CN114680047B - Tissue culture rapid propagation method taking field tree spinach stem as explant - Google Patents
Tissue culture rapid propagation method taking field tree spinach stem as explant Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention discloses a tissue culture rapid propagation method taking a field tree spinach stem as an explant. The method of the invention takes the stem section of the field tree spinach as the explant, successfully obtains the regeneration plant of the tree spinach in vitro through the steps of sterilization, axillary bud starting and proliferation, adventitious bud elongation and rooting, acclimatization and transplantation, and the like, successfully carries out the tissue culture and rapid propagation of the tree spinach for the first time, and establishes a new propagation mode of the tree spinach. Compared with the prior other spinach tree propagation methods, the method has the advantages of simpler operation, wider material selection, more efficient propagation expansion and capability of keeping the excellent characters of the female parent. The tissue culture seedling transplanting survival rate of the spinacia oleracea cultivated by the method reaches over 90 percent, and high-quality seedling guarantee can be provided for large-scale planting of the spinacia oleracea.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture, and particularly relates to a tissue culture rapid propagation method taking a field tree spinach stem section as an explant.
Background
Spinach (Zu Fang (A)Cnidoscolus aconitifolius) Has high comprehensive utilization value, and is a special economic forest tree species with low initial investment, long benefit time, short growth period and quick income. The fresh leaves of the tree spinach not only have rich protein, crude fiber and vitamin contents, but also contain various amino acids and mineral elements which are necessary for human bodies, and are a new resource food with high nutritive value, and the relatively low moisture content in the leaves ensures that the leaves can better resist the infection of microorganisms, thereby effectively prolonging the storage life of the leaves. It can be used as staple food by people in native place, and can be used as green leaf vegetableThe vegetables are the main sources and are deeply loved as health-care vegetables and health-care beverages. The tree spinach has rich medicinal effects. The hydroalcoholic extract of the tree spinach leaves contains various abundant phenolic chemical substances, has strong oxidation resistance, and is an excellent source of natural antioxidants; and the ethanol extracts of roots, stems and leaves of the bacillus subtilis and the escherichia coli show obvious inhibiting effect, wherein the stem extract has the widest antibacterial spectrum, and all the extracts show better antibacterial effect compared with common antibiotics such as tetracycline. The spinach tree extract also has the potential of reducing blood sugar, protecting stomach, preventing precancerous lesion of colon cancer, protecting liver and the like. In addition, the spinach granules added in the traditional feed can obviously improve the content of nutrient components and the digestibility of crude protein in the feed, and is beneficial to solving the problem of the shortage of feed resources in the world. Spinach is therefore a versatile economic tree species that is receiving increasing attention worldwide.
At present, researches on tree spinach mainly focus on nutrition, biochemical functions and animal feeding effects, and rapid propagation is an important guarantee for industrial development of the tree spinach. The spinach tree seeds are rare, the seedling production of the spinach tree at present mostly depends on cuttage, but the cuttage propagation has the problems of short service life, weak root system, easy crown deviation, great damage to mother plants and the like. The tissue culture has the advantages of high efficiency, high propagation coefficient, capability of keeping the excellent properties of woody plants and the like, and the tissue culture of the tree spinach has the potential of helping to promote the generation of more secondary metabolites with medicinal effects. The tissue culture of the spinacia oleracea L.can lay a foundation for researches on polyploid breeding, genetic transformation and the like in the future while ensuring the efficient regeneration of the spinacia oleracea L.. In order to help ensure the large-scale production and utilization of the tree spinach, the research of tissue culture in-vitro regeneration of the tree spinach is necessary. However, as the planting time of the spinacia oleracea in the field is prolonged, the in vivo endophytes are gradually increased, the explant sterilization difficulty is higher, and rich phenolic substances of the spinacia oleracea also increase the difficulty in establishing a sterile system, and no relevant report is available at home and abroad at present.
Disclosure of Invention
In order to overcome the defects of the conventional propagation mode of the spinacia oleracea L.A.Merr, the invention aims to provide an efficient tissue culture and rapid propagation method taking a stem section of the spinacia oleracea L.in the field as an explant. The method takes the stem section of the field tree spinach as an explant, successfully obtains the in vitro regeneration plant of the tree spinach through the steps of sterilization, axillary bud starting and proliferation, adventitious bud elongation and rooting, acclimatization and transplantation and the like, and establishes a new propagation mode of the tree spinach. Compared with the prior other spinach tree propagation methods, the method has the advantages of simpler operation, wider material selection, more efficient propagation expansion and capability of keeping the excellent characters of the female parent.
The invention provides a tissue culture rapid propagation method taking a field tree spinach stem as an explant, which comprises the following steps:
s1. explant sterilization: selecting a stem section of a field tree spinach with a bud point for two years as an explant, and sterilizing to obtain a sterilized stem section;
s2. axillary bud initiation and adventitious bud proliferation induction: inoculating the stem sections sterilized in the step S1 to an axillary bud starting culture medium in an axial vertical mode, cutting the axillary buds after the axillary buds grow to be more than 2 cm in length, and transferring the cut axillary buds to an adventitious bud proliferation induction culture medium for culture until adventitious buds are induced to form;
s3, elongation and rooting culture: after the adventitious bud grows to be more than 1 cm in length, cutting the adventitious bud and inoculating the adventitious bud to an adventitious bud elongation culture medium for elongation culture before rooting; transferring the adventitious bud to a rooting culture medium after the adventitious bud grows to be 1.5-2 cm long, and carrying out induced culture until the adventitious bud grows to root to form a regenerated seedling;
s4, hardening and transplanting: and (3) putting the regenerated seedlings which grow to be more than 3 cm in length and have good rooting into natural light for hardening, then transplanting into a culture medium, transplanting into a field after hardening, and obtaining the regenerated seedlings which are transplanted to survive.
Preferably, the sterilization treatment in step S1 specifically includes:
washing with running water for 120-150 min for pretreatment, soaking with 75% ethanol water solution by volume fraction for 50 s, then washing with sterile water for 3 times, soaking with 0.1% mercuric chloride solution by mass fraction for 10-15 min, and then washing with sterile water for 5 times.
Preferably, the axillary bud initiation medium contains PPM per liter TM 2～4 mL, 0.5-1.0 mg of 6-BA, 30 g of cane sugar and 6.0 g of agar, the balance being 1/2 MS, and the pH value being 5.8.
Preferably, the adventitious bud proliferation induction culture medium contains 2.0-4.0 mg of 6-BA, 0.1-0.5 mg of IBA, 30 g of sucrose and 6.0 g of agar per liter, and the balance is 1/2 MS and pH is 5.8.
Preferably, the adventitious bud elongation culture medium contains 0.1-0.2 mg of 6-BA, 1.0-2.0 mg of IAA, 30 g of sucrose and 6.0 g of agar per liter, and the balance is 1/2 MS and pH is 5.8.
Preferably, each liter of the rooting medium contains 0.1-0.5 mg of NAA, 20 g of sucrose and 4.0 g of agar, and the balance is 1/2 MS, and the pH value is 5.8.
Preferably, the culture conditions in step S2 are 25-28 deg.C, 12 h/12 h of light/dark, and 2500 lx of light intensity.
Preferably, in step S3, the culture conditions on the adventitious bud elongation medium are: the temperature is 25-28 ℃, the illumination time is 12 h/dark time is 12 h, and the illumination intensity is 1500 lx; transferring the adventitious bud to a rooting culture medium, culturing in full dark at 25-28 deg.C for 3 d, and culturing under the conditions of 25-28 deg.C, 12 h/12 h in dark and 1500 lx illumination intensity.
Preferably, in the step S4, the seedling training time is 5-7 d, and the domestication time is 2-3 weeks.
More preferably, the sterilization treatment in step S1 specifically includes: washing with running water for 120 min for pretreatment, soaking in 75% ethanol water solution by volume fraction for 50 s, washing with sterile water for 3 times, soaking in 0.1% mercuric chloride solution by mass fraction for 10 min, and washing with sterile water for 5 times; the axillary bud initiation culture medium contains PPM per liter TM 3 mL, 0.5 mg of 6-BA, 30 g of cane sugar and 6.0 g of agar, the balance being 1/2 MS, and the pH value being 5.8; the adventitious bud proliferation induction culture medium contains 4.0 mg of 6-BA, 0.2 mg of IBA, 30 g of cane sugar and 6.0 g of agar per liter, and the balance is 1/2 MS, and the pH value is 5.8; the adventitious bud elongation culture medium contains 0.1 mg of 6-BA, 1.5 mg of IAA, 30 g of cane sugar and 6.0 g of agar per liter, and the balance is 1/2 MS, and the pH value is 5.8; the rooting culture medium contains 0.1 mg of NAA, 20 g of cane sugar and 4.0 g of agar per liter, and the balance is1/2 MS, pH 5.8.
Compared with the prior art, the invention has the beneficial effects that:
(1) according to the invention, a set of complete in-vitro tissue culture and rapid propagation system is constructed by researching the influence of factors such as the treatment time of a sterilizing agent, the concentration of antibiotics, the pretreatment mode, the sampling time, the type of a culture medium, the type and concentration of exogenous hormones and the like on the sterilization and regeneration efficiency of explants. The seeds of the tree spinach are rare, the tree spinach is mostly bred by adopting a cutting mode at present, the invention fills up the blank of the research on the tissue culture and rapid propagation of the tree spinach, enriches the propagation modes of the tree spinach, only needs about 60 days in the total process from an explant to the formation of a complete plant, and the transplanting survival rate can reach more than 90 percent.
(2) The system breaks the conventional way, takes the two-year young stem section with the bud point of the field spinach as the explant, the spinach tree grows rapidly, the two-year seedlings can be used for early selection, excellent provenance, family or single plant is selected, the first established in-vitro tissue culture rapid propagation system of the field spinach by taking the two-year young stem section with the bud point as the explant has small genetic variation in the propagation process, and the excellent characters of the female parent can be fully ensured while the high-efficiency rapid propagation is ensured.
(3) The invention considers factors influencing the explant sterilization effect in many aspects, overcomes the problem that the field tree spinach has poor sterilization effect along with the prolonged planting time and rich phenolic substances in the early pre-experiment, and establishes a sterile system of the field tree spinach stem.
(4) The invention takes the stem section as the explant, and has the advantages of convenient material acquisition, easy operation, wide source, being beneficial to the repeated operation of regeneration and genetic transformation, and the like. Therefore, the invention takes the stem section of the tree spinach as the explant, successfully obtains the in vitro regeneration plant of the tree spinach through the processes of sterilization, axillary bud starting and proliferation, adventitious bud rooting, seedling hardening, transplanting and the like, establishes the high-efficiency regeneration system of the tree spinach, has important significance for the rapid propagation and large-area popularization of excellent varieties of the tree spinach and the promotion of the industrial development of the tree spinach, and lays a foundation for the construction of the genetic transformation system of the tree spinach, thereby providing a platform for the molecular biological research of gene function and the like in the tree spinach.
The invention utilizes plant tissue culture technology to carry out large-scale production of tree spinach seedlings, establishes a tissue culture rapid propagation method taking field tree spinach stems as explants, and has the characteristics of simplicity, easy implementation, high efficiency and economy. The transplanting survival rate of the tissue culture seedlings of the tree spinach cultivated by the method reaches over 90 percent, and high-quality seedling guarantee can be provided for large-scale planting of the tree spinach.
Drawings
FIG. 1 shows the sterilization effect of stem explant; wherein A is a contaminated stem explant, B is an uncontaminated but dead stem explant, and C is an uncontaminated and viable stem explant.
FIG. 2 is axillary bud induction of stem segments on different media; wherein the medium in A was the axillary bud initiation medium used in comparative example 8 (containing 0.5 mg of 6-BA, 30 g of sucrose and 6.0 g of agar per liter, the balance being MS, pH 5.8), the medium in B was the axillary bud initiation medium used in example 1 (containing 0.5 mg of 6-BA, 30 g of sucrose and 6.0 g of agar per liter, the balance being 1/2 MS, pH 5.8), and the medium in C was the axillary bud initiation medium used in comparative example 9 (containing 0.5 mg of 6-BA, 30 g of sucrose and 6.0 g of agar per liter, the balance being B5, pH 5.8).
FIG. 3 shows the proliferation of adventitious buds on media containing different concentrations of 6-BA; wherein the medium in A was the adventitious bud growth induction medium used in comparative example 11 (containing 6-BA 1.0 mg, IBA 0.2 mg, sucrose 30 g and agar 6.0 g per liter, the balance being 1/2 MS, pH 5.8), the medium in B was the adventitious bud growth induction medium used in example 4 (containing 6-BA 2.0 mg, IBA 0.2 mg, sucrose 30 g and agar 6.0 g per liter, the balance being 1/2 MS, pH 5.8), the medium in C was the adventitious bud growth induction medium used in example 1 (containing 6-BA 4.0 mg, IBA 0.2 mg, sucrose 30 g and agar 6.0 g per liter, the balance being 1/2 MS, pH 5.8), the medium in D was the adventitious bud growth induction medium used in comparative example 16 (containing 6-BA 5.0 mg per liter, sucrose 30 g and agar 6.0 g per liter, and the balance being 1/2 MS, pH 5.8), and the medium in D was the adventitious bud growth induction medium used in comparative example 16, IBA 0.2 mg, sucrose 30 g and agar 6.0 g, the balance 1/2 MS, pH 5.8).
FIG. 4 shows the elongation of adventitious buds on media containing different concentrations of IAA; wherein the medium in A is the adventitious bud elongation medium used in example 3 (containing 0.1 mg of 6-BA, 1.0 mg of IAA, 30 g of sucrose and 6.0 g of agar per liter, the balance being 1/2 MS, pH 5.8), the medium in B is the adventitious bud elongation medium used in example 1 (containing 0.1 mg of 6-BA, 1.5 mg of IAA, 30 g of sucrose and 6.0 g of agar per liter, the balance being 1/2 MS, pH 5.8), and the medium in C is the adventitious bud elongation medium used in example 2 (containing 0.1 mg of 6-BA, 2.0 mg of IAA, 30 g of sucrose and 6.0 g of agar per liter, the balance being 1/2 MS, pH 5.8).
FIG. 5 is the rooting of adventitious buds on media containing different concentrations of NAA; wherein the medium in A is the rooting medium used in example 4 (containing NAA 0 mg, sucrose 20 g and agar 4.0 g per liter, the balance 1/2 MS, pH 5.8), the medium in B is the rooting medium used in example 1 (containing NAA 0.1 mg, sucrose 20 g and agar 4.0 g per liter, the balance 1/2 MS, pH 5.8), the medium in C is the rooting medium used in example 2 (containing NAA 0.2 mg, sucrose 20 g and agar 4.0 g per liter, the balance 1/2 MS, pH 5.8), and the medium in D is the rooting medium used in example 3 (containing NAA 0.5 mg, sucrose 20 g and agar 4.0 g per liter, the balance 1/2 MS, pH 5.8).
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
The MS culture Medium referred to in the following examples And comparative examples refers to a general Medium of a formula known in the art, the composition And the configuration of which are described in Toshio Murashige, Folke Skoog (1962) A recycled Medium For Rapid Growth And old Bio Assays With Tobacco Tissue cultures, Physiollogian Plantarum, 15: 473-497; 1/2 the MS culture medium is the MS culture medium with half of macroelements and unchanged other components. 6-BA means 6-benzylamino adenine; NAA is naphthylacetic acid; IBA refers to indolebutyric acid. PPM TM Is PPM used as Plant tissue culture antibacterial agent Plant regenerative mix TM Is ppm-100 (cat #) from Plant Cell technology corporation.
Example 1
(1) And (3) explant sterilization: cutting tender stem segments (each length is about 3 cm) with bud points of two-year growth of field trees, namely spinach, as explants for sterilization; the sterilization treatment comprises the steps of washing with running water for 120 min for pretreatment, soaking with 75% ethanol water solution by volume fraction for 50 s, washing with sterile water for 3 times, soaking with 0.1% mercuric chloride solution by mass fraction for 10 min, and washing with sterile water for 5 times.
(2) Axillary bud initiation and proliferation induction: inoculating the stem sections sterilized in the step (1) to an axillary bud initiation culture medium in an axial vertical mode, culturing under the conditions that the temperature is 25 ℃, the illumination is 12 h/dark 12 h and the illumination intensity is 2500 lx, cutting the axillary buds (shown in figure 2) with the length of more than 2 cm after 15 d, transferring the axillary buds to an adventitious bud proliferation induction culture medium, and culturing under the conditions of the same temperature and illumination until adventitious buds are induced to form (shown in figure 3). The axillary bud initiation culture medium contains PPM per liter TM 3 mL, 0.5 mg of 6-BA, 30 g of sucrose and 6.0 g of agar, the balance being 1/2 MS, and pH being 5.8. The adventitious bud proliferation induction culture medium contains 4.0 mg of 6-BA, 0.2 mg of IBA, 30 g of sucrose and 6.0 g of agar per liter, and the balance is 1/2 MS, and the pH value is 5.8.
(3) And (3) elongation and rooting culture: cutting the adventitious bud growing to about 1 cm or more in the step (2) and inoculating the cut adventitious bud to an adventitious bud elongation culture medium for elongation culture before rooting (FIG. 4), wherein the culture conditions are as follows: the temperature is 25 ℃, the illumination is 12 h/dark 12 h, the illumination intensity is 1500 lx, then the adventitious bud growing to the length of 1.5-2 cm is transferred to a rooting medium for culture, after inoculation, the adventitious bud is cultured in full dark for 3 d under the condition of 25 ℃, and then induced and cultured under the conditions of illumination of 12 h/dark 12 h and the illumination intensity of 1500 lx until the adventitious bud grows to root to form a regeneration seedling (figure 5). The adventitious bud elongation culture medium contains 0.1 mg of 6-BA, 1.5 mg of IAA, 30 g of cane sugar and 6.0 g of agar per liter, and the balance is 1/2 MS, and the pH value is 5.8. The rooting culture medium contains 0.1 mg of NAA, 20 g of cane sugar and 4.0 g of agar per liter, and the balance is 1/2 MS, and the pH value is 5.8.
(4) Hardening and transplanting seedlings: unscrewing the bottle cap of a culture bottle of a regenerated seedling which grows to be more than 5 cm in length and well takes root, hardening the seedling for 5 days in natural light, carefully removing the regenerated seedling from the culture bottle, and carefully washing off the culture medium on the root by using clear water; then transplanting the regenerated seedlings into a wet and sterilized culture medium, and covering a preservative film on a culture pot where the regenerated seedlings are located so as to keep high humidity; after 2 weeks of acclimation, transplanting the regenerated seedlings into a field for continuous culture, and marking the survival of the transplanted tissue culture seedlings when new terminal buds of the regenerated seedlings grow again.
Example 2
(1) And (3) explant sterilization: cutting tender stem segments (each length is about 3 cm) with bud points of two-year growth of field trees, namely spinach, as explants for sterilization; the sterilization treatment comprises the steps of washing with running water for 150 min for pretreatment, soaking with 75% ethanol water solution by volume fraction for 50 s, washing with sterile water for 3 times, soaking with 0.1% mercuric chloride solution by mass fraction for 10 min, and washing with sterile water for 5 times.
(2) Axillary bud initiation and proliferation induction: inoculating the stem sections sterilized in the step (1) to an axillary bud initiation culture medium in an axial vertical mode, culturing under the conditions that the temperature is 25 ℃, the illumination is 12 h/dark 12 h and the illumination intensity is 2500 lx, cutting the axillary buds with the length of more than 2 cm after 15 d, transferring the cut axillary buds to an adventitious bud proliferation induction culture medium, and culturing under the conditions of the same temperature and illumination until adventitious buds are induced to form. The axillary bud initiation culture medium contains PPM per liter TM 3 mL, 1.0 mg of 6-BA, 30 g of sucrose and 6.0 g of agar, the balance being 1/2 MS, and pH being 5.8. The adventitious bud proliferation induction culture medium contains 4.0 mg of 6-BA, 0.5 mg of IBA, 30 g of sucrose and 6.0 g of agar per liter, and the balance is 1/2 MS, and the pH value is 5.8.
(3) And (3) elongation and rooting culture: cutting the adventitious bud growing to about 1 cm or more in the step (2), inoculating the cut adventitious bud into an adventitious bud elongation culture medium, and performing elongation culture before rooting under the culture conditions: the temperature is 25 ℃, the illumination is 12 h/dark 12 h, the illumination intensity is 1500 lx, then the adventitious bud growing to the length of 1.5-2 cm is transferred to a rooting culture medium for culture, after inoculation, the adventitious bud is cultured in full dark for 3 d under the condition of 25 ℃, and then the adventitious bud is induced and cultured under the conditions of illumination of 12 h/dark 12 h and the illumination intensity of 1500 lx until the adventitious bud grows to root to form a regenerated seedling. The adventitious bud elongation culture medium contains 0.1 mg of 6-BA, 2.0 mg of IAA, 30 g of cane sugar and 6.0 g of agar per liter, and the balance is 1/2 MS, and the pH value is 5.8. The rooting culture medium contains 0.2 mg of NAA, 20 g of cane sugar and 4.0 g of agar per liter, and the balance is 1/2 MS, and the pH value is 5.8.
(4) Hardening and transplanting seedlings: unscrewing the bottle cap of a culture bottle of a regenerated seedling which grows to be more than 5 cm in length and well takes root, hardening the seedling for 6 days in natural light, carefully removing the regenerated seedling from the culture bottle, and carefully washing off the culture medium on the root by using clear water; then transplanting the regenerated seedlings into a wet and sterilized culture medium, and covering a preservative film on a culture pot where the regenerated seedlings are located so as to keep high humidity; after 2 weeks of acclimation, transplanting the regenerated seedlings into a field for continuous culture, and marking the survival of the transplanted tissue culture seedlings when new terminal buds of the regenerated seedlings grow again.
Example 3
(1) And (3) explant sterilization: cutting tender stem sections (each length is about 3 cm) with bud points of two years of spinach in a field tree as explants for sterilization; the sterilization treatment comprises the steps of washing with running water for 120 min for pretreatment, soaking with 75% ethanol water solution by volume fraction for 50 s, washing with sterile water for 3 times, soaking with 0.1% mercuric chloride solution by mass fraction for 12 min, and washing with sterile water for 5 times.
(2) Axillary bud initiation and proliferation induction: inoculating the stem sections sterilized in the step (1) to an axillary bud initiation culture medium in an axial vertical mode, culturing under the conditions that the temperature is 25 ℃, the illumination is 12 h/dark 12 h and the illumination intensity is 2500 lx, cutting the axillary buds with the length of more than 2 cm after 15 d, transferring the cut axillary buds to an adventitious bud proliferation induction culture medium, and culturing under the conditions of the same temperature and illumination until adventitious buds are induced to form. The axillary bud initiation culture medium contains PPM per liter TM 2 mL, sucrose 30 g and agar 6.0 g, the balance 1/2 MS, pH 5.8. The adventitious bud proliferation induction culture medium contains 4.0 mg of 6-BA, 0.1 mg of IBA, 30 g of sucrose and 6.0 g of agar per liter, and the balance is 1/2 MS, and the pH value is 5.8.
(3) And (3) elongation and rooting culture: cutting the adventitious bud growing to about 1 cm or more in the step (2), inoculating the cut adventitious bud into an adventitious bud elongation culture medium, and performing elongation culture before rooting under the culture conditions: the temperature is 25 ℃, the illumination is 12 h/dark 12 h, the illumination intensity is 1500 lx, then the adventitious bud growing to the length of 1.5-2 cm is transferred to a rooting culture medium for culture, after inoculation, the adventitious bud is cultured in full dark for 3 d under the condition of 25 ℃, and then the adventitious bud is induced and cultured under the conditions of illumination of 12 h/dark 12 h and the illumination intensity of 1500 lx until the adventitious bud grows to root to form a regenerated seedling. The adventitious bud elongation culture medium contains 0.1 mg of 6-BA, 1.0 mg of IAA, 30 g of cane sugar and 6.0 g of agar per liter, and the balance is 1/2 MS, and the pH value is 5.8. The rooting culture medium contains 0.5 mg of NAA, 20 g of cane sugar and 4.0 g of agar per liter, and the balance is 1/2 MS, and the pH value is 5.8.
(4) Hardening and transplanting seedlings: unscrewing the bottle cap of a culture bottle of a regenerated seedling which grows to be more than 5 cm in length and well takes root, hardening the seedling for 7 d in natural light, carefully removing the regenerated seedling from the culture bottle, and carefully washing off the culture medium on the root by using clear water; then transplanting the regenerated seedlings into a wet and sterilized culture medium, and covering a preservative film on a culture pot where the regenerated seedlings are located so as to keep high humidity; after 2 weeks of acclimation, transplanting the regenerated seedlings into a field for continuous culture, and marking the survival of the transplanted tissue culture seedlings when new terminal buds of the regenerated seedlings grow again.
Example 4
(1) And (3) explant sterilization: cutting tender stem segments (each length is about 3 cm) with bud points of two-year growth of field trees, namely spinach, as explants for sterilization; the sterilization treatment comprises the steps of washing with running water for 120 min for pretreatment, soaking with 75% ethanol water solution by volume fraction for 50 s, washing with sterile water for 3 times, soaking with 0.1% mercuric chloride solution by mass fraction for 15 min, and washing with sterile water for 5 times.
(2) Axillary bud initiation and proliferation induction: inoculating the stem sections sterilized in the step (1) to an axillary bud initiation culture medium in an axial vertical mode, culturing under the conditions that the temperature is 25 ℃, the illumination is 12 h/dark 12 h and the illumination intensity is 2500 lx, cutting the axillary buds with the length of more than 2 cm after 15 d, transferring the cut axillary buds to an adventitious bud proliferation induction culture medium, and culturing under the conditions of the same temperature and illumination until adventitious buds are induced to form. The axillary bud initiation culture medium contains PPM per liter TM 4 mL, sucrose 30 g and agar 6.0 g, the balance 1/2 MS, pH 5.8. The adventitious bud proliferation induction culture medium contains 2.0 mg of 6-BA and IBA per liter0.2 mg, 30 g of sucrose and 6.0 g of agar, the balance being 1/2 MS, pH 5.8.
(3) And (3) elongation and rooting culture: cutting the adventitious bud growing to about 1 cm or more in the step (2), inoculating the cut adventitious bud into an adventitious bud elongation culture medium, and performing elongation culture before rooting under the culture conditions: the temperature is 25 ℃, the illumination is 12 h/dark 12 h, the illumination intensity is 1500 lx, then the adventitious bud growing to the length of 1.5-2 cm is transferred to a rooting culture medium for culture, after inoculation, the adventitious bud is cultured in full dark for 3 d under the condition of 25 ℃, and then the adventitious bud is induced and cultured under the conditions of illumination of 12 h/dark 12 h and the illumination intensity of 1500 lx until the adventitious bud grows to root to form a regenerated seedling. The adventitious bud elongation culture medium contains 0.2 mg of 6-BA, 1.5 mg of IAA, 30 g of cane sugar and 6.0 g of agar per liter, and the balance is 1/2 MS, and the pH value is 5.8. The rooting medium contains 20 g of sucrose and 4.0 g of agar per liter, and the balance is 1/2 MS, and the pH value is 5.8.
(4) Hardening and transplanting seedlings: unscrewing the bottle cap of a culture bottle of a regenerated seedling which grows to be more than 3 cm in length and well takes root, hardening the seedling for 7 d in natural light, carefully removing the regenerated seedling from the culture bottle, and carefully washing off the culture medium on the root by using clear water; then transplanting the regenerated seedlings into a wet and sterilized culture medium, and covering a preservative film on a culture pot where the regenerated seedlings are located so as to keep high humidity; after 2 weeks of acclimation, transplanting the regenerated seedlings into a field for continuous culture, and marking the survival of the transplanted tissue culture seedlings when new terminal buds of the regenerated seedlings grow again.
The total period from the explant to the formation of the whole plant of the regenerated seedling of the stem of the tree spinach of example 1-example 4 is about 60 days.
Comparative example 1
The difference from example 1 is that: PPM contained in axillary bud initiation Medium TM Is 1 mL/L, and the concentrations of the other components are the same as those in example 1.
Comparative example 2
The difference from example 1 is that: axillary bud initiation culture medium does not contain PPM TM The concentrations of the remaining components were the same as in example 1.
Comparative example 3
The difference from example 1 is that: the sterilization treatment is not the pretreatment of washing with running water, and is directly soaking with 75% ethanol water solution by volume fraction for 50 s, then washing with sterile water for 3 times, soaking with 0.1% mercuric chloride solution by mass fraction for 10 min, and then washing with sterile water for 5 times.
Comparative example 4
The difference from example 1 is that: the sterilization treatment comprises the steps of washing with running water for 30 min for pretreatment, soaking with 75% ethanol water solution by volume fraction for 50 s, washing with sterile water for 3 times, soaking with 0.1% mercuric chloride solution by mass fraction for 10 min, and washing with sterile water for 5 times.
Comparative example 5
The difference from example 1 is that: the sterilization treatment comprises the steps of washing with running water for 120 min for pretreatment, soaking with 75% ethanol water solution by volume fraction for 50 s, washing with sterile water for 3 times, soaking with 0.1% mercuric chloride solution by mass fraction for 8 min, and washing with sterile water for 5 times. Axillary bud initiation culture medium does not contain PPM TM The concentrations of the remaining components were the same as in example 1.
Comparative example 6
The difference from example 1 is that: the sterilization treatment comprises the steps of washing with running water for 120 min for pretreatment, soaking with 75% ethanol water solution by volume fraction for 50 s, washing with sterile water for 3 times, soaking with 0.1% mercuric chloride solution by mass fraction for 5 min, and washing with sterile water for 5 times. Axillary bud initiation culture medium does not contain PPM TM The concentrations of the remaining components were the same as in example 1.
Comparative example 7
The difference from example 1 is that: the sterilization treatment is not the pretreatment of washing with running water, but the pretreatment of soaking with 0.1% carbendazim for 20 min, soaking with 75% ethanol water solution by volume fraction for 50 s, then washing with sterile water for 3 times, soaking with 0.1% mercuric chloride solution by mass fraction for 10 min, and then washing with sterile water for 5 times.
Comparative example 8
The difference from example 1 is that: 1/2 MS in axillary bud initiation medium was changed to MS. The axillary bud initiation medium used in this comparative example contained PPM per liter TM 3 mL, 0.5 mg of 6-BA, 30 g of sucrose and 6.0 g of agar, the balance being MS and pH 5.8.
Comparative example 9
The difference from example 1 is that: 1/2 MS was changed to B5 in axillary bud initiation medium. The axillary bud initiation medium used in this comparative example contained PPM per liter TM 3 mL, 0.5 mg of 6-BA, 30 g of sucrose and 6.0 g of agar, the balance being B5, and the pH value being 5.8.
Comparative example 10
The difference from example 1 is that: the adventitious bud growth inducing medium used in this comparative example contained 1.0 mg of 6-BA, 0.1 mg of IBA, 30 g of sucrose and 6.0 g of agar per liter, with the balance being 1/2 MS, pH 5.8.
Comparative example 11
The difference from example 1 is that: the adventitious bud growth inducing medium used in this comparative example contained 1.0 mg of 6-BA, 0.2 mg of IBA, 30 g of sucrose and 6.0 g of agar per liter, with the balance being 1/2 MS, pH 5.8.
Comparative example 12
The difference from example 1 is that: the adventitious bud growth inducing medium used in this comparative example contained 1.0 mg of 6-BA, 0.5 mg of IBA, 30 g of sucrose and 6.0 g of agar per liter, with the balance being 1/2 MS, pH 5.8.
Comparative example 13
The difference from example 1 is that: the adventitious bud growth inducing medium used in this comparative example contained 2.0 mg of 6-BA, 0.1 mg of IBA, 30 g of sucrose and 6.0 g of agar per liter, with the balance being 1/2 MS, pH 5.8.
Comparative example 14
The difference from example 1 is that: the adventitious bud growth inducing medium used in this comparative example contained 2.0 mg of 6-BA, 0.5 mg of IBA, 30 g of sucrose and 6.0 g of agar per liter, with the balance being 1/2 MS, pH 5.8.
Comparative example 15
The difference from example 1 is that: the adventitious bud growth inducing medium used in this comparative example contained 5.0 mg of 6-BA, 0.1 mg of IBA, 30 g of sucrose and 6.0 g of agar per liter, with the balance being 1/2 MS, pH 5.8.
Comparative example 16
The difference from example 1 is that: the adventitious bud growth induction medium used in this comparative example contained 5.0 mg of 6-BA, 0.2 mg of IBA, 30 g of sucrose and 6.0 g of agar per liter, with the remainder being 1/2 MS, pH 5.8.
Comparative example 17
The difference from example 1 is that: the adventitious bud growth inducing medium used in this comparative example contained 5.0 mg of 6-BA, 0.5 mg of IBA, 30 g of sucrose and 6.0 g of agar per liter, with the balance being 1/2 MS, pH 5.8.
Comparative example 18
The difference from example 1 is that: the adventitious bud elongation medium used in this comparative example contained 1.0 mg of IAA, 30 g of sucrose and 6.0 g of agar per liter, with the balance being 1/2 MS, pH 5.8.
Comparative example 19
The difference from example 1 is that: the adventitious bud elongation medium used in this comparative example contained 1.5 mg of IAA, 30 g of sucrose and 6.0 g of agar per liter, with the balance being 1/2 MS, pH 5.8.
Comparative example 20
The difference from example 1 is that: the adventitious bud elongation medium used in this comparative example contained 0.2 mg of 6-BA, 1.0 mg of IAA, 30 g of sucrose and 6.0 g of agar per liter, the balance being 1/2 MS, pH 5.8.
The regeneration effect of the regeneration bottles of the explants of the stem segments of spinach as the field trees obtained in examples 1 to 4 and comparative examples 1 to 18 is shown in Table 1. Wherein 30 specimens were inoculated per treatment and repeated 3 times for the study of the sterilization effect (examples 1-4, comparative examples 1-7) and axillary bud initiation (examples 1-4, comparative examples 8-9); for adventitious bud proliferation (comparative examples 10-17), adventitious bud elongation (comparative examples 18-20), and rooting and acclimatization transplanting, 15 specimens were inoculated per treatment and repeated 3 times.
As can be seen from table 1, in examples 1 to 4 of the present invention, the pollution rate of the field tree spinach stem explant is as low as 40%, the adventitious bud induction rate is as high as 40%, the multiplication coefficient is as high as 3.5, and the final transplanting survival rate is as high as 90%.
PPM in comparative examples 1 and 2, in comparison with example 1 TM Is changed to 1 ml/L and 0 ml-L, the rinsing time of the pretreated flowing water in comparative example 3 and comparative example 4 was changed to 0 min and 30 min, respectively, and the sterilization time of the mercuric chloride solution with the mass fraction of 0.1% in comparative example 5 and comparative example 6 was changed to 8 min and 5 min, respectively, without adding PPM TM And the pretreatment mode in the comparative example 7 is changed to 0.1 percent carbendazim soaking for 20 min, so that the obtained field tree spinach stem segment explants have high pollution rate and undesirable sterilization effect (figure 1, table 1). The basic culture mediums in the axillary bud initiation culture mediums of comparative example 8 and comparative example 9 are MS and B5 respectively, and the obtained axillary buds are short in length and not beneficial to later-stage proliferation (figure 2 and table 1); the concentrations of 6-BA in the adventitious bud proliferation induction culture media in the comparative examples 10 to 14 are low, the obtained adventitious bud proliferation induction rate is low, the number of induced adventitious buds is small, and the proliferation coefficient is small (figure 3, table 1); the adventitious bud proliferation induction culture medium in the comparative example 15-the comparative example 17 has higher 6-BA, the obtained axillary bud proliferation induction rate is low, the number of the induced adventitious buds is small, the proliferation coefficient is small, the induced adventitious buds have shorter length and high aberration rate, and the later rooting is not facilitated (figure 3 and table 1); 6-BA is not added in the adventitious bud elongation culture medium in the comparative example 18 and the comparative example 19, the elongation effect of the adventitious bud is weaker, the adventitious bud elongation culture medium 6-BA in the comparative example 20 is higher, the elongation effect of the adventitious bud is not ideal, and the rooting at the later stage is not facilitated.
Examples 1 to 4 were carried out in the following manner: washing with running water for 120-150 min for pretreatment, soaking with 75% ethanol water solution by volume fraction for 50 s, washing with sterile water for 3 times, soaking with 0.1% mercuric chloride solution by mass fraction for 10 min, and washing with sterile water for 5 times; and 2-4 ml/L PPM is added into the axillary bud initiation culture medium TM (ii) a The sterilization treatment ensures that the bottle-entering stem section of the tree spinach has low pollution rate and high survival rate; the adventitious bud proliferation induction culture medium (each liter contains 2.0-4.0 mg of 6-BA, 0.1-0.5 mg of IBA, 30 g of cane sugar and 6.0 g of agar, and the balance is 1/2 MS, pH 5.8) of the examples 1-4 has high proliferation rate and high proliferation coefficient, and the induced adventitious bud has normal growth vigor, can be used for late stage elongation and rooting, and has high utilization rate. In conclusion, the method of example 1 has the best effect, low pollution rate, the length of axillary buds is as long as 2.47 cm, the proliferation induction rate is 80 percent, and the proliferation coefficient of adventitious buds5.69, and the rooting rate reaches 82%.
The invention establishes a tissue culture rapid propagation method taking the stem section of the field tree spinach as the explant, has the characteristics of simplicity, easy implementation, economy and capability of keeping the excellent characters of woody plants, and provides a high-quality seedling guarantee for the large-scale planting of the tree spinach.
Claims (2)
1. A tissue culture rapid propagation method taking a field tree spinach stem as an explant is characterized by comprising the following steps:
s1. explant sterilization: selecting a stem section of a field tree spinach with a bud point for two years as an explant, and sterilizing to obtain a sterilized stem section; the sterilization treatment comprises the following steps: washing with running water for 120 min for pretreatment, soaking in 75% ethanol water solution by volume fraction for 50 s, washing with sterile water for 3 times, soaking in 0.1% mercuric chloride solution by mass fraction for 10 min, and washing with sterile water for 5 times;
s2. axillary bud initiation and adventitious bud proliferation induction: inoculating the stem sections sterilized in the step S1 to an axillary bud starting culture medium in an axial vertical mode for culture, cutting the axillary buds after the axillary buds grow to be more than 2 cm in length, and transferring the cut axillary buds to an adventitious bud proliferation induction culture medium for culture until adventitious buds are formed through induction; the axillary bud initiation culture medium contains PPM per liter TM 3 mL, 0.5 mg of 6-BA, 30 g of cane sugar and 6.0 g of agar, the balance being 1/2 MS, and the pH value being 5.8; the adventitious bud proliferation induction culture medium contains 4.0 mg of 6-BA, 0.2 mg of IBA, 30 g of cane sugar and 6.0 g of agar per liter, and the balance is 1/2 MS, and the pH value is 5.8; the culture conditions are that the temperature is 25-28 ℃, the illumination is 12 h/dark 12 h, and the illumination intensity is 2500 lx;
s3, elongation and rooting culture: after the adventitious bud grows to be more than 1 cm in length, cutting the adventitious bud and inoculating the adventitious bud to an adventitious bud elongation culture medium for elongation culture before rooting; the culture conditions were: the temperature is 25-28 ℃, the illumination time is 12 h/dark time is 12 h, and the illumination intensity is 1500 lx; transferring the adventitious bud to a rooting culture medium after the adventitious bud grows to be 1.5-2 cm long, performing full-dark culture at 25-28 ℃ for 3 days, then performing culture under the conditions of 25-28 ℃, 12 h/12 h of illumination and 1500 lx of illumination intensity, and performing induced culture until the adventitious bud grows to root to form a regenerated seedling; the adventitious bud elongation culture medium contains 0.1 mg of 6-BA, 1.5 mg of IAA, 30 g of cane sugar and 6.0 g of agar per liter, and the balance is 1/2 MS, and the pH value is 5.8; the rooting culture medium contains 0.1 mg of NAA, 20 g of cane sugar and 4.0 g of agar per liter, and the balance is 1/2 MS, and the pH value is 5.8;
s4, hardening and transplanting: and (3) putting the regenerated seedlings which grow to be more than 3 cm in length and have good rooting into natural light for hardening, then transplanting into a culture medium, transplanting into a field after hardening, and obtaining the regenerated seedlings which are transplanted to survive.
2. The method according to claim 1, wherein in step S4, the hardening-off time is 5-7 days and the acclimatization time is 2-3 weeks.
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| Effect of rooting media and number of nodes on growth and leaf yield of chaya (Cnidoscolus aconitifolius McVaugh) at Dire Dawa, Eastern Ethiopia;Yesuf Fantahun et al;《COGENT FOOD & AGRICULTURE》;20210513;第7卷(第1期);第1-14页 * |
| 不同处理因子对卡亚扦插生根的影响;魏秋兰等;《广西林业科学》;20210630;第50卷(第3期);第314-318页 * |
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