CN107135945B - Tissue culture medium of linden tree and rapid propagation method thereof - Google Patents
Tissue culture medium of linden tree and rapid propagation method thereof Download PDFInfo
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- CN107135945B CN107135945B CN201710316354.0A CN201710316354A CN107135945B CN 107135945 B CN107135945 B CN 107135945B CN 201710316354 A CN201710316354 A CN 201710316354A CN 107135945 B CN107135945 B CN 107135945B
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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
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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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
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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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
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
Abstract
The invention discloses a tissue culture medium of linden and a rapid propagation method thereof, aiming at providing a method for propagating commercial tissue culture seedlings of the linden capable of rapidly propagating, the technical scheme is that the method comprises an induction culture medium, a propagation culture medium, a rooting culture medium, an explant disinfection method, an environment control and an operation process, wherein the induction culture medium comprises the following components: MS culture medium: 4630-4640 mg; thidiazuron TDZ: 0.001-0.010 mg; gibberellin: 2-3 mg; agar: 5-8 g; sucrose: 25-40 g; inositol: 90-110 mg, adding water to make up to 1 liter, and the multiplication culture medium comprises: MS culture medium: 4630-4640 mg; thidiazuron TDZ: 0.001-0.010 mg; gibberellin: 2-3 mg; agar: 5-8 g; sucrose: 25-40 g; inositol: 90-110 mg, adding water to complement to 1 liter, wherein the rooting medium comprises: 2315-2320 mg; indole butyric acid IBA: 1.0mg to 2.0 mg; naphthylacetic acid NAA: 1.0mg to 2.0 mg; agar: 5-8 g; sucrose: 10-20 g; water was added to make up to 1 liter.
Description
Technical Field
The invention relates to the technical field of plant propagation, in particular to a tissue culture medium of linden and a rapid propagation method thereof.
Background
The European young leaf forge (TiliaGreenstand pire) belongs to the genus Tilia in the family Tiliaceae, the leaves of the Tilia can change color, are relatively rough, have strong capability of resisting smoke dust and toxic gas and excellent wood, and are one of the most popular shade trees and shade trees in the world, but most of the Tilia in China is wild at present, the Tilia is still in the starting stage as the shade trees, the artificial propagation and cultivation are few, and the demand of the Tilia seedlings in China is large and the supply is not in demand at present along with the development of the forest industry in towns and gardens.
The germination rate of the linden seeds is low and the seedlings emerge unevenly due to the impermeability of hard bone substances of seed coats, embryo dormancy and the existence of inhibitors in the seeds, and meanwhile, the flowers of the linden are almost sterile due to the first maturity of male stamens of the linden flowers, cross pollination is carried out, the setting rate is low and is generally not more than 10%. In addition, in China and abroad, the tissue culture research of the linden tree is less, and the linden tree cannot adapt to industrial large-scale production.
The tissue culture technology is utilized to breed the linden seedlings, the breeding period is short, the efficiency is high, the cost is low, and a large amount of high-quality seedlings can be industrially produced in a short time, so that the proper culture temperature, illumination and other conditions are determined by selecting a proper culture medium type and matching with a reasonable plant growth regulator, and the tissue culture technology has important significance for establishing a linden tissue culture and rapid propagation system.
At present, chinese patent publication No. CN105746106A discloses a propagation method of tilia miqueliana, which includes five steps to complete propagation, step (i): collecting seeds when the seeds are mature, and treating the seeds by adopting a 'pushing and drying threshing' method after the seeds are collected; step (II): the seeds are stored by adopting three methods of 'ventilation dry storage', 'low-temperature storage' and 'sand wetting storage'; step (three): accelerating germination of seeds, namely soaking the seeds in water at the temperature of 10-20 ℃ for 4-6 hours, soaking the seeds in a potassium permanganate solution with the content of 0.5% for 1-2 hours before sowing, and then sowing the seeds at intervals of 30 times 15 centimeters in a drilling manner; step (IV): inserting the treated twigs into a cutting bed by using the prior art, wherein the cutting bed is arched, covering with a plastic film kettle, shielding the outside of a greenhouse and spraying water for cooling, the light intensity in the greenhouse is 20 to 40 percent of the total sunlight, and the relative humidity in the greenhouse is kept between 80 and 90 percent; step (V): transplanting the sapling whose growth period is half a year into field.
Although the propagation method of tilia miqueliana is used for solving the problems of low vitality and low germination rate of seeds in the growth process through five steps, the propagation method of tilia miqueliana still cannot solve the problems of low vitality and low germination rate of seeds in the growth process, so that a proper culture medium and environment regulation are required, the tilia miqueliana can be rapidly propagated and grown, the problems of difficult propagation and seedling culture, difficult induction and difficult rooting in the growth process of the tilia are solved, and the large-scale and industrial seedling culture can be carried out.
Disclosure of Invention
The invention aims to provide a tissue culture medium of a linden tree, which has the advantages that the tissue culture technology of the dormant axillary buds of the linden tree for inducing cluster buds is utilized to carry out rapid culture and propagation on the linden tree, the propagation rate is high, the rooting rate is good, the cost can be effectively saved, large-scale rapid propagation can be carried out, and the large-scale factory production of the linden tree can be carried out.
The technical purpose of the invention is realized by the following technical scheme:
a tissue culture medium of linden trees comprises an induction culture medium, a proliferation culture medium and a rooting culture medium, wherein the induction culture medium comprises the following components: MS culture medium: 4630-4640 mg; thidiazuron TDZ: 0.001-0.010 mg; gibberellin: 2-3 mg; agar: 5-8 g; sucrose: 25-40 g; inositol: 90-110 mg, adding water to make up to 1 liter, wherein the multiplication medium consists of the following components: MS culture medium: 4630-4640 mg; thidiazuron TDZ: 0.001-0.010 mg; gibberellin: 2-3 mg; agar: 5-8 g; sucrose: 25-40 g; inositol: 90-110 mg, adding water to complement to 1 liter, wherein the rooting medium consists of the following components: MS culture medium: 2315-2320 mg; indole butyric acid IBA: 1.0mg to 2.0 mg; naphthylacetic acid NAA: 1.0mg to 2.0 mg; agar: 5-8 g; sucrose: 10-20 g; water was added to make up to 1 liter.
Preferably, the method comprises the following steps: the induction culture medium consists of the following components: MS culture medium: 4636.43 mg; thidiazuron TDZ: 0.005 mg; gibberellin: 2.5 mg; agar: 6g of a mixture; sucrose: 30g of the total weight of the mixture; inositol: 100mg, adding water to make up to 1 liter; the proliferation culture medium consists of the following components: MS culture medium: 4636.43 mg; thidiazuron TDZ: 0.005 mg; gibberellin: 2.0 mg; agar: 6g of a mixture; sucrose: 30g of the total weight of the mixture; inositol: 100mg, adding water to make up to 1 liter; the rooting culture medium comprises the following components: MS culture medium: 2318.22 mg; indole butyric acid IBA: 1.5 mg; naphthylacetic acid NAA: 1.5 mg; agar: 6g of a mixture; sucrose: 15g of the total weight of the mixture; water was added to make up to 1 liter.
By adopting the technical scheme, the MS culture medium is the most common culture medium used at present, has higher inorganic salt concentration, can ensure mineral nutrition required by tissue growth and can accelerate the growth of callus. Because the ion concentration in the formula is high, even if some components are slightly mixed in and out in the processes of preparation, storage, disinfection and the like, the balance among ions can not be influenced, and the TDZ serving as a plant growth regulator has very strong cytokinin activity which is dozens of to hundreds of times higher than that of a common plant growth regulator, so that the regeneration and propagation of plant buds can be promoted, the eyes of the buds can be broken, the seed germination can be promoted, the callus growth can be promoted, the plant aging can be delayed and the like. And can regulate the growth and development process of plants by acting on other plant hormones and physiologically active substances. Gibberellin is mainly used for accelerating cell growth and improving the content of auxin in plants, the auxin directly regulates the cell growth, promotes cell division and can promote cell expansion, and besides, the gibberellin also has physiological effects of inhibiting maturation, lateral bud dormancy, senescence and tuber formation. Agar acts as a coagulant to convert a liquid medium into a solid or semi-solid medium. Compared with a liquid culture medium, the solid culture medium added with the agar has the advantages of simple and convenient operation, easy solution of aeration problem and convenient frequent observation and research, and the cane sugar is used as a standard carbon source of the culture medium and supplies nutrients required by cell growth. The key of the initiation of the growth of the explant is mainly the hormone proportion and concentration of a culture medium, generally higher-concentration cytokinin and lower-concentration auxin are used, the inhibition effect of apical dominance can be relieved, and the generation of cluster buds is induced. Callus is easily generated when the concentration of auxin is too high; the stem tip grows upwards to form a rootless seedling if the hormone proportion is proper. And inducing the regeneration of the cell callus through the induction culture medium, growing the terminal bud of the explant through the multiplication culture medium, and finally rooting the terminal bud through the rooting culture medium so as to ensure survival.
Another object of the present invention is to provide a rapid propagation method of tilia amurensis, which has the advantages of short propagation period, high efficiency, low cost, and capability of industrially producing a large amount of high quality seedlings in a short time by using tissue culture technique to propagate tilia amurensis seedlings.
The technical purpose of the invention is realized by the following technical scheme:
a rapid propagation method of linden trees comprises the following culture steps:
s1: selecting an explant, wherein the explant selects a full and plump semi-lignified branch stem tip part of a bud of a current-year branch of a healthy linden tree;
s2: the method comprises the following steps of (1) sterilizing and primary culture, cutting leaves of a linden explant, washing the leaves with clear water, putting the washed explant into 75% alcohol on a workbench for sterilizing for 30 seconds, washing the explant with sterile water for 3-4 times, soaking the explant in a disinfectant for 60 minutes, and inoculating the explant into an induction culture medium after the sterilization is finished; culturing at 16 deg.C under dark light for 1 week under illumination intensity of 1500Lx, and culturing at 23 deg.C for 16 h. The culture was carried out for 6 weeks.
S3: subculturing: transferring the explant into a proliferation culture medium 30-40 days later when the shoot tip of the explant appears in the induction culture medium, and transferring the explant into a new proliferation culture medium every four weeks in a certain culture environment;
s4: rooting culture: and (3) when the explant of the tissue culture seedling of the linden tree after subculture has terminal buds, taking the terminal buds of 2-3 cm, and putting the terminal buds into a rooting culture medium until the rooting culture is finished.
By adopting the technical scheme, the stem tip is an explant commonly used for plant tissue culture. The reason is that the stem tip has high growth speed and high propagation rate, genetic variation is not easy to generate, and the method is an effective way for obtaining virus-free seedlings, so that the explant selects the stem tip part of the semi-lignified branch of healthy linden with plump current-year branch buds. The selected explant, the whole experimental environment and an appliance used by the explant are effectively cleaned and disinfected, the bacterial infection of bacteria and viruses in the air and the bacterial infection of the explant brought by operation are reduced, so that the success rate of an experiment is influenced, the explant is effectively disinfected by soaking in alcohol and a disinfectant, when a new shoot appears in the explant, the explant is placed into a multiplication culture medium to multiply cells, so that a terminal bud grows, a strong tree tissue culture seedling is subcultured, 2-3 cm terminal buds are taken and placed into a rooting culture medium to complete rooting culture, and as the pH value of body fluid of a plant is about 5.80, the pH value of the solution is controlled to about 5.80 when the culture medium is adjusted, agar added under the pH value becomes good in white coagulability, if the pH value is too low, the culture medium cannot be coagulated, and if the pH value is too high, the coagulation is too hard.
Further setting: the disinfectant comprises the following components: bleaching water 1 part and sterile water 3 parts.
Through adopting above-mentioned technical scheme setting, through the antiseptic solution that uses white cat bleaching water preparation, can effectually disinfect to the explant.
Further setting: the culture environment in the S3 is that the illumination intensity is 1500Lx, the temperature is 25 ℃, the illumination time is 16 hours, and the culture environment is placed for 8 hours in the dark condition, the temperature is 23 ℃.
By adopting the technical scheme, the photoperiod is continuously illuminated for 16h and is dark for 8h, thereby being beneficial to stem tip culture and bud differentiation and proliferation. The enhancement of illumination is beneficial to the rooting of the test-tube plantlet, and has good effect on the transplantation of the test-tube plantlet, but the illumination intensity is too strong, and once the roots are directly irradiated by strong light for a long time, the growth of the roots can be inhibited.
Further setting: the culture medium is sterilized, the prepared culture medium is placed in a sterilization pot, and sterilization is carried out for 15-30 min under the conditions that the pressure is 0.105MPa and the temperature is 121 DEG C
Through adopting above-mentioned technical scheme setting, not only need carry out effectual sterilization to the explant, more need carry out sterilization to the culture medium, reduce because the bacterial contamination culture medium that is infected with in the operation process.
Further setting: the culture medium is also added with an antioxidant which is cysteine or ascorbic acid.
Through adopting the technical scheme, in the culture medium, the browning phenomenon of a plurality of explants can be effectively avoided or reduced by using antioxidants such as cysteine, ascorbic acid and the like.
Further setting: the culture medium is also added with 0.1-0.5 g of activated carbon.
Through adopting above-mentioned technical scheme setting, the brown stain phenomenon of the reduction explant that can be better because its own is the adsorbent that the adsorptivity is stronger.
In conclusion, the invention has the following beneficial effects: the method has good disinfection success rate, and simultaneously, the explant germination success rate is high by matching with an induction culture medium, a sterile regeneration system can be effectively established, and the tissue culture seedling of the linden tree finally obtained by the method has high proliferation success rate and developed root system.
Drawings
Fig. 1 is a flow chart of a rapid propagation method of linden tree.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings.
Example 1: a tissue culture medium of linden tree is shown in figure 1 and comprises an induction culture medium, a proliferation culture medium and a rooting culture medium, wherein the induction culture medium consists of the following components: MS culture medium: 4630 mg; thidiazuron TDZ: 0.001 mg; gibberellin: 2g of the total weight of the mixture; agar: 5g of the total weight of the mixture; sucrose: 25g of the total weight of the mixture; inositol: 90mg, water is added to make up to 1 litre, the multiplication medium consists of the following components: MS culture medium: 4630 mg; thidiazuron TDZ: 0.001 mg; gibberellin: 2 mg; agar: 5g of the total weight of the mixture; sucrose: 25g of the total weight of the mixture; inositol: 90mg, adding water to make up to 1 liter, wherein the rooting medium consists of the following components: MS culture medium: 2315 mg; indole butyric acid IBA: 1.0 mg; naphthylacetic acid NAA: 1.0 mg; agar: 5g of the total weight of the mixture; sucrose: 10g of a mixture; water was added to make up to 1 liter.
A rapid propagation method of linden tree is shown in figure 1:
s1: selecting an explant, wherein the explant selects a stem tip part of a half-lignified branch with a plump bearing shoot bud of a healthy linden tree;
s2: the method comprises the following steps of (1) sterilizing and primary culture, cutting leaves of a linden explant, washing the leaves with clear water, putting the washed explant into 75% alcohol on a workbench for sterilizing for 30 seconds, washing the explant with sterile water for 3-4 times, soaking the explant in a disinfectant for 60 minutes, and inoculating the explant into an induction culture medium after the sterilization is finished; culturing at 16 deg.C under dark light for 1 week under illumination intensity of 1500Lx, culturing at 23 deg.C under illumination time of 16h for 6 weeks.
S3: subculturing: transferring the explant into a proliferation culture medium 30-40 days later when the shoot tip of the explant appears in the induction culture medium, and transferring the explant into a new proliferation culture medium every four weeks in a certain culture environment;
s4: rooting culture: and (3) when the explant of the tissue culture seedling of the linden tree after subculture has terminal buds, taking the terminal buds of 2-3 cm, and putting the terminal buds into a rooting culture medium until the rooting culture is finished.
Example 2: a tissue culture medium of linden trees comprises an induction culture medium, a proliferation culture medium and a rooting culture medium, wherein the induction culture medium comprises the following components: MS culture medium: 4640 mg; thidiazuron TDZ: 0.010 mg; gibberellin: 3 mg; agar: 8g of the total weight of the mixture; sucrose: 40g of the total weight of the mixture; inositol: 110mg, water is added to make up to 1 liter, and the multiplication medium consists of the following components: MS culture medium: 4640 mg; thidiazuron TDZ: 0.010 mg; gibberellin: 3 mg; agar: 8g of the total weight of the mixture; sucrose: 40g of the total weight of the mixture; inositol: 110mg, adding water to make up to 1 liter, wherein the rooting medium consists of the following components: MS culture medium: 2320 mg; indole butyric acid IBA: 2.0 mg; naphthylacetic acid NAA: 2.0 mg; agar: 8g of the total weight of the mixture; sucrose: 20g of the total weight of the mixture; water was added to make up to 1 liter.
A rapid propagation method of linden tree is shown in figure 1:
s1: selecting an explant, wherein the explant selects a stem tip part of a half-lignified branch with a plump bearing shoot bud of a healthy linden tree;
s2: the method comprises the following steps of (1) sterilizing and primary culture, cutting leaves of a linden explant, washing the leaves with clear water, putting the washed explant into 75% alcohol on a workbench for sterilizing for 30 seconds, washing the explant with sterile water for 3-4 times, soaking the explant in a disinfectant for 60 minutes, and inoculating the explant into an induction culture medium after the sterilization is finished; culturing at 16 deg.C under dark light for 1 week under illumination intensity of 1500Lx, culturing at 23 deg.C under illumination time of 16h for 6 weeks.
S3: subculturing: transferring the explant into a proliferation culture medium 30-40 days later when the shoot tip of the explant appears in the induction culture medium, and transferring the explant into a new proliferation culture medium every four weeks in a certain culture environment;
s4: rooting culture: and (3) when the explant of the tissue culture seedling of the linden tree after subculture has terminal buds, taking the terminal buds of 2-3 cm, and putting the terminal buds into a rooting culture medium until the rooting culture is finished.
Example 3: a tissue culture medium of linden trees comprises an induction culture medium, a proliferation culture medium and a rooting culture medium, wherein the induction culture medium comprises the following components: MS culture medium: 4636.43 mg; thidiazuron TDZ: 0.005 mg; gibberellin: 2.5 mg; agar: 6g of a mixture; sucrose: 30g of the total weight of the mixture; inositol: 100mg, adding water to make up to 1 liter, wherein the proliferation medium consists of the following components: MS culture medium: 4636.43 mg; thidiazuron TDZ: 0.005 mg; gibberellin: 2.0 mg; agar: 6g of a mixture; sucrose: 30g of the total weight of the mixture; inositol: 100mg, adding water to complement to 1 liter, wherein the rooting medium consists of the following components: MS culture medium: 2318.22 mg; indole butyric acid IBA: 1.5 mg; naphthylacetic acid NAA: 1.5 mg; agar: 6g of a mixture; sucrose: 15g of the total weight of the mixture; water was added to make up to 1 liter.
A rapid propagation method of linden tree is shown in figure 1:
s1: selecting an explant, wherein the explant selects a stem tip part of a half-lignified branch with a plump bearing shoot bud of a healthy linden tree;
s2: the method comprises the following steps of (1) sterilizing and primary culture, cutting leaves of a linden explant, washing the leaves with clear water, putting the washed explant into 75% alcohol on a workbench for sterilizing for 30 seconds, washing the explant with sterile water for 3-4 times, soaking the explant in a disinfectant for 60 minutes, and inoculating the explant into an induction culture medium after the sterilization is finished; culturing at 16 deg.C under dark light for 1 week under illumination intensity of 1500Lx, culturing at 23 deg.C under illumination time of 16h for 6 weeks.
S3: subculturing: transferring the explant into a proliferation culture medium 30-40 days later when the shoot tip of the explant appears in the induction culture medium, and transferring the explant into a new proliferation culture medium every four weeks in a certain culture environment;
s4: rooting culture: and (3) when the explant of the tissue culture seedling of the linden tree after subculture has terminal buds, taking the terminal buds of 2-3 cm, and putting the terminal buds into a rooting culture medium until the rooting culture is finished.
Example 4: a tissue culture medium of linden trees comprises an induction culture medium, a proliferation culture medium and a rooting culture medium, wherein the induction culture medium comprises the following components: MS culture medium: 4636.43 mg; thidiazuron TDZ: 0.008 mg; gibberellin: 2.9 g; agar: 6g of a mixture; sucrose: 30g of the total weight of the mixture; inositol: 100mg, adding water to make up to 1 liter, wherein the proliferation medium consists of the following components: MS culture medium: 4636.43 mg; thidiazuron TDZ: 0.008 mg; gibberellin: 2.9 mg; agar: 6g of a mixture; sucrose: 30g of the total weight of the mixture; inositol: 100mg, adding water to complement to 1 liter, wherein the rooting medium consists of the following components: MS culture medium: 2318.22 mg; indole butyric acid IBA: 1.8 mg; naphthylacetic acid NAA: 1.8 mg; agar: 6g of a mixture; sucrose: 15g of the total weight of the mixture; water was added to make up to 1 liter.
A rapid propagation method of linden tree is shown in figure 1:
s1: selecting an explant, wherein the explant selects a stem tip part of a half-lignified branch with a plump bearing shoot bud of a healthy linden tree;
s2: the method comprises the following steps of (1) sterilizing and primary culture, cutting leaves of a linden explant, washing the leaves with clear water, putting the washed explant into 75% alcohol on a workbench for sterilizing for 30 seconds, washing the explant with sterile water for 3-4 times, soaking the explant in a disinfectant for 60 minutes, and inoculating the explant into an induction culture medium after the sterilization is finished; culturing at 16 deg.C under dark light for 1 week under illumination intensity of 1500Lx, culturing at 23 deg.C under illumination time of 16h for 6 weeks.
S3: subculturing: transferring the explant into a proliferation culture medium 30-40 days later when the shoot tip of the explant appears in the induction culture medium, and transferring the explant into a new proliferation culture medium every four weeks in a certain culture environment;
s4: rooting culture: and (3) when the explant of the tissue culture seedling of the linden tree after subculture has terminal buds, taking the terminal buds of 2-3 cm, and putting the terminal buds into a rooting culture medium until the rooting culture is finished.
Example 5: a tissue culture medium of linden trees comprises an induction culture medium, a proliferation culture medium and a rooting culture medium, wherein the induction culture medium comprises the following components: MS culture medium: 4636.43 mg; thidiazuron TDZ: 0.002 mg; gibberellin: 2.1 g; agar: 6g of a mixture; sucrose: 30g of the total weight of the mixture; inositol: 100mg, adding water to make up to 1 liter, wherein the proliferation medium consists of the following components: MS culture medium: 4636.43 mg; thidiazuron TDZ: 0.002 mg; gibberellin: 2.1 mg; agar: 6g of a mixture; sucrose: 30g of the total weight of the mixture; inositol: 100mg, adding water to complement to 1 liter, wherein the rooting medium consists of the following components: MS culture medium: 2318.22 mg; indole butyric acid IBA: 1.1 mg; naphthylacetic acid NAA: 1.1 mg; agar: 6g of a mixture; sucrose: 15g of the total weight of the mixture; water was added to make up to 1 liter.
A rapid propagation method of linden tree is shown in figure 1:
s1: selecting an explant, wherein the explant selects a stem tip part of a half-lignified branch with a plump bearing shoot bud of a healthy linden tree;
s2: the method comprises the following steps of (1) sterilizing and primary culture, cutting leaves of a linden explant, washing the leaves with clear water, putting the washed explant into 75% alcohol on a workbench for sterilizing for 30 seconds, washing the explant with sterile water for 3-4 times, soaking the explant in a disinfectant for 60 minutes, and inoculating the explant into an induction culture medium after the sterilization is finished; culturing at 16 deg.C under dark light for 1 week under illumination intensity of 1500Lx, culturing at 23 deg.C under illumination time of 16h for 6 weeks.
S3: subculturing: transferring the explant into a proliferation culture medium 30-40 days later when the shoot tip of the explant appears in the induction culture medium, and transferring the explant into a new proliferation culture medium every four weeks in a certain culture environment;
s4: rooting culture: and (3) when the explant of the tissue culture seedling of the linden tree after subculture has terminal buds, taking the terminal buds of 2-3 cm, and putting the terminal buds into a rooting culture medium until the rooting culture is finished.
Example 6: a tissue culture medium of linden trees comprises an induction culture medium, a proliferation culture medium and a rooting culture medium, wherein the induction culture medium comprises the following components: MS culture medium: 4636.43 mg; thidiazuron TDZ: 0.005 mg; gibberellin: 2.0 g; agar: 6g of a mixture; sucrose: 30g of the total weight of the mixture; inositol: 100mg, adding water to make up to 1 liter, wherein the proliferation medium consists of the following components: MS culture medium: 4636.43 mg; thidiazuron TDZ: 0.005 mg; gibberellin: 2.0 mg; agar: 6g of a mixture; sucrose: 30g of the total weight of the mixture; inositol: 100mg, adding water to complement to 1 liter, wherein the rooting medium consists of the following components: MS culture medium: 2318.22 mg; indole butyric acid IBA: 1.5 mg; naphthylacetic acid NAA: 2 mg; agar: 6g of a mixture; sucrose: 15g of the total weight of the mixture; water was added to make up to 1 liter.
A rapid propagation method of linden tree is shown in figure 1:
s1: selecting an explant, wherein the explant selects a stem tip part of a half-lignified branch with a plump bearing shoot bud of a healthy linden tree;
s2: the method comprises the following steps of (1) sterilizing and primary culture, cutting leaves of a linden explant, washing the leaves with clear water, putting the washed explant into 75% alcohol on a workbench for sterilizing for 30 seconds, washing the explant with sterile water for 3-4 times, soaking the explant in a disinfectant for 60 minutes, and inoculating the explant into an induction culture medium after the sterilization is finished; culturing at 16 deg.C under dark light for 1 week under illumination intensity of 1500Lx, culturing at 23 deg.C under illumination time of 16h for 6 weeks.
S3: subculturing: transferring the explant into a proliferation culture medium 30-40 days later when the shoot tip of the explant appears in the induction culture medium, and transferring the explant into a new proliferation culture medium every four weeks in a certain culture environment;
s4: rooting culture: and (3) when the explant of the tissue culture seedling of the linden tree after subculture has terminal buds, taking the terminal buds of 2-3 cm, and putting the terminal buds into a rooting culture medium until the rooting culture is finished.
Example 7: a tissue culture medium of Tilia Miqueliana Maxim comprises induction culture medium, proliferation culture medium and rooting culture medium,
the induction culture medium consists of the following components: MS culture medium: 4636.43 mg; thidiazuron TDZ: 0.005 mg; gibberellin: 2.5 g; agar: 6g of a mixture; sucrose: 30g of the total weight of the mixture; inositol: 100mg, adding water to make up to 1 liter, wherein the proliferation medium consists of the following components: MS culture medium: 4636.43 mg; thidiazuron TDZ: 0.005 mg; gibberellin: 2.0 mg; agar: 6g of a mixture; sucrose: 30g of the total weight of the mixture; inositol: 100mg, adding water to complement to 1 liter, wherein the rooting medium consists of the following components: MS culture medium: 2318.22 mg; indole butyric acid IBA: : 2 mg; naphthylacetic acid NAA: 1.5 mg; agar: 6g of a mixture; sucrose: 15g of the total weight of the mixture; water was added to make up to 1 liter.
A rapid propagation method of linden tree is shown in figure 1:
s1: selecting an explant, wherein the explant selects a stem tip part of a half-lignified branch with a plump bearing shoot bud of a healthy linden tree;
s2: the method comprises the following steps of (1) sterilizing and primary culture, cutting leaves of a linden explant, washing the leaves with clear water, putting the washed explant into 75% alcohol on a workbench for sterilizing for 30 seconds, washing the explant with sterile water for 3-4 times, soaking the explant in a disinfectant for 60 minutes, and inoculating the explant into an induction culture medium after the sterilization is finished; culturing at 16 deg.C under dark light for 1 week under illumination intensity of 1500Lx, culturing at 23 deg.C under illumination time of 16h for 6 weeks.
S3: subculturing: transferring the explant into a proliferation culture medium 30-40 days later when the shoot tip of the explant appears in the induction culture medium, and transferring the explant into a new proliferation culture medium every four weeks in a certain culture environment;
s4: rooting culture: and (3) when the explant of the tissue culture seedling of the linden tree after subculture has terminal buds, taking the terminal buds of 2-3 cm, and putting the terminal buds into a rooting culture medium until the rooting culture is finished.
The virus detection is carried out after the explants are disinfected in the above examples 1 to 7, meanwhile, the growth condition of the young shoots is observed after the explants are placed in the induction culture medium, the proliferation rate is increased after the explants are placed in the proliferation culture medium, and the rooting rate and the rooting coefficient of the terminal buds placed in the rooting culture medium are analyzed and compared, and the results are shown in the following table:
according to the above table, the linden can be rapidly propagated by the induction culture medium, the multiplication culture medium and the rooting culture medium which are relatively proper in proportion, the rooting rate of the linden reaches more than 90%, the rooting coefficient reaches more than 4, meanwhile, the root system is developed and robust through observation, the multiplication rate is about 3-5 times, the method is completely suitable for industrial production of the linden, and the method is favorable for large-scale production of high-quality linden seedlings.
The present embodiment is only for explaining the present invention, and it is not limited to the present invention, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present invention.
Claims (7)
1. A tissue culture medium of linden trees comprises an induction culture medium, a proliferation culture medium and a rooting culture medium, and is characterized in that: the induction culture medium consists of the following components: MS culture medium: 4630-4640 mg, thidiazuron TDZ: 0.001-0.010 mg, gibberellin: 2-3 mg, agar: 5-8 g, sucrose: 25-40 g, inositol: 90-110 mg, adding water to make up to 1 liter, wherein the multiplication medium consists of the following components: MS culture medium: 4630-4640 mg, thidiazuron TDZ: 0.001-0.010 mg, gibberellin: 2-3 mg, agar: 5-8 g, sucrose: 25-40 g, inositol: 90-110 mg, adding water to complement to 1 liter, wherein the rooting medium consists of the following components: MS culture medium: 2315-2320 mg, indolebutyric acid IBA: 1.0 mg-2.0 mg, NAA: 1.0mg to 2.0mg, agar: 5-8 g, sucrose: 10-20 g, adding water to make up to 1 liter.
2. The tissue culture medium of linden tree of claim 1, wherein: the induction culture medium consists of the following components: MS culture medium: 4636.43mg, thidiazuron TDZ: 0.005mg, gibberellin: 2.5mg, agar: 6g, sucrose: 30g, inositol: 100mg, adding water to make up to 1 liter, wherein the proliferation medium consists of the following components: MS culture medium: 4636.43mg, thidiazuron TDZ: 0.005mg, gibberellin: 2.0mg, agar: 6g, sucrose: 30g, inositol: 100mg, adding water to complement to 1 liter, wherein the rooting medium consists of the following components: 2318.22mg, indolebutyric acid IBA: 1.5mg, NAA naphthalene acetate: 1.5mg, agar: 6g, sucrose: 15g, add water to make up to 1 liter.
3. A rapid propagation method of linden trees is characterized in that: comprises the following culture steps:
s1: selecting an explant, wherein the explant selects the full semi-lignified shoot tip part of the bud of the current-year branch of the healthy linden tree;
s2: the method comprises the following steps of (1) disinfection and primary culture, cutting leaves of a linden explant, washing the leaves with clear water, putting the washed explant into 75% alcohol on a workbench for disinfection for 30 seconds, washing the explant with sterile water for 3-4 times, soaking the explant in a disinfectant for 60 minutes, inoculating the explant into an induction culture medium according to claim 1 after disinfection, culturing the explant in a dark light at 16 ℃ for 1 week and then at 23 ℃ for 16 hours under the condition of illumination intensity of 1500Lx, and culturing the explant for 6 weeks; the disinfectant comprises the following components: white cat bleaching water 1 part and sterile water 3 parts;
s3: subculturing: transferring the explant into the proliferation culture medium according to claim 1 when a shoot of the stem tip of the explant appears 30-40 days in the induction culture medium, and transferring the explant into a new proliferation culture medium every four weeks under a certain culture environment;
s4: rooting culture: when the explant of the tissue culture seedling of the linden tree after subculture has terminal buds, taking the terminal buds of 2-3 cm, and putting the terminal buds into the rooting culture medium according to claim 1 until rooting is finished.
4. The rapid propagation method of tilia amurensis as claimed in claim 3, wherein: in the step S3, the culture environment is that the illumination intensity is 1500Lx, the temperature is 25 ℃, the illumination time is 16 hours, and the culture environment is placed for 8 hours in the dark condition, the temperature is 23 ℃.
5. The rapid propagation method of tilia amurensis as claimed in claim 3, wherein: the culture medium is sterilized, the prepared culture medium is placed in a sterilization pot, and sterilization is carried out for 15-30 min under the conditions that the pressure is 0.105MPa and the temperature is 121 ℃.
6. The rapid propagation method of tilia amurensis as claimed in claim 3, wherein: an antioxidant is also added into the culture medium, and the antioxidant is cysteine.
7. The rapid propagation method of tilia amurensis as claimed in claim 3, wherein: the culture medium is also added with 0.1-0.5 g of activated carbon.
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| Publication number | Priority date | Publication date | Assignee | Title |
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Non-Patent Citations (3)
| Title |
|---|
| 南京椴组织培养快繁体系的建立;汤诗杰等;《江苏农业科学》;20081231(第6期);第87-89页 * |
| 日本椴的组织培养与快速繁殖;陈罡等;《植物生理学通讯》;20070430;第43卷(第2期);第327页第3-4部分 * |
| 紫椴的组织培养技术研究;王彦彬等;《防护林科技》;20020331(第2期);第37-39页 * |
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