C, 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 the medium is 5.75-5.80, the culture condition is dark culture at 27-29°C, and subculture 1-1 month per month. 2 times, a total of 2 to 3 generations.

Preferably, in the somatic embryo germination and culture stage, the pH value of the medium is 5.75-5.80, the culture conditions are 27-29° C., the photoperiod is 12-18 h/d, and the subculture is performed 1-2 times per month until the plant is regenerated.

Preferably, in the cultivation stage of the regenerated plants, the pH value of the medium is 5.75-5.80, the culture conditions are 27-29° C., the photoperiod is 12-18 h/d, and the subcultures are carried out 1 to 2 times per month, for a total of 2 subcultures. ~ 3 times.

Preferably, in the stage of transplanting and cultivating the tissue culture seedlings, the culture conditions are 27-29°C, the photoperiod is 12-18 h/d, the tissue culture seedlings are kept moist with a moisturizing cover, and the moisturizing cover is opened every day to expose the tissue culture seedlings to the external environment for 1-10 h/d. 2h, the exposure time was extended day by day until the exposure time was 24h every day, after which it could be transplanted to the nursery.

Preferably, the adopted coconut varieties are selected from Hainan local high species, Wenye No. 2, Wenye No. 3, Wenye No. 4, Brown Dwarf or Mawa.

Compared with the prior art, the beneficial effects of the present invention are:

(1) The present invention strictly controls the medium formulation and the culture method at each stage of tissue culture, and effectively improves the explant callus induction rate, somatic embryo induction rate, somatic embryo regeneration rate and transplant survival rate; the highest callus induction rate can be 98.0%, the highest somatic embryo induction rate can reach 98.4%, the highest somatic embryo regeneration rate can reach 98.7%, and the transplant survival rate can reach 98.1%.

(2) As known in the art, due to the large difference between the genotypes of coconut tissue culture, the same medium formula, the same culture method, some varieties respond, and some do not respond. The culture medium and the culture method provided by the present invention have better broad-spectrum, and can be applied to the tissue culture of Hainan local high species, Wenye No. 2, Wenye No. 3 and other varieties at the same time. In addition, it can be seen from Table 1 that As shown, each group repeats the test 50 times, there is no obvious deviation in the results of each test in the embodiment, and the results of the comparative examples are quite different, indicating that the present invention has excellent repeatability and stable method.

(3) From the test results of Example 1 and Examples 4 to 8 of the present invention, it can be seen that using the medium and the culture method of the present invention, the callus induction rate, the induction rate of somatic embryos, the regeneration rate of somatic embryos and the survival rate of transplanting The best results were obtained when applied to the tissue culture of Wenye No. 2. The callus induction rate, somatic embryo induction rate, somatic embryo regeneration rate and transplant survival rate all reached 98%, higher than other varieties.

(4) the present invention effectively shortens the tissue culture cycle, which can be shortened by about 5 months compared with the conventional tissue culture method;

(5) the present invention is applicable to the coconut tissue culture of explants with mature zygotic embryos;

(6) Based on the technical scheme of the present invention, it is beneficial for coconut tissue culture seedlings to carry out factory and commercial production.

Detailed ways

The present invention will be further described in detail below through specific embodiments.

The experimental methods used in the embodiments of the present invention are conventional methods unless otherwise specified.

Materials, reagents, etc. used in the examples of the present invention can be obtained from commercial sources unless otherwise specified.

The meanings of some abbreviations in the present invention are:

6-BA: 6-benzyl adenine

2,4-D: 2,4-Dichlorophenoxyacetic acid

GA3: Gibberellin

ABA: Abscisic Acid

NAA: Naphthalene Acetic Acid

2iP: Dimethacrylopurine

The Y3 medium of the present invention is a medium containing the following components: ammonium chloride 535mg/L, potassium nitrate 2020mg/L, magnesium sulfate 247mg/L, calcium chloride 294mg/L, potassium chloride 1492mg/L, diphosphate diphosphate Ammonium hydrogen 312mg/L, potassium iodide 8.3mg/L, boric acid 3.1mg/L, manganese sulfate 11.2mg/L, zinc sulfate 7.2mg/L, copper sulfate 0.25mg/L, cobalt chloride 0.24mg/L, sodium molybdate 0.24mg/L, nickel chloride 0.024mg/L, ferrous sulfate 27.8mg/L, sodium chelate 37.3mg/L, pyridoxine hydrochloride 1mg/L thiamine hydrochloride 1mg/L hydrochloride 1mg/L, muscle Alcohol 102mg/L, L-glutamine 100mg/L, L-arginine 121mg/L, L-asparagine 88mg/L.

Example 1

A method for culturing coconut mature zygotic embryo tissue, comprising the following steps:

The mature zygotic embryos of Wenye 2 were taken as explants; the callus induction stage, callus propagation stage, somatic embryo induction stage, somatic embryo maturation stage, somatic embryo germination stage, regenerated plant root-strengthening stage and group Cultivated in the transplanting culture stage;

1.1 Callus induction stage:

The callus induction medium includes the following components: 110 μM 2,4-D, 8.8 μM 6-BA, 4 g/L phytogel, 3 g/L activated carbon and 40 g/L sucrose were added to Y3 medium;

The pH value of the medium was 5.75, and the culture condition was 28°C dark culture, and the culture time was 1 month.

1.2 Callus expansion stage:

The callus propagation medium is the same as the callus induction medium;

The pH value of the medium was 5.75, and the culture conditions were dark culture at 28°C.

1.3 Somatic embryo induction culture stage, somatic embryo maturation culture stage, somatic embryo germination culture stage

The somatic embryo induction medium includes the following components: 16μМ2,4-D, 5μМABA, 4g/L phytogel, 3g/L activated carbon and 40g/L sucrose were added to the WPM medium;

The somatic embryo maturation medium includes the following components: 4g/L phytogel, 3g/L activated carbon and 40g/L sucrose are added to the WPM medium;

The somatic embryo germination medium includes the following components: add 10μМNAA, 5μМABA, 4g/L phytogel, 3g/L activated carbon and 40g/L sucrose in WPM medium;

In the somatic embryo induction culture stage and the somatic embryo maturation culture stage, the pH value of the medium was 5.75, and the culture condition was dark culture at 28°C, and the cells were subcultured once every 1 month, for a total of 2 subcultures.

In the somatic embryo germination culture stage, the pH value of the medium was 5.75, the culture condition was 28°C, the photoperiod was 16h/d, and the subculture was performed once a month until the plant regenerated.

1.4 Stage of strong root of regenerated plants

The root-strengthening medium for regenerated plants includes the following components: add 0.46 μM GA3, 2g/L phytogel, 3g/L activated carbon and 45g/L sucrose in 1/2CRI72 medium; the 1/2CRI72 medium includes the following components: 10 mM ammonium nitrate, 10 mM potassium nitrate, 1 mM sodium dihydrogen phosphate, 1.5 mM calcium chloride, 1.5 mM magnesium sulfate, 150 μM boric acid, 100 μM manganese sulfate, 40 μM zinc sulfate, 1.5 μM copper sulfate, 1 μM sodium molybdate, 1 μM cobalt chloride , 5 μM potassium iodide, 100 μM ferrous sulfate, 100 μM Na ₂ EDTA, 650 μM sodium sulfate, 600 μM inositol, 40 μM niacin, 40 μM thiamine hydrochloride, 1 μM biotin, 5 μM calcium D-pantothenate, 6 μM pyridoxine hydrochloride, 10 μM riboflavin 10 μM ascorbic acid, 120 μM L-cysteine hydrochloride and 50 μM glycine; at the stage of root-strengthening of regenerated plants, the pH value of the medium was 5.75, the culture condition was 28 °C, the photoperiod was 16 h/d, and the subculture was performed once a month. , a total of 2 generations.

1.5 Tissue culture seedling transplanting culture stage

The tissue culture seedling transplanting medium includes the following components: by mass ratio, vermiculite: coconut bran: perlite=2:1:1.

In the transplanting culture stage of the tissue culture seedlings, the culture condition is 28°C, the photoperiod is 16h/d, the tissue culture seedlings are kept moisturizing with a moisturizing cover, and the moisturizing cover is opened every day to expose the tissue culture seedlings to the external environment for 1 hour, and the exposure time is prolonged day by day until every day Exposure to 24h, and then transplanted to the nursery.

Example 2

A method for culturing coconut mature zygotic embryo tissue, comprising the following steps:

The mature zygotic embryos of Wenye 2 were taken as explants; the callus induction stage, callus propagation stage, somatic embryo induction stage, somatic embryo maturation stage, somatic embryo germination stage, regenerated plant root-strengthening stage and Cultivated in the transplanting culture stage;

1.1 Callus induction stage:

The callus induction medium includes the following components: 100 μM 2,4-D, 5 μM 6-BA, 2 g/L phytogel, 1 g/L activated carbon and 20 g/L sucrose were added to Y3 medium;

The pH value of the medium was 5.75, and the culture condition was 27°C dark culture for 1 month.

1.2 Callus expansion stage:

The callus propagation medium is the same as the callus induction medium;

The pH value of the medium was 5.75, and the culture condition was dark culture at 27°C, and the cells were subcultured once every 1 month, for a total of 2 subcultures.

1.3 Somatic embryo induction culture stage, somatic embryo maturation culture stage, somatic embryo germination culture stage

Somatic embryo induction medium includes the following components: add 10μМ2,4-D, 2μМABA, 2g/L phytogel, 1g/L activated carbon and 20g/L sucrose in WPM medium;

The somatic embryo maturation medium includes the following components: add 2g/L phytogel, 1g/L activated carbon and 20g/L sucrose in WPM medium;

The somatic embryo germination medium includes the following components: add 8μМNAA, 2μМABA, 2g/L phytogel, 1g/L activated carbon and 20g/L sucrose in WPM medium;

In the somatic embryo induction culture stage and the somatic embryo maturation culture stage, the pH value of the medium was 5.75, and the culture condition was dark culture at 27°C, and the cells were subcultured once every 1 month, for a total of 2 subcultures.

In the somatic embryo germination culture stage, the pH value of the medium was 5.75, the culture condition was 27 °C, the photoperiod was 12 h/d, and the subculture was performed once a month until the plant regenerated.

1.4 Stage of strong root of regenerated plants

The root-strengthening medium for regenerated plants includes the following components: 0.20 μM GA3, 1 g/L phytogel, 1 g/L activated carbon and 20 g/L sucrose are added to 1/2 CRI72 medium; the 1/2 CRI72 medium includes the following components: 5 mM ammonium nitrate, 5 mM potassium nitrate, 0.5 mM sodium dihydrogen phosphate, 0.5 mM calcium chloride, 0.5 mM magnesium sulfate, 100 μM boric acid, 80 μM manganese sulfate, 20 μM zinc sulfate, 1.0 μM copper sulfate, 0.5 μM sodium molybdate, 0.5 μM Cobalt chloride, 2 μM potassium iodide, 80 μM ferrous sulfate, 80 μM Na ₂ EDTA, 500 μM sodium sulfate, 500 μM inositol, 20 μM niacin, 20 μM thiamine hydrochloride, 0.5 μM biotin, 2 μM calcium D-pantothenate, 3 μM pyridoxine hydrochloride , 5 μM riboflavin, 5 μM ascorbic acid, 100 μM L-cysteine hydrochloride and 30 μM glycine;

In the stage of root-strengthening culture of regenerated plants, the pH value of the medium was 5.75, the culture condition was 27°C, the photoperiod was 12h/d, and the subculture was performed once a month, for a total of 2 subcultures.

1.5 Tissue culture seedling transplanting culture stage

The tissue culture seedling transplanting medium includes the following components: by mass ratio, vermiculite: coconut bran: perlite=1:1:1.

In the transplanting culture stage of the tissue culture seedlings, the culture condition is 27°C, the photoperiod is 12h/d, the tissue culture seedlings are kept moisturizing with a moisturizing cover, and the moisturizing cover is opened every day to expose the tissue culture seedlings to the external environment for 1 hour, and the exposure time is prolonged day by day until every day Exposure to 24h, and then transplanted to the nursery.

Example 3

A method for culturing coconut mature zygotic embryo tissue, comprising the following steps:

The mature zygotic embryos of Wenye 2 were taken as explants; the callus induction stage, callus propagation stage, somatic embryo induction stage, somatic embryo maturation stage, somatic embryo germination stage, regenerated plant root-strengthening stage and Cultivated in the transplanting culture stage;

1.1 Callus induction stage:

The callus induction medium includes the following components: 150 μM 2,4-D, 10 μM 6-BA, 8 g/L phytogel, 8 g/L activated carbon and 50 g/L sucrose were added to Y3 medium;

The pH value of the medium was 5.80, and the culture condition was 29°C dark culture, and the culture time was 3 months.

1.2 Callus expansion stage:

The callus propagation medium is the same as the callus induction medium;

The pH value of the medium was 5.80, the culture condition was 29°C dark culture, and the cells were subcultured 2 times every 1 month, for a total of 3 subcultures.

1.3 Somatic embryo induction culture stage, somatic embryo maturation culture stage, somatic embryo germination culture stage

Somatic embryo induction medium includes the following components: add 20μМ2,4-D, 8μМABA, 8g/L phytogel, 8g/L activated carbon and 50g/L sucrose in WPM medium;

Somatic embryo maturation medium includes the following components: add 8g/L phytogel, 8g/L activated carbon and 50g/L sucrose in WPM medium;

The somatic embryo germination medium includes the following components: add 16μМNAA, 8μММABA, 8g/L phytogel, 8g/L activated carbon and 50g/L sucrose in WPM medium;

In the somatic embryo induction culture stage and the somatic embryo maturation culture stage, the pH value of the medium was 5.80, the culture condition was 29°C dark culture, and the cells were subcultured 2 times every 1 month, for a total of 3 subcultures.

In the somatic embryo germination culture stage, the pH value of the medium was 5.80, the culture condition was 29°C, the photoperiod was 18h/d, and the cells were subcultured twice a month until the plants were regenerated.

1.4 Stage of strong root of regenerated plants

The root-strengthening medium for regenerated plants includes the following components: adding 0.60μM GA3, 8g/L phytogel, 8g/L activated carbon and 60g/L sucrose in WPM medium;

In the stage of root-strengthening culture of regenerated plants, the pH value of the medium was 5.80, the culture condition was 29 °C, the photoperiod was 18 h/d, and the subcultures were carried out twice a month, for a total of three subcultures.

1.5 Tissue culture seedling transplanting culture stage

The tissue culture seedling transplanting medium includes the following components: by mass ratio, vermiculite: coconut bran: perlite=6:1:1.

In the transplanting culture stage of the tissue culture seedlings, the culture condition is 29°C, the photoperiod is 18h/d, the tissue culture seedlings are kept moisturizing with a moisturizing cover, and the moisturizing cover is opened every day to expose the tissue culture seedlings to the external environment for 2 hours, and the exposure time is prolonged day by day until every day Exposure to 24h, and then transplanted to the nursery.

Example 4 to Example 8

The varieties selected in Examples 4 to 8 are Hainan native high species, Wenye No. 3, Wenye No. 4, Brown Dwarf, and Mawa. Other operations are the same as in Example 1.

Comparative Example 1

The difference between Comparative Example 1 and Example 1 is:

The callus induction medium includes the following components: 80 μM 2,4-D, 3 μM 6-BA, 2 g/L phytogel, 4 g/L activated carbon and 40 g/L sucrose were added to Y3 medium;

The somatic embryo induction medium includes the following components: 8 μM 2,4-D, 1 μM ABA, 2 g/L phytogel, 4 g/L activated carbon and 40 g/L sucrose were added to WPM medium;

The somatic embryo germination medium includes the following components: 6μM NAA, 1μM ABA, 2g/L phytogel, 4g/L activated carbon and 40g/L sucrose were added to WPM medium;

The root-strengthening medium for regenerated plants includes the following components: add 0.15 μM GA3, 1 g/L phytogel, 0.5 g/L activated carbon and 20 g/L sucrose to 1/2 CRI72 medium; the 1/2 CRI72 medium includes the following components : 10 mM ammonium nitrate, 10 mM potassium nitrate, 1 mM sodium dihydrogen phosphate, 1.5 mM calcium chloride, 1.5 mM magnesium sulfate, 150 μM boric acid, 100 μM manganese sulfate, 40 μM zinc sulfate, 1.5 μM copper sulfate, 1 μM sodium molybdate, 1 μM chloride Cobalt, 5 μM potassium iodide, 100 μM ferrous _sulfate , 100 μM Na2EDTA, 650 μM sodium sulfate, 600 μM inositol, 40 μM niacin, 40 μM thiamine hydrochloride, 1 μM biotin, 5 μM calcium D-pantothenate, 6 μM pyridoxine hydrochloride, 3 μM nuclear Flavin, 3 μM ascorbic acid, 80 μM L-cysteine hydrochloride, and 20 μM glycine;

The tissue culture seedling transplanting medium includes the following components: by mass ratio, vermiculite: coconut bran: perlite=1:2:1.

Comparative Example 2

The difference between Comparative Example 2 and Example 1 is:

The callus induction medium includes the following components: 180 μM 2,4-D, 12 μM 6-BA, 2 g/L phytogel, 4 g/L activated carbon and 40 g/L sucrose were added to Y3 medium;

Somatic embryo induction medium includes the following components: 25μM 2,4-D, 8μM ABA, 2g/L phytogel, 4g/L activated carbon and 40g/L sucrose were added to WPM medium;

The somatic embryo germination medium includes the following components: 20μM NAA, 10μM ABA, 2g/L phytogel, 4g/L activated carbon and 40g/L sucrose were added to WPM medium;

The root-strengthening medium for regenerated plants includes the following components: 0.80μM GA3, 8g/L phytogel, 10g/L activated carbon and 50g/L sucrose are added to 1/2CRI72 medium; the 1/2CRI72 medium includes the following components: 10 mM ammonium nitrate, 10 mM potassium nitrate, 1 mM sodium dihydrogen phosphate, 1.5 mM calcium chloride, 1.5 mM magnesium sulfate, 150 μM boric acid, 100 μM manganese sulfate, 40 μM zinc sulfate, 1.5 μM copper sulfate, 1 μM sodium molybdate, 1 μM cobalt chloride , 5 μM potassium iodide, 100 μM ferrous sulfate, 100 μM Na ₂ EDTA, 650 μM sodium sulfate, 600 μM inositol, 40 μM niacin, 40 μM thiamine hydrochloride, 1 μM biotin, 5 μM calcium D-pantothenate, 6 μM pyridoxine hydrochloride, 12 μM riboflavin 12 μM ascorbic acid, 150 μM L-cysteine hydrochloride and 80 μM glycine;

The tissue culture seedling transplanting medium includes the following components: by mass ratio, vermiculite: coconut bran: perlite=7:1:2.

Comparative Example 3

The difference between Comparative Example 3 and Example 1 is:

The callus induction medium also contained 5 μM 2iP;

Somatic embryo induction medium and somatic embryo maturation medium also contain 30 μM pyrimidinol;

Somatic embryo germination medium does not contain NAA;

The tissue culture seedling transplanting medium includes the following components: by mass ratio, vermiculite: coconut bran=2:1.

Comparative Example 4

The difference between Comparative Example 4 and Example 1 is that the WPM medium described in the somatic embryo induction medium, the somatic embryo maturation medium, and the somatic embryo germination medium was replaced by the Y3 medium, and the regeneration plant described in the strong root medium. 1/2 of CRI72 medium was changed to Y3 medium.

Test example:

Coconut tissue culture was carried out using the methods of Examples and Comparative Examples respectively, and the test was repeated 50 times for each group, and the callus induction rate, somatic embryo induction rate, somatic embryo regeneration rate and transplant survival rate were counted. The results are shown in Table 1.

Callus induction rate=number of explants with callus induced/number of inoculated explants×100%

Somatic embryo induction rate=number of somatic embryos/number of inoculated callus blocks×100%

Somatic embryo regeneration rate = number of somatic embryos induced to produce complete plants/number of somatic embryos inoculated × 100%

Survival rate of transplanting = number of transplanted survival / total number of transplants × 100%

Table 1

Callus induction rate/% Somatic embryo induction rate/% Somatic embryo regeneration rate/% Transplanting survival rate/% 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 results show that the coconut mature zygotic embryos are used as explants, and the callus induction rate, somatic embryo induction rate, somatic embryo regeneration rate and transplant survival rate can all reach more than 90% after tissue culture, and the callus induction rate is the highest. It can reach 98.0%, the highest somatic embryo induction rate can reach 98.4%, the highest somatic embryo regeneration rate can reach 98.7%, and the highest transplant survival rate can reach 98.1%.

The culture medium formula and method of the invention has a broader spectrum, is suitable for Hainan local high species, Wenye No. 2, Wenye No. 3 and other varieties, has good repeatability and short cycle, and is conducive to industrialization and commercialization. .

The above content is a further detailed description of the present invention in conjunction with specific embodiments, and it cannot be considered that the specific implementation of the present invention is limited to these descriptions. For those of ordinary skill in the technical field to which the present invention pertains, some simple deductions or substitutions can also be made without departing from the concept of the present invention.

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 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 MNa₂EDTA, 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).

2. The tissue culture method of coconut mature zygotic embryo according to claim 1,

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 somatic embryo induction culture medium comprises the following components: 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 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 active carbon and 40g/L sucrose into WPM culture medium;

the regenerated plant strong root culture medium comprises the following components: adding 0.46 mu MGA3, 2g/L plant gel, 3g/L activated carbon and 45g/L sucrose into 1/2CRI72 culture medium or WPM culture medium;

the tissue culture seedling transplanting culture medium comprises the following components: 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 can be 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.