CN113575417B - Method for in vitro rapid propagation of gorgon fruit - Google Patents
Method for in vitro rapid propagation of gorgon fruit Download PDFInfo
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Abstract
The invention relates to a method for quickly propagating gordon euryale seeds in vitro, firstly, selecting seeds, selecting newly harvested gordon euryale seeds in the current year; performing seed shell treatment, then performing explant treatment, then performing seed germination acceleration culture, then establishing a gordon euryale seed sterile rapid propagation system, and finally performing tissue culture and rooting seedling domestication and transplantation on the gordon euryale seeds; timely breaking the dormancy of the gordon euryale seeds, removing seed shells and exoendosperm of the seeds, and accelerating germination of embryo by using a tissue culture technology to obtain gordon euryale seeds; completely establishing a gordon euryale seed sterile rapid propagation system from the initial generation of seeds to the transplantation of tissue culture rooting seedlings; the dormancy of the gordon euryale seeds can be broken at any time all the year round for the first time, and the restriction of the seasons on the research, development and production of the gordon euryale seeds is thoroughly broken through.
Description
Technical Field
The invention relates to a gorgon fruit in-vitro rapid propagation method.
Background
Gorgon fruit (Euryale ferox Salisb.) belongs to Nymphaeaceae family, genus Euryale, and is also called gorgon fruit, cockscomb, gorgon fruit, etc., and is mostly distributed in lakes, ponds and beaches in China and southeast Asia. One of the gorgon euryale seed named as Suzhou water eight immortals is a high-quality genuine medicinal material in Suzhou.
Gordon euryale seed contains a large amount of protein, calcium, phosphorus, iron, fat, starch, vitamin B1, vitamin B2, vitamin C, crude fiber, carotene, amino acid and the like, and contains up to 18 kinds of amino acid, wherein threonine, phenylalanine, lysine and the like are necessary amino acid for human body, so that the gordon euryale seed has high nutritional value and has the food therapy effects of nourishing blood, soothing nerves, tonifying kidney, securing essence, eliminating dampness, tonifying spleen, stopping diarrhea, stopping leukorrhagia and the like. The gorgon fruit can be used as both medicine and food, contains various functional components such as sterol compounds, flavonoid compounds, cyclic peptide compounds, sesqui-neolignanoid compounds, cerebroside compounds, phenolic compounds and the like, has pharmacological effects of resisting oxidation, resisting fatigue, reducing blood sugar, delaying senility, resisting cancer and the like, and is often applied to clinically treating diseases such as early diabetic kidney diseases, chyluria, stroke sequelae, chronic enteritis and the like. As the gorgon fruit can play a positive role in food therapy and health care, the gorgon fruit is more and more accepted and favored by people.
The gordon euryale seeds are divided into the gordon euryale seeds and the gordon euryale seeds, the gordon euryale seeds are cultivated by domestication, have few thorns, large seeds, large rice kernels, glutinous nature, good quality, high yield and high economic benefit, are precious high-quality resources in Suzhou, and are also widely cultivated, the gordon euryale seeds mainly comprise the traditional local varieties of the gordon euryale seeds and the gorgon euryale seeds, and the hybrid breeding varieties comprise the gordon euryale seeds of safflower, the gordon euryale seeds of No. 1, the gordon euryale seeds of No. 2, the gordon euryale seeds of No. 3, the gordon euryale seeds of No. 4 and the like. From 8 to 10 months of each year, the fresh semen euryales seeds are not in demand, and the selling price is 100-200 Yuan/jin. The breeding and development prospect of the new gordon euryale seed variety is more and more extensive.
The gordon euryale seeds are propagated by seeds, but the seeds can finish after-ripening only after a period of dormancy after being ripe, and can germinate only in spring of the next year after being stored under natural conditions. Experimental study by vermilion et al: concentrated H 2 SO 4 The dormancy of the gordon euryale seeds cannot be rapidly released by methods such as treatment, mechanical treatment, hormone treatment and the like. The physiological after-ripening of the gordon euryale seeds still needs a certain time after the gordon euryale seeds are separated from the matrix, the dormancy is an after-ripening dormancy, the low-temperature treatment can promote the release of the after-ripening dormancy, and the low-temperature treatment possibly can be a biological adaptability to environmental conditions and seasonal changes obtained by the long-term evolution of the gordon euryale seeds. plum-Yanlian et al studied the tissue culture and rapid propagation technique of gordon euryale seed, but did not mention the problem of breaking seed dormancy, and did not completely realize tissue culture, seedling growth and transplantation.
The seed shell of the Suqian is thick and uneven in thickness, difficult to process and low in meat yield, and completely depends on manual shelling, so that new products must be continuously pushed to completely meet the requirements of agricultural efficiency improvement and income increase of farmers. At present, the breeding of new gorgon fruit varieties completely depends on natural mutagenesis and crossbreeding, the character of the gorgon fruit offspring obtained by the method is extremely unstable, the workload and the difficulty of breeding workers are greatly increased, however, the gorgon fruit in-vitro regeneration optimization system can be widely applied to rapid seedling propagation and genetic transformation, a rapid and effective method is provided for the resource preservation of gorgon fruit seeds, the dormancy of the gorgon fruit seeds is broken, the restriction of seasons on the research and development and production of the gorgon fruit is broken, and the market needs are met.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a gorgon fruit in-vitro rapid propagation method.
The purpose of the invention is realized by the following technical scheme:
a gorgon fruit in vitro rapid propagation method is characterized in that: the method comprises the following steps:
1) selecting seeds, namely selecting newly harvested gordon euryale seeds in the current year, soaking the seeds in pure water, sealing and storing the seeds in an environment at 0-5 ℃, cleaning once and replacing the pure water within 3-5 days, and taking out the seeds for use after 1 month;
2) seed shell treatment, namely peeling off the seed shells of the selected seeds, and reserving complete seed embryos, endosperm and a transparent protective film on the surface; the peeled seed with the whole seed shell removed is soaked in sterile water, and the environmental temperature is 18-25 ℃;
3) explant treatment, continuous washing of the whole seed without seed shell with water, then displacement of a superclean workbench, and the disinfection sequence is as follows in sequence: soaking and sterilizing for 30 s-1 min by 75% alcohol, washing for 3-5 times by sterile water, and then using 0.1% HgCl 2 Soaking and disinfecting the solution for 8-12 min, washing the tissue culture bottle for 3-5 times by using sterile water, continuously shaking the tissue culture bottle during the washing, and finally sucking surface water by using sterile filter paper;
4) accelerating germination of seeds, wherein the surface of the sterilized de-shelled complete seeds is free of moisture and the internal moisture is not less than 50%, the seeds are taken off on an ultra-clean workbench by using a scalpel and a forceps, the seeds with the diameter of more than 2mm are selected and immediately inoculated into a germination culture medium, the germination culture medium is an MS basic culture medium, 1.0-2.0 mg/L, NAA0.1 of 6-BA, 0.1-0.2 mg/L of sucrose, 7-7.5 g/L of agar and 20g/L of ground endosperm, and the pH value is 5.8-6.0; placing the inoculated gordon euryale seed embryo under illumination for culturing, wherein the culture conditions are as follows: the temperature is 25 +/-1 ℃, and the illumination intensity is 30-100 mu mol.m -2 ·s -1 The illumination time is 12-14 h/d;
5) establishing a gordon euryale seed sterile rapid propagation system, continuing to perform germination induction culture, transplanting into a secondary rooting culture medium when the seed embryo germinates to 2cm, wherein the culture formula is 1/2MS basic culture medium, and adding 0.2mg/L NAA, 20-30 g/L sucrose, 7-7.5 g/L agar and 5% AC activated carbon; after the aseptic seedling takes root, 5ml of aseptic water is injected into the culture bottle in a super clean bench to promote the growth of the rooted seedling;
6) domesticating and transplanting the gorgon fruit tissue culture rooted seedlings, and selecting the rooted tissue culture seedlings with more than 3 leaves, more than 3cm plant height and robust root systems in the continuous culture process; the cover is opened in the culture chamber, sterile water is added, and acclimatization culture is continued.
Further, in the method for in-vitro rapid propagation of the gordon euryale seeds, step 1), the gordon euryale seeds are sequentially divided into six grades of yellow, big shoulder, small flower coats, embryo peeling, big sonorous and old seeds according to the grade of tender to old maturity, and the small flower coats, the embryo peeling, the big sonorous and the old seeds are selected, and the full and complete seeds with the diameter of more than 1.0cm are obtained.
Further, the method for the in vitro rapid propagation of the gordon euryale seeds comprises the step 2), the peeled whole seeds without the seed shells are soaked in sterile water for no more than 24 hours.
Further, the method for the isolated rapid propagation of the gorgon fruit comprises the step 3) of continuously washing the seed-coat-removed complete seeds for 2-6 hours by using water.
Further, the method for the isolated rapid propagation of the gorgon fruit comprises the step 4) of timely cleaning polluted plant materials during the germination accelerating culture period.
Further, the method for the in vitro rapid propagation of the gorgon fruit comprises the step 6), after 10-20 days of acclimatization culture, transferring to a transplanting water area, then acclimatizing culture for more than two weeks, and then planting.
Compared with the prior art, the invention has obvious advantages and beneficial effects, and is embodied in the following aspects:
the method breaks the dormancy of the gordon euryale seeds in time, removes seed shells and exoendosperm of the seeds, and accelerates germination of embryo by using a tissue culture technology so as to obtain gordon euryale seed seedlings; completely establishing a gordon euryale seed sterile rapid propagation system from the initial generation of seeds to the transplantation of tissue culture rooting seedlings; the dormancy of the gorgon fruit seeds can be broken at any time all year round for the first time, and the restriction of seasons on the research and development and production of the gorgon fruit is thoroughly broken;
secondly, establishing a gorgon fruit sterile rapid propagation system, realizing annual seedling raising of gorgon fruit and providing a foundation for gorgon fruit breeding research; because of the own biological characteristics of the gorgon fruit, seedling raising can only be completed once a year under natural conditions, and the characters of the seeds cannot be identified efficiently by traditional means such as cross breeding, mutation breeding and the like, but the method can be applied to the preliminary identification of new varieties, so that the breeding efficiency is greatly improved, and the breeding period is shortened; the euryale ferox is still blank in the field of transgenic breeding, and the aseptic seedling of the euryale ferox in the method is the most appropriate receptor in the transgenic breeding, so that a solid foundation is provided for the research on the breeding of the euryale ferox;
Thirdly, a rapid and effective method is provided for gorgon fruit germplasm resource preservation, the dormancy of gorgon fruit seeds is broken, the restriction of seasons on the research and development and production of gorgon fruit is broken, a large number of gorgon fruit seedlings with the same character are rapidly propagated, for the breeding of new gorgon fruit varieties, offspring generated through cross breeding are rapidly propagated through the method, excellent characters are perfectly copied, and the breeding period can be greatly shortened;
fourthly, for physical mutagenesis, chemical mutagenesis, polyploid breeding and transgenic breeding, the method can rapidly propagate offspring, and the aseptic seedling of the gordon euryale seed is the most suitable receptor; the method can be used as a necessary means for traditional breeding and transgenic breeding.
Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by the practice of the invention. The objectives and other advantages of the invention may be realized and attained by the structure particularly pointed out in the written description.
Detailed Description
In order to more clearly understand the technical features, objects, and effects of the present invention, specific embodiments will now be described in detail.
1) Selecting seeds, namely selecting newly harvested gordon euryale seeds in the current year, sequentially dividing the gordon euryale seeds into six grades of yolk, big shoulder, small flower coat, embryo peeling, big sound and old grains according to the biological uniform regulation, selecting full and complete seeds with the diameters of more than 1.0cm, soaking the seeds in pure water, sealing and storing in an environment at 0-5 ℃, cleaning once in 3-5 days, replacing the pure water, and taking out for use after 1 month;
2) Processing seed shells, namely carefully peeling off the hard seed shells by using a shelling finger protector for selected seeds, and reserving complete seed embryos, endosperm and a protective film with a transparent surface; the peeled seed with the whole seed shell removed is soaked in sterile water, the environment temperature is 18-25 ℃, and the soaking time is not more than 24 hours;
3) treating explants, wherein hard seed shells and thick endosperm are one of main reasons for gordon euryale seed dormancy, continuously washing the whole seed without seed shells for 2-6 hours with water, and then moving the clean bench to remove the seedThe toxic sequence is as follows: soaking and sterilizing for 30 s-1 min by 75% alcohol, washing for 3-5 times by sterile water, and then using 0.1% HgCl 2 Soaking and disinfecting the solution for 8-12 min, washing the tissue culture bottle for 3-5 times by using sterile water, continuously shaking the tissue culture bottle during the washing, and finally sucking surface water by using sterile filter paper;
4) accelerating germination of seeds, wherein the surface of the sterilized de-shelled intact seeds is free of water and the internal water content is not less than 50%, the seeds are taken down on an ultra-clean workbench by using a scalpel and a forceps, the diameter is more than 2mm, the seeds are directly discarded when the seeds do not reach the standard in size, and the seeds are immediately inoculated into a germination culture medium, wherein the germination culture medium is an MS basic culture medium (containing major elements, trace elements, iron salts and organic components), 6-BA (6-benzylaminopurine) 1.0-2.0 mg/L, NAA (naphthylacetic acid) 0.1-0.2 mg/L, sucrose 20-30 g/L, agar 7-7.5 g/L and ground endosperm 20g/L, and the pH is 5.8-6.0; placing the inoculated gordon euryale seed embryo under illumination for culturing, wherein the culture conditions are as follows: the temperature is 25 +/-1 ℃, and the illumination intensity is 30-100 mu mol.m -2 ·s -1 The illumination time is 12-14 h/d; during the period, the growth condition of the bottle seedlings is observed every day, polluted plant materials which are not disinfected thoroughly are found in time, and the polluted tissue culture bottle seedlings are immediately moved out of a culture room for disinfection and cleaning;
5) the method comprises the steps of establishing a gordon euryale seed sterile rapid propagation system, continuing to perform germination induction culture, transplanting into a subculture rooting culture medium when the seed embryo germinates to 2cm, wherein the culture and formula is 1/2MS basic culture medium (the content of major elements is half of that of MS), adding 0.2mg/L NAA, 20-30 g/L sucrose, 7-7.5 g/L agar and 5% AC activated carbon, and if the transfer is too early, the seedling is easy to stop growing, and if the transfer is too late, the seedling can be browned; after the aseptic seedling takes root, 5ml of aseptic water is injected into the culture bottle in a super clean bench to promote the growth of the rooted seedling;
6) domesticating and transplanting the gorgon fruit tissue culture rooted seedlings, and selecting the rooted tissue culture seedlings with more than 3 leaves, more than 3cm plant height and robust root systems in the continuous culture process; opening a cover in a culture room, adding sterile water, continuing acclimatization culture, moving to a side of a transplanting water area after 10-20 days, then acclimatizing culture for more than two weeks, and then planting.
Example 1
Treating explants, taking 100 gordon euryale embryo-peeled seeds (full and complete and disease-free seeds with the diameter of more than 1.0 cm), cleaning, performing germination induction culture, soaking the seeds in pure water, sealing and storing in a refrigerator at 4 ℃, cleaning once and replacing the pure water in 3-5 days, taking out after 1 month, shelling by using a special gordon euryale seed sheller, peeling off the selected seeds, reserving complete embryos, endosperms and transparent protective films on the surfaces, washing the peeled and shelled complete seeds respectively with tap water, soaking in 800 times of 50% carbendazim solution for 20min, then washing with running water for 1 h-2 h, transferring into a sterile tissue culture bottle, transferring to a super-clean workbench, sterilizing with 75% alcohol for 45s in sequence, washing with sterile water for 3-5 times, sterilizing with 0.1% HgCl2 solution for 12min, cleaning with sterile water for 6-8 times, shaking the tissue culture bottle continuously in the period, and finally sucking dry surface water in the sterile tissue culture bottle, and drying the surface moisture by using sterile air.
The method comprises the steps of seed dormancy breaking and germination accelerating treatment, cutting thick endosperm with a scalpel, carefully peeling off the embryo part in the seed, inoculating the seed into a germination culture medium (20-30 g/L of cane sugar, 6-8 g/L of agar, 20g/L of euryale ferox exoendosperm and 5.8 of PH are added into an MS culture medium for culture), and inoculating only 1 seed embryo in each bottle of culture medium during inoculation, so that observation and statistics are facilitated, and cross infection among explants can be avoided. Because the embryo does not need to experience hard and hard seed shells and thick endosperm under the natural state, the method greatly improves the germination accelerating speed, the average germination time of the gordon euryale seed embryo is 3-8 days, the endosperm is propped open, then the epicotyl is extended, the embryo is extended, the 1 st linear leaf is formed after continuous extension, the average culture time is 10-20 days when the 1 st leaf is extended to about 1cm, the germination rate is kept above 80% in the whole germination accelerating induction process, and the pollution rate is lower than 15%.
A gordon euryale seed sterile rapid propagation system is established, after germination acceleration of gordon euryale seeds is completed, the 1 st leaf is stretched to more than 1cm, subculture is carried out, and the formula of a culture medium is that 20-30 g/L of cane sugar and 6-8 g/L of agar, 1.0-2.0 mg/L of 6-BA, 0.1-0.2 mg/L of NAA and 0.5% of active carbon are added into an MS culture medium. Then, the 2 nd and 3 rd leaves are extracted, the 1 st leaf is stretched to be more than 2cm, the average time is 10-15 days, the bud growth state is strong, and the petiole is thick. Transplanting the first leaf to a rooting culture medium when the length of the first leaf reaches 2cm, wherein the culture formula is 1/2MS basic culture medium, and 0.2-1.0 mg/L NAA, 20-30 g/L sucrose, 7-7.5 g/L agar and 5.8 PH are added. If the transfer is too early, the seedlings are easy to stop growing, and if the transfer is too late, the seedlings are browned. After the aseptic seedling takes root, 5ml of aseptic water is injected into the culture bottle in a super clean bench, so that the growth of the rooted seedling can be promoted. In the process of rooting culture of the aseptic seedlings, the rooting rate is up to more than 85%.
Domesticating and transplanting the gordon euryale tissue culture rooting seedlings, and selecting the rooting tissue culture seedlings with more than 3 leaves, more than 3cm of plant height and robust root systems in the continuous culture process after completing the rooting culture of the gordon euryale aseptic seedlings. Opening a cover in a culture room, adding quantitative sterile water, continuing acclimatization culture, transferring to a side of a transplanting water area after 10-20 days, then acclimatizing culture for more than two weeks, then field planting, and obtaining the transplanting survival rate which is more than 90% after 30 days.
Example 2
Treating explants, taking 100 gordon euryale embryo-peeled seeds (more than 1.0cm in diameter, full and complete and disease-free), cleaning, performing germination induction culture, soaking the seeds in pure water, sealing and storing in a 0 ℃ refrigerator, cleaning once in 3-5 days and replacing the pure water, taking out after 1 month, respectively washing with tap water, soaking in 800 times of 50% carbendazim solution for 20min, then washing with running water for 1 h-2 h, transferring into a sterile tissue culture bottle, transferring to a super-clean workbench, sterilizing for 45s with 75% alcohol in sequence, washing with sterile water for 3-5 times, sterilizing with 0.1% HgCl2 solution for 12min, cleaning with sterile water for 6-8 times, shaking the tissue culture bottle continuously in the period, and finally, drying surface moisture by sterile air after absorbing the surface moisture.
The method comprises the steps of breaking seed dormancy and accelerating germination, shelling by a special gordon euryale seed shucker, removing the shell of the complete part (embryo and complete exoendosperm) after the shell removal, inoculating the complete part into a germination culture medium (20-30 g/L of cane sugar, 6-8 g/L of agar, 20g/L of exoendosperm of gordon euryale seed and pH 5.8 added into an MS culture medium) for culture, and inoculating only 1 seed of the shell removal per bottle of the culture medium during inoculation, so that observation and statistics are facilitated, and cross infection among explants can be avoided. As the embryo does not need to experience hard and hard seed shells and thick endosperm under the natural state, the method greatly improves the germination accelerating speed, the average germination time of the gordon euryale seed embryo is 25-35 days, the embryo firstly stretches out 4 white papilla, then the endosperm is stretched, then the epicotyl is stretched, the embryo stretches out, the embryo continues to stretch to form the 1 st linear leaf, the average culture time is 8-15 days when the 1 st leaf stretches to about 1cm, the germination rate is kept above 70% in the whole germination accelerating induction process, and the pollution rate is 15-25%.
The method comprises the steps of establishing a gordon euryale seed sterile rapid propagation system, after germination acceleration of gordon euryale seeds is completed, and after the 1 st leaf is stretched to be more than 1cm, transferring a complete bud body and a perisperm to be subjected to subculture, wherein a culture medium formula comprises an MS culture medium added with 20-30 g/L of sucrose and 6-8 g/L of agar, 1.0-2.0 mg/L of 6-BA, 0.1-0.2 mg/L of NAA and 0.5% of activated carbon. And then, extracting the 2 nd and 3 th leaves, and extending the 1 st leaf to be more than 2cm, wherein the average time is 6-12 days, the bud growth state is strong, and the leaf stalk is thick. Transplanting the first leaf to a rooting culture medium when the length of the first leaf reaches 2cm, wherein the culture formula is 1/2MS basic culture medium, and 0.2-1.0 mg/L NAA, 20-30 g/L sucrose, 7-7.5 g/L agar and 5.8 PH are added. If the transfer is too early, the seedlings are easy to stop growing, and if the transfer is too late, the seedlings are browned. After the aseptic seedling takes root, 5ml of aseptic water is injected into the culture bottle in a super clean bench, so that the growth of the rooted seedling can be promoted. In the process of rooting culture of the aseptic seedlings, the rooting rate is up to over 95 percent.
Domesticating and transplanting the gordon euryale tissue culture rooting seedlings, and selecting the rooting tissue culture seedlings with more than 3 leaves, more than 3cm of plant height and robust root systems in the continuous culture process after completing the rooting culture of the gordon euryale aseptic seedlings. Opening a cover in a culture room, adding quantitative sterile water, continuing acclimatization culture, transferring to a side of a transplanting water area after 10-20 days, then acclimatizing culture for more than two weeks, then field planting, and transplanting the seedlings with a survival rate of more than 95% after 30 days.
Example 3
Treating explants, taking 100 gordon euryale embryo-peeled seeds (full and complete and disease-free seeds with the diameter of more than 1.0 cm), cleaning, performing germination induction culture, soaking the seeds in pure water, sealing and storing in a refrigerator at 5 ℃, cleaning once and replacing the pure water in 3-5 days, taking out after 1 month, shelling by using a special gordon euryale seed sheller, peeling off the selected seeds, reserving complete embryos, endosperms and transparent protective films on the surfaces, washing the peeled and shelled complete seeds respectively with tap water, soaking in 800 times of 50% carbendazim solution for 20min, then washing with running water for 1 h-2 h, transferring into a sterile tissue culture bottle, transferring to a super-clean workbench, sterilizing with 75% alcohol for 45s in sequence, washing with sterile water for 3-5 times, sterilizing with 0.1% HgCl2 solution for 12min, cleaning with sterile water for 6-8 times, shaking the tissue culture bottle continuously in the period, and finally sucking dry surface water in the sterile tissue culture bottle, and drying the surface moisture by using sterile air.
Seed dormancy breaking and germination accelerating treatment, cutting thick endosperm with a scalpel, carefully peeling off the embryo part in the seed, inoculating into a germination culture medium (the MS culture medium is added with 20-30 g/L of cane sugar, 6-8 g/L of agar and 5.8 of PH) for culture, and inoculating only 1 seed embryo in each bottle of culture medium during inoculation, thereby facilitating observation and statistics and avoiding cross infection among explants. As the embryo does not need to experience hard and hard seed shells and thick endosperm under the natural state, the method greatly improves the germination accelerating speed, the average germination time of the gordon euryale seed embryo is 3-8 days, then the endosperm is propped open, then the epicotyl is extended, the embryo is extended out, the embryo continues to be extended to form the 1 st linear leaf, the average culture time is 10-20 days when the 1 st leaf is extended to about 1cm, in the whole germination accelerating induction process, the germination percentage is 50-60%, and the pollution rate is lower than 15%.
The method comprises the steps of establishing a gordon euryale seed sterile rapid propagation system, after germination acceleration of gordon euryale seeds is completed, transferring to perform subculture when the 1 st leaf is stretched to be more than 1cm, and adding 20-30 g/L of cane sugar and 6-8 g/L of agar into an MS culture medium according to the formula of a culture medium, 1.0-2.0 mg/L of 6-BA, 0.1-0.2 mg/L of NAA and 0.5% of activated carbon. And then, extracting the 2 nd and 3 th leaves, and extending the 1 st leaf to be more than 2cm, wherein the average time is 10-15 days, the bud is long and healthy in growth state, and the leaf stalk is thick and strong. Transplanting the first leaf to a rooting culture medium when the length of the first leaf reaches 2cm, wherein the culture formula is 1/2MS basic culture medium, and 0.2-1.0 mg/L NAA, 20-30 g/L sucrose, 7-7.5 g/L agar and 5.8 PH are added. If the transfer is too early, the seedlings are easy to stop growing, and if the transfer is too late, the seedlings can be browned. After the aseptic seedling takes root, 5ml of aseptic water is injected into the culture bottle in a super clean bench, so that the growth of the rooted seedling can be promoted. In the process of rooting culture of the aseptic seedlings, the rooting rate is up to more than 85%.
Domesticating and transplanting the gordon euryale tissue culture rooting seedlings, and selecting the rooting tissue culture seedlings with more than 3 leaves, more than 3cm of plant height and robust root systems in the continuous culture process after completing the rooting culture of the gordon euryale aseptic seedlings. Opening a cover in a culture room, adding quantitative sterile water, continuing acclimatization culture, transferring to a side of a transplanting water area after 10-20 days, then acclimatizing culture for more than two weeks, then field planting, and obtaining the transplanting survival rate which is more than 90% after 30 days.
Example 4
Treating explants, taking 100 gordon euryale embryo-peeled seeds (full and complete and disease-free seeds with the diameter of more than 1.0 cm), cleaning, performing germination induction culture, soaking the seeds in pure water, sealing and storing in a refrigerator at 2 ℃, cleaning once and replacing the pure water in 3-5 days, taking out after 1 month, shelling by using a special gordon euryale seed sheller, peeling off the selected seeds, reserving complete embryos, endosperms and transparent protective films on the surfaces, washing the peeled and shelled complete seeds respectively with tap water, soaking in 800 times of 50% carbendazim solution for 20min, then washing with running water for 1 h-2 h, transferring into a sterile tissue culture bottle, transferring to a super-clean workbench, sterilizing with 75% alcohol for 45s in sequence, washing with sterile water for 3-5 times, sterilizing with 0.1% HgCl2 solution for 6min, cleaning with sterile water for 6-8 times, shaking the tissue culture bottle continuously in the period, and finally sucking dry surface water in the sterile tissue culture bottle, and drying the surface moisture by using sterile air.
Seed dormancy breaking and germination accelerating treatment, cutting off thick endosperm with a scalpel, carefully peeling off the embryo part in the seed, inoculating the seed into a germination culture medium (the MS culture medium is added with 20-30 g/L of sucrose, 6-8 g/L of agar, 20g/L of euryale ferox exoendosperm and 5.8 of PH) for culture, and inoculating only 1 seed embryo in each bottle of culture medium during inoculation, thereby facilitating observation and statistics and avoiding cross infection among explants. As the embryo does not need to experience hard and hard seed shells and thick endosperm under the natural state, the method greatly improves the germination accelerating speed, the average germination time of the gordon euryale seed embryo is 3-8 days, then the endosperm is propped open, then the epicotyl is extended, the embryo is extended out, the embryo continues to be extended to form the 1 st linear leaf, the average culture time is 10-20 days when the 1 st leaf is extended to about 1cm, in the whole germination accelerating induction process, the germination percentage is 15-20%, and the pollution rate is 65-75%.
A gordon euryale seed sterile rapid propagation system is established, after germination acceleration of gordon euryale seeds is completed, the 1 st leaf is stretched to more than 1cm, subculture is carried out, and the formula of a culture medium is that 20-30 g/L of cane sugar and 6-8 g/L of agar, 1.0-2.0 mg/L of 6-BA, 0.1-0.2 mg/L of NAA and 0.5% of active carbon are added into an MS culture medium. And then, extracting the 2 nd and 3 th leaves, and extending the 1 st leaf to be more than 2cm, wherein the average time is 10-15 days, the bud is long and healthy in growth state, and the leaf stalk is thick and strong. Transplanting the first leaf to a rooting culture medium when the length of the first leaf reaches 2cm, wherein the culture formula is 1/2MS basic culture medium, and 0.2-1.0 mg/L NAA, 20-30 g/L sucrose, 7-7.5 g/L agar and 5.8 PH are added. If the transfer is too early, the seedlings are easy to stop growing, and if the transfer is too late, the seedlings can be browned. After the aseptic seedling takes root, 5ml of aseptic water is injected into the culture flask in an ultra-clean workbench, so that the growth of the rooted seedling can be promoted. In the process of rooting culture of the aseptic seedlings, the rooting rate is up to more than 85%.
Domesticating and transplanting the gordon euryale tissue culture rooting seedlings, and selecting the rooting tissue culture seedlings with more than 3 leaves, more than 3cm of plant height and robust root systems in the continuous culture process after completing the rooting culture of the gordon euryale aseptic seedlings. Opening a cover in a culture room, adding quantitative sterile water, continuing acclimatization culture, transferring to a side of a transplanting water area after 10-20 days, then acclimatizing culture for more than two weeks, then field planting, and obtaining the transplanting survival rate which is more than 90% after 30 days.
Example 5
Treating explants, taking 100 gordon euryale embryo-peeled seeds (full and complete and disease-free seeds with the diameter of more than 1.0 cm), cleaning, performing germination induction culture, soaking the seeds in pure water, sealing and storing in a refrigerator at 1 ℃, cleaning once and replacing the pure water in 3-5 days, taking out after 1 month, shelling by using a special gordon euryale seed sheller, peeling off the selected seeds, reserving complete embryos, endosperms and transparent protective films on the surfaces, washing the peeled and shelled complete seeds respectively with tap water, soaking in 800 times of 50% carbendazim solution for 20min, then washing with running water for 1 h-2 h, transferring into a sterile tissue culture bottle, transferring to a super-clean workbench, sterilizing with 75% alcohol for 45s in sequence, washing with sterile water for 3-5 times, sterilizing with 0.1% HgCl2 solution for 12min, washing with sterile water for 6-8 times, shaking the tissue culture bottle continuously in the period, and finally sucking dry surface water in the sterile tissue culture bottle, and drying the surface moisture by using sterile air.
Seed dormancy breaking and germination accelerating treatment are carried out, thick endosperm is cut by a scalpel, the part of an embryo in the seed is carefully peeled off, and the embryo is inoculated in a germination culture medium (20-30 g/L of cane sugar, 6-8 g/L of agar, 20g/L of euryale ferox exoendosperm and 5.8 of PH added in an MS culture medium) for culture, only 1 embryo is inoculated in each bottle of culture medium during inoculation, so that observation and statistics are facilitated, and cross infection among explants can be avoided. As the embryo does not need to go through hard and hard seed shells and thick endosperm under the natural state, the method greatly improves the germination accelerating speed, the average germination time of the gordon euryale seed embryo is 3-8 days, the endosperm is propped open, then the epicotyl is extended, the embryo is extended out, the embryo is extended to form a1 st linear leaf after continuous extension, the average culture time is 10-20 days when the 1 st leaf is extended to about 1cm, the germination percentage is kept above 80% in the whole germination accelerating induction process, and the pollution rate is lower than 15%.
The method comprises the steps of establishing a gordon euryale seed sterile rapid propagation system, after germination acceleration of gordon euryale seeds is completed, transferring to perform subculture when the 1 st leaf is stretched to be more than 1cm, and adding 20-30 g/L of cane sugar and 6-8 g/L of agar, 1.0-2.0 mg/L of 6-BA and 0.1-0.2 mg/L of NAA into an MS culture medium. And then, drawing out 2 nd and 3 th leaves, extending the 1 st leaf to be more than 2cm, taking 15-20 days on average, enabling the bud growth state to be normal, and enabling the withered and dead rate of plants to be higher than 50-70%. Transplanting the first leaf to a rooting culture medium when the length of the first leaf reaches 2cm, wherein the culture formula is 1/2MS basic culture medium, and 0.2-1.0 mg/L NAA, 20-30 g/L sucrose, 7-7.5 g/L agar and 5.8 PH are added. If the transfer is too early, the seedlings are easy to stop growing, and if the transfer is too late, the seedlings can be browned. After the aseptic seedling takes root, 5ml of aseptic water is injected into the culture flask in an ultra-clean workbench, so that the growth of the rooted seedling can be promoted. In the process of rooting culture of the aseptic seedlings, the rooting rate is up to more than 70%.
Domesticating and transplanting the gordon euryale tissue culture rooting seedlings, and selecting the rooting tissue culture seedlings with more than 3 leaves, more than 3cm of plant height and robust root systems in the continuous culture process after completing the rooting culture of the gordon euryale aseptic seedlings. Opening a cover in a culture room, adding quantitative sterile water, continuing acclimatization culture, transferring to a side of a transplanting water area after 10-20 days, then acclimatizing culture for more than two weeks, then field planting, and obtaining the transplanting survival rate which is more than 90% after 30 days.
Example 6
Treating explants, taking 100 gordon euryale embryo-peeled seeds (full and complete and disease-free seeds with the diameter of more than 1.0 cm), cleaning, performing germination induction culture, soaking the seeds in pure water, sealing and storing in a refrigerator at 4 ℃, cleaning once and replacing the pure water in 3-5 days, taking out after 1 month, shelling by using a special gordon euryale seed sheller, peeling off the selected seeds, reserving complete embryos, endosperms and transparent protective films on the surfaces, washing the peeled and shelled complete seeds respectively with tap water, soaking in 800 times of 50% carbendazim solution for 20min, then washing with running water for 1 h-2 h, transferring into a sterile tissue culture bottle, transferring to a super-clean workbench, sterilizing with 75% alcohol for 45s in sequence, washing with sterile water for 3-5 times, sterilizing with 0.1% HgCl2 solution for 12min, cleaning with sterile water for 6-8 times, shaking the tissue culture bottle continuously in the period, and finally sucking dry surface water in the sterile tissue culture bottle, and drying the surface moisture by aseptic air.
Seed dormancy breaking and germination accelerating treatment are carried out, thick endosperm is cut by a scalpel, the part of an embryo in the seed is carefully peeled off, and the embryo is inoculated in a germination culture medium (20-30 g/L of cane sugar, 6-8 g/L of agar, 20g/L of euryale ferox exoendosperm and 5.8 of PH added in an MS culture medium) for culture, only 1 embryo is inoculated in each bottle of culture medium during inoculation, so that observation and statistics are facilitated, and cross infection among explants can be avoided. Because the embryo does not need to experience hard and hard seed shells and thick endosperm under the natural state, the method greatly improves the germination accelerating speed, the average germination time of the gordon euryale seed embryo is 3-8 days, the endosperm is propped open, then the epicotyl is extended, the embryo is extended, the 1 st linear leaf is formed after continuous extension, the average culture time is 10-20 days when the 1 st leaf is extended to about 1cm, the germination rate is kept above 80% in the whole germination accelerating induction process, and the pollution rate is lower than 15%.
A gordon euryale seed sterile rapid propagation system is established, after germination acceleration of gordon euryale seeds is completed, the 1 st leaf is stretched to more than 1cm, subculture is carried out, and the formula of a culture medium is that 20-30 g/L of cane sugar and 6-8 g/L of agar, 1.0-2.0 mg/L of 6-BA, 0.1-0.2 mg/L of NAA and 0.5% of active carbon are added into an MS culture medium. And then, extracting the 2 nd and 3 th leaves, and extending the 1 st leaf to be more than 2cm, wherein the average time is 10-15 days, the bud is long and healthy in growth state, and the leaf stalk is thick and strong. Transplanting the first leaf to a rooting culture medium when the length of the first leaf reaches 2cm, wherein the culture formula is 1/2MS basic culture medium, and 0.2-1.0 mg/L NAA, 20-30 g/L sucrose, 7-7.5 g/L agar and 5.8 PH are added. If the transfer is too early, the seedlings are easy to stop growing, and if the transfer is too late, the seedlings can be browned. In the process of rooting culture of aseptic seedlings, the rooting rate is up to more than 85%, the petioles of the seedlings are short, and the leaves are abnormal in shape.
Domesticating and transplanting the gordon euryale tissue culture rooting seedlings, and selecting the rooting tissue culture seedlings with more than 3 leaves, more than 3cm of plant height and robust root systems in the continuous culture process after completing the rooting culture of the gordon euryale aseptic seedlings. Opening a cover in a culture room, adding quantitative sterile water, continuing acclimatization culture, transferring to a transplanting water area after 10-20 days, then acclimatizing culture for more than two weeks, then field planting, and obtaining the transplanting survival rate which reaches 65-75% after 30 days.
The method breaks dormancy of the gordon euryale seeds to promote germination, the gordon euryale seeds naturally germinate in March under natural conditions, and the gordon euryale seeds are in a dormant state in half a year.
And (3) establishing a gordon euryale aseptic rapid propagation system, completing annual seedling raising of gordon euryale, and providing a foundation for gordon euryale breeding research. Because of the own biological characteristics of the gordon euryale seeds, the gordon euryale seeds can only be bred once a year under natural conditions, and the characters of the gordon euryale seeds cannot be identified efficiently by the seeds obtained by traditional means such as crossbreeding, mutation breeding and the like. The euryale ferox aseptic seedling is the most suitable receptor in transgenic breeding, and a solid foundation is provided for the research of the euryale ferox breeding.
The method provides a quick and effective method for gorgon fruit germplasm resource preservation, breaks through gorgon fruit seed dormancy, breaks through the restriction of seasons on gorgon fruit research and development and production, quickly breeds a large number of gorgon fruit seedlings with the same characters, and for gorgon fruit new variety breeding, offspring generated through cross breeding are quickly bred through the method, excellent characters are perfectly duplicated, and the breeding period can be greatly shortened.
For physical mutagenesis, chemical mutagenesis, polyploid breeding and transgenic breeding, the method can rapidly propagate offspring, and the aseptic seedling of the gordon euryale seed is the most suitable receptor; the method can be used as a necessary means for traditional breeding and transgenic breeding.
In conclusion, the invention completely establishes a gordon euryale seed sterile rapid propagation system from the initial generation of seeds to the transplantation of tissue culture rooting seedlings; the dormancy of the gorgon fruit seeds can be broken at any time all the year around for the first time, and the restriction of seasons on the research and development and production of the gorgon fruit is thoroughly broken.
It should be noted that: the above description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention; while the foregoing description will be apparent to those skilled in the relevant art and it is intended to cover in the appended claims all such modifications and changes as fall within the true spirit of the invention.
Claims (6)
1. A gordon euryale seed in-vitro rapid propagation method is characterized in that: the method comprises the following steps:
1) selecting seeds, namely selecting newly harvested gordon euryale seeds in the current year, soaking the seeds in pure water, sealing and storing the seeds in an environment at 0-5 ℃, cleaning once and replacing the pure water within 3-5 days, and taking out the seeds for use after 1 month;
2) processing seed shells, namely peeling off the seed shells of the selected seeds, and reserving complete seed embryos, endosperm and a protective film with a transparent surface; the peeled seed with the whole seed shell removed is soaked in sterile water, and the environmental temperature is 18-25 ℃;
3) at the position of the explantAnd (3) cleaning, namely continuously washing the seed husking complete seeds with water, then moving the superclean workbench, and sequentially carrying out disinfection: soaking and sterilizing for 30 s-1 min by 75% alcohol, washing for 3-5 times by sterile water, and then using 0.1% HgCl 2 Soaking and disinfecting the solution for 8-12 min, washing the tissue culture bottle for 3-5 times by using sterile water, continuously shaking the tissue culture bottle during the washing, and finally sucking surface water by using sterile filter paper;
4) accelerating germination of seeds, wherein the surface of the sterilized de-shelled complete seeds is free of water and the internal water content is not less than 50%, the seeds are taken off on an ultra-clean workbench by using a scalpel and a forceps, the seeds with the diameter of more than 2mm are selected and immediately inoculated into a germination culture medium, the germination culture medium is an MS basic culture medium, 1.0-2.0 mg/L, NAA0.1 of 6-BA, 0.1-0.2 mg/L of sucrose, 7-7.5 g/L of agar and 20g/L of ground exoendosperm, the pH is 5.8-6.0, and 20g/L of ground exoendosperm is 20g/L of gorgon fruit exoendosperm; placing the inoculated gordon euryale seed embryo under illumination for culturing, wherein the culture conditions are as follows: the temperature is 25 +/-1 ℃, and the illumination intensity is 30-100 mu mol.m -2 ·s -1 The illumination time is 12-14 h/d;
5) establishing a gordon euryale seed sterile rapid propagation system, continuing to perform germination induction culture, transplanting into a subculture rooting culture medium when the seed embryo germinates to 2cm, wherein the culture and formula is 1/2MS basic culture medium, and adding 0.2-1.0 mg/L NAA, 20-30 g/L sucrose, 7-7.5 g/L agar and 5% AC activated carbon; after the aseptic seedling takes root, 5ml of aseptic water is injected into the culture bottle in a super clean bench to promote the growth of the rooted seedling;
6) domesticating and transplanting the gorgon fruit tissue culture rooted seedlings, and selecting plants with more than 3 leaves, more than 3cm of plant height and robust root systems in the rooted tissue culture seedlings in the continuous culture process; the cover is opened in the culture chamber, sterile water is added, and acclimatization culture is continued.
2. The method for rapid in vitro propagation of gorgon fruit according to claim 1, which is characterized in that: step 1), dividing the gordon euryale seeds into six grades of yellow, big shoulder tree, small flower coat, embryo peeling, big sound and old seed in sequence according to the grade of tender to old maturity, and selecting the full and complete seeds with the diameter of more than 1.0cm, wherein the small flower coat, the embryo peeling, the big sound and the old seed are selected.
3. The method for rapid in-vitro propagation of gordon euryale seed according to claim 1, wherein the method comprises the following steps: step 2), the peeled seed-coat-removed complete seeds are soaked in sterile water for no more than 24 hours.
4. The method for rapid in vitro propagation of gorgon fruit according to claim 1, which is characterized in that: and 3) continuously washing the de-shelled complete seeds for 2-6 hours by using water.
5. The method for rapid in-vitro propagation of gordon euryale seed according to claim 1, wherein the method comprises the following steps: and 4) timely cleaning polluted plant materials during the germination accelerating culture period.
6. The method for rapid in vitro propagation of gorgon fruit according to claim 1, which is characterized in that: and 6), after 10-20 days of acclimatization culture, transferring to a side of a transplanting water area, then acclimatizing culture for more than two weeks, and then planting.
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