CN114766364B

CN114766364B - Rapid breeding method of areca tissue culture seedlings suitable for multiple explants

Info

Publication number: CN114766364B
Application number: CN202210466450.4A
Authority: CN
Inventors: 李志瑛; 王永; 张大鹏; 石鹏; 黄丽云; 周焕起; 刘立云
Original assignee: Coconut Research Institute of Chinese Academy of Tropical Agricultural Sciences
Current assignee: Coconut Research Institute of Chinese Academy of Tropical Agricultural Sciences
Priority date: 2022-04-29
Filing date: 2022-04-29
Publication date: 2023-04-07
Anticipated expiration: 2042-04-29
Also published as: CN114766364A

Abstract

The invention belongs to the technical field of plant tissue culture, and relates to a rapid propagation method of areca tissue culture seedlings suitable for various explants. The method is suitable for culturing the complete areca tissue culture seedling by taking an areca material as an explant through a somatic embryogenesis way, is suitable for various explants, has the advantages of simple disinfection method, less added hormone types, simplified culture medium formula and short period, has important significance for industrial production of the areca tissue culture seedling, provides assistance for development of areca industry, and lays a foundation for research on areca genetic transformation, molecular breeding and the like.

Description

Rapid breeding method of areca tissue culture seedlings suitable for multiple explants

Technical Field

The invention belongs to the technical field of plant tissue culture, and relates to a rapid breeding method of areca tissue culture seedlings, in particular to a method for obtaining complete regeneration plants by taking the stem tips of areca big trees, mature zygotic embryos and aseptic seedling radicles as explants through a somatic embryogenesis way.

Background

The Areca (Areca catechu) is a perennial tree of Areca of Palmae, is a typical tropical economic crop, is one of four southern medicines (Areca, fructus alpiniae oxyphyllae, fructus amomi and morinda officinalis) in China, has a planting area of 233.7 ten thousand acres (accounting for more than 95% of the national planting area) in Hainan, has a planting and primary processing yield of about 287.3 billion yuan, is one of the pillar industries in the hot work industry in Hainan, has become a main economic source of over 200 million farmers in Hainan, and plays a role in building tropical characteristic efficient agriculture.

At present, the production of areca seed seedlings is mainly seed seedling, the seedling raising period is long, the seedling quality is difficult to ensure, the problems of disease carrying, character separation, poor disease and pest resistance and the like are easy to occur, and the orderly and benign development of the areca industry is severely restricted, so that the specific areca seed planting material is bred by a tissue culture technology, the specific material progeny is produced in a large scale, the seedling quality is improved, and the method is a new way for solving the key problem restricting the development of the areca industry. Meanwhile, a genetic transformation system of the betel nuts can be established on the basis of a tissue culture technology, and a platform can be provided for basic researches such as betel nut molecular biology and the like.

Disclosure of Invention

The invention aims to provide a rapid breeding method of areca tissue culture seedlings suitable for various explants, which takes areca materials as explants to culture complete areca tissue culture seedlings through a somatic embryogenesis way, is suitable for various explants, has less added hormone types, high speed of obtaining somatic embryos and short period, provides assistance for the development of areca industry, and lays a foundation for researches on areca genetic transformation, molecular breeding and the like.

The technical scheme adopted by the invention is as follows:

a rapid breeding method of betel nut tissue culture seedlings suitable for various explants comprises the following steps:

1. callus induction culture

Taking an areca material as an explant, inoculating the areca material into a callus induction proliferation culture medium for culture; the culture conditions are as follows: dark culture, wherein the culture temperature is 25-28 ℃, and the humidity is 40-55%; subculturing once every 20-30 days for 2-3 times to obtain callus, and continuously subculturing for 2-3 times to proliferate to obtain a large amount of callus. The callus induction proliferation culture medium is Y3+ 20-100 mg/L2, 4-D +4g/L plant gel +2g/L active carbon +30g/L cane sugar, and the pH of the culture medium before sterilization is 5.8. The areca material is the stem tip of a big areca tree, a mature zygote embryo or a germ of an aseptic seedling.

2. Somatic embryo induction culture

Inoculating the obtained callus into a somatic embryo induction culture medium for culture, wherein the culture conditions are as follows: dark culture is carried out, the culture temperature is 25-28 ℃, and the humidity is 40-55%. Subculturing once every 20-30 days for 1-2 times to obtain the somatic embryo. The somatic embryo induction culture medium is Y3+ 1-10 mg/L2, 4-D + 1-10 mg/L6-BA +30g/L sucrose +4g/L plant gel +2g/L active carbon, and the pH value is 5.8 before sterilization.

3. Induced culture of regenerated bud

Transferring the obtained mature somatic embryos into a regeneration bud induction culture medium for induction culture, wherein the culture conditions are as follows: culturing under weak light, wherein the illumination intensity is 500-800 lx, the illumination time per day for the first 15 days is 2-4 hours/day, the illumination time per day for the last 15 days is 8-16 hours/day, the culture temperature is 28-32 ℃, and the humidity is 55-65%. Subculturing once every 20-30 days, and subculturing for 2-4 times to obtain the regeneration bud. The regenerated bud induction culture medium is Y3+ 1-5 mg/L6-BA + 1-5 mg/L NAA +30g/L sucrose +4g/L plant gel +2g/L active carbon, and the pH value is 5.8 before sterilization.

4. Rooting culture

Inoculating the obtained regeneration bud into a rooting culture medium for rooting induction. The culture conditions are as follows: light culture, the illumination intensity is 1000-1200 lx, the illumination duration is 8-16 hours/day, the culture temperature is 28-32 ℃, and the humidity is 65-85%. Subculturing once every 20-30 days, and rooting after 2-4 times of subculturing. The rooting medium is Y3+ 0.1-1 mg/L6-BA + 0.1-1 mg/L NAA +30g/L sucrose +4g/L plant gel +2g/L active carbon, and the pH value is 5.8 before sterilization.

5. Test-tube seedling domestication

After rooting, the tissue culture seedlings grow to the number of leaves of 1-3 and the root length of 1-3 cm, the tissue culture seedlings are transferred to a strong seedling culture medium for culture, and each seedling is independently inoculated in a culture bottle. The culture conditions are as follows: light culture with illumination intensity of 1500-2000 lx, illumination time of 14-16 hours/day, culture temperature of 30-35 ℃ and humidity of 55-85%, subculturing once every 25-35 days, and subculturing 2-4 times to obtain complete regeneration plants. The strong seedling culture medium is Y3+1 mg/L6-BA +1mg/L NAA +4g/L plant gel +2g/L active carbon +30g/L cane sugar, and the pH value of the culture medium before sterilization is 5.8.

Further, the stem tip of the big areca nut tree is obtained by taking an areca nut tree which is free of plant diseases and insect pests and grows robustly for 5-8 years as a sampling parent, stripping old leaves on the outer layer until young leaves are exposed, cutting off the areca nut tree 10 cm below the joint of the young leaves and the stem, spraying 75% alcohol on the whole, wrapping the areca nut tree with tinfoil paper, bringing the areca nut tree back to a tissue culture room, putting the areca nut tree into a superclean bench, stripping the young leaves on the outer layer until the stem tip material is completely exposed, and longitudinally cutting and inoculating the areca nut tree.

Furthermore, the mature zygotic embryo is prepared by taking mature areca nuts of 10-11 months old as materials, taking out the zygotic embryo under the condition that the periphery is wrapped by endosperm after cleaning, soaking and sterilizing the zygotic embryo by 75% alcohol for 10-20 minutes, cleaning the zygotic embryo by sterile water for 3-5 times, removing the endosperm on sterile filter paper to obtain complete zygotic embryo, and inoculating after slitting.

Furthermore, the aseptic seedling radicle is a parent of the aseptic seedling, and a robust radicle is selected and cut into 2-3cm small sections to be directly inoculated without disinfection.

Compared with the prior art, the invention has the beneficial effects that:

1. the invention completely provides a rapid breeding method of areca tissue culture seedlings suitable for various explants (stem tips of big trees, mature zygotic embryos and aseptic seedling radicles), has the advantages of multiple explants, simple disinfection method, simplified culture medium formula and short culture period, has practical significance for breeding of germplasm with excellent or special characteristics of areca and shortening the variety breeding period by applying the germplasm to biotechnology breeding, and has promotion effect on the development of areca industry.

2. On the premise of reducing the use of the disinfectant, the sterile explant is obtained by using only 75% alcohol and sterile water, and the 75% alcohol does not directly contact the explant material, so that the damage of the explant is avoided.

3. The Y3 is used as a basic culture medium, the formula contains few components, the formula is simplified, the preparation of the culture medium is rapid, and the efficiency is high.

4. Compared with the seedling raising of the betel nut seeds, the seedling raising is not limited by seasons, a large number of seedlings can be propagated in a short time, and the conventional betel nut seedling breeding mode can be replaced after further curing.

Drawings

FIG. 1 is a flow chart of tissue culture of Areca catechu L. A. The stem tip of the big tree; B. zygotic embryo; C. young roots; D. callus tissue; E. a somatic embryo; F. rooting the regenerated buds; G. and (5) tissue culture seedling.

Detailed Description

The following examples are given to further illustrate embodiments of the present invention. The following examples are intended to illustrate the invention, but are not intended to limit the scope of the invention. The experimental procedures for which specific conditions are not noted in the following examples are generally conducted under conventional conditions or conditions recommended by the manufacturer.

In the quantitative tests in the following examples and comparative examples, three replicates were set, and the results were averaged.

1. Betel nut seedling breeding

The first embodiment is as follows:

1. explant selection and disinfection

(1) Stem tip of big tree: selecting a betelnut tree which is free of plant diseases and insect pests and strong and has the age of 5 years as a sampling parent, stripping outer old leaves until young leaves are exposed, cutting off the betelnut tree 10 cm below the joint of the young leaves and the stem, spraying 75% alcohol on the whole, wrapping the betelnut tree with tinfoil paper, bringing the betelnut tree back to a tissue culture room, putting the betelnut tree into a superclean workbench, stripping outer young leaves until stem tip materials are completely exposed (figure 1A), and longitudinally cutting the betelnut tree into multiple parts for later use.

(2) Mature zygotic embryo: taking mature Arecae semen (10-11 months old). After the surface of the areca nut is cleaned, most endosperm is removed, part of endosperm is kept to wrap the zygotic embryo, and the areca nut is soaked in 75% alcohol for 15 minutes for disinfection. After sterilization, the tube was washed 3 times with sterile water. The endosperm was removed on sterile filter paper to obtain a complete zygotic embryo (FIG. 1B), which was cut longitudinally along the embryo into multiple portions for use.

(3) Aseptic seedling radicle: and (3) inoculating the zygotic embryos sterilized in the step (2) to a culture medium (Y3 +30g/L sucrose +4g/L plant gel +2g/L active carbon) to induce the embryos to germinate and root. After 4 months of illumination culture, selecting Arecae semen tissue culture seedling with vigorous root system and 2-3 leaves, taking secondary root with lateral root (figure 1C), and cutting into 2-3cm segments.

2. Callus induction proliferation culture

The explants are inoculated in a callus induction proliferation culture medium (Y3 +20 mg/L2, 4-D +4g/L plant gel +2g/L active carbon +30g/L sucrose, and the pH of the culture medium is 5.8 before sterilization). The culture conditions are dark culture, the culture temperature is 27 ℃, the humidity is 40% -55%, subculture is carried out once every 30 days, the callus can be obtained after 2 subcultures, and the subculture is continued for 2 times for proliferation, so that a large amount of proliferated callus is obtained (figure 1D).

3. Somatic embryo induction culture

The obtained callus is inoculated in a somatic embryo induction culture medium (Y3 +10 mg/L2, 4-D +3 mg/L6-BA +4g/L plant gel +2g/L active carbon +30g/L sucrose, the pH of the culture medium before sterilization is 5.8) for culture under the dark culture condition, the culture temperature is 27 ℃, the humidity is 40-55%, subculture is carried out once every 25 days, and the somatic embryo can be obtained after 2 subcultures (figure 1E).

4. Induced culture of regenerated bud

Transferring the obtained mature somatic embryos into a regeneration bud induction culture medium (Y3 +5 mg/L6-BA +5mg/LNAA +4g/L plant gel +30g/L sucrose +2g/L active carbon, the pH of the culture medium before sterilization is 5.8) for regeneration bud induction culture under the condition of weak light culture, wherein the illumination intensity is 500lx, the illumination time length is 4 hours/day in the first 15 days per day, the illumination time length is 8 hours/day after 15 days, the culture temperature is 28 ℃, the humidity is 55-65%, subculture is carried out once every 30 days, and the regeneration buds can be obtained after 2 subcultures.

5. Rooting culture

The obtained regeneration bud is inoculated into a rooting culture medium (Y3 +0.3 mg/L6-BA +1mg/L NAA +4g/L plant gel +30g/L sucrose +2g/L active carbon, the pH of the culture medium is 5.8 before sterilization) for rooting induction. The culture conditions are light culture, the illumination intensity is 1100lx, the illumination time is 10 hours/day, the culture temperature is 30 ℃, the humidity is 65-75%, the subculture is carried out once every 30 days, and the rooting can be carried out after 2 subcultures (figure 1F).

6. Test-tube seedling domestication

Transferring the rooted tissue culture seedlings (the number of leaves is 3, the root length is 3 cm) to a strong seedling culture medium (Y3 +1 mg/L6-BA +1mg/L NAA +4g/L plant gel +30g/L sucrose +2g/L active carbon, the pH of the culture medium before sterilization is 5.8) for culture, and independently inoculating each seedling in a culture bottle. The culture conditions are light culture, the illumination intensity is 1500x, the illumination time is 14 hours/day, the culture temperature is 32-35 ℃, the humidity is 65% -85%, subculture is carried out once every 30 days, and a complete regeneration plant can be obtained after 3 subcultures (figure 1G).

Example two:

1. explant selection and disinfection

(1) Stem tip of the big tree: selecting a betelnut tree which is free of plant diseases and insect pests and strong in growth and is 7 years old as a sampling parent, stripping outer old leaves until young leaves are exposed, cutting off the betelnut tree 10 cm below a joint of the young leaves and a stem, spraying 75% alcohol on the whole, wrapping the betelnut tree with tinfoil paper, bringing the betelnut tree back to a tissue culture room, putting the betelnut tree into a superclean workbench, stripping outer young leaves until stem tip materials are completely exposed, and longitudinally cutting the betelnut tree into multiple parts for later use.

(2) Mature zygotic embryo: taking mature Arecae semen (10-11 months old). After the surface of the areca nut is cleaned, most endosperm is removed, part of endosperm is reserved to wrap the zygotic embryo, and the areca nut is soaked in 75% alcohol for 18 minutes for disinfection. After disinfection, the mixture is washed for 3 times by using sterile water. And (4) removing endosperm from the sterile filter paper to obtain a complete zygotic embryo, and longitudinally cutting the zygotic embryo into multiple parts along the embryo for later use.

(3) Aseptic seedling radicle: and (3) inoculating the zygotic embryos sterilized in the step (2) to a culture medium (Y3 +30g/L sucrose +4g/L plant gel +2g/L active carbon) to induce the embryos to germinate and root. After 5 months of illumination culture, selecting areca tissue culture seedling with vigorous root system and 2-3 leaves, taking secondary roots with lateral roots, and cutting into 2-3cm segments for later use.

2. Callus induction proliferation culture

The explants were inoculated into callus induction proliferation medium (Y3 +60 mg/L2, 4-D +4g/L plant gel +2g/L activated carbon +30g/L sucrose, pH 5.8 of medium before sterilization) for culture. The culture condition is dark culture, the culture temperature is 27 ℃, the humidity is 40% -55%, subculture is carried out once every 25 days, 3 times of subculture can obtain the callus, and subculture is carried out for 2 times to proliferate, so that a large amount of proliferated callus is obtained.

3. Somatic embryo induction culture

Inoculating the obtained callus into a somatic embryo induction culture medium (Y3 +7 mg/L2, 4-D +10 mg/L6-BA +4g/L plant gel +2g/L active carbon +30g/L sucrose, and the pH of the culture medium before sterilization is 5.8) for culture under the dark culture condition, wherein the culture temperature is 26 ℃, the humidity is 40-55%, and the somatic embryo can be obtained by subculture once every 20 days and 2 times.

4. Induced culture of regenerated bud

And transferring the obtained mature somatic embryos into a regeneration bud induction culture medium (Y3 +2 mg/L6-BA +4mg/LNAA +4g/L plant gel +30g/L sucrose +2g/L active carbon, and the pH of the culture medium is 5.8 before sterilization) to perform regeneration bud induction culture under the conditions of weak light culture, wherein the illumination intensity is 500lx, the illumination duration is 4 hours/day in the first 15 days, the illumination duration is 10 hours/day after 15 days, the culture temperature is 30 ℃, the humidity is 55-65%, subculture is performed once every 20 days, and the regeneration buds can be obtained after 3 subcultures.

5. Rooting culture

The obtained regenerated bud is inoculated into a rooting culture medium (Y3 +1 mg/L6-BA +0.5mg/L NAA +4g/L plant gel +30g/L sucrose +2g/L active carbon, and the pH of the culture medium before sterilization is 5.8) for rooting induction. The culture conditions are light culture, the illumination intensity is 1000lx, the illumination time is 14 hours/day, the culture temperature is 30 ℃, the humidity is 65-75%, the subculture is carried out once every 20 days, and the rooting can be carried out after 3 subcultures.

6. Test-tube seedling domestication

Transferring the rooted tissue culture seedlings (the number of leaves is 2, the root length is 3 cm) to a strong seedling culture medium (Y3 +1 mg/L6-BA +1mg/L NAA +4g/L plant gel +30g/L sucrose +2g/L active carbon, the pH of the culture medium before sterilization is 5.8) for culture, and independently inoculating each seedling in a culture bottle. The culture conditions are light culture, the illumination intensity is 1800x, the illumination time is 14 hours/day, the culture temperature is 30-32 ℃, the humidity is 65% -75%, subculture is carried out once every 28 days, and 3 times of subculture can obtain a complete regeneration plant.

Example three:

1. explant selection and disinfection

(1) Stem tip of the big tree: selecting a betelnut tree which is free of plant diseases and insect pests and strong in growth and is 8 years old as a sampling parent, stripping outer old leaves until young leaves are exposed, cutting off the betelnut tree 10 cm below a joint of the young leaves and a stem, spraying 75% alcohol on the whole, wrapping the betelnut tree with tinfoil paper, bringing the betelnut tree back to a tissue culture room, putting the betelnut tree into a superclean workbench, stripping outer young leaves until stem tip materials are completely exposed, and longitudinally cutting the betelnut tree into multiple parts for later use.

(2) Mature zygotic embryo: taking mature Arecae semen (10-11 months old). After the surface of the areca nut is cleaned, most endosperm is removed, part of endosperm is reserved to wrap the zygotic embryo, and the areca nut is soaked in 75% alcohol for 20 minutes for disinfection. After disinfection, the mixture is washed for 3 times by using sterile water. And (4) removing endosperm from the sterile filter paper to obtain a complete zygotic embryo, and longitudinally cutting the zygotic embryo into multiple parts along the embryo for later use.

(3) Aseptic seedling and radicle: inoculating the zygotic embryos sterilized in the step (2) to a culture medium (Y3 +30g/L of sucrose +4g/L of plant gel +2g/L of activated carbon) to induce the embryos to germinate and root. After 6 months of illumination culture, selecting betel nut tissue culture seedlings with 2-3 leaves and vigorous root systems, taking secondary roots with lateral roots, and cutting into 2-3cm segments for later use.

2. Callus induction proliferation culture

The explants are inoculated in a callus induction proliferation culture medium (Y3 +100 mg/L2, 4-D +4g/L plant gel +2g/L active carbon +30g/L sucrose, and the pH of the culture medium is 5.8 before sterilization). The culture condition is dark culture, the culture temperature is 28 ℃, the humidity is 40% -55%, subculture is carried out once every 30 days, the callus can be obtained after 2 subcultures, and the callus which is proliferated in large quantity can be obtained after 2 subcultures.

3. Somatic embryo induction culture

Inoculating the obtained callus into a somatic embryo induction culture medium (Y3 +3 mg/L2, 4-D +5 mg/L6-BA +4g/L plant gel +2g/L active carbon +30g/L sucrose, and the pH of the culture medium before sterilization is 5.8) for culture under the dark culture condition, wherein the culture temperature is 28 ℃, the humidity is 40-55%, and the somatic embryo can be obtained by subculture once every 30 days and 1 time.

4. Induced culture of regenerated bud

Transferring the obtained mature somatic embryos into a regeneration bud induction culture medium (Y3 +4 mg/L6-BA +3mg/LNAA +4g/L plant gel +30g/L sucrose +2g/L active carbon, the pH of the culture medium before sterilization is 5.8) for regeneration bud induction culture under the condition of weak light culture, wherein the illumination intensity is 800lx, the illumination time length is 3 hours/day in the first 15 days per day, the illumination time length is 14 hours/day after 15 days, the culture temperature is 30 ℃, the humidity is 55-65%, subculture is carried out once every 25 days, and 3 times of subculture are carried out to obtain regeneration buds.

5. Rooting culture

The obtained regenerated bud is inoculated into a rooting culture medium (Y3 +0.3 mg/L6-BA +0.7mg/L NAA +4g/L plant gel +30g/L sucrose +2g/L active carbon, and the pH of the culture medium before sterilization is 5.8) for rooting induction. The culture conditions are light culture, the illumination intensity is 1200lx, the illumination duration is 16 hours/day, the culture temperature is 30 ℃, the humidity is 65-75%, subculture is carried out once every 25 days, and rooting can be carried out after 3 subcultures.

6. Test-tube seedling domestication

Transferring the rooted tissue culture seedlings (the number of leaves is 3, the root length is 3 cm) to a strong seedling culture medium (Y3 +1 mg/L6-BA +1mg/L NAA +4g/L plant gel +30g/L sucrose +2g/L active carbon, the pH of the culture medium before sterilization is 5.8) for culture, and independently inoculating each seedling in a culture bottle. The culture conditions are light culture, the illumination intensity is 1500x, the illumination time is 15 hours/day, the culture temperature is 32-35 ℃, the humidity is 65% -75%, the subculture is carried out once every 35 days, and the subculture is carried out for 2 times to obtain a complete regeneration plant.

2. Identification of tissue culture effect of betel nut

1. Identifying the callus induction effect:

the first comparative example is as follows: taking the stem tip of a big areca nut tree, a mature zygotic embryo and a sterile seedling radicle as explants respectively, and inoculating the explants in a culture medium (Y3 +5 mg/L2, 4-D +4g/L plant gel +2g/L active carbon +30g/L cane sugar, the pH of the culture medium before sterilization is 5.8) for callus induction. Other conditions were the same as in example one.

Comparative example two: the content of 2,4-D was adjusted to 140mg/L, and the other conditions were the same as in comparative example one.

In order to examine the induction condition of areca callus, the induction rate was calculated by observing and counting the number of explants inoculated and the number of calli induced in the above examples and the control examples. The results are shown in Table 1.

TABLE 1 Effect of different 2,4-D concentrations on callus induction from different explants

Note: induction rate/% = number of calli formed explants/number of inoculated explants 100% >

As can be seen from Table 1, different concentrations of 2,4-D have significant differences in callus induction efficiency. 2,4-D at high concentration (140 mg/L) caused tissue death, and 2,4-D at low concentration had low induction rate. When the content of 2,4-D is 60mg/L, the callus induction effect is best, the callus induction rate is 56.7% at most, and the proliferation coefficient is higher.

2. And (3) somatic embryo induction culture identification:

comparative example one: the callus was inoculated into a medium (Y3 +10 mg/L2, 4-D +4g/L plant gel +2g/L activated carbon +30g/L sucrose, pH 5.8 of the medium before sterilization) for somatic embryo induction. Other conditions were the same as in example one.

Comparative example two: the callus was inoculated into a medium (Y3 +10 mg/L6-BA +4g/L plant gel +2g/L activated carbon +30g/L sucrose, pH of the medium before sterilization was 5.8) for somatic embryo induction. Other conditions were the same as in example one.

In order to examine the somatic embryo induction effect, the somatic embryo occurrence conditions of the above examples and the comparative examples were observed, the inoculation number and the somatic embryo number were counted, and the somatic embryo induction rate was calculated, and the results are shown in table 2.

TABLE 2 Effect of different hormone combinations on Areca catechu somatic embryo Induction

Item	Callus inoculation number (piece)	Number of embryos	Inductivity (%)
				Example one	300	198	66.0
Example two	300	233	77.7
				EXAMPLE III	300	213	71.0
Comparison example 1	300	87	29.0
				Comparative example two	300	73	24.3

Note: induction rate/% = number of calli having somatic embryogenesis/number of inoculated calli%

As can be seen from Table 2, different hormones have significant differences in induction rate of the areca somatic embryo. The combination of 2,4-D and 6-BA has better somatic embryo induction effect than the single use of 2,4-D or 6-BA, and the highest somatic embryo induction rate is 77.7 percent.

3. And (3) identifying the regenerated bud through induction culture:

the first comparative example is as follows: the mature somatic embryos are inoculated in a culture medium (Y3 +5 mg/L6-BA +4g/L plant gel +30g/L sucrose +2g/L active carbon, the pH value of the culture medium before sterilization is 5.8) for regeneration bud induction culture.

Other conditions were the same as in example one.

Comparative example two: the mature somatic embryos are inoculated in a culture medium (Y3 +5mg/L NAA +4g/L plant gel +30g/L cane sugar +2g/L active carbon, the pH of the culture medium before sterilization is 5.8) for regeneration bud induction culture.

Other conditions were the same as in example one.

To examine the effect of inducing regenerated shoots, the examples and the control examples were observed for the occurrence of regenerated shoots, the number of somatic embryos inoculated and the number of regenerated shoots were counted, and the induction rate of regenerated shoots was calculated, and the results are shown in Table 3.

TABLE 3 Effect of different hormone combinations on induction of areca nut regenerated shoots

Item	Somatic embryo inoculation number	Regenerated bud number	Inductivity (%)
				Example one	200	145	72.5
Example two	200	162	81.0
				EXAMPLE III	200	157	78.5
Comparison example 1	100	25	25.0
				Comparative example two	100	29	29.0

Note: induction rate/% = number of somatic embryos forming regenerated shoots/number of inoculum somatic embryos 100%

As can be seen from table 3, different hormones have significant differences in induction rate of areca regeneration buds. The combination of 6-BA and NAA has better effect than the single use of 6-BA or NAA on inducing the regeneration bud, and the highest somatic embryo induction rate is 81.0 percent.

4. And (3) rooting induction culture identification:

the first comparative example is as follows: the regenerated bud is inoculated in a culture medium (Y3 +1 mg/L6-BA +4g/L plant gel +30g/L cane sugar +2g/L active carbon, the pH value of the culture medium before sterilization is 5.8) for rooting induction culture. Other conditions were the same as in example one.

Comparative example two: the regenerated bud is inoculated in a culture medium (Y3 +1mg/L NAA +4g/L plant gel +30g/L cane sugar +2g/L active carbon, the pH of the culture medium before sterilization is 5.8) for rooting induction culture. Other conditions were the same as in example one.

In order to examine the rooting induction effect of the regenerated buds, the rooting occurrence conditions of the regenerated buds in the above examples and the comparative examples were observed, the inoculation number and the rooting number of the regenerated buds were counted, and the rooting induction rate was calculated, and the results are shown in table 4.

TABLE 4 Effect of different hormone combinations on induction of areca nut regenerated shoots

Item	Regeneration bud grafting seed number	Root number (number)	Induction rate (%)
				Example one	100	83	83.0
Example two	100	91	91.0
				EXAMPLE III	100	87	87.0
Comparison example 1	100	41	41.0
				Comparative example II	100	56	56.0

As can be seen from Table 4, different hormones have significant differences in induction of rooting of the areca nut somatic embryos. The 6-BA and NAA are combined to achieve better rooting induction effect of the regenerated buds than the regenerated buds obtained by singly using the 6-BA or NAA, and the rooting induction rate of the regenerated buds is up to 91.0 percent.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, it is possible to make various improvements and modifications without departing from the technical principle of the present invention, and these improvements and modifications should also be considered as the protection scope of the present invention.

Claims

1. A rapid propagation method of areca tissue culture seedlings suitable for various explants is characterized by comprising the following steps:

1) Callus induction culture

Taking an areca material as an explant, and inoculating the areca material into a callus induction proliferation culture medium for culture; the culture conditions are as follows: dark culture, wherein the culture temperature is 25-28 ℃, and the humidity is 40-55%; subculture once every 20-30 days; the callus induction proliferation culture medium is Y3+ 20-100 mg/L2, 4-D +4g/L plant gel +2g/L active carbon +30g/L cane sugar, and the pH of the culture medium before sterilization is 5.8; the areca material is the stem tip of a big areca tree, mature zygotic embryo or aseptic seedling radicle;

2) Somatic embryo induction culture

Inoculating the obtained callus into a somatic embryo induction culture medium for culture, wherein the culture conditions are as follows: dark culture, wherein the culture temperature is 25-28 ℃, and the humidity is 40-55%; subculturing once every 20-30 days; the somatic embryo induction culture medium is Y3+ 1-10 mg/L2, 4-D + 1-10 mg/L6-BA +30g/L sucrose +4g/L plant gel +2g/L active carbon, and the pH value is 5.8 before sterilization;

3) Induced culture of regenerated bud

Transferring the obtained mature somatic embryos into a regeneration bud induction culture medium for induction culture, wherein the culture conditions are as follows: culturing in weak light with the illumination intensity of 500-800 lx, the illumination time of 2-4 hours/day for the first 15 days per day, the illumination time of 8-16 hours/day after 15 days, the culture temperature of 28-32 ℃ and the humidity of 55-65%; subculturing once every 20-30 days; the regenerated bud induction culture medium is Y3+ 1-5 mg/L6-BA + 1-5 mg/L NAA +30g/L sucrose +4g/L plant gel +2g/L active carbon, and the pH value is 5.8 before sterilization;

4) Rooting culture

Inoculating the obtained regeneration bud into a rooting culture medium for rooting induction; the culture conditions are as follows: light culture, wherein the illumination intensity is 1000-1200 lx, the illumination time is 8-16 h/day, the culture temperature is 28-32 ℃, and the humidity is 65-85%; subculture once every 20-30 days; the rooting medium is Y3+ 0.1-1 mg/L6-BA + 0.1-1 mg/L NAA +30g/L sucrose +4g/L plant gel +2g/L active carbon, and the pH value is 5.8 before sterilization;

5) Test-tube seedling domestication

After rooting, the tissue culture seedlings grow to the number of leaves of 1-3 and the root length of 1-3 cm, and are transferred into a strong seedling culture medium for culture, and each seedling is independently inoculated into a culture bottle; the culture conditions are as follows: light culture, wherein the illumination intensity is 1500-2000 lx, the illumination time is 14-16 h/day, the culture temperature is 30-35 ℃, and the humidity is 55% -85%; subculturing once every 25-35 days until a complete regeneration plant is obtained; the strong seedling culture medium is Y3+1 mg/L6-BA +1mg/L NAA +4g/L plant gel +2g/L active carbon +30g/L cane sugar, and the pH value of the culture medium before sterilization is 5.8.

2. The rapid propagation method of tissue culture seedlings of betel nuts according to claim 1, characterized in that: the stem tip of the big areca tree is prepared by taking an areca nut tree which is free of plant diseases and insect pests and grows robustly for 5-8 years as a sampling parent, stripping outer old leaves until young leaves are exposed, cutting off the areca nut tree 10 cm below a joint of the young leaves and the stem, spraying 75% alcohol on the whole, wrapping the areca nut tree with tinfoil paper, bringing the areca nut tree back to a tissue culture room, putting the areca nut tree into a superclean bench, stripping outer young leaves until the stem tip material is completely exposed, and longitudinally cutting and inoculating the areca nut tree.

3. The rapid propagation method of tissue culture seedlings of betel nuts according to claim 1, characterized in that: the mature zygotic embryo is prepared by taking mature areca nuts of 10-11 months old as a material, cleaning, taking out the zygotic embryo under the condition that the periphery of the mature areca nuts is wrapped by endosperm, soaking and sterilizing the zygotic embryo in 75% alcohol for 10-20 minutes, cleaning the zygotic embryo with sterile water for 3-5 times, removing the endosperm on sterile filter paper to obtain complete zygotic embryo, and longitudinally cutting and inoculating.

4. The rapid propagation method of tissue culture seedlings of betel nuts according to claim 1, characterized in that: the aseptic seedling radicle is a parent for sampling the aseptic seedling, and a robust radicle is selected, cut into 2-3cm small sections and then directly inoculated without disinfection.