CN111955345A - Method and culture medium for culturing elaeagnus plant tissues by using leaves - Google Patents
Method and culture medium for culturing elaeagnus plant tissues by using leaves Download PDFInfo
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- CN111955345A CN111955345A CN202010697957.1A CN202010697957A CN111955345A CN 111955345 A CN111955345 A CN 111955345A CN 202010697957 A CN202010697957 A CN 202010697957A CN 111955345 A CN111955345 A CN 111955345A
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- 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
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- A01H4/008—Methods for regeneration to complete plants
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Abstract
The invention relates to the field of plant tissue culture, and provides a method for performing tissue culture on elaeagnus plants by using leaves, and a corresponding regeneration culture medium. The method comprises the following steps: obtaining leaves from a plant of the genus elaeagnus, wherein the plant of the genus elaeagnus is a sterile plant; cutting the blade, thereby obtaining a blisk; the leaf discs are placed on a regeneration medium for culturing, thereby obtaining regenerated buds. The tissue culture method of the elaeagnus plants with high efficiency and high survival rate can be used for culturing the elaeagnus plants in large batch.
Description
Technical Field
The invention relates to the field of plant tissue culture, in particular to a method for carrying out tissue culture on elaeagnus plants by using leaves and a corresponding regeneration culture medium.
Background
Elaeagnus angustifolia L is a plant of the genus Elaeagnus (Elaeagnus), originating in North America and Europe, and naturally distributed in the Caucasian and middle Asia regions. The leaves of the oleaster are rich in nutrition and can be used as food of livestock, the flower fragrance can be used as a source of nectar, the fruits can be used as substitute food, the kernel contains more than 20% of oil, and the root system of the oleaster is symbiotic with rhizobium so that the oleaster can fix nitrogen from soil, so the oleaster is considered as a sapling as a food and oil source by many people and is beneficial to ecology. Elaeagnus angustifolia is also an environmentally important species that grows rapidly and is tolerant to drought, salt and alkali, and sand burial. Planting oleaster is an ideal choice for controlling desertification of land (e.g., desertification in the northwest region of china), and increasing the income of local farmers.
In order to be able to grow such trees efficiently and economically, it is critical to provide efficient regeneration and breeding methods to produce plant material and to effect genetic transformation of the plant material to improve tree traits.
Compared with the traditional breeding method, the tissue culture method can be used for the rapid production and planting of elaeagnus angustifolia. However, the existing tissue culture method of Elaeagnus angustifolia has the defects of small quantity of expanding culture, low proliferation rate, rooting rate and survival rate and the like, and has the problems of vitrification and the like in the culture process. Therefore, a tissue culture method of elaeagnus angustifolia with high efficiency and high survival rate is urgently needed in the field.
Disclosure of Invention
The invention aims to provide a method for tissue culture of elaeagnus plants and a corresponding regeneration medium.
An aspect of the application provides a tissue culture method for elaeagnus plants, the method comprising: (a) obtaining leaves from a plant of the genus elaeagnus, wherein the plant of the genus elaeagnus is a sterile plant; (b) cutting the blade, thereby obtaining a blisk; (c) the leaf discs are placed on a regeneration medium for culturing, thereby obtaining regenerated buds.
In some embodiments, the elaeagnus plant is elaeagnus angustifolia.
In some embodiments, the leaf discs have a size of 0.6 to 1.5cm by 0.6 to 1.5cm, preferably about 1cm by 1 cm.
In some embodiments, the blade is severed from the petiole. In some embodiments, the length of the petiole to which the blade remains attached after the blade is cut from the petiole is about 1.0mm to 3.5mm, 1.5mm to 3.0mm, 2.0mm to 2.5mm, or about 2 mm.
In some embodiments, the cutting is further performed at a main vein of the leaf. In some embodiments, the cutting is performed at one, two, three, or four locations on the main veins of the leaf.
In some embodiments, the regeneration medium comprises a basal medium, agar, and at least one phytohormone.
In some embodiments, the basal medium in the regeneration medium is WPM medium.
In some embodiments, the agar in the regeneration medium is at a concentration of 5g/L to 20g/L, 8g/L to 15g/L, 8g/L to 12g/L, or 6g/L to 10 g/L.
In some embodiments, the concentration of said phytohormone in the regeneration medium is from 0.01mg/L to 5mg/L, 0.02mg/L to 4mg/L, 0.03mg/L to 3mg/L, 0.05mg/L to 2.5mg/L, 0.08mg/L to 2.5mg/L, 0.1mg/L to 2.5mg/L, 0.2mg/L to 2.5mg/L, 0.5mg/L to 2.2mg/L, 0.8mg/L to 2.2mg/L, or 1mg/L to 2mg/L (w/v). In some embodiments, the phytohormone in the regeneration medium is selected from the group consisting of an auxin, a cytokinin, a gibberellin, or a combination thereof. In some embodiments, the regeneration medium does not include gibberellin. In some embodiments, the phytohormone in the regeneration medium is a combination of auxin and cytokinin or cytokinin alone. In some embodiments, the cytokinin in the regeneration medium comprises 6-benzyladenine. In some embodiments, the concentration of the 6-benzyladenine in the regeneration medium is 0.01mg/L to 5mg/L, 0.02mg/L to 4mg/L, 0.03mg/L to 3mg/L, 0.05mg/L to 2.5mg/L, 0.08mg/L to 2mg/L, 0.1mg/L to 1.8mg/L, 0.2mg/L to 1.5mg/L, 0.3mg/L to 1.2mg/L, 0.4mg/L to 1mg/L, 0.4mg/L to 0.8mg/L, or 0.4mg/L to 0.6 mg/L. In some embodiments, said plant auxin in the regeneration medium comprises indole-3-butyric acid. In some embodiments, the indole-3-butyric acid in the regeneration medium is at a concentration of 0.01mg/L to 1mg/L, 0.02mg/L to 0.8mg/L, 0.03mg/L to 0.5mg/L, 0.05mg/L to 0.4mg/L, 0.06mg/L to 0.3mg/L, 0.08mg/L to 0.2mg/L, or 0.1 mg/L.
In some embodiments, the regeneration medium further comprises hydrolyzed protein. In some embodiments, the hydrolyzed protein is hydrolyzed casein. In some embodiments, the concentration of the hydrolyzed protein in the regeneration medium is 0.1g/L to 5g/L, 0.2g/L to 3g/L, 0.5g/L to 2g/L, 0.8g/L to 1.5g/L, 0.9g/L to 1.2g/L, or 1 g/L.
In some embodiments, the regeneration medium further comprises sucrose. In some embodiments, the sucrose concentration in the regeneration medium is from 10g/L to 50g/L, 15g/L to 40g/L, 20g/L to 40g/L, or 10g/L to 30 g/L.
In some embodiments, activated carbon is not included in the regeneration medium.
In some embodiments, the regeneration medium has a pH of 4.5-6.5, 5-6, 5.5-6, or 5.8.
In some embodiments, the regeneration medium comprises WPM salt, WPM organic, 10g/L to 30g/L sucrose, 0.1g/L to 1.5g/L hydrolyzed casein, 6g/L to 10g/L agar, 0.4mg/L to 0.6 mg/L6-benzyladenine, and 0.08mg/L to 0.2mg/L indole-3-butyric acid.
In some embodiments, step (c) comprises subculture. In some embodiments, the regeneration medium is renewed at least once or at least twice. In some embodiments, each subculture is separated by a period of 1 week to 1 month.
In some embodiments, the container used for culturing in step (c) is provided with air-permeable pores, preferably the lid of the container is provided with air-permeable pores, more preferably the lid of the container has a pore sieve with a pore size of 0.2-0.45 μm.
In some embodiments, the culturing in step (c) is performed at a temperature of 23 ℃ to 27 ℃, preferably at a temperature of 26 ℃.
In some embodiments, the culturing in step (c) is performed under conditions of 16 hours light (3000lux) per day, followed by 8 hours of darkness.
In some embodiments, the method further comprises: (d) transferring the material from step (c) together with the culture medium into a culture flask and culturing after adding sterile water, thereby obtaining an acclimatized seedling.
In some embodiments, the culture flask of step (d) is semi-open. In some embodiments, the culture flask of step (d) is a bottle cap. In some embodiments, the sterile water of step (d) submerges the culture medium. In some embodiments, the sterile water is 0.5cm to 2cm, 0.5cm to 1.5cm, 0.5cm to 1cm, 1cm to 2cm, or about 1cm above the culture medium. In some embodiments, said culturing in step (d) is performed at a temperature of 23 ℃ to 27 ℃, preferably at a temperature of 26 ℃. In some embodiments, the culturing in step (d) is performed under conditions of 10 hours light (4000lux) per day followed by 14 hours dark. In some embodiments, the culturing in step (d) is for at least 1 day, 2 days, 3 days, or 4 days.
In some embodiments, the method further comprises: (e) and (3) planting the acclimatized seedlings into a mixture mixed by vermiculite and perlite in a volume ratio of 3 to 1. In some embodiments, the acclimatized seedling is subjected to step (e) after being removed from the culture flask and washed with running water to remove the culture medium.
In another aspect of the present invention, there is provided a regeneration medium for regenerating leaves in tissue culture of an elaeagnus plant, the regeneration medium comprising a basal medium, agar and at least one phytohormone.
In some embodiments, the basal medium is WPM medium.
In some embodiments, the concentration of the phytohormone is 0.01mg/L to 5mg/L, 0.02mg/L to 4mg/L, 0.03mg/L to 3mg/L, 0.05mg/L to 2.5mg/L, 0.08mg/L to 2.5mg/L, 0.1mg/L to 2.5mg/L, 0.2mg/L to 2.5mg/L, 0.5mg/L to 2.2mg/L, 0.8mg/L to 2.2mg/L, or 1mg/L to 2mg/L (w/v). In some embodiments, the plant hormone is selected from an auxin, a cytokinin, a gibberellin, or a combination thereof. In some embodiments, the regeneration medium does not include gibberellin. In some embodiments, the plant hormone is a combination of auxin and cytokinin or cytokinin alone. In some embodiments, the cytokinin comprises 6-benzyladenine. In some embodiments, the concentration of 6-benzyladenine is 0.01mg/L to 5mg/L, 0.02mg/L to 4mg/L, 0.03mg/L to 3mg/L, 0.05mg/L to 2.5mg/L, 0.08mg/L to 2mg/L, 0.1mg/L to 1.8mg/L, 0.2mg/L to 1.5mg/L, 0.3mg/L to 1.2mg/L, 0.4mg/L to 1mg/L, 0.4mg/L to 0.8mg/L, or 0.4mg/L to 0.6 mg/L. In some embodiments, the plant auxin comprises indole-3-butyric acid. In some embodiments, the indole-3-butyric acid has a concentration of 0.01mg/L to 1mg/L, 0.02mg/L to 0.8mg/L, 0.03mg/L to 0.5mg/L, 0.05mg/L to 0.4mg/L, 0.06mg/L to 0.3mg/L, 0.08mg/L to 0.2mg/L, or 0.1 mg/L.
In some embodiments, the regeneration medium further comprises hydrolyzed protein. In some embodiments, the hydrolyzed protein is hydrolyzed casein. In some embodiments, the hydrolyzed protein has a concentration of 0.1g/L to 5g/L, 0.2g/L to 3g/L, 0.5g/L to 2g/L, 0.8g/L to 1.5g/L, 0.9g/L to 1.2g/L, or 1 g/L.
In some embodiments, the regeneration medium further comprises sucrose. In some embodiments, the sucrose concentration is from 10g/L to 50g/L, from 15g/L to 40g/L, from 20g/L to 40g/L, or from 10g/L to 30 g/L.
In some embodiments, activated carbon is not included in the regeneration medium.
In some embodiments, the agar has a concentration of 5g/L to 20g/L, 8g/L to 15g/L, 8g/L to 12g/L, or 6g/L to 10 g/L.
In some embodiments, the regeneration medium has a pH of 4.5-6.5, 5-6, 5.5-6, or about 5.8.
In some embodiments, the regeneration medium comprises WPM salt, WPM organic, 10g/L to 30g/L sucrose, 0.1g/L to 1.5g/L hydrolyzed casein, 6g/L to 10g/L agar, 0.4mg/L to 0.6 mg/L6-benzyladenine, and 0.08mg/L to 0.2mg/L indole-3-butyric acid.
Drawings
Fig. 1 shows the appearance of a blisk obtained by cutting a blade according to the method of example 1 of the present application.
Fig. 2 is a schematic view of a blade cut according to the method of the present application to obtain a full leaf disc and a half leaf disc, respectively. Wherein, the plate A is a schematic diagram of a whole-leaf blade disc obtained by cutting the blades according to the method of the application, wherein after the whole blades are cut along the solid line and the shadow part is discarded, the rest part is the whole-leaf blade disc; and the plate B is a schematic diagram of a half-blade disc obtained by cutting blades according to the method, wherein after the full-blade disc is obtained according to the mode shown by the plate A, the full-blade disc is cut along a dotted line and a shadow part is discarded, and the rest part is the half-blade disc.
FIG. 3 shows the growth of Elaeagnus angustifolia at various stages during the cultivation of the leaf discs in regeneration medium according to the method of example 1 of the present application. Wherein panel A shows the outgrowth of regeneration shoots from the callus of leaf discs; panel B shows that regenerated shoots begin to extract and form stems; panel C showed further elongation of the stem; panel D shows tissue culture seedlings as viewed from the top when the regenerative culture is completed; panel E shows the roots of the tissue culture plantlets as seen from the bottom when the regeneration culture was completed.
FIG. 4 shows a comparison of growth after culturing leaf disks in regeneration medium containing different kinds and concentrations of phytohormones in example 2 of the present application. Wherein, the growth of leaf discs after culturing with regeneration medium containing 0.5 mg/L6-benzyladenine and 0.1mg/L indole-3-butyric acid, 0.5 mg/L6-benzyladenine and 0.1mg/L alpha-naphthylacetic acid, and 0.3 mg/L6-benzyladenine and 0.05mg/L alpha-naphthylacetic acid is shown from left to right.
Fig. 5 shows a comparison of growth using half-leaf discs and using full-leaf discs as starting materials in example 2 of the present application. Among them, a whole-leaf disk was used in the No. 4 dish, and a half-leaf disk was used in the No. 1-3 dish.
Detailed Description
The present application provides methods and corresponding media for tissue culture of elaeagnus plants.
In one aspect, the present application provides a tissue culture method for elaeagnus plants, the method comprising: (a) obtaining leaves from a plant of the genus elaeagnus, wherein the plant of the genus elaeagnus is a sterile plant; (b) cutting the blade, thereby obtaining a blisk; (c) the leaf discs are placed on a regeneration medium for culturing, thereby obtaining regenerated buds.
Plant of Elaeagnus
The term "Elaeagnus plant" as used in this application refers to any plant that can be classified as Elaeagnus (Elaeagnus) in plant taxonomy. Plants of the genus elaeagnus include, but are not limited to: elaeagnus angustifolia, elaeagnus taiwan elaeagnus, elaeagnus hongkongensis, elaeagnus lancifolia, acerola elaeagnus, elaeagnus acontana, elaeagnus wushanensis, elaeagnus within elaeagnus, elaeagnus angustifolia, elaeagnus pungens, elaeagnus angustifolia, elaeagnus quinata, elaeagnus longifolia, elaeagnus quinata, elaeagnus angustifolia, elaeagnus gracilifolia, elaeagnus angustifolia, elaeag. In some embodiments, the elaeagnus plant is elaeagnus angustifolia.
Explant
The term "explant" as used in this application refers to a segment of an organ or tissue that is the material of ex vivo culture in plant tissue culture. In the present application, the explants used are derived from leaves.
Because the tolerance of the leaves to the degerming agent is generally poor, after the leaves are subjected to effective degerming treatment, the leaves are difficult to keep strong enough activity, so that the survival rate of the leaves in the subsequent tissue culture process is difficult to guarantee. Thus, leaves directly from sterile plants are used as explants in this application. In some embodiments, the sterile elaeagnus plants provided for use in the methods described herein are plants obtained by sterile tissue culture.
Leaves can be obtained from sterile elaeagnus plants using methods conventional in the art. For example, the desired leaf can be separated from the plant by cutting (e.g., at the petiole) using a sterile bladed instrument. Sterile bladed instruments that may be used include, but are not limited to, sterile blades, sterile scalpels, and sterile scissors.
In the method of the present application, cutting of the blade to obtain a blisk is included. In some embodiments, the cutting of the present application is a substantially straight cutting. In some embodiments, the method of the present application uses a full leaf disc, i.e. obtained by cutting the leaf substantially according to the shape of the inscribed rectangle of the complete leaf. In some embodiments, the methods of the present application use a half-leaf disc, i.e. obtained by further cutting a full-leaf disc in a direction substantially perpendicular to the main veins. A schematic of the whole leaf disc and half leaf disc cutting method can be seen in fig. 2.
In some embodiments, the leaf discs have a size of 0.6 to 1.5cm x 0.6 to 1.5cm or about 1cm x 1 cm.
In some embodiments, the blisk includes at least a portion of a petiole. In some embodiments, the blisk comprises a petiole having a length of about 1.0mm to 3.5mm, 1.5mm to 3.0mm, 2.0mm to 2.5mm, or about 2 mm.
In some embodiments, the wound may be further created on the leaf disc by cutting to induce callus formation, for example, cutting at one, two, three or four of the main veins, wherein the cuts formed by the cutting intersect the main veins but not the edges of the leaf disc, preferably the cuts formed by the cutting are substantially perpendicular to the main veins. In some embodiments, the length of the incision is no more than 0.5mm, no more than 1mm, no more than 1.5mm, no more than 2mm, no more than 2.5mm, no more than 3mm, no more than 3.5mm, no more than 4mm, no more than 4.5mm, no more than 5mm, no more than 5.5mm, no more than 6mm, no more than 6.5mm, no more than 7mm, no more than 7.5mm, no more than 8mm, no more than 8.5mm, no more than 9mm, no more than 9.5mm, no more than 10mm, no more than 10.5mm, no more than 11mm, no more than 11.5mm, no more than 12 mm.
Regenerative culture
In the application, the potential totipotency of cells in a leaf disc is restored by culturing the leaf disc with wounds in a regeneration medium, so that callus is formed, and regeneration buds are differentiated from the callus to grow into tissue culture seedlings. The term "tissue culture seedling" as used herein refers to an explant that has been grown to a whole plant by tissue culture.
In some embodiments, after the plant tissue is cultured in the regeneration medium for a period of time, the regeneration medium is reconstituted fresh and the plant tissue cultured in the original regeneration medium is transferred to the freshly prepared regeneration medium, i.e., subcultured. In some embodiments, the subculture is performed at least once, at least twice, at least three times, at least four times, at least five times, or at least six times. In some embodiments, each subculture is separated by a period of 1 week to 1 month, e.g., 1 week, 2 weeks, 10 days, 15 days, 20 days, or 1 month.
In some embodiments, the leaf disks described herein are cultured in the regeneration medium described herein for a total time of at least 20 days, at least 30 days, at least 40 days, at least 50 days, at least 60 days, at least 70 days, at least 80 days, at least 90 days, or at least 100 days.
In some embodiments, the container used for culturing in the methods described herein is vented. In some embodiments, the lid of the container is vented. In some embodiments, the lid of the container has a pore screen with a pore size of 0.2 to 0.45 microns. The container used for culturing in the methods described herein may be a petri dish, a test tube, a triangular flask, an eppendorf tube, a children's food bottle, a can bottle, or any other container capable of supporting growth of elaeagnus tissue according to the methods of the invention.
In some embodiments, the culturing in the methods described herein is performed at a temperature of 23 ℃ to 27 ℃. In some embodiments, the culturing in the methods described herein is performed at a temperature of 26 ℃. In some embodiments, the culturing in the methods described herein is performed under conditions of 16 hours light (3000lux) per day, followed by 8 hours of darkness.
The regenerative culture described herein is performed in a sterile environment.
Regeneration medium
In some embodiments, the regeneration medium for the tissue culture methods of the present application comprises a basal medium, agar, and at least one phytohormone.
The basic culture medium refers to a culture medium which provides inorganic salts and organic substances required for plant growth. The inorganic salts include a large amount of salt, Fe salt and trace amount of salt. A wide variety of salts include, but are not limited to, potassium-containing, calcium-containing, sodium-containing, magnesium-containing, nitrogen-containing, phosphorus-containing, and/or sulfur-containing inorganic salts, particularly soluble inorganic salts. Fe salts refer to iron-containing inorganic salts such as, but not limited to, ferrous sulfate, ferric EDTA, and the like. Trace salts include, but are not limited to, inorganic salts, particularly soluble inorganic salts, containing manganese, zinc, copper, molybdenum, cobalt, boron, iodine, and/or chlorine, and the like. The organic matter in the basal medium comprises vitamins and amino acids, wherein the vitamins include but are not limited to vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin C, vitamin D, vitamin E, vitamin K, nicotinic acid, biotin, folic acid and pantothenic acid; wherein the amino acids include, but are not limited to, alanine, arginine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and the like. In some embodiments, the organic matter in the basal medium comprises inositol, niacin, vitamin B6, vitamin B1, glycine, folic acid, and biotin. Basic media conventional in the art can be selected for use in the various media of the present application. In some embodiments, the basal medium in the media described herein is MS medium, WPM medium, B5 medium, Ni medium, GD medium, NN medium, MT medium, or N6 medium. A representative recipe for each of the basal media described above is shown in Table 1.
In some embodiments, the inorganic salts and organic matter in the basal medium are used in normal amounts in the media described herein. In some embodiments, reduced or increased amounts of inorganic salts and organic matter in the basal medium are used in the media described herein, for example 1/5, 1/4, 1/3, 1/2, 2/3, or 3/4 amounts of inorganic salts and organic matter in the basal medium or 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 times the amount of inorganic salts and organic matter in the basal medium.
In some embodiments, the basal medium in the media described herein is MS medium. MS medium is a medium formulation developed by Murashige and Skoog in 1962 (see Murashige T.&Skoog F., A recycled Medium for Rapid Growth and Bioassays with Tobacco Tissue culture plant Physiol 15: 473-. Representative formulations are shown in table 1). MS medium is commercially available. In some embodiments, the MS medium used in the present application is purchased from PhytoTechnology
Item number L467. The term "MS salt" as used in this application means 1650mg/L NH4NO3、1900mg/L KNO3、440mg/L CaCl2·2H2O、370mg/L MgSO4·7H2O、170mg/L KH2PO4、37.3mg/L Na2-EDTA、27.8mg/L FeSO4·7H2O、0.83mg/L KI、6.2mg/L H3BO3、22.3mg/L MnSO4·4H2O、8.6mg/L ZnSO4·7H2O、0.25mg/LNa2MoO4·2H2O、0.025mg/L CuSO4·5H2O and 0.025mg/L CoCl2·6H2A combination of O. The term "MS organics" as used in this application refers to a combination of 100mg/L inositol, 0.5mg/L niacin, 0.5mg/L vitamin B6, 0.1mg/L vitamin B1, and 2mg/L glycine. The term "1/2 MS salt" as used in this application refers to an MS salt in which the concentration of each salt is halved; "1/2 MS organics" refers to MS organics in which the concentration of each organic is halved.
In some embodiments, the basal medium in the media described herein is WPM medium. WPM medium is a medium formulation developed by Lloyd and McCown in 1980 (see Lloyd G).&McCown, commercial-Feasible Micropropagation of Mountain Laurel, Kalmia Latifolia, by Use of shot-Tip Culture, B., int.plant Prop.Soc.Proc.30,421 (1980). Representative formulations are shown in table 1). WPM medium is commercially available. In some embodiments, the WPM media used in the present application is purchased from PhytoTechnology
Item number L449. The term "WPM salts" as used in this application"means 400mg/L NH4NO3、72.5mg/L CaCl2、386mg/L Ca(NO3)2、180.7mg/L MgSO4、170mg/L KH2PO4、990mg/L K2SO4、37.3mg/L N2-EDTA、27.85mg/L FeSO4·7H2O、6.2mg/L H3BO3、22.3mg/L MnSO4·H2O、8.6mg/L ZnSO4·7H2O、0.25mg/L Na2MoO4·2H2O and 0.25mg/L CuSO4·5H2A combination of O. The term "WPM organic" as used in this application refers to a combination of 100mg/L inositol, 0.5mg/L niacin, 0.5mg/L vitamin B6, 1mg/L vitamin B1, and 2mg/L glycine. The term "1/2 WPM salt" as used in this application refers to a WPM salt wherein the concentration of each salt is halved; "1/2 WPM organics" refers to WPM organics in which the concentration of each organic is halved.
In some embodiments, the basal medium in the media described herein is B5 medium. The B5 medium is a medium formulation developed by Gamborg et al in 1968 (see Gamborg et al, Nutrient requirements of Cultures of Soybean Root Cells, exp. cell Res.,50,151 (1968.) representative formulations are given in Table 1). The B5 medium is commercially available. In some embodiments, the B5 medium used in the present application is purchased from PhytoTechnology
The good number is G398. The term "B5 salt" as used in this application refers to KNO at 2500mg/L3、150mg/L CaCl2·2H2O、250mg/L MgSO4·7H2O、134mg/L(NH4)2SO4、150mg/L NaH2PO4·H2O、37.3mg/L Na2-EDTA、27.8mg/L FeSO4·7H2O、0.75mg/L KI、3mg/L H3BO3、10mg/L MnSO4·H2O、2mg/L ZnSO4·7H2O、0.25mg/L Na2MoO4·2H2O、0.025mg/LCuSO4·5H2O and 0.025mg/LCoCl2·6H2A combination of O. The term "B5 organic" as used herein refers to a combination of 100mg/L inositol, 1mg/L niacin, 1mg/L vitamin B6, and 10mg/L vitamin B1.
Ni media is a media formulation developed by Nielsen in 1960 (see Nielsen LW, immunization of the Internal core Virus by Culturing applications for fermented Sweet spots, Phytopathology 50:840-841 (1960.) representative formulations are given in Table 1). GD Medium is a medium formulation developed by Gresshoff and Doy in 1972 (see Gresshoff P.M.et. al., Haploid Arabidopsis Thaliana plus and Plants from Heat Culture, Aust.J.biol.Sci,25,259 (1972); Gresshoff P.M.et. al., Derivation of a Haploid Cell Line from View and the Import of the Stage of biological Development of Heat for Haploid Culture of This Othera, Z.Pflanzenysiol.73, 132-141 (1974.) representative formulations are shown in Table 1). NN medium is a medium formulation developed by Nitsch and Nitsch in 1969 (see Nitsch J.P. & Nitsch C., Haploid Plants from Pollen Grains, Science 169,85 (1969); representative formulations of Nitsch J.P.Experimental agriculture in Nicotiana, Phytophoromy 19,389(1969) are shown in Table 1). MT medium is a medium formulation developed by Murashage and Tucker in 1969 (see Murashige T & Tucker DPH, Growth Factor Requirements of Citrus Tissue Culture, Pp.1155-1161in Chapman II D. (ed.). Proc.1st int. Citrus Symp. Vol.3, Univ.Calif., Riverside Publication (1969). representative formulations are shown in Table 1). The N6Medium is a Medium formulation developed by Chu in 1978 (see Chu C.C., The N6Medium and its Application to other Culture of Central Crops, Proc. Symp. plant Tissue Culture, Peking,43 (1978); Chu C.C.et al., expression of Rice Through comprehensive Experiments on The Nitrogen Source Sound, science silicon, 18,659(1975) for representative formulations, see Table 1).
Table 1: representative basal Medium formulations
Agar is a high molecular weight polysaccharide substance comprising 1, 3-linked β -D-galactopyranose linked to 1, 4-linked 3, 6-lacto- α -L-galactopyranose in repeating alternating chains. Agar acts primarily as a solidifying medium and does not provide any nutrients by itself. Too low an agar concentration may lead to vitrification of the plant, while too high an agar concentration may lead to too hard a medium, which may affect the absorption of nutrients by the plant. In some embodiments, the agar in the regeneration medium described herein is at a concentration of 5g/L to 20g/L, 8g/L to 15g/L, 8g/L to 12g/L, or 6g/L to 10 g/L.
Phytohormones described herein include auxins, cytokinins and gibberellins.
The term "auxin" as used in this application refers to a plant hormone which promotes elongation of plant shoots and controls other specific growth effects. The auxin can be used singly or in combination. In some embodiments, the auxin is indoleacetic acid, indolepropionic acid, indolebutyric acid, abscisic acid, picloram, dicamba, 2, 4-dichlorophenoxyacetic acid, naphthylacetic acid, or a combination thereof.
The term "cytokinin" as used in this application refers to a plant hormone that promotes root cell elongation. Cytokinins may be used singly or in combination. In some embodiments, the cytokinin is benzyladenine, kinetin, isopentenyladenine, N- (2-chloro-4-pyridine) -N' phenylurea, N-benzyl 1-9 (2-tetrahydropyran) adenine, thidiazuron, zeatin, 6- (y-y-dimethylallyl acid) purine, DL-dihydrozeatin, t-zeatin riboside, or a combination thereof.
In some embodiments, the phytohormone is present in the regeneration medium described herein at a concentration of 0.01mg/L to 5mg/L, 0.02mg/L to 4mg/L, 0.03mg/L to 3mg/L, 0.05mg/L to 2.5mg/L, 0.08mg/L to 2.5mg/L, 0.1mg/L to 2.5mg/L, 0.2mg/L to 2.5mg/L, 0.5mg/L to 2.2mg/L, 0.8mg/L to 2.2mg/L, or 1mg/L to 2mg/L (w/v).
In some embodiments, the regeneration medium does not include gibberellin. In some embodiments, the phytohormone in the regeneration medium is a combination of auxin and cytokinin or cytokinin alone. In some embodiments, the cytokinin comprises 6-benzyladenine. In some embodiments, the concentration of 6-benzyladenine is 0.01mg/L to 5mg/L, 0.02mg/L to 4mg/L, 0.03mg/L to 3mg/L, 0.05mg/L to 2.5mg/L, 0.08mg/L to 2mg/L, 0.1mg/L to 1.8mg/L, 0.2mg/L to 1.5mg/L, 0.3mg/L to 1.2mg/L, 0.4mg/L to 1mg/L, 0.4mg/L to 0.8mg/L, or 0.4mg/L to 0.6 mg/L. In some embodiments, the plant auxin in the regeneration medium described herein comprises indole-3-butyric acid. In some embodiments, the indole-3-butyric acid in the regeneration medium is at a concentration of 0.01mg/L to 1mg/L, 0.02mg/L to 0.8mg/L, 0.03mg/L to 0.5mg/L, 0.05mg/L to 0.4mg/L, 0.06mg/L to 0.3mg/L, 0.08mg/L to 0.2mg/L, or 0.1 mg/L.
In some embodiments, the regeneration medium herein further comprises a carbon source. Carbon sources include, but are not limited to, glucose, fructose, sucrose. In some embodiments, the carbon source of the regeneration medium herein is sucrose. In some embodiments, the sucrose concentration is from 10g/L to 50g/L, from 15g/L to 40g/L, from 20g/L to 40g/L, or from 10g/L to 30 g/L.
In some embodiments, the regeneration medium described herein further comprises a hydrolyzed protein, which in some embodiments is hydrolyzed casein. In some embodiments, the hydrolyzed protein has a concentration of 0.1g/L to 5g/L, 0.2g/L to 3g/L, 0.5g/L to 2g/L, 0.8g/L to 1.5g/L, 0.9g/L to 1.2g/L, or 1 g/L.
In some embodiments, activated carbon is not included in the regeneration media described herein.
In some embodiments, the regeneration medium described herein has a pH of 4.5-6.5, 5-6, 5.5-6, or about 5.8. In some embodiments, the regeneration medium described herein has a pH of 5.8.
In some embodiments, the regeneration medium described herein comprises WPM salt, WPM organic, 10g/L to 30g/L sucrose, 0.1g/L to 1.5g/L hydrolyzed casein, 6g/L to 10g/L agar, 0.4mg/L to 0.6 mg/L6-benzyladenine, and 0.08mg/L to 0.2mg/L indole-3-butyric acid.
Hardening off seedlings
In order to improve the viability of the plants after transplantation into the natural environment, a step of gradually adapting the plants in the process of gradually changing from the sterile environment to the bacteria-carrying environment is required. Thus, in some embodiments, the tissue culture methods described herein for elaeagnus plants further comprise the step of refining the material obtained by culturing in regeneration medium. In some embodiments, the material is transferred to a culture flask along with a culture medium for culturing, thereby obtaining an acclimatized seedling. In some embodiments, the material used to obtain the acclimatized seedling has rooted.
In some embodiments, sterile water is added to the culture flask after the material is transferred to the flask along with the culture medium. In some embodiments, the sterile water in the culture flask submerges the culture medium. In some embodiments, the sterile water in the culture flask is 0.5cm-2cm, 0.5cm-1.5cm, 0.5cm-1cm, 1cm-2cm, or about 1cm above the culture medium. In some embodiments, the culture flask used in the acclimatization step is semi-open. In some embodiments, the cap of the culture flask used in the acclimatization step is a dummy cap. In some embodiments, the culturing in the acclimatization step is performed at a temperature of 23 ℃ to 27 ℃. In some embodiments, the culturing in the acclimatization step is performed at a temperature of 26 ℃. In some embodiments, the culturing in the acclimatization step is performed under conditions of 10 hours light (4000lux) per day followed by 14 hours dark. In some embodiments, the culturing in the exercising step is for at least 1 day, 2 days, 3 days, or 4 days.
Transplantation
In some embodiments, the tissue culture method for elaeagnus plants described herein further comprises the step of transplanting the acclimatized plantlets.
In some embodiments, the acclimatized plantlets described herein are transplanted after removal from the culture flask and after rinsing with running water to remove the culture medium.
In some embodiments, the transplanting is performed by transplanting the acclimatized seedling into a mixture of vermiculite and perlite.
In some embodiments, after the acclimatized plantlets are planted in a mixture of vermiculite and perlite, watering is carried out until saturation.
In some embodiments, after the acclimatized plantlets are planted in the mixture of vermiculite and perlite, the acclimatized plantlets are shielded with a translucent container. In some embodiments, the translucent container is of a plastic material. In some embodiments, the translucent container is a plastic cup. In some embodiments, after the acclimatized seedlings grow new leaves, the translucent container covering the acclimatized seedlings is removed.
In some embodiments, the volume of vermiculite and perlite in the mixture of vermiculite and perlite is 2 to 1 to 2.5 to 1, 2.5 to 1 to 3 to 1, 3 to 1 to 3.5 to 1, 3.5 to 1 to 4 to 1, or about 3 to 1.
In another aspect, the present application provides a regeneration medium as described above for regenerating leaves in tissue culture of an elaeagnus plant.
The following examples, in conjunction with the above descriptions, are intended to better illustrate some embodiments of the invention and should not be construed as limiting the scope of the invention. All of the specific compositions, materials and methods described below, in whole or in part, are within the scope of the invention. These specific compositions, materials and methods are not intended to limit the invention, but are merely illustrative of specific embodiments within the scope of the invention. Those skilled in the art may develop equivalent compositions, materials, and methods without adding inventive step and without departing from the scope of the invention. It will be appreciated that various modifications to the method of the invention may still be included within the scope of the invention. The inventors intend such variations to be included within the scope of the present invention.
Examples
Example 1 regeneration and propagation of sterile leaves
Regeneration
Leaves from sterile Elaeagnus angustifolia plants were used as explants and were cut at the petioles and main veins to make full leaf disks of approximately 1cm by 1 cm.
The leaf disks were cultured in the selected regeneration medium (see example 2 for details of the selection process). The regeneration medium comprises WPM salt, WPM organic matter, 20g/L sucrose, 1g/L hydrolyzed casein, 8g/L agar, 0.5mg/L BA and 0.1mg/L IBA, and the pH is 5.8.
Subculture was performed every two weeks, and callus was formed after about 1 month, and the callus induction rate was 85%. After about 20 days, regenerated shoots were produced with a differentiation rate of 90%. The regenerated shoots were transferred to freshly prepared regeneration medium for further culture and further shoot elongation was followed for 20 days, in which step more shoots were produced and the elongated shoots also produced roots in this step. The regenerated shoots can grow into well-grown tissue culture seedlings with normal roots. FIG. 3 shows the growth of Elaeagnus angustifolia at various stages during the cultivation of the leaf discs in regeneration medium.
All conditions of culture in regeneration medium were as follows: at 25 ℃. + -. 2 ℃ under 16 hours light (3000lux) and then 8 hours dark daily, the tissue culture bottle used was fitted with a plastic cap provided with a gas-permeable vent with a gas filtration membrane, with a pore size of 0.22. mu.M.
Hardening and transplanting seedlings
The tissue culture seedlings after the regeneration step were cultured in a semi-open culture flask for 4 days in a growth chamber under culture conditions of 10 hours light (4000Lux) and then 14 hours dark at 26 ℃ per day, and then the tissue culture seedlings were taken out from the tissue culture flask, washed with tap water to remove the medium coagulant, and the root system was not damaged as much as possible.
The tissue culture seedlings are planted in plastic flowerpots, the pots are filled with a mixture of vermiculite and perlite (wherein the volume ratio of the vermiculite to the perlite is 3: 1), and the pots are watered until the pots are saturated. The tissue culture seedlings are covered by a transparent disposable cup. And taking the plastic cup away after new blades are generated. Followed by weekly watering. Plants of one month of age may be prepared for transplantation into the field. The survival rate (number of surviving seedlings/total number of transplanted seedlings x 100%) was 95%.
Example 2 selection of regeneration Medium
Using the same method as in example 1, the culture results of different formulations of regeneration medium and different explants were compared, and the data are as follows:
table 2: regeneration medium and explant screening test results
In the above table, BA represents 6-benzyladenine; NAA represents alpha-naphthylacetic acid; ZT represents zeatin; IBA stands for indole-3-butyric acid.
From the data in Table 2 and FIGS. 4 (corresponding to experiment No. 5 in Table 2 for screening basal medium) and 5 (corresponding to experiment No. 6 in Table 2 for screening composition of phytohormones), it can be seen that the regeneration medium in experiment No. 5 (WPM salt + WPM organic + sucrose (20g/L) + Casein hydrolysate (1g/L) + agar (8g/L) + BA (0.5mg/L) + IBA (0.1mg/L)) can regenerate leaves very well compared to other regeneration media.
The percentages in the examples of the present application are weight/volume percentages unless otherwise specifically indicated.
While the disclosure has been described with reference to specific embodiments thereof, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the disclosure. In addition, many modifications may be made to adapt a particular situation, material, composition, process, or step, to the objective, spirit and scope of the present disclosure. Such variations are intended to fall within the scope of the present disclosure.
All publications, patents, patent applications, and sequence accession numbers mentioned in this application are herein incorporated in their entirety by reference into the present application as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.
Claims (10)
1. A tissue culture method for plants of the genus elaeagnus, the method comprising:
(a) obtaining leaves from a plant of the genus elaeagnus, wherein the plant of the genus elaeagnus is a sterile plant;
(b) cutting the blade, thereby obtaining a blisk;
(c) the leaf discs are placed on a regeneration medium for culturing, thereby obtaining regenerated buds.
2. The method of claim 1, wherein the elaeagnus plant is elaeagnus angustifolia.
3. The method of claim 1, wherein in step (b) the petioles of the blades are severed.
4. The method of claim 3, wherein step (b) further comprises cutting at the main veins of the leaflets.
5. The method of claim 1, wherein said regeneration medium comprises basal medium, agar, and at least one phytohormone.
6. The method of claim 1, wherein step (c) comprises subculture.
7. The method of claim 1, further comprising:
(d) transferring the material from step (c) together with the culture medium into a culture flask and culturing after adding sterile water, thereby obtaining an acclimatized seedling.
8. The method of claim 7, further comprising:
(e) and (3) implanting the acclimatized seedling into a mixture of vermiculite and perlite, wherein the volume ratio of the vermiculite to the perlite is 2: 1 to 2.5: 1, 2.5: 1 to 3: 1, 3: 1 to 3.5: 1, 3.5: 1 to 4: 1, or 3: 1.
9. A regeneration medium for regenerating leaves in tissue culture of an elaeagnus plant, comprising a basal medium, agar and at least one phytohormone.
10. The regeneration medium of claim 9, wherein the medium comprises WPM salts, WPM organics, 10-30 g/L sucrose, 0.1-1.5 g/L hydrolyzed casein, 6-10 g/L agar, 0.4-0.6 mg/L6-benzyladenine, and 0.08-0.2 mg/L indole-3-butyric acid.
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