CN104186320B - A kind of method of Bulbus Lycoridis longitubae seed cultured in vitro - Google Patents
A kind of method of Bulbus Lycoridis longitubae seed cultured in vitro Download PDFInfo
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Abstract
The present invention discloses a kind of method of Bulbus Lycoridis longitubae seed cultured in vitro, comprises the following steps: step one, fertilization the choosing and sterilization of ovary: retain the complete fertilization ovary of Bulbus Lycoridis longitubae flower in full bloom；Sterilizing is carried out by being placed on superclean bench after fertilization ovary epidermis washes clean；Step 2, the cultured in vitro of fertilization ovary: the fertilization ovary after sterilization is put into and cultivates in the first culture medium until fertilization ovary development becomes full capsule；Step 3, the cultured in vitro of seed: full capsule is put into and continues in the second culture medium to cultivate until seed maturity；Step 4, the sprouting of seed: removed the seed coat of mature seed, then put into the seed removing seed coat and cultivate until seed germination in the 3rd culture medium；The invention have the advantage that and fertilization ovary in-vitro breeding can be become mature seed and seedling, thus substantially increase the breeding potential of Bulbus Lycoridis longitubae, moreover it is possible to be effectively improved the success rate of distant hybridization.
Description
Technical field
The present invention relates to vegetable seeds cultured in vitro field, a kind of method particularly relating to Bulbus Lycoridis longitubae seed cultured in vitro.
Background technology
Bulbus Lycoridis longitubae belongs to Amaryllidaceae Lycoris, and for herbaceos perennial, petal white, flower-shape is like lily, and flower appearance is beautiful, and pattern variation is relatively big, and ornamental value is higher.There is spherical bulb Bulbus Lycoridis longitubae underground, the multiple alkaloids such as contained galanthamine, lycoramine, lycorine, there is certain active anticancer, and can anti-inflammatory, antipyretic, calm and emetic, can be used for treating paralysis, the illness such as myasthenia gravis that the central paralysis such as senile dementia, polio causes, it is important ornamental plant and medicinal plant, thus the market demand of this plant is bigger.
In its natural state, the breeding of Bulbus Lycoridis longitubae is typically based on bulb separation breeding, but its breeding potential is relatively low, split every year on average can only mitogenetic go out 1～2 bulbec, each bulbec is it is generally required to about 4 years just can grow into split.In addition to bulb separation is bred, Bulbus Lycoridis longitubae can also pass through seminal propagation, although Bulbus Lycoridis longitubae has higher setting percentage, but restricted by growing environment, ripe and rudiment the seed of energy is few, adds the biological characteristics of its uniqueness, and seed is bloomed the needs time of 4～5 years from rudiment to kind of ball.Additionally, kind for distant hybridization, not affinity characteristic is hybridized owing to existing, thus be fertilized after hybridization the plant embryos stage of development the most in early days the most abortion in ovary or degeneration, seed cannot be ripe, substantially gathering in the crops less than ripe hybrid seed, thus the success rate obtaining the seed of ripe also energy rudiment after distant hybridization is the lowest, this will limit the research and development of new varieties.
Summary of the invention
Solve the technical problem that needed for the present invention and be: a kind of method that Bulbus Lycoridis longitubae seed cultured in vitro is provided, fertilization ovary cultured in vitro in the environment of aseptic can be made to become mature seed and seedling, thus substantially increase the breeding potential of Bulbus Lycoridis longitubae, in addition can also be effectively improved the success rate of distant hybridization.
For solving the problems referred to above, the technical solution used in the present invention is: the method for described a kind of Bulbus Lycoridis longitubae seed cultured in vitro, comprises the following steps:
Step one, fertilization the choosing and sterilization of ovary: choose Bulbus Lycoridis longitubae flower in full bloom and tapel and the holder of Bulbus Lycoridis longitubae flower are removed, only retained complete fertilization ovary；Sterilizing is carried out: rinse 30 ± 5 seconds with the alcohol that mass percent concentration is 75 ± 2% by being placed on superclean bench after complete fertilization ovary epidermis washes clean；It is then used by sterile water wash 3～5 times, every all over rinsing 30 ± 5 seconds；Again with the aseptic water washing 4 containing sodium hypochlorite～5 times, every all over rinsing 20～30 seconds, finally with aseptic filter paper by clean for the moisture removal of fertilization ovary epidermis；The mass percent concentration of the described sodium hypochlorite in the sterilized water containing sodium hypochlorite is 1 ± 0.2%；
Step 2, the cultured in vitro of fertilization ovary: the fertilization ovary after sterilization is put into and in the first culture medium, cultivates the capsule becoming full to ovary development of being fertilized: cultivation temperature is 25 ± 1 DEG C, intensity of illumination 1000～3000lx, the light application time of every day is 12 ± 1 hours, and incubation time is 30～40 days；
Step 3, the cultured in vitro of seed: full capsule is put into and continues in the second culture medium to cultivate to seed maturity: cultivation temperature is 25 ± 1 DEG C, intensity of illumination 1000～3000lx, and the light application time of every day is 12 ± 1 hours；
Step 4, the sprouting of seed: the seed coat of full mature seed is removed, then the seed removing seed coat is put in the 3rd culture medium and cultivate to seed germination: cultivation temperature is 25 ± 1 DEG C, intensity of illumination 1000～3000lx, the light application time of every day is 12 ± 1 hours；
Further, the method of aforesaid a kind of Bulbus Lycoridis longitubae seed cultured in vitro, wherein, in step one by the method for fertilization ovary epidermis washes clean being: fertilization ovary is placed in the cleaning solution in shaking table concussion washing 15～20min, described cleaning solution is the Amway cleaning solution of dilution 1000 times.
Further, the method of aforesaid a kind of Bulbus Lycoridis longitubae seed cultured in vitro, wherein, the formula of the first culture medium described in step 2 is: be added with the 6-benzyladenine of 6 ± 0.2mg/L, the methyl α-naphthyl acetate of 0.5 ± 0.1mg/L and the sucrose of 30 ± 2g/L in MS minimal medium；The pH value of the first culture medium is 5.8～6.
Further, the method of aforesaid a kind of Bulbus Lycoridis longitubae seed cultured in vitro, wherein, the formula of the second culture medium described in step 3 is: be added with the 6-benzyladenine of 6 ± 0.2mg/L, the methyl α-naphthyl acetate of 0.5 ± 0.1mg/L and the sucrose of 30 ± 2g/L in MS minimal medium；The pH value of the second culture medium is 5.8～6.
Further, the method of aforesaid a kind of Bulbus Lycoridis longitubae seed cultured in vitro, wherein, the formula of the 3rd culture medium described in step 4 is: being added with the 6-benzyladenine of the gibberellin of 0.3 ± 0.1g/L, 1.5 ± 0.2mg/L in 1/2MS minimal medium, the pH value of the 3rd culture medium is 5.8～6.
The invention has the beneficial effects as follows: can by fertilization ovary be placed in gnotobasis cultivate into mature seed and and then cultivate into seedling, mature seed and seedling are not easy to be contaminated by bacterial, thus substantially increase the breeding potential of Bulbus Lycoridis longitubae, and the research and development of the success rate of distant hybridization, beneficially new varieties can be effectively improved.
Detailed description of the invention
Below in conjunction with embodiment, technical solutions according to the invention are described in further detail.
Embodiment one
The method of the Bulbus Lycoridis longitubae seed cultured in vitro described in the present embodiment, comprises the following steps:
Step one, fertilization the choosing and sterilization of ovary: choosing Bulbus Lycoridis longitubae flower in full bloom and tapel and the holder of Bulbus Lycoridis longitubae flower removed, only retain complete fertilization ovary, now fertilization ovary is immature seed；Sterilizing is carried out: rinse 30 seconds with the alcohol that mass percent concentration is 75% by being placed on superclean bench after complete fertilization ovary epidermis washes clean；It is then used by sterile water wash 3 times, every all over rinsing 35 seconds；Again with the aseptic water washing containing sodium hypochlorite 4 times, every all over rinsing 30 seconds, finally with aseptic filter paper by clean for the moisture removal of fertilization ovary epidermis；The mass percent concentration of the described sodium hypochlorite in the sterilized water containing sodium hypochlorite is 1%.In the present embodiment, by the method for fertilization ovary epidermis washes clean it is: fertilization ovary is placed in the cleaning solution in shaking table concussion washing 15min, described cleaning solution is the Amway cleaning solution of dilution 1000 times, Amway cleaning solution described in the present embodiment is that the Amway LOC that net content is 1 liter produced by Anli (China) Daily Article Co., Ltd. concentrates multifunctional cleaning agent, and this kind of cleaning solution commercially can directly buy.
Step 2, the cultured in vitro of fertilization ovary: the fertilization ovary after sterilization is put into and in the first culture medium, cultivates the capsule becoming full to ovary development of being fertilized: cultivation temperature is 25 ± 1 DEG C, intensity of illumination 1000～3000lx, the light application time of every day is 12 ± 1 hours, and incubation time is 30～40 days；The formula of the first described culture medium is: be added with the 6-benzyladenine (6-BA) of 6mg/L, the methyl α-naphthyl acetate (NAA) of 0.6mg/L and the sucrose of 30g/L in MS minimal medium；The pH value of the first culture medium is 6.
Step 3, the cultured in vitro of seed: full capsule is put into and continues in the second culture medium to cultivate to seed maturity: cultivation temperature is 25 ± 1 DEG C, intensity of illumination 1000～3000lx, and the light application time of every day is 12 ± 1 hours；The formula of the second described culture medium is: be added with the 6-benzyladenine (6-BA) of 6.2mg/L, the methyl α-naphthyl acetate (NAA) of 0.5mg/L and the sucrose of 30g/L in MS minimal medium；The pH value of the second culture medium is 5.9.
Step 4, the sprouting of seed: the seed coat of full mature seed is removed, then the seed removing seed coat is put in the 3rd culture medium and cultivate to seed germination: cultivation temperature is 25 ± 1 DEG C, intensity of illumination 1000～3000lx, the light application time of every day is 12 ± 1 hours；The formula of the 3rd described culture medium is: be added with the gibberellin (GA of 0.3g/L in 1/2MS minimal medium3), the 6-benzyladenine (6-BA) of 1.3mg/L, the pH value of the 3rd culture medium is 5.8.
Embodiment two
The method of the Bulbus Lycoridis longitubae seed cultured in vitro described in the present embodiment, comprises the following steps:
Step one, fertilization the choosing and sterilization of ovary: choose Bulbus Lycoridis longitubae flower in full bloom and tapel and the holder of Bulbus Lycoridis longitubae flower are removed, only retained complete fertilization ovary；Sterilizing is carried out: rinse 35 seconds with the alcohol that mass percent concentration is 73% by being placed on superclean bench after complete fertilization ovary epidermis washes clean；It is then used by sterile water wash 5 times, every all over rinsing 30 seconds；Again with the aseptic water washing containing sodium hypochlorite 4 times, every all over rinsing 20 seconds, finally with aseptic filter paper by clean for the moisture removal of fertilization ovary epidermis；The mass percent concentration of the described sodium hypochlorite in the sterilized water containing sodium hypochlorite is 1.2%.In the present embodiment, by the method for fertilization ovary epidermis washes clean being: fertilization ovary is placed in the cleaning solution in shaking table concussion washing 18min, described cleaning solution is the Amway cleaning solution of dilution 1000 times.
Step 2, the cultured in vitro of fertilization ovary: the fertilization ovary after sterilization is put into and in the first culture medium, cultivates the capsule becoming full to ovary development of being fertilized: cultivation temperature is 25 ± 1 DEG C, intensity of illumination 1000～3000lx, the light application time of every day is 12 ± 1 hours, and incubation time is 30～40 days；The formula of the first described culture medium is: be added with the 6-benzyladenine (6-BA) of 6.2mg/L, the methyl α-naphthyl acetate (NAA) of 0.5mg/L and the sucrose of 32g/L in MS minimal medium；The pH value of the first culture medium is 6.
Step 3, the cultured in vitro of seed: full capsule is put into and continues in the second culture medium to cultivate to seed maturity: cultivation temperature is 25 ± 1 DEG C, intensity of illumination 1000～3000lx, and the light application time of every day is 12 ± 1 hours；The formula of the second described culture medium is: be added with the 6-benzyladenine (6-BA) of 6mg/L, the methyl α-naphthyl acetate (NAA) of 0.4mg/L and the sucrose of 32g/L in MS minimal medium；The pH value of the second culture medium is 5.8.
Step 4, the sprouting of seed: the seed coat of full mature seed is removed, then the seed removing seed coat is put in the 3rd culture medium and cultivate to seed germination: cultivation temperature is 25 ± 1 DEG C, intensity of illumination 1000～3000lx, the light application time of every day is 12 ± 1 hours；The formula of the 3rd described culture medium is: be added with the gibberellin (GA of 0.4g/L in 1/2MS minimal medium3), the 6-benzyladenine (6-BA) of 1.5mg/L, the pH value of the 3rd culture medium is 5.8.
Embodiment three
The method of the Bulbus Lycoridis longitubae seed cultured in vitro described in the present embodiment, comprises the following steps:
Step one, fertilization the choosing and sterilization of ovary: choose Bulbus Lycoridis longitubae flower in full bloom and tapel and the holder of Bulbus Lycoridis longitubae flower are removed, only retained complete fertilization ovary；Sterilizing is carried out: rinse 25 seconds with the alcohol that mass percent concentration is 77% by being placed on superclean bench after complete fertilization ovary epidermis washes clean；It is then used by sterile water wash 4 times, every all over rinsing 25 seconds；Again with the aseptic water washing containing sodium hypochlorite 5 times, every all over rinsing 28 seconds, finally with aseptic filter paper by clean for the moisture removal of fertilization ovary epidermis；The mass percent concentration of the described sodium hypochlorite in the sterilized water containing sodium hypochlorite is 0.8%.In the present embodiment, by the method for fertilization ovary epidermis washes clean being: fertilization ovary is placed in the cleaning solution in shaking table concussion washing 20min, described cleaning solution is the Amway cleaning solution of dilution 1000 times.
Step 2, the cultured in vitro of fertilization ovary: the fertilization ovary after sterilization is put into and in the first culture medium, cultivates the capsule becoming full to ovary development of being fertilized: cultivation temperature is 25 ± 1 DEG C, intensity of illumination 1000～3000lx, the light application time of every day is 12 ± 1 hours, and incubation time is 30～40 days；The formula of the first described culture medium is: be added with the 6-benzyladenine (6-BA) of 5.8mg/L, the methyl α-naphthyl acetate (NAA) of 0.4mg/L and the sucrose of 28g/L in MS minimal medium；The pH value of the first culture medium is 5.9.
Step 3, the cultured in vitro of seed: full capsule is put into and continues in the second culture medium to cultivate to seed maturity: cultivation temperature is 25 ± 1 DEG C, intensity of illumination 1000～3000lx, and the light application time of every day is 12 ± 1 hours；The formula of the second described culture medium is: be added with the 6-benzyladenine (6-BA) of 5.8mg/L, the methyl α-naphthyl acetate (NAA) of 0.6mg/L and the sucrose of 28g/L in MS minimal medium；The pH value of the second culture medium is 5.9.
Step 4, the sprouting of seed: removed by the seed coat of mature seed, then put into the seed removing seed coat in the 3rd culture medium and cultivate to seed germination: cultivation temperature is 25 ± 1 DEG C, intensity of illumination 1000～3000lx, and the light application time of every day is 12 ± 1 hours；The formula of the 3rd described culture medium is: be added with the gibberellin (GA of 0.2g/L in 1/2MS minimal medium3), the 6-benzyladenine (6-BA) of 1.7mg/L, the pH value of the 3rd culture medium is 5.8.
The invention have the advantage that fertilization ovary to be placed in gnotobasis and cultivate into mature seed and seedling, mature seed and seedling are not easy to be contaminated by bacterial, and after distant hybridization can be effectively improved, obtain the success rate of the seed of ripe also energy rudiment, can be by seed immature in open flower by fertilization ovary in-vitro breeding after distant hybridization, thus fertilization ovary is cultivated into mature seed and seedling, it is prevented effectively from plant embryos stage of development abortion or the degeneration in early days in fertilization ovary, it is capable of embryo rescue, the fertilization ovary that will be unable to maturation cultivates into mature seed or seedling, this is conducive to the research and development of new varieties.In addition, fertilization ovary can form the callus of several more than seed volume times during in-vitro breeding, callus can be formed the bud of some sproutings, the part having formed rudiment on callus can be separated by cultivating process with overall callus, i.e. individually cultivate into seedling after plant division, the callus not forming rudiment of remainder still can continue to cultivate thus forms bigger callus, and then rudiment, plant division can be carried out the most again, thus a callus can carry out repeatedly plant division in cultivating process, which greatly enhances the breeding potential of Bulbus Lycoridis longitubae.
Claims (2)
1. the method for a Bulbus Lycoridis longitubae seed cultured in vitro, it is characterised in that: comprise the following steps:
Step one, fertilization the choosing and sterilization of ovary: choose Bulbus Lycoridis longitubae flower in full bloom and tapel and the holder of Bulbus Lycoridis longitubae flower are removed, only retained complete fertilization ovary；Sterilizing is carried out: rinse 30 ± 5 seconds with the alcohol that mass percent concentration is 75 ± 2% by being placed on superclean bench after complete fertilization ovary epidermis washes clean；It is then used by sterile water wash 3～5 times, every all over rinsing 30 ± 5 seconds；Again with the aseptic water washing 4 containing sodium hypochlorite～5 times, every all over rinsing 20～30 seconds, finally with aseptic filter paper by clean for the moisture removal of fertilization ovary epidermis；The mass percent concentration of the described sodium hypochlorite in the sterilized water containing sodium hypochlorite is 1 ± 0.2%；
Step 2, the cultured in vitro of fertilization ovary: the fertilization ovary after sterilization is put into and in the first culture medium, cultivates the capsule becoming full to ovary development of being fertilized: cultivation temperature is 25 ± 1 DEG C, intensity of illumination 1000～3000lx, the light application time of every day is 12 ± 1 hours, and incubation time is 30～40 days；The first described culture medium is made up of MS minimal medium, 6-benzyladenine, methyl α-naphthyl acetate and sucrose, and concrete proportioning is: be added with the 6-benzyladenine of 6 ± 0.2mg/L, the methyl α-naphthyl acetate of 0.5 ± 0.1mg/L and the sucrose of 30 ± 2g/L in MS minimal medium；The pH value of the first culture medium is 5.8～6；
Step 3, the cultured in vitro of seed: full capsule is put into and continues in the second culture medium to cultivate to seed maturity: cultivation temperature is 25 ± 1 DEG C, intensity of illumination 1000～3000lx, and the light application time of every day is 12 ± 1 hours；The second described culture medium is made up of MS minimal medium, 6-benzyladenine, methyl α-naphthyl acetate and sucrose, and concrete proportioning is: be added with the 6-benzyladenine of 6 ± 0.2mg/L, the methyl α-naphthyl acetate of 0.5 ± 0.1mg/L and the sucrose of 30 ± 2g/L in MS minimal medium；The pH value of the second culture medium is 5.8～6；
Step 4, the sprouting of seed: the seed coat of full mature seed is removed, then the seed removing seed coat is put in the 3rd culture medium and cultivate to seed germination: cultivation temperature is 25 ± 1 DEG C, intensity of illumination 1000～3000lx, the light application time of every day is 12 ± 1 hours；The 3rd described culture medium is made up of 1/2MS minimal medium, gibberellin, 6-benzyladenine, concrete proportioning is: being added with the 6-benzyladenine of the gibberellin of 0.3 ± 0.1g/L, 1.5 ± 0.2mg/L in 1/2MS minimal medium, the pH value of the 3rd culture medium is 5.8～6.
2. according to the method for a kind of Bulbus Lycoridis longitubae seed cultured in vitro described in claim 1, it is characterized in that: in step one by the method for fertilization ovary epidermis washes clean be: fertilization ovary is placed in the cleaning solution in shaking table concussion washing 15～20min, described cleaning solution is the Amway cleaning solution of dilution 1000 times.
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