CN114831025B - Rapid induction method of konjak polyploid - Google Patents
Rapid induction method of konjak polyploid Download PDFInfo
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Abstract
The invention belongs to the field of plant breeding, and particularly relates to a rapid induction method of konjak polyploid, which comprises the following steps: selecting seeds of different periods as an induction material, and cleaning and sterilizing the induction material; and sequentially carrying out primary culture, mutagenesis and secondary culture to obtain seedlings; obtaining an original seed after seedling culture, wherein the immature seeds and the mature seeds are used in different periods, when the immature seeds are subjected to primary culture, callus is obtained, and when the mature seeds are subjected to primary culture, seeds after germination acceleration are obtained; according to the invention, when the konjak seeds are immature, young embryo is adopted for induction, after the seeds are mature, embryo is adopted for induction, so that the utilization rate of the konjak materials of the same plant is increased, and the induction rate of the homozygous polyploid is respectively 7.93% and 9.07%, which is obviously higher than that of other methods, so that a new way is opened for konjak breeding, inbred line establishment and the like.
Description
Technical Field
The invention relates to the field of plant breeding, in particular to a rapid induction method of konjak polyploid.
Background
Konjak (Amorphophalms konjac) is also called konjak, is perennial herb plant of konjak genus of Araceae family, is one of original places in China, is mainly distributed in hilly area of mountain areas of various provinces in the south of Qinling, is a special economic crop capable of providing glucomannan in large quantity in nature, and is widely used in the fields of food industry, medical industry, daily chemical industry, light spinning, printing and dyeing, electronic industry, petroleum drilling, feed industry and the like because of being rich in various effective components. Through development for more than 30 years, konjak has become an important cash crop in mountain areas in the middle and west of China, and the rich source of Yunnan, the partial areas of Sichuan and Chongqing and the konjak producing area in the south of Shaanxi form an industry integrating departments, agriculture, industry, commerce and trade. Over 300 konjak manufacturers exist in China, and China has become the largest country for konjak production and export in the world. The Shaanxi province is one of the four main product provinces of konjak in the whole country, is a dominant characteristic industry in the region of the China, is a new industry with development potential after grain, fruits, livestock, vegetables and tea, and plays an important role in promoting the economic development of the region and promoting the happiness of the rural industry.
Polyploid plants are ubiquitous in nature, have stronger adaptability and larger plasticity in physiology and genetics than diploid plants, have slightly thicker stems, deeper leaves, obvious veins, obviously improved nutrient content, enlarged organs and increased medicinal active ingredients, and simultaneously enhance the ecological adaptability and stress resistance of the polyploid plants, and the polyploid plants are obtained by various methods by various medicinal plants. However, the research on konjak polyploid is relatively few, only Liu Haoxia and the like have performed experimental exploration on konjak polyploid in 2006, yang Pei is equal to 2013, and experimental research on konjak polyploid is performed, and all the experimental researches are only in the laboratory research stage. The research of konjak polyploid, firstly, aiming at varieties and strains with low corm expansion coefficient, such as white konjak and offspring materials obtained by hybridization by taking white konjak as a parent, the commodity of the corm can be improved by utilizing a polyploid induction technology; secondly, aiming at varieties with super-strong resistance but triploid ploidy such as the bulbil type mueller konjak, the genetic ploidy of the varieties is changed by utilizing a polyploid induction technology so as to facilitate the hybridization between konjak seeds; thirdly, aiming at the variety characteristics of the konjac, such as poor resistance, low propagation coefficient, good commodity property and the like, the polyploidy technology is utilized to improve the variety resistance and the propagation property of the konjac.
In addition, the polyploid induction rate is low by soaking or wrapping and wetting the konjak bulbs, rhizomes and the like, and most of the polyploid induction rate is low by chimeras, and the induction treatment is performed by using cluster buds, so that the chimeras occupy higher proportion of homozygotes. The problem of low embryogenic callus induction rate and polyploid induction rate is also found in double treatment of directly induced callus by bulbs, rhizomes, petioles or solid seeds.
In summary, the prior art has the problem of low induction rate of konjak polyploid. Therefore, the invention explores a rapid induction method of konjak polyploid.
Disclosure of Invention
In order to solve the technical problems, the invention provides a rapid induction method of konjak polyploid, which comprises the following steps:
s1, selecting seeds of different periods as an induction material, and cleaning and sterilizing the induction material;
s2, performing primary culture on the treated induction material to obtain a primary culture;
s3, using a mutagen to mutagenize the primary culture to obtain a mutagenized product;
s4, carrying out second generation culture on the mutation product to obtain seedlings;
s5, obtaining stock seeds after seedling culture;
the real seeds in different periods are immature real seeds and mature real seeds;
when the immature seeds are subjected to primary culture, the primary culture is callus, and when the mature seeds are subjected to primary culture, the primary culture is germinated seeds.
Preferably, when the inducing material is immature seeds, the specific method for inducing the seeds comprises the following steps:
s1, cleaning the seeds, and performing primary culture;
the primary culture is as follows: inoculating young embryo of the seedling seed on a callus induction culture medium for culturing to obtain a primary culture;
the callus induction culture medium takes MS as a basic culture medium, and 1.0-2.0mg of 6-BA, 0.1-0.2mg of KT, 0.1-0.2mg of 2,4-D and 30g of sucrose are added into each liter of MS culture medium;
s2, adding a mutagen into the callus subculture medium, uniformly mixing to obtain a mutagenesis medium, and inoculating the primary culture on the mutagenesis medium for mutagenesis to obtain a mutagenesis product;
the callus subculture medium takes MS as a basic culture medium, and 0.5-1.0mg of 6-BA or 0.1-0.2mg of KT, 0.1-0.2mg of NAA and 30g of sucrose are added into each liter of MS culture medium;
the mutagen is a mixed solution of colchicine with the mass fraction of 0.05-0.1% and dimethyl sulfoxide solution with the mass fraction of 0.1-0.2%;
s3, carrying out second generation culture on the mutation product, wherein the second generation culture comprises the steps of treating the mutation product to obtain clustered buds and treating the clustered buds to obtain seedlings.
Preferably, in S1, the conditions for the primary culture are: the temperature is 25-28 ℃, the humidity is 30-40%, and the dark culture is carried out for 15-18 days; in S2, the condition of mutagenesis is dark culture for 7-10 days, the temperature is 25-28 ℃, and the humidity is 30% -40%.
Preferably, the pH of the callus induction medium and the pH of the callus subculture medium are both 5.8-6.0.
Preferably, the specific process of the second generation culture is as follows:
s1, inoculating a mutagenesis product into an induced bud differentiation culture medium, and culturing to obtain cluster buds;
the bud differentiation induction culture medium takes MS as a basic culture medium, and 2.0-4.0mg of 6-BA, 0.5-1.0mg of KT, 0.1-0.2mg of NAA and 20g of sucrose are added into each liter of MS, wherein the pH value of the bud differentiation induction culture medium is 5.8-6.0;
s2, inoculating the cluster buds into a secondary culture medium to induce root primordia;
the secondary culture medium takes MS as a basic culture medium, 0.5-1.0mg of 6-BA, 0.5-1.0mg of NAA and 20g of sucrose are added into each liter of MS, and the pH value of the secondary culture medium is 5.8-6.0;
s3, transferring the single buds inducing root primordia into an induced seedling differentiation medium to differentiate seedlings;
the seedling differentiation induction culture medium takes 1/2MS as a basic culture medium, 1.0-2.0mg KT, 0.2-0.5mg 6-BA, 1.0-1.5mg NAA and 20g sucrose are added into each liter of 1/2MS, and the pH value of the seedling differentiation induction culture medium is 5.8-6.0.
Preferably, the culture conditions in S1 are: the illumination intensity is 1000-1200lx, the illumination time is 8/16h, the temperature is 25-28 ℃, and the humidity is 40% -60%; the induction conditions in S2 are: the illumination intensity is 1000-1200lx, the illumination time is 8/16h, the temperature is 25-28 ℃, and the humidity is 40% -60%; the conditions for differentiation in S3 were: the illumination intensity is 1500-2000lx, the illumination time is 12/12h, the temperature is 25-28 ℃, the humidity is 40-60%, and the culture is carried out for 4-5 weeks.
Preferably, when the inducing material is mature seeds, the specific inducing method comprises the following steps:
s1, cleaning, dormancy breaking, disinfection and primary culture are carried out on young embryos to obtain seeds after germination acceleration;
the break dormancy: soaking the seeds in a mixed solution containing 10-20mg KT, 500-800mg GA3 and 5-10mg 2,4-D in each liter of solution for a certain period of time, taking out, cleaning and airing;
the primary culture: accelerating germination of seeds under the conditions of 25-28 ℃ and 60-70% humidity and wetting;
s2, carrying out mutation culture on the seeds subjected to germination acceleration by using a mutagen to obtain a mutation product;
the mutagen is a mixed solution of 0.005-0.008% of trifluralin or 0.01-0.03mol/L of pendimethalin;
s3, carrying out second generation culture on the mutation product, namely inoculating the mutation product into a subculture medium to induce bud growth and differentiation, and culturing to obtain seedlings;
the secondary culture medium takes MS as a basic culture medium, and 1.0-2.0mg KT, 0.2-0.5mg 6-BA, 1.0-1.5mg NAA and 20g sucrose are added into each liter of MS.
Preferably, in S2, the mutagenesis culture is performed by a mixed culture-titration-encapsulation method.
Preferably, in S2, the mutagenesis condition is that the temperature is 25-28 ℃ and the humidity is 50% -60%; in S3, the conditions of the second generation culture are as follows: the illumination intensity is 1500-2000lx, the illumination time is 8/16h, the temperature is 25-28 ℃, and the humidity is 40-60%.
Preferably, the pH of the secondary culture medium is 5.8-6.0.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention provides a rapid induction method of konjak polyploid, which is characterized in that young embryo is adopted for induction when konjak seeds are immature, embryo is adopted for induction after the seeds are mature, the utilization rate of the same konjak material is increased, the induction rate with higher homozygote/chimera ratio is obtained, and the rapid induction method has important significance for later konjak breeding, quality improvement and the like.
2. The induction rates of the obtained polyploids are 7.93%, 26.64%, 9.07% and 20.19% respectively, and are obviously better than those of polyploids obtained by a dipping method, a dropping method, a packaging method, an injection method and a spraying method.
3. The invention uses the callus induced by the in vitro culture of the young embryo of the konjak seed to carry out polyploid mutagenesis, compared with other tissues and organs, the embryogenic callus can be efficiently induced, and the young embryo is tender, loose and easy to separate compared with the callus induced by other tissues and organs, and is more sensitive to mutagens, so that the polyploid induction effect is more remarkable; polyploid mutagenesis is performed by utilizing the embryo of the mature konjak seed, compared with polyploid mutagenesis by utilizing konjak corms, konjak whips, cluster buds and the like, the mutagenesis efficiency of homozygote can be remarkably improved, the seedling rate is high, and the process is simple and easy to operate;
4. the invention utilizes the characteristic that the callus induced by young embryo is highly sensitive to the selected mutagen, and adopts a mixed culture method for culture; meanwhile, by utilizing the characteristic of selective pre-bud treatment of the embryo on the existence of the selected mutagen, culturing by adopting a mixed culture-titration-wrapping method; both can obviously reduce the browning death rate of the material caused by inhibition or poisoning of the mutagen, and can obviously improve the overall mutagenesis effect.
Drawings
FIG. 1 is a diagram of the mutagenized products of young embryos after mutagenesis;
FIG. 2 shows seedlings obtained after the embryo has been subjected to a second generation of culture;
FIG. 3 is a polyploid plant obtained by immature embryo callus induction;
FIG. 4 shows polyploid plants obtained by embryo induction.
Detailed Description
The following detailed description of specific embodiments of the invention is, but it should be understood that the invention is not limited to specific embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. The experimental methods described in the examples of the present invention are conventional methods unless otherwise specified.
Example 1
When the inducing material is immature embryo of immature seed, the inducing method comprises the following steps:
s1, selecting 3-year flowering konjak corms, and selecting young embryos from the late 6 months to the early July, wherein the ears are full, the pericarps are green and the embryos are tender, so that callus induction is facilitated;
s2, drying or airing the collected seed in the shade at a ventilation position, peeling off a layer of pericarp outside the seed coat, and airing the surface of the seed coat at a shade position;
s3, cleaning the surface of the seed coat under running water, soaking the seed coat in a sodium hypochlorite solution with the mass fraction of 0.1% on an ultra-clean bench for 8min, taking out the seed coat, and cleaning the seed coat with sterile water for 4 times; soaking in 70% alcohol for 30s, taking out, cleaning with sterile water for 3 times, and finally sucking water on the surface of seed coat with sterile filter paper for use;
s4, clamping the seeds on the culture dishes by using tweezers, slightly cutting the protruding point at the top of one end of the young embryo of the concave surface of the seeds by using an inoculating blade, obliquely downwards extruding the young embryo by using a knife tip, downwards pouring one end of the incision of the young embryo on a callus induction culture medium, uniformly filling each culture dish, and sealing by using a sealing film; culturing in incubator at 25deg.C and humidity of 30%, and culturing in dark for 15 days to induce callus; the callus induction medium is: MS+1.0mg/L6-BA+0.1mg/LKT+0.1mg/L2,4-D+30g/L sucrose, pH5.8; all tool pieces and culture medium were autoclaved;
s5, selecting embryogenic callus which is uniform in growth vigor, loose in structure, compact in particles and milky white to carry out mutagenesis; filtering and sterilizing the mutagen solution on an ultra-clean bench, then filling the mutagen solution into a brown volumetric flask, and fixing the volume of sterile water to the required concentration; preparing a callus subculture medium, adding a mutagen solution when the temperature of the culture medium is cooled to 30 ℃, uniformly mixing, and pouring into a plate; after the culture medium is solidified, lightly inoculating the selected callus incision ends downwards on the culture medium, inoculating 10 callus incision ends on each culture dish, sealing a sealing film of the culture dish, placing the culture dish in an incubator for dark culture for 7 days, and setting the temperature to 25 ℃ and the humidity to 30%; the mutagen is a mixed solution of colchicine with the mass fraction of 0.05% and dimethyl sulfoxide with the mass fraction of 0.1%, when the mixed solution is prepared, a little 95% ethanol is used for dissolving the colchicine and the dimethyl sulfoxide, filtering and sterilizing are carried out on an ultra-clean bench, and then sterile water is used for fixing the volume to the required concentration. Callus subculture medium: MS+1.0mg/L6-BA+0.1mg/L NAA+30g/L sucrose, pH5.8;
s6, inducing bud differentiation: selecting embryogenic callus which is dark-cultured for 7 days, transferring the embryogenic callus into an induced bud differentiation culture medium, and differentiating cluster buds in 22 days at the temperature of 25 ℃ and the humidity of 40% under the illumination intensity of 1000lx and the illumination time of 8/16 h; inducing cluster bud differentiation culture medium: MS+2.0mg/L6-BA+0.5mg/LKT+0.1mg/LNAA+20g/L sucrose, pH5.8-6.0
S7, single bud subculture: and (3) selecting cluster buds which are uniform in differentiation, have bud length of 0.5cm and are light green in bud heads, cutting the cluster buds according to single buds, reserving partial callus at the base of each single bud, and transferring the cluster buds into a subculture medium. Setting the illumination intensity to 1000lx, the illumination time to 8/16h, the temperature to 25 ℃, the humidity to 40%, and the root primordia can be differentiated after 10 days of single bud basal callus; subculture medium: MS+0.5mg/L6-BA+0.5mg/LNAA+20g/L sucrose, pH5.8;
s8, transferring the single buds inducing root primordia into an induced seedling differentiation medium, wherein the illumination intensity is 1500lx, the illumination time is 12/12h, the temperature is 25 ℃, the humidity is 40%, and the seedlings can be differentiated after 4 weeks of culture; inducing seedling differentiation culture medium: 1/2MS+1.0mg/LKT+0.2mg/L6-BA+1.0mg/LNAA+20g/L sucrose, pH5.8;
s9, after strengthening and hardening the tissue culture seedlings, culturing the tissue culture seedlings in a water culture room by utilizing a field planting basket mist culture technology, and harvesting the stock seeds about 2 months
Example 2
When the inducing material is immature embryo of immature seed, the inducing method comprises the following steps:
s1, selecting 4-year-old flowering konjak bulbs, and selecting young embryos from the late 6 months to the early July, wherein the ears are full, the pericarps are green and the embryos are tender, so that callus induction is facilitated;
s2, drying or airing the collected seed in the shade at a ventilation position, peeling off a layer of pericarp outside the seed coat, and airing the surface of the seed coat at a shade position;
s3, cleaning the surface of the seed coat under running water, soaking the seed coat in a sodium hypochlorite solution with the concentration of 0.15% on an ultra-clean bench for 10min, taking out the seed coat, and cleaning the seed coat with sterile water for 5 times; soaking in 70% alcohol for 30s, taking out, cleaning with sterile water for 3 times, and finally sucking water on the surface of seed coat with sterile filter paper for use;
s4, clamping the seeds on the culture dishes by using tweezers, slightly cutting the protruding point at the top of one end of the young embryo of the concave surface of the seeds by using an inoculating blade, obliquely downwards extruding the young embryo by using a knife tip, downwards pouring one end of the incision of the young embryo on a callus induction culture medium, uniformly filling each culture dish, and sealing by using a sealing film; culturing in incubator at 28deg.C and humidity of 40%, and culturing in dark for 18 days to induce callus; the callus induction medium is: MS+2.0mg/L6-BA+0.2mg/LKT+0.2mg/L2,4-D+30g/L sucrose, pH6.0; all tool pieces and culture medium were autoclaved;
s5, selecting embryogenic callus which is uniform in growth vigor, loose in structure, compact in particles and milky white to perform mutagenesis, wherein the mutagenesis result is shown in figure 1; filtering and sterilizing the mutagen solution on an ultra-clean bench, then filling the mutagen solution into a brown volumetric flask, and fixing the volume of sterile water to the required concentration; preparing a callus subculture medium, adding a mutagen solution when the temperature of the culture medium is cooled to 40 ℃, uniformly mixing, and pouring into a plate; after the culture medium is solidified, lightly inoculating the selected callus incision ends downwards on the culture medium, inoculating 10 callus incision ends on each culture dish, sealing a sealing film of the culture dish, placing the culture dish in an incubator for dark culture for 10 days, and setting the temperature to be 28 ℃ and the humidity to be 40%; the mutagen is a mixed solution of colchicine with the mass fraction of 0.1% and dimethyl sulfoxide with the mass fraction of 0.2%, when the mixed solution is prepared, a little 95% ethanol is used for dissolving the colchicine and the dimethyl sulfoxide, filtering and sterilizing are carried out on an ultra-clean bench, and then sterile water is used for fixing the volume to the required concentration. Callus subculture medium: MS+0.1mg/LKT+0.2mg/L NAA+30g/L sucrose, pH6.0;
s6, inducing bud differentiation: selecting embryogenic callus which is dark-cultured for 10 days, transferring the embryogenic callus into an induced bud differentiation culture medium, and differentiating cluster buds in 25 days at the temperature of 28 ℃ and the humidity of 60% under the illumination intensity of 1200lx and the illumination time of 8/16 h; inducing cluster bud differentiation culture medium: MS+4.0mg/L6-BA+1.0mg/LKT+0.2mg/LNAA+20g/L sucrose, pH6.0;
s7, single bud subculture: and (3) selecting cluster buds which are uniform in differentiation, 1cm in bud length and light green in bud heads, cutting the cluster buds according to single buds, reserving partial calli at the base of each single bud, and transferring the cluster buds into a subculture medium. Setting the illumination intensity to 1200lx, and the illumination time to 8/16h, wherein the temperature is 28 ℃, the humidity is 60%, and the root primordia can be differentiated after the single bud basal callus is healed in 15 days; subculture medium: MS+1.0mg/L6-BA+1.0mg/LNAA+20g/L sucrose, pH6.0;
s8, transferring the single buds inducing root primordia into an induced seedling differentiation medium, wherein the illumination intensity is 2000lx, the illumination time is 12/12h, the temperature is 28 ℃, the humidity is 60%, and the seedlings can be differentiated after culturing for 5 weeks; inducing seedling differentiation culture medium: 1/2MS+2.0mg/LKT+0.5mg/L6-BA+1.5mg/LNAA+20g/L sucrose, pH6.0;
s9, after strengthening and hardening the tissue culture seedlings, culturing the tissue culture seedlings in a water culture room by utilizing a field planting basket mist culture technology, and harvesting the stock seeds about 2 months.
Example 3
When the induction material is the embryo of mature seed, the specific induction method comprises the following steps:
s1, selecting 3-4-year-old flowering konjak corms, and selecting germs in the middle and late 9-10 middle and upper ten days after artificial pollination, wherein the ears are mature, the pericarps are orange-red, and the germs are developed and mature, so that germ induction is facilitated.
S2, after the harvested seed is dried in a shade and dry place, peeling off a layer of pericarp outside the seed coat, cleaning the surface of the seed coat under running water, and then sun-drying the surface of the seed coat under the sun (under the condition of being not suitable for insolation and good weather, sun-drying at 9-11 am).
S3, preparing a mixed solution of 10mg/LKT+500mg/L GA3+5 mg/L2,4-D, placing the mixed solution into a glass jar of a frosted bottle stopper with a brown color tape, selecting solid seeds which are uniform in size, full in seeds, black and brown in seed coats and glossy, placing the solid seeds into a mesh bag, soaking the solid seeds in the solution, shaking the solid seeds for 24 hours at a shaking table 120r/min in a dark place at a room temperature of 16 ℃, taking out the solid seeds, washing the solid seeds with running water for 5 times, and airing the solid seeds to break dormancy. This step aims at breaking seed dormancy.
S4, soaking the seed coats in a sodium hypochlorite solution with the concentration of 0.1% on an ultra-clean bench for 20min, taking out, cleaning with sterile water for 4 times, soaking in 70% alcohol for 1min, taking out, cleaning with sterile water for 3 times, and finally sucking water on the surfaces of the seed coats with sterile filter paper for later use.
S5, spreading a breathable cotton net at the bottom of a culture dish, uniformly wetting the cotton net with sterile water (the cotton net is not excessively wet, so that seeds are in a semi-soaked state and easy to crack and rot at embryo ends of the seeds), uniformly placing the seeds on the cotton net by forceps, wherein the embryo ends are obliquely placed in the cotton net downwards, covering a cover, sealing by a sealing film, performing dark culture in an illumination incubator, setting the temperature to 25 ℃ and the humidity to 60%, keeping the cotton net in a wet state, and exposing the seeds after 4 days.
The used instruments, the cotton net, the water and the like are all subjected to high-pressure sterilization treatment so as to prevent the seeds from mildewing and rotting in the germination accelerating process.
S6, taking out the exposed seeds, cleaning the seeds with sterile water for 4 times, removing the slimy surface of the seed coats, and then airing the seeds on sterile filter paper (the exposed bud points cannot be touched or damaged in the cleaning process); the whole process is carried out in a dark environment by adopting a mixed culture-titration-wrapping method, namely agar is added into deionized water, the temperature is cooled to 30 ℃ after high-pressure sterilization, a filtered degerming mutagen is added, the mixture is uniformly mixed, then, the seeds are clamped by forceps, the bud points face upwards, a small amount of agar is picked by a small concave spoon for inoculation, the agar is dripped on the bud points, and the whole bud point positions are wrapped after the agar is solidified. Finally, placing the mixture in a culture dish with sterile absorbent cotton paved at the bottom, inoculating 20 grains per culture dish, sealing by a sealing film, placing the culture dish in an incubator for dark culture, and setting the temperature to 25 ℃ and the humidity to 50%. Thus, the agar-mutagen surrounding the bud is titrated once per day at 8 am for 5-7 days.
The mutagen is a mixed solution of 0.005% by mass and 0.01mol/L by mass of trifluralin and pendimethalin; when the mixed solution is prepared, a little 95% ethanol is used for dissolving the trifluralin and the pendimethalin, the solution is filtered and sterilized on an ultra-clean bench, and then sterile water is used for fixing the volume to the required concentration, and the solution is preserved in a dark place for later use;
s7, selecting the real seeds which grow or survive from the germs or the bud sheaths after the mutagenesis culture, obliquely downwards picking one end of the germs on an ultra-clean bench by using the tip of an inoculating knife, cutting off endosperm carried on the base part of the picked germs, and transferring the germs into a subculture medium for inducing the growth and differentiation of the buds. The illumination intensity is 1500lx, the illumination time is 8/16h, the temperature is 25 ℃, the humidity is 40%, and the seedlings can be differentiated after 2 weeks of culture. Subculture medium: MS+1.0mg/LKT+0.2mg/L6-BA+1.0mg/LNAA+20g/L sucrose, pH5.8. The differentiated seedlings are shown in FIG. 2.
S8, after hardening seedlings, culturing the seedlings in a water culture room by utilizing a field planting basket mist culture technology, and harvesting stock seeds about 2 months
Example 4
When the induction material is the embryo of mature seed, the specific induction method comprises the following steps:
s1, selecting 4-year-old flowering konjak corms, and selecting germs in the middle and late 9-10 middle and upper ten days after artificial pollination, wherein the ears are mature, the pericarps are orange-red, and the germs are developed and mature, so that germ induction is facilitated.
S2, after the harvested seed is dried in a shade and dry place, peeling off a layer of pericarp outside the seed coat, cleaning the surface of the seed coat under flowing water, and then sun-drying the surface of the seed coat under the sun, wherein under the condition of being not suitable for insolation and good weather, sun-drying is carried out at 9-11 am.
S3, preparing a mixed solution of 20mg/LKT+800mg/L GA3+10 mg/L2,4-D, placing the mixed solution into a glass jar of a frosted bottle stopper with a brown color tape, selecting solid seeds which are uniform in size, full in seeds, black and brown in seed coats and glossy, placing the solid seeds into a mesh bag, soaking the solid seeds in the solution, shaking the solid seeds for 24 hours at a shaking table 120r/min in a dark place, taking out the solid seeds, washing the solid seeds with flowing water for 6 times at the temperature of between 16 and 20 ℃, and airing the solid seeds to break dormancy. This step aims at breaking seed dormancy.
S4, soaking the seed coats in a sodium hypochlorite solution with the concentration of 0.15% on an ultra-clean bench for 25min, taking out, cleaning with sterile water for 5 times, soaking in 70% alcohol for 1min, taking out, cleaning with sterile water for 3 times, and finally sucking water on the surfaces of the seed coats with sterile filter paper for later use.
S5, spreading a breathable cotton net at the bottom of a culture dish, uniformly wetting the cotton net with sterile water (the cotton net is not excessively wet, so that seeds are in a semi-soaked state and easy to crack and rot at embryo ends of the seeds), uniformly placing the seeds on the cotton net by forceps, wherein the embryo ends are obliquely placed in the cotton net downwards, covering a cover, sealing by a sealing film, performing dark culture in an illumination incubator, setting the temperature to be 28 ℃ and the humidity to be 70%, keeping the cotton net in a wet state during the period, and exposing the seeds to white after 5 days.
The used instruments, the cotton net, the water and the like are all subjected to high-pressure sterilization treatment so as to prevent the seeds from mildewing and rotting in the germination accelerating process.
S6, taking out the exposed seeds, cleaning the seeds with sterile water for 5 times, removing the slimy surface of the seed coats, and then airing the seeds on sterile filter paper (in the cleaning process, exposed bud points cannot be touched or damaged); the whole process is carried out in a dark environment by adopting a mixed culture-titration-wrapping method, namely agar is added into deionized water, the temperature is cooled to 40 ℃ after high-pressure sterilization, a filtered degerming mutagen is added, the mixture is uniformly mixed, then the seeds are clamped by forceps, the bud points face upwards, a small amount of agar is picked by a small concave spoon for inoculation, the agar is dripped on the bud points, and the whole bud point positions are wrapped after the agar is solidified. Finally, placing the mixture in a culture dish with sterile absorbent cotton paved at the bottom, inoculating 20 grains per culture dish, sealing by a sealing film, placing the culture dish in an incubator for dark culture, and setting the temperature to 28 ℃ and the humidity to 50% -60%. Thus, the agar-mutagen surrounding the bud was titrated once per day at 8 am for 7 days.
The mutagen is a mixed solution of the amisulpride with the mass fraction of 0.008 percent and the pendimethalin with the mass fraction of 0.03 mol/L; when the mixed solution is prepared, a little 95% ethanol is used for dissolving the trifluralin and the pendimethalin, the solution is filtered and sterilized on an ultra-clean bench, and then sterile water is used for fixing the volume to the required concentration, and the solution is preserved in a dark place for later use;
s7, selecting the real seeds which grow or survive from the germs or the bud sheaths after the mutagenesis culture, obliquely downwards picking one end of the germs on an ultra-clean bench by using the tip of an inoculating knife, cutting off endosperm carried on the base part of the picked germs, and transferring the germs into a subculture medium for inducing the growth and differentiation of the buds. The light intensity is 2000lx, the light time is 8/16h, the temperature is 28 ℃, the humidity is 60%, and the seedlings can be differentiated after 3 weeks of culture. Subculture medium: MS+2.0mg/LKT+0.5mg/L6-BA+1.5mg/LNAA+20g/L sucrose, pH6.0.
S8, after hardening seedlings, culturing the seedlings in a water culture room by utilizing a field planting basket mist culture technology, and harvesting stock seeds about 2 months.
As shown in FIG. 3, polyploid plants induced by young embryo callus are shown in FIG. 3 (A) with multiple main branches, few branches, big leaves, compact plants and the like, FIG. 3 (B) with multiple leaves, big leaves, few main branches and the like, FIG. 3 (C) with big leaves, compact plants, side branches and the like, FIG. 3 (D) with big leaves, dwarfing and the like, FIG. 3 (E) with big leaves, multiple branches and compact plants and the like, and FIG. 3 (F) with slender leaf shape, side branches and loose plant types.
As shown in fig. 4, in polyploid plants induced by embryo, fig. 4 (a) shows multi-branched, multi-lobular and other traits, fig. 4 (B) shows multi-branched, large-lobular, long-elliptic and other traits of main branches, fig. 4 (C) shows multi-branched, large-lobular, loose plant and large She Xiuchang and other traits, fig. 4 (D) shows thicker and coarser multi-branched, large-lobular and leaves and other traits, fig. 4 (E) shows multi-branched, multi-branched and other traits of main branches, fig. 4 (F) shows dwarfed, thickened leaves and darkened color and other traits.
In summary, konjak polyploids are successfully induced by using konjak induction materials at different positions and at different periods.
In order to verify the induction effect of the rapid induction method of konjak polyploid provided by the invention, the following test is carried out:
1. the young embryo in example 1 was replaced with bulb cutting, taro penis cutting, petiole cutting, seed endosperm and anther, and the procedure was carried out in the same manner as in example 1 to obtain seedlings, and the statistical induction rate was calculated, and the results are shown in Table 1.
2. The same procedure as in example 3 was carried out to obtain seedlings by replacing germs in example 3 with corm top buds, corm adventitious buds, taro whip top buds, taro whip adventitious buds and tissue culture cluster buds, respectively, and the results are shown in Table 2.
3. The same procedure as in example 3 was carried out except that the mixed culture-titration-wrapping method in example 3 was replaced with the dipping method, the dropping method, the wrapping method, the injection method and the injection method, respectively, to obtain seedlings, and the statistical induction rate was calculated, and the results are shown in Table 3.
The problem of chimeras is a hurdle in polyploid breeding, chimeras require repeated cutting culture and are difficult to purify, so that a larger homozygote/chimera ratio indicates a more efficient induction method. As shown in Table 1, polyploid induction of callus obtained by in vitro culture of young embryo of konjak seed is significantly different from that of callus induced by konjak bulb, konjak whip, leaf stalk, endosperm of seedling seed and anther in terms of ease of embryogenic callus induction, length of induction time, homozygote/chimera ratio, etc. The ratio of homozygote/chimera induced in example 1 was 29.77% as high as the sum of the overall induction rate and homozygote induction rate.
As shown in Table 2, polyploid induction by using mature konjak seed germ is highest in the overall induction rate, homozygote/chimera ratio, etc., as compared with konjak corm top bud, corm adventitious bud, konjak whip top bud, konjak whip adventitious bud, and konjak tissue culture cluster bud.
As shown in Table 3, the use of the culture-titration-wrapping method for germ mutagenesis showed the best results in terms of controlling the browning mortality, contamination rate, overall induction rate, homozygote/chimera ratio, etc., as compared with the dipping method, direct dripping method, foreign matter wrapping method, injection method, spraying method, etc.
TABLE 1 comparison of polyploid inductances of different types of calli
TABLE 2 comparison of the induction rates of different types of bud polyploids
TABLE 3 comparison of different induction methods for embryo
It should be noted that, when the claims refer to numerical ranges, it should be understood that two endpoints of each numerical range and any numerical value between the two endpoints are optional, and the present invention describes the preferred embodiments for preventing redundancy.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (7)
1. The rapid induction method of konjak polyploid is characterized by comprising the following steps:
s1, selecting seeds of different periods as an induction material, and cleaning and sterilizing the induction material;
s2, performing primary culture on the treated induction material to obtain a primary culture;
s3, using a mutagen to mutagenize the primary culture to obtain a mutagenized product;
s4, carrying out second generation culture on the mutation product to obtain seedlings;
s5, obtaining stock seeds after seedling culture;
the real seeds in different periods are immature real seeds or mature real seeds;
when the immature seeds are subjected to primary culture, the primary culture is callus, and when the mature seeds are subjected to primary culture, the primary culture is germinated seeds;
when the induction material is immature seeds, the specific induction method comprises the following steps:
s1, carrying out primary culture after cleaning and sterilizing treatment on the seeds;
the primary culture is as follows: inoculating young embryo of the seedling seed on a callus induction culture medium for culturing to obtain callus;
the callus induction culture medium takes MS as a basic culture medium, and 1.0-2.0mg of 6-BA, 0.1-0.2mg of KT, 0.1-0.2mg of 2,4-D and 30g of sucrose are added into each liter of MS culture medium;
s2, adding a mutagen into the callus subculture medium, uniformly mixing to obtain a mutagenesis medium, and inoculating the callus on the mutagenesis medium for mutagenesis to obtain a mutagenesis product;
the callus subculture medium takes MS as a basic culture medium, and 0.5-1.0mg of 6-BA or 0.1-0.2mg of KT, 0.1-0.2mg of NAA and 30g of sucrose are added into each liter of MS culture medium;
the mutagen is a mixed solution of colchicine with the mass fraction of 0.05-0.1% and dimethyl sulfoxide solution with the mass fraction of 0.1-0.2%;
s3, carrying out second generation culture on the mutation product, wherein the second generation culture comprises the steps of treating the mutation product to obtain clustered buds and treating the clustered buds to obtain seedlings;
the specific process of the second generation culture is as follows:
s1, inoculating a mutagenesis product into an induced bud differentiation culture medium, and culturing to obtain cluster buds;
the bud differentiation induction culture medium takes MS as a basic culture medium, and 2.0-4.0mg of 6-BA, 0.5-1.0mg of KT, 0.1-0.2mg of NAA and 20g of sucrose are added into each liter of MS, wherein the pH value of the bud differentiation induction culture medium is 5.8-6.0;
s2, inoculating the cluster buds into a secondary culture medium to induce root primordia;
the secondary culture medium takes MS as a basic culture medium, 0.5-1.0mg of 6-BA, 0.5-1.0mg of NAA and 20g of sucrose are added into each liter of MS, and the pH value of the secondary culture medium is 5.8-6.0;
s3, transferring the single buds inducing root primordia into an induced seedling differentiation medium to differentiate seedlings;
the seedling differentiation induction culture medium takes 1/2MS as a basic culture medium, 1.0-2.0mg KT, 0.2-0.5mg 6-BA, 1.0-1.5mg NAA and 20g sucrose are added into each liter of 1/2MS, and the pH value of the seedling differentiation induction culture medium is 5.8-6.0;
when the induction material is mature seeds, the specific induction method comprises the following steps:
s1, cleaning the seeds, breaking dormancy, sterilizing and primary culturing to obtain seeds after germination acceleration;
the break dormancy: soaking the seeds in a mixed solution containing 10-20mg KT, 500-800mg GA3 and 5-10mg 2,4-D in each liter of solution for a certain period of time, taking out, cleaning and airing;
the primary culture: accelerating germination of seeds under the conditions of 25-28 ℃ and 60-70% humidity and wetting;
s2, using a mutagen to mutagenize the seeds after germination to obtain a mutagenic product;
the mutagen is a mixed solution of 0.005-0.008% of amisulpride and 0.01-0.03mol/L of pendimethalin;
s3, carrying out second generation culture on the mutation product, namely inoculating the mutation product into a subculture medium to induce bud growth and differentiation, and culturing to obtain seedlings;
the secondary culture medium takes MS as a basic culture medium, and 1.0-2.0mg KT, 0.2-0.5mg 6-BA, 1.0-1.5mg NAA and 20g sucrose are added into each liter of MS.
2. The rapid induction method of konjak polyploidy according to claim 1, wherein when the induction material is immature seeds, the conditions of the primary culture in S1 are: the temperature is 25-28 ℃, the humidity is 30-40%, and the dark culture is carried out for 15-18 days; in S2, the condition of mutagenesis is dark culture for 7-10 days, the temperature is 25-28 ℃, and the humidity is 30% -40%.
3. The rapid induction method of konjak polyploidy according to claim 1, wherein when the induction material is immature seeds, the pH of the callus induction medium and the pH of the callus subculture medium are both 5.8-6.0.
4. The rapid induction method of konjak polyploidy according to claim 1, wherein when the induction material is immature seeds, the specific process of the second generation culture is that the culture conditions in S1 are: the illumination intensity is 1000-1200lx, the illumination time is 8/16h, the temperature is 25-28 ℃, and the humidity is 40% -60%; the induction conditions in S2 are: the illumination intensity is 1000-1200lx, the illumination time is 8/16h, the temperature is 25-28 ℃, and the humidity is 40% -60%; the conditions for differentiation in S3 were: the illumination intensity is 1500-2000lx, the illumination time is 12/12h, the temperature is 25-28 ℃, the humidity is 40-60%, and the culture is carried out for 4-5 weeks.
5. The rapid induction method of konjak polyploidy according to claim 1, wherein when the induction material is mature seed, the mutagenesis culture is performed by a mixed culture-titration-encapsulation method in S2.
6. The rapid induction method of konjak polyploid according to claim 1, wherein when the induction material is mature seed, the condition of mutagenesis in S2 is that the temperature is 25-28 ℃ and the humidity is 50% -60%; in S3, the conditions of the second generation culture are as follows: the illumination intensity is 1500-2000lx, the illumination time is 8/16h, the temperature is 25-28 ℃, and the humidity is 40-60%.
7. The rapid induction method of konjak polyploidy according to claim 1, wherein the pH of the secondary culture medium is 5.8-6.0 when the induction material is mature solid seeds.
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