CN109601378B - Low-temperature preservation method and cultivation method for seed source of sugarcane tissue culture seedling - Google Patents
Low-temperature preservation method and cultivation method for seed source of sugarcane tissue culture seedling Download PDFInfo
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- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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Abstract
The invention relates to the field of preservation of sugarcane seedlings, in particular to a low-temperature preservation method and a cultivation method of a sugarcane tissue culture seedling seed source. A low-temperature preservation method for seed sources of sugarcane tissue culture seedlings comprises the following steps: cutting the cluster buds, soaking the cluster buds in a sodium alginate solution, then putting the cluster bud blocks into a calcium chloride solution, and embedding the cluster bud blocks by using a gel substance generated by the reaction of the sodium alginate and the calcium chloride; and (4) storing the embedded cluster buds at low temperature. According to the low-temperature storage method of the seed source of the sugarcane tissue culture seedling, provided by the invention, the cluster bud blocks are embedded by adopting the gel substance generated by the reaction of sodium alginate and calcium chloride, and then the embedded cluster bud blocks are stored at low temperature, so that the cluster buds are changed into the low-temperature storage sub-period of 3-4 months from the original sub-period of 20-30 days, the sub-period is greatly prolonged, the sub-period is reduced, and the propagation can be carried out as required.
Description
Technical Field
The invention relates to the field of preservation of sugarcane seedlings, in particular to a low-temperature preservation method and a cultivation method of a sugarcane tissue culture seedling seed source.
Background
Sugarcane is an important sugar crop in China, cane sugar accounts for more than 85% of sugar yield in China, and the enhancement of the protection and the utilization of sugarcane germplasm resources has important significance for promoting the improvement of sugarcane varieties, increasing the yield of cane sugar and ensuring the sugar safety in China. The protection, development and utilization of germplasm resources are the basis for cultivating excellent varieties, particularly specific resistant germplasm with high biotic stress or abiotic adversity stress, and have great application potential in the aspect of coping with future natural disasters. However, because of the biological characteristics of high sugarcane plants, long growth period and the like, the existing technology using the nutritive bodies as conservation is difficult to preserve a large amount of germplasm, and a plurality of specific materials selected in the breeding process cannot be discarded. The main reasons that the current sugarcane conservation technology is difficult to preserve a large amount of germplasm are as follows: (1) sugarcane is a genome complex formed by the hybridization of tropical seeds, wild species with thin stems and other species, the seeds of the sugarcane are not homozygote,but the sugarcane is highly separated, and the genotypes of each seed are different, so that the sugarcane cannot be preserved by the seeds, the seed stem breeding and the preservation are generally carried out by seed stem breeding, and the seed stem breeding is also a general method for preserving the germplasm of the sugarcane at home and abroad; (2) the sugarcane plants are tall and big, generally can reach more than 3 meters, in addition, the tillering capacity of the sugarcane is strong, each cluster of sugarcane generally has more than 5 tillers, and the conservation of single-part germplasm is usually required to be 3.0m2The area of more than 2 meters of each germplasm is increased according to the row spacing of 1.5 meters, and the area of the sugarcane conservation land is large; (3) the growth period of the sugarcane is long, generally about 12 months, and the labor cost for field conservation is high; (4) sugarcane can only be suitable for growing in tropical zone and subtropical zone generally, and the condition of flowering is very harsh, only in China is suitable for flowering and hybridization in Hainan, so even sugarcane germplasm is conserved in other areas, because the flowering characteristic of the conserved germplasm cannot be obtained, the exploitation value is difficult to develop, and the significance of conservation is not great. Because the difficulty of conservation of sugarcane germplasm is high, the conservation quantity of each sugarcane germplasm resource bank in China is only thousands of parts generally, and is far behind the conservation quantity of tens of thousands of parts or even hundreds of thousands of parts of other crops.
The number of seedlings sown in a breeding unit in China is more than 10 ten thousand per year, and the excellent genotypes with different characteristics selected per year can reach dozens or even hundreds. Besides the very individual lines which can be popularized in a large area, other lines often have to be eliminated due to the insufficient conservation capacity. Therefore, the establishment of a novel germplasm conservation technology and the expansion of the number of conservation germplasm have important significance for promoting the breeding improvement of the sugarcane.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first purpose of the invention is to provide a method for preserving the seed source of the sugarcane tissue culture seedling at low temperature, which changes the original subculture period of the cluster buds from 20-30 days to the subculture period of the low-temperature preservation for 3-4 months.
The second purpose of the invention is to provide a method for cultivating the seed source of the sugarcane tissue culture seedling, which can recover cluster buds stored at low temperature.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
a low-temperature preservation method for seed sources of sugarcane tissue culture seedlings comprises the following steps: cutting the cluster buds, soaking the cluster buds in a sodium alginate solution, then putting the cluster bud blocks into a calcium chloride solution, and embedding the cluster bud blocks by using a gel substance generated by the reaction of the sodium alginate and the calcium chloride;
and (4) storing the embedded cluster buds at low temperature.
The method for preserving the seed source of the sugarcane tissue culture seedling at the low temperature provided by the invention is characterized in that a gel substance generated by the reaction of sodium alginate and calcium chloride is adopted to embed cluster bud blocks, wherein the sodium alginate has certain hardness and can be rapidly solidified into transparent small rubber balls in the calcium chloride solution, so that the supporting and protecting effects can be realized; and then the embedded cluster buds are stored at low temperature, so that the cluster buds are changed from the original subculture period of 20-30 days to the subculture period of low-temperature storage of 3-4 months, the subculture period is greatly prolonged, the subculture frequency is reduced, and the propagation can be carried out as required.
The concentration of the sodium alginate solution and the concentration of the calcium chloride solution are both too high and too low, which is unfavorable for storage, and the generated gel substances prevent cell respiration and can not be stored for a long time; if the amount is too low, an effective protective layer cannot be formed, and the storage time is greatly reduced.
Further, the mass concentration of the sodium alginate solution is 4% +/-0.5%.
Further, cutting the cluster buds, and soaking in a sodium alginate solution for 2-3 min.
The cluster buds are soaked by sodium alginate with proper concentration, so that the sodium alginate is soaked into the surfaces of the cluster buds, in the subsequent step, the cluster buds are placed into calcium chloride solution, the sodium alginate reacts with the calcium chloride, and the generated gel substances are attached to the surfaces of the cluster buds, so that the cluster buds are embedded, and the cluster buds in the state are stored at low temperature for a long time.
Further, the mass concentration of the calcium chloride solution is 2.5% + -0.5%.
Calcium chloride with proper concentration reacts with sodium alginate to generate proper gel substances, which is a good foundation for improving the preservation of cluster buds.
Further, after embedding the cluster bud blocks with the gel substance, washing the cluster bud blocks with sterile water for 3-4 times, placing the cluster bud blocks on sterile paper to absorb surface water, and then storing the cluster bud blocks at low temperature.
By washing out other unreacted material, a good basis is provided for the preservation of the clumped buds.
Further, the culture temperature of the low-temperature preservation is 14-18 ℃, the culture illumination is 800-.
Further, the low-temperature preservation culture medium takes 1/2MS as a solvent and comprises the following components: 0.20-0.80 mg/L of 6-benzylamino adenine, 0.02-0.1 mg/L of paclobutrazol, 10-20 g/L of cane sugar, 6-7 g/L of agar and 5.8-6.2 of pH.
Further, the cluster buds are prepared by the following method: selecting strong and disease-free sugarcane plants, selecting tender and strong-growing-capacity stem buds, cutting into sections and keeping 5-7 cm of single buds, cleaning with water, carrying out water bath at 52 +/-2 ℃ for 2 hours, taking out, culturing at 32 +/-2 ℃ for 10-14 days, selecting strong and strong buds, disinfecting explants under the aseptic condition, peeling to growing points, and carrying out cluster bud proliferation.
Further, the culture conditions for the proliferation of the multiple shoots are as follows: the temperature is 28-31 ℃, the illumination intensity is 1800-2100 lx, and the illumination time is 15-17 hours/day.
Further, inoculating the culture medium into an axillary bud initiation culture medium for proliferating cluster buds, wherein the axillary bud initiation culture medium takes MS culture solution as a solvent and comprises the following components: 1.0-2.0 mg/L of 6-benzylamino adenine, 0.2-0.6 mg/L of indolebutyric acid, 20-30 g/L of cane sugar and 5.5-7 g/L of agar, and the pH value is 5.8-6.2.
Further, when the cluster buds are propagated to the 2 nd to 3 rd generations on the axillary bud initiation culture medium, 3 to 5 buds exist in each explant, 3 to 4 samples are taken for virus immunological detection of mosaic disease, and strains without germs are detected for seed source low-temperature preservation.
The invention also provides a cultivation method of the seed source of the sugarcane tissue culture seedling, when the seed source of the sugarcane tissue culture seedling obtained by the low-temperature preservation method is transferred into a growth recovery culture medium to be cultivated for 15-20 days, the embedding body for recovering growth is inoculated into a new growth recovery culture medium to carry out proliferation and rapid propagation, and the cluster bud is obtained.
Further, the culture conditions in the recovery growth medium are: the temperature is 26-31 ℃, the illumination intensity is 1800-2100 lx, and the illumination time is 15-17 hours/day.
Further, the recovery growth medium takes MS culture solution as a solvent and comprises the following components: 1.0-2.0 mg/L of 6-benzylamino adenine, 0.2-0.6 mg/L of indolebutyric acid, 20-30 g/L of cane sugar and 5.5-7 g/L of agar, and the pH value is 5.8-6.2.
Further, the cluster buds are subjected to rooting culture to obtain test-tube plantlets, and the rooting culture step is as follows: and cutting the obtained cluster buds into cluster buds with 2-3 buds, transferring the cluster buds into a rooting culture medium for rooting culture, wherein the culture temperature is 28-31 ℃, the illumination intensity is 1800-2100 lx, and the illumination time is 15-17 hours/day until the cluster buds grow into test-tube plantlets.
The cultivation method of the sugarcane tissue culture seedling seed source provided by the invention is a process of restoring and cultivating the stored tissue culture seedling seed source, namely in the storage process, when a test-tube seedling is needed, the cluster bud stored at low temperature can be restored to grow and subcultured to obtain the cluster bud, and the storage time of the cluster bud is prolonged. After the cluster buds are subjected to rooting culture, a large number of test-tube plantlets are obtained in a short time, and an effective way is provided for timely production of sugarcane seedlings.
By using the method provided by the invention, each germplasm only needs to occupy 0.002m3The volume (calculated according to 6 culture bottles and 50 seedlings) is reduced, and the growth speed is slowed down, the culture time is prolonged, the subculture times are reduced, and the cost is obviously reduced by regulating and controlling the culture conditions.
Compared with the prior art, the invention has the beneficial effects that:
(1) the invention adopts a unique embedding technology to carry out low-temperature preservation treatment on cluster buds (seed sources) after embedding, thereby prolonging the subculture period of the cluster buds.
(2) The invention also carries out low-temperature preservation treatment on the cluster buds (seed sources) by matching with proper culture conditions such as culture temperature, culture illumination and the like, so that the cluster buds are changed from the original subculture period of 20-30 days to the subculture period of low-temperature preservation for 3-4 months.
(3) The invention establishes a method for preserving the sugarcane germplasm at low temperature by applying the tissue culture seedlings through technical innovation and combining the biological characteristics of the sugarcane, and the method for preserving the sugarcane germplasm by using the technology has the characteristics of small occupied space, large preservation quantity and capability of expanding propagation at any time.
(4) The method has the advantages of low storage cost, simple operation, good storage effect and the like, when the seedlings need to grow in a large scale, the seed source stored at low temperature can be recovered for growth and subcultured for proliferation to obtain cluster buds in a short time, and a large number of test-tube seedlings can be obtained in a short time after the cluster buds are subjected to rooting culture, so that an effective way is provided for the timely production of the sugarcane seedlings.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Example 1
1. Preparing a solution for later use:
sodium alginate solution: the mass concentration is 4%.
Calcium chloride solution: the mass concentration is 2.5%.
The culture medium stored at low temperature takes 1/2MS as a solvent and comprises the following components: 0.80mg/L of 6-benzylamino adenine, 0.1mg/L of paclobutrazol, 20g/L of cane sugar, 6g/L of agar and 5.8-6.2 of pH.
The axillary bud initiation culture medium takes MS culture solution as a solvent and comprises the following components: 2.0mg/L of 6-benzylamino adenine, 0.6mg/L of indolebutyric acid, 30g/L of cane sugar and 5.5g/L of agar, and the pH value is 5.8-6.2.
The growth recovery culture medium takes MS culture solution as a solvent and comprises the following components: 2.0mg/L of 6-benzylamino adenine, 0.6mg/L of indolebutyric acid, 30g/L of cane sugar and 5.5g/L of agar, and the pH value is 5.8-6.2.
2. The low-temperature preservation method comprises the following steps:
(1) seedling collection: selecting strong and disease-free sugarcane plants.
(2) Inducing cluster buds: selecting tender stem buds with strong growing power, cutting into sections and reserving a single bud by 5-7 cm, washing with water, placing at 52 ℃, carrying out water bath for 2 hours, taking out, culturing at 32 ℃ for 10-14 days, selecting robust buds, disinfecting explants with 75% alcohol under an aseptic condition, peeling to growing points, inoculating to an axillary bud starting culture medium, carrying out cluster bud proliferation on the axillary bud starting culture medium to 2-3 generations, wherein each explant has 3-5 buds, sampling 3-4 buds to carry out virus immunological detection of mosaic disease, and detecting strains without germs to be used for low-temperature preservation of seed sources.
(3) Embedding cluster buds: cutting the cluster buds obtained by tissue culture of sugarcane, putting the cut cluster buds into a 4% sodium alginate solution, fully mixing, soaking for 2-3 min, absorbing 1 cluster bud block each time by using a rubber dropper, and dropping into a 2.5% calcium chloride solution. The formed embedding body is washed by sterile water for 3-4 times and placed on sterile paper to absorb surface water.
(4) And (4) low-temperature preservation: and inoculating the embedded body into a culture medium stored at a low temperature, wherein the culture temperature is 14-18 ℃, the culture illumination is 1000lx, and the illumination time is 10 hours/day, and the embedded body is used for storing seed sources of sugarcane tissue culture seedlings.
The seed source of the sugarcane tissue culture seedling stored at low temperature can be stored for 3-4 months at the longest, and finally, recovery culture is carried out according to needs.
3. The recovery culture steps are as follows:
and (3) transferring the embedding body stored at low temperature to a growth recovery culture medium for culturing for 15-20 days, and inoculating the embedding body recovered from growth to a new growth recovery culture medium for proliferation and rapid propagation to obtain cluster buds. The culture conditions are as follows: the temperature is 26-31 ℃, the illumination intensity is 2100lx, and the illumination time is 15 hours/day. And (4) carrying out conventional rooting culture on the cluster buds to obtain test-tube plantlets.
Wherein, the rooting culture step is as follows: cutting the cluster buds subjected to rapid propagation in a new growth recovery culture medium into cluster buds carrying 2-3 buds, transferring the cluster buds into a rooting culture medium for rooting culture, wherein the culture temperature is 28-31 ℃, the illumination intensity is 2100lx, and the illumination time is 15 hours/day until the cluster buds grow into test-tube plantlets.
Example 2
1. Preparing a solution for later use:
sodium alginate solution: the mass concentration is 4%.
Calcium chloride solution: the mass concentration is 2.5%.
The culture medium stored at low temperature takes 1/2MS as a solvent and comprises the following components: 0.20mg/L of 6-benzylamino adenine, 0.02mg/L of paclobutrazol, 10g/L of cane sugar, 7g/L of agar and 5.8-6.2 of pH.
The axillary bud initiation culture medium takes MS culture solution as a solvent and comprises the following components: 1.0mg/L of 6-benzylamino adenine, 0.2mg/L of indolebutyric acid, 20g/L of cane sugar and 7g/L of agar, and the pH value is 5.8-6.2.
The growth recovery culture medium takes MS culture solution as a solvent and comprises the following components: 1.0mg/L of 6-benzylamino adenine, 0.2mg/L of indolebutyric acid, 20g/L of cane sugar and 7g/L of agar, and the pH value is 5.8-6.2.
2. The low-temperature preservation method comprises the following steps:
(1) seedling collection: selecting strong and disease-free sugarcane plants.
(2) Inducing cluster buds: selecting tender stem buds with strong growing power, cutting into sections and reserving a single bud by 5-7 cm, washing with water, placing at 52 ℃, carrying out water bath for 2 hours, taking out, culturing at 32 ℃ for 10-14 days, selecting robust buds, disinfecting explants with 75% alcohol under an aseptic condition, peeling to growing points, inoculating to an axillary bud starting culture medium, carrying out cluster bud proliferation on the axillary bud starting culture medium to 2-3 generations, wherein each explant has 3-5 buds, sampling 3-4 buds to carry out virus immunological detection of mosaic disease, and detecting strains without germs to be used for low-temperature preservation of seed sources.
(3) Embedding cluster buds: cutting the cluster buds obtained by tissue culture of sugarcane, putting the cut cluster buds into a 4% sodium alginate solution, fully mixing, soaking for 2-3 min, absorbing 1 cluster bud block each time by using a rubber dropper, and dropping into a 2.5% calcium chloride solution. The formed embedding body is washed by sterile water for 3-4 times and placed on sterile paper to absorb surface water.
(4) And (4) low-temperature preservation: and inoculating the embedded body into a culture medium stored at a low temperature, wherein the culture temperature is 14-18 ℃, the culture illumination is 900lx, and the illumination time is 12 hours/day, and the embedded body is used for storing seed sources of sugarcane tissue culture seedlings.
The seed source of the sugarcane tissue culture seedling stored at low temperature can be stored for 3-4 months at the longest, and finally, recovery culture is carried out according to needs.
3. The recovery culture steps are as follows:
and (3) transferring the embedding body stored at low temperature to a growth recovery culture medium for culturing for 15-20 days, and inoculating the embedding body recovered from growth to a new growth recovery culture medium for proliferation and rapid propagation to obtain cluster buds. The culture conditions are as follows: the temperature is 26-31 ℃, the illumination intensity is 2000lx, and the illumination time is 16 hours/day. And (4) carrying out conventional rooting culture on the cluster buds to obtain test-tube plantlets.
Wherein, the rooting culture step is as follows: cutting the cluster buds subjected to rapid propagation in a new growth recovery culture medium into cluster buds carrying 2-3 buds, transferring the cluster buds into a rooting culture medium for rooting culture at the culture temperature of 28-31 ℃, the illumination intensity of 2000lx and the illumination time of 16 hours/day until the cluster buds grow into test-tube plantlets.
Example 3
1. Preparing a solution for later use:
sodium alginate solution: the mass concentration is 4%.
Calcium chloride solution: the mass concentration is 2.5%.
The culture medium stored at low temperature takes 1/2MS as a solvent and comprises the following components: 0.50mg/L of 6-benzylamino adenine, 0.07mg/L of paclobutrazol, 15g/L of cane sugar and 6g/L of agar, and the pH value is 5.8-6.2.
The axillary bud initiation culture medium takes MS culture solution as a solvent and comprises the following components: 1.5mg/L of 6-benzylamino adenine, 0.4mg/L of indolebutyric acid, 25g/L of sucrose and 6g/L of agar, and the pH value is 5.8-6.2.
The growth recovery culture medium takes MS culture solution as a solvent and comprises the following components: 1.5mg/L of 6-benzylamino adenine, 0.4mg/L of indolebutyric acid, 25g/L of sucrose and 6g/L of agar, and the pH value is 5.8-6.2.
2. The low-temperature preservation method comprises the following steps:
(1) seedling collection: selecting strong and disease-free sugarcane plants.
(2) Inducing cluster buds: selecting tender stem buds with strong growing power, cutting into sections and reserving a single bud by 5-7 cm, washing with water, placing at 52 ℃, carrying out water bath for 2 hours, taking out, culturing at 32 ℃ for 10-14 days, selecting robust buds, disinfecting explants with 75% alcohol under an aseptic condition, peeling to growing points, inoculating to an axillary bud starting culture medium, carrying out cluster bud proliferation on the axillary bud starting culture medium to 2-3 generations, wherein each explant has 3-5 buds, sampling 3-4 buds to carry out virus immunological detection of mosaic disease, and detecting strains without germs to be used for low-temperature preservation of seed sources.
(3) Embedding cluster buds: cutting the cluster buds obtained by tissue culture of sugarcane, putting the cut cluster buds into a 4% sodium alginate solution, fully mixing, soaking for 2-3 min, absorbing 1 cluster bud block each time by using a rubber dropper, and dropping into a 2.5% calcium chloride solution. The formed embedding body is washed by sterile water for 3-4 times and placed on sterile paper to absorb surface water.
(4) And (4) low-temperature preservation: and inoculating the embedded body into a culture medium stored at a low temperature, wherein the culture temperature is 14-18 ℃, the culture illumination is 800lx, and the illumination time is 12 hours/day, and the embedded body is used for storing seed sources of sugarcane tissue culture seedlings.
The seed source of the sugarcane tissue culture seedling stored at low temperature can be stored for 3-4 months at the longest, and finally, recovery culture is carried out according to needs.
3. The recovery culture steps are as follows:
and (3) transferring the embedding body stored at low temperature to a growth recovery culture medium for culturing for 15-20 days, and inoculating the embedding body recovered from growth to a new growth recovery culture medium for proliferation and rapid propagation to obtain cluster buds. The culture conditions are as follows: the temperature is 26-31 ℃, the illumination intensity is 1800lx, and the illumination time is 17 hours/day. And (4) carrying out conventional rooting culture on the cluster buds to obtain test-tube plantlets.
Wherein, the rooting culture step is as follows: cutting the cluster buds subjected to rapid propagation in a new growth recovery culture medium into cluster buds carrying 2-3 buds, transferring the cluster buds into a rooting culture medium for rooting culture at the culture temperature of 28-31 ℃, the illumination intensity of 1800lx and the illumination time of 17 hours/day until the cluster buds grow into test-tube plantlets.
Comparative example 1
The conventional culture method comprises the following steps:
(1) seedling collection: selecting strong and disease-free sugarcane plants.
(2) Inducing cluster buds: selecting tender stem buds with strong growing power, cutting into sections and reserving 5-7 cm of single buds, washing with water, placing at 52 ℃, carrying out water bath for 2 hours, taking out, culturing at 32 ℃ for 10-14 days, selecting robust buds, disinfecting explants with 75% alcohol under an aseptic condition, peeling to growing points, inoculating to an axillary bud starting culture medium, carrying out cluster bud proliferation, and carrying out cluster bud proliferation on the axillary bud starting culture medium until 2-3 generations, wherein each explant has 3-5 buds.
(3) Subculturing: the subculture medium takes MS culture solution as a solvent and comprises the following components: 1.5mg/L of 6-benzylamino adenine, 25g/L of cane sugar and 6g/L of agar, and the pH value is 5.8-6.2. The obtained cluster buds were cultured on a subculture medium and subcultured every 15 days.
(4) Rooting culture medium: the rooting culture medium takes MS culture solution as a solvent and comprises the following components: 2.0mg/L of naphthylacetic acid, 25g/L of cane sugar and 6g/L of agar, and the pH value is 5.8-6.2. And (4) culturing the clump seedlings after 6-8 times of subculture in a rooting culture medium, and obtaining more roots in 15-20 days. And then hardening and heeling in the seedlings after root promotion.
According to the conventional method, one generation of tissue culture seedlings can only be kept for about 2 weeks, and continuous subculture is needed, otherwise, the tissue culture seedlings are yellow and killed.
After the cluster buds stored by the low-temperature storage method of the seed source of the sugarcane tissue culture seedling provided by the invention are subjected to recovery culture, the performance of the tissue culture seedling after the cluster buds are recovered and cultured and the final cultured seedling have no obvious difference with the cluster buds stored by the conventional method.
In the present invention, the components of the MS medium and 1/2MS medium are as follows:
while particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.
Claims (6)
1. A low-temperature preservation method for seed sources of sugarcane tissue culture seedlings is characterized by comprising the following steps: cutting the cluster buds, soaking the cluster buds in a sodium alginate solution, then putting the cluster bud blocks into a calcium chloride solution, and embedding the cluster bud blocks by using a gel substance generated by the reaction of the sodium alginate and the calcium chloride;
storing the embedded cluster buds at low temperature;
the mass concentration of the sodium alginate solution is 4% +/-0.5%;
cutting the cluster buds, and soaking in a sodium alginate solution for 2-3 min;
the mass concentration of the calcium chloride solution is 2.5% +/-0.5%;
the culture temperature of the low-temperature preservation is 14-18 ℃, the culture illumination is 800-;
the culture medium for low-temperature preservation takes 1/2MS as a solvent and comprises the following components: 0.20-0.80 mg/L of 6-benzylamino adenine, 0.02-0.1 mg/L of paclobutrazol, 10-20 g/L of cane sugar, 6-7 g/L of agar and 5.8-6.2 of pH.
2. The method for preserving the seed source of the sugarcane tissue culture seedling as claimed in claim 1, wherein the gel substance is used for embedding the cluster buds for 3-4 times, is washed with sterile water, is placed on sterile paper to absorb surface water, and is preserved at low temperature.
3. The method for cryopreservation of seed sources of sugarcane tissue culture seedlings as claimed in claim 1 or 2, wherein the cluster buds are prepared by the following method: selecting strong and disease-free sugarcane plants, selecting tender and strong-growing-capacity stem buds, cutting into sections and keeping 5-7 cm of single buds, cleaning with water, carrying out water bath at 52 +/-2 ℃ for 2 hours, taking out, culturing at 32 +/-2 ℃ for 10-14 days, selecting strong and strong buds, disinfecting explants under the aseptic condition, peeling to growing points, and carrying out cluster bud proliferation.
4. The method for cryopreservation of seed sources of sugarcane tissue culture seedlings as claimed in claim 3, wherein the culture conditions for the propagation of the multiple shoots are as follows: the temperature is 28-31 ℃, the illumination intensity is 1800-2100 lx, and the illumination time is 15-17 hours/day;
inoculating the mixture into an axillary bud initiation culture medium for cluster bud proliferation, wherein the axillary bud initiation culture medium takes MS culture solution as a solvent and comprises the following components: 1.0-2.0 mg/L of 6-benzylamino adenine, 0.2-0.6 mg/L of indolebutyric acid, 20-30 g/L of cane sugar and 5.5-7 g/L of agar, and the pH value is 5.8-6.2.
5. The method for low-temperature preservation of seed sources of sugarcane tissue culture seedlings according to claim 4, wherein 3-5 buds are obtained from each explant when cluster buds are propagated to 2-3 generations on the axillary bud initiation medium, 3-4 samples are taken for virus immunological detection of mosaic disease, and strains without pathogenic bacteria are detected for low-temperature preservation of seed sources.
6. A cultivation method of a sugarcane tissue culture seedling seed source is characterized in that when the sugarcane tissue culture seedling seed source obtained by the low-temperature preservation method of the sugarcane tissue culture seedling seed source in any one of claims 1 to 5 is transferred to a growth recovery culture medium for cultivation for 15 to 20 days, an embedding body for recovering growth is inoculated to a new growth recovery culture medium for proliferation and rapid propagation to obtain cluster buds;
culture conditions in the recovery growth medium: the temperature is 26-31 ℃, the illumination intensity is 1800-2100 lx, and the illumination time is 15-17 hours/day;
the growth recovery culture medium takes MS culture solution as a solvent and comprises the following components: 1.0-2.0 mg/L of 6-benzylamino adenine, 0.2-0.6 mg/L of indolebutyric acid, 20-30 g/L of cane sugar, 5.5-7 g/L of agar and 5.8-6.2 of pH;
the cluster buds are subjected to rooting culture to obtain test-tube plantlets, and the rooting culture step is as follows: and cutting the obtained cluster buds into cluster buds with 2-3 buds, transferring the cluster buds into a rooting culture medium for rooting culture, wherein the culture temperature is 28-31 ℃, the illumination intensity is 1800-2100 lx, and the illumination time is 15-17 hours/day until the cluster buds grow into test-tube plantlets.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1252685A (en) * | 1997-02-21 | 2000-05-10 | 莱拉·扎卡里亚·阿布德拉曼 | Sugar cane production |
| CN102217537A (en) * | 2011-04-20 | 2011-10-19 | 中国科学院华南植物园 | Low-temperature conservation method of banana tissue culture plantlet provenances |
| CN105165606A (en) * | 2015-08-04 | 2015-12-23 | 江门市新会区林业科学研究所 | Preparation method of A.melanoxylon artificial seeds |
| CN105746351A (en) * | 2016-03-09 | 2016-07-13 | 上海市农业生物基因中心 | Method for ultralow-temperature storage and renewal cultivation of wild rice stem tips |
-
2018
- 2018-11-30 CN CN201811453475.0A patent/CN109601378B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1252685A (en) * | 1997-02-21 | 2000-05-10 | 莱拉·扎卡里亚·阿布德拉曼 | Sugar cane production |
| EP1007629A2 (en) * | 1997-02-21 | 2000-06-14 | Layla Zakaria Abdelrahman | Sugar cane production |
| CN102217537A (en) * | 2011-04-20 | 2011-10-19 | 中国科学院华南植物园 | Low-temperature conservation method of banana tissue culture plantlet provenances |
| CN105165606A (en) * | 2015-08-04 | 2015-12-23 | 江门市新会区林业科学研究所 | Preparation method of A.melanoxylon artificial seeds |
| CN105746351A (en) * | 2016-03-09 | 2016-07-13 | 上海市农业生物基因中心 | Method for ultralow-temperature storage and renewal cultivation of wild rice stem tips |
Non-Patent Citations (2)
| Title |
|---|
| 植物种质资源的保存——离体保存;张巧仙、范冬梅;《太原师范学院学报(自然科学版)》;20050331;第4卷(第1期);82-85 * |
| 甘蔗人工种子的研究;文颖等;《甘蔗糖业》;19951231(第1期);8-13 * |
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