CN114287345B - Tissue culture method and culture medium for witloof mosla - Google Patents
Tissue culture method and culture medium for witloof mosla Download PDFInfo
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Abstract
The invention discloses a tissue culture method and a culture medium for witloof moss, and belongs to the technical field of plant tissue culture. The method comprises the following steps: the method comprises the steps of taking tender leaves of witloof moss as explants to be pretreated, wherein the pretreatment method comprises the steps of washing the explants for 6-24 hours by using running water, and then placing the explants at a low temperature of 3-7 ℃ to be treated for 6-24 hours; and sterilizing the pretreated explants, and then sequentially carrying out primary induction culture, secondary multiplication culture and rooting culture. The method is simple and easy to operate, and can realize mass production of the chirita cinnabarina seedlings.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a tissue culture method and a culture medium for witloof moss.
Background
Lawsonia inermis (Calcareobeboea coccinea) is a perennial herb of the genus Lawsonia of the family Gesneriaceae. The rootstock is up to 1 cm thick. The leaves are all grown; the leaf is leather or hard paper, is elliptical, narrow oval or long round, has slightly unequal two sides, slightly pointed top, blunt, round or wide wedge-shaped base, small teeth at the edge and flowers in inflorescence; the bracts are dense, and the bracts are in a narrow egg shape or in a needle shape and are short and prone to hair; is pale yellow and soft. The calyx lobe is in the shape of a narrow thread and is in the shape of a needle, the corolla is vermilion, the outer surface is densely covered, stamen filaments grow from the base part of the corolla, a few glands exist, and anthers are hairless; the degenerated stamen grows from the base of the crown, with wavy edges. The ovary has no hair, and the style has a few small glandular hairs. Capsule shape, flowering in 4-6 months. Distributed in southeast and west of Guangxi provinces of China, and grown on limestone mountain forest at an elevation of 1000-1460 m.
The witloof moss is a unique single species in China, is a witloof moss plant with extremely high ornamental value, is not listed in the name of national protection plants, but is actually distributed in a limited way, and the field quantity is very small, so the witloof moss plant is also protected. In recent years, due to the damage of the habitat, the wild resource of the witloof moss is gradually reduced, and the solution of the propagation problem of the witloof moss is urgent. At present, the tissue culture research on the witloof mosla is not reported. Due to species and habitat particularity of the witloof, the fructification is difficult, the fructification rate is low, and therefore, the seed material is less. The leaves are the best explant material, but the witloof leaves are easy to brown after being disinfected, and the browning degree is serious, so that the development of the in vitro rapid propagation technology is seriously influenced.
Disclosure of Invention
The invention aims to provide a tissue culture method of witloof moss. The method is simple to operate, can be used for producing the seedlings of the witchweed moseri in batches, and lays a foundation for intensive planting of the witchweed moseri in the later period.
The invention provides a tissue culture method of witloof moss, which comprises the following steps:
the method comprises the steps of taking tender leaves of witloof as explants to be pretreated, wherein the pretreatment method comprises the steps of washing the explants for 6-24 h with running water, and then placing the explants at 3-7 ℃ for low-temperature treatment for 6-24 h; sterilizing the pretreated explant, and sequentially performing primary induction culture, secondary multiplication culture and rooting culture;
the primary induction culture medium takes 1/2MS as a basic culture medium and also comprises 0.1-1.0 mg.L of 6-BA -1 、IBA0.01～0.1mg·L -1 5.0-20.0 mg.L of vitamin C -1 1.0-2.0 g.L of active carbon -1 20 to 30 g.L of sucrose -1 And agar 3-4 g.L -1 ，pH5.0～5.8；
The subculture multiplication medium takes MS as a basic culture medium and also comprises 0.2-1.0 mg.L of 6-BA -1 、IBA0.02～0.1mg·L -1 5% -10% of banana and 20-30 g.L of cane sugar -1 And agar 3-4 g.L -1 ，pH5.0～5.8；
The rooting culture medium takes 1/2MS as a basic culture medium and also comprises 0.1-1.0 mg.L of NAA -1 20 to 30 g.L of sucrose -1 And agar 3-4 g.L -1 ，pH5.0～5.8。
In the method, the disinfection comprises the steps of soaking the witloof leaves in washing powder water for 5-10 min, brushing and washing the leaves with running waterSurface, surface-sterilized with 75% by volume of alcohol for 30-60s, 0.1% 2 And (5) disinfecting for 3-8 min.
In the method of the invention, the sterilized explant is cut into small sections of 1-1.5 cm and inoculated in the primary induction culture medium.
In the method of the invention, the primary induction culture comprises a dark culture stage and a light culture stage, and the dark culture time is 7-14 days.
In the method, the sterile seedlings obtained by the primary induction culture are cut into stem sections with 1 or 2 axillary buds and are transferred into a subculture multiplication medium for illumination culture.
In the method, the clustered seedlings obtained by the subculture proliferation culture are divided and transferred into a rooting culture medium for illumination culture.
In the method, the temperature of the primary induction culture, the secondary multiplication culture and the rooting culture is 25-31 ℃.
In the above method of the present invention, the illumination conditions of the primary induction culture, the secondary proliferation culture and the rooting culture are as follows: the illumination time is 10 to 15 h.d -1 The illumination intensity is 35 to 45 mu mol/m -2 ·s -1 。
In the method, the time of primary induction culture is 30-50 d, the time of secondary multiplication culture is 30-60 d, and the time of rooting culture is 10-20 d.
The invention also provides a culture medium for tissue culture of the witloof moss, which comprises an initial generation induction culture medium, a secondary generation multiplication culture medium and a rooting culture medium;
the primary induction culture medium takes 1/2MS as a basic culture medium and also comprises 0.1-1.0 mg.L of 6-BA -1 、IBA0.01～0.1mg·L -1 5.0-20.0 mg.L of vitamin C -1 1.0-2.0 g.L of active carbon -1 20 to 30 g.L of sucrose -1 And agar 3-4 g.L -1 ，pH5.0～5.8；
The subculture multiplication medium takes MS as a basic culture medium and also comprises 0.2-1.0 mg.L of 6-BA -1 、IBA0.02～0.1mg·L -1 5% -10% of banana and cane sugar20～30g·L -1 And agar 3-4 g.L -1 ，pH5.0～5.8；
The rooting culture medium takes 1/2MS as a basic culture medium and also comprises 0.1-1.0 mg.L of NAA -1 20 to 30 g.L of sucrose -1 And agar 3-4 g.L -1 ，pH5.0～5.8。
Compared with the prior art, the invention has the following technical effects:
according to the method, young leaves of the witloof moss are used as explants, leaf browning after disinfection of the witloof moss leaves is effectively prevented by reasonable pretreatment, aseptic seedlings are induced by a proper culture medium formula and subjected to subculture proliferation culture and rooting culture, and a large number of excellent seedlings can be obtained in a short time. The time required for obtaining aseptic bud seedlings by primary induction of explant leaves is 30-50 d; the time for carrying out subculture proliferation culture on the stem section with the axillary bud is 30-60 days; the time for obtaining the rooting seedlings by culturing the bud seedlings in a rooting culture medium is 10-20 d, each plant has 5-8 long roots, the root length is 4-5 cm, and the rooting rate is 98%. The tissue culture method of the witloof moss lays a foundation for conservation, sustainable utilization and large-scale planting of witloof moss germplasm resources.
Detailed Description
The invention provides a tissue culture method of witloof moss, which comprises the following steps:
the method comprises the steps of taking tender leaves of witloof moss as explants to be pretreated, wherein the pretreatment method comprises the steps of washing the explants for 6-24 hours by using running water, and then placing the explants at a low temperature of 3-7 ℃ to be treated for 6-24 hours; sterilizing the pretreated explant, and sequentially performing primary induction culture, secondary multiplication culture and rooting culture;
the primary induction culture medium takes 1/2MS as a basic culture medium and also comprises 0.1-1.0 mg.L of 6-BA -1 、IBA0.01～0.1mg·L -1 5.0-20.0 mg.L of vitamin C -1 1.0-2.0 g.L of active carbon -1 20-30 g.L of cane sugar -1 And agar 3-4 g.L -1 ，pH5.0～5.8；
The subculture multiplication medium takes MS as a basic culture medium and also comprises 0.2-1.0 mg.L of 6-BA -1 、IBA0.02～0.1mg·L -1 5% -10% of banana and 20-30 g.L of cane sugar -1 And agar 3-4 g.L -1 ，pH5.0～5.8；
The rooting culture medium takes 1/2MS as a basic culture medium and also comprises 0.1-1.0 mg.L of NAA -1 20 to 30 g.L of sucrose -1 And agar 3-4 g.L -1 ，pH5.0～5.8。
When the leaves are injured, the polyphenol substances in the cell vacuoles are contacted with polyphenol oxidase and the like in cytoplasm due to cell damage, the phenol substances are oxidized into quinone under the condition of oxygen, the quinone substances are subjected to a series of dehydration and polymerization reactions, and finally, blackish brown substances are formed, so that browning is caused. Browning is the most common and first-to-be-solved problem in the research of the in vitro rapid propagation of witloof mosses. The method provided by the invention has the advantages that the young and tender leaves of the witloof mosses which grow well and are free of diseases and insect pests are cut and washed by running water, so that juice containing a large amount of phenolic substances at wounds can be removed, and the content of the phenolic substances of substrates is reduced, so that browning can be effectively prevented; meanwhile, the flowing water can also wash off the microorganisms apparently attached to the explant, thereby reducing the risk of fungus pollution.
The washed explant is put in a refrigerator with the temperature of 3-7 ℃ for low-temperature treatment for 6-24 h. The preferred low temperature treatment temperature is 4 deg.C, 5 deg.C, 6 deg.C. The low-temperature treatment time is preferably 10 to 20 hours, more preferably 12 to 16 hours. The explant can be pretreated at low temperature to reduce the oxidative browning of tissues, possibly to reduce the activity of enzymes (such as polyphenol oxidase, peroxidase, phenylalanine ammonia lyase and the like) causing browning at low temperature, or to reduce the flow rate of fluid containing phenolic substances and related oxidase in cells at low temperature, so that the contact time of related substances is prolonged, and the enzymatic browning reaction is influenced. In the invention, the running water washing and low-temperature treatment time cannot be too long, and the low-temperature treatment temperature cannot be too low, so that the damage to the explants is avoided.
The invention disinfects the pretreated leaves. In the present invention, the method of sterilization comprises the steps of: selecting the leaves which are green in color and have no damage after pretreatment, soaking in washing powder water, and brushingWashing and washing the surface of the blade by running water; then sequentially using alcohol and HgCl 2 Sterilizing, rinsing with sterile water, and drying surface water. The mass percentage of the washing powder water used by the invention is preferably 0.1-0.3%, and the soaking time is preferably 5-10 min, and more preferably 7-9 min. The disinfection of the invention is carried out by disinfecting the surface of alcohol with 75 percent of volume percentage for 30 to 60 seconds, and more preferably for 45 to 50 seconds. 0.1% HgCl for the present invention 2 Sterilizing for 3-8 min, preferably 4-6 min. In the present invention, the brushing is preferably performed by lightly brushing the surface with a soft brush pen or absorbent cotton, taking care not to damage the leaves. In the present invention, the sterilization process requires constant shaking. In the present invention, the number of rinsing with the sterile water is preferably 3 to 5. In the present invention, the blotting is preferably performed using a sterile filter paper. The explant disinfection method is remarkable in alcohol and HgCl pairs of witloof leaves 2 Is relatively sensitive and therefore the treatment time is not suitable to be too long.
The invention cuts the disinfected young leaf of the witloof into small sections of 1-1.5 cm, and inoculates the small sections in the primary induction culture medium for primary induction culture to obtain the regeneration bud. The primary induction culture medium takes 1/2MS as a basic culture medium and also comprises 0.1-1.0 mg.L of 6-BA -1 、IBA0.01～0.1mg·L -1 5.0-20.0 mg.L of vitamin C (Vc) -1 1.0-2.0 g.L of active carbon -1 20 to 30 g.L of sucrose -1 And agar 3-4 g.L -1 pH5.0-5.8; preferably, the primary induction culture medium takes 1/2MS as a basic culture medium and also comprises 0.4-0.6 mg.L of 6-BA -1 、IBA0.04～0.06mg·L -1 Vitamin C (Vc) 5.0-10.0 mg.L -1 1.0-2.0 g.L of active carbon -1 20-30 g.L of cane sugar -1 And agar 3-4 g.L -1 pH5.0-5.8. More preferably, the primary induction medium is a 1/2 MS-based medium, and further comprises 6-BA0.5mg.L -1 、IBA0.05mg·L -1 Vitamin C (Vc) 10.0 mg. L -1 1.0 g.L of activated carbon -1 20 g.L of sucrose -1 And agar 3.5 g.L -1 pH5.0-5.8. The witloof leaf histiocyte may be rich in a large amount of phenolic substances, so the witloof leaf histiocyte is easy to be seriously sterilized after disinfectionHeavy browning, in order to prevent the cut leaves from browning, the invention can reduce the phenol overflow by reducing the concentrations of inorganic salt and sucrose in the culture medium, thereby reducing the browning degree. The selection of proper types and contents of the antioxidant and the absorbent are important factors for preventing the browning of the explant, and the vitamin C and the active carbon are used as the antioxidant and the absorbent, so that the browning degree of the witloof moss is obviously improved, the induction rate of the clustered buds is high, and the witloof moss grows well.
In the invention, dark culture is carried out for a period of time after explant inoculation, and the dark culture time of primary induction culture is preferably 7-14 days; more preferably 8 to 10d. The temperature of the primary induction culture is 25-31 ℃, and the illumination conditions are preferably as follows: the illumination time is 10 to 15 h.d -1 The illumination intensity is 35 to 45 mu mol/m -2 ·s -1 。
The invention cuts the aseptic seedling into stem segments with 1 or 2 axillary buds, transfers the stem segments into a subculture multiplication medium, and obtains the cluster seedling through illumination culture. The subculture multiplication medium takes MS as a basic culture medium and also comprises 0.2-1.0 mg.L of 6-BA -1 、IBA0.02～0.1mg·L -1 5% -10% of banana and 20-30 g.L of cane sugar -1 And agar 3-4 g.L -1 pH5.0-5.8; preferably, the subculture multiplication medium takes MS as a basic medium and also comprises 0.2-0.4 mg.L of 6-BA -1 、IBA0.02～0.6mg·L -1 5% -10% of banana and 20-30 g.L of cane sugar -1 And agar 3-4 g.L -1 And pH5.0-5.8. More preferably, the subculture multiplication medium takes MS as a basic medium and further comprises 6-BA0.2mg.L -1 、IBA0.05mg·L -1 10% banana and cane sugar 30 g.L -1 And agar 3.5 g. L -1 pH5.8. In the present invention, the temperature of the subculture is 25 to 31 ℃, and the illumination conditions are preferably: the illumination time is 10 to 15 h.d -1 The illumination intensity is 35 to 45 mu mol.m -2 ·s -1 . In the subculture multiplication stage, bananas with proper concentration are added, so that the growth is promoted, the strong seedlings are facilitated, and the number of effective seedlings is increased.
The invention divides the obtained cluster seedlings into plants and transfers the plants into rootsAnd (5) in a culture medium, performing illumination culture to obtain the rooted seedlings of the witloof moss. The rooting culture medium takes 1/2MS as a basic culture medium and also comprises 0.1-1.0 mg.L of NAA -1 20-30 g.L of cane sugar -1 And agar 3-4 g.L -1 pH5.0-5.8. In the invention, preferably, the rooting culture medium takes 1/2MS as a basic culture medium and also comprises 0.1-0.2 mg.L of NAA -1 20 g.L of sucrose -1 And agar 3-4 g.L -1 pH5.0-5.8. More preferably, the rooting culture medium takes 1/2MS as a basic culture medium and also comprises NAA0.2mg.L -1 20 g.L of sucrose -1 And agar 3.5 g.L -1 pH5.8. In the invention, the temperature of rooting culture is 25-31 ℃, and the illumination conditions are preferably as follows: the illumination time is 10 to 15 h.d -1 The illumination intensity is 35 to 45 mu mol.m -2 ·s -1 . The rooting culture medium and the culture conditions of the invention can obtain rooting seedlings suitable for transplanting after about 10-20 days of culture, thereby improving the survival rate of transplanting.
In the invention, the time required for obtaining aseptic bud seedlings by primary induction of the explant leaves is 30-50 days; the time for carrying out subculture multiplication culture on the stem sections with axillary buds is 30-60 days; the time for obtaining the rooting seedlings by culturing the bud seedlings in a rooting culture medium is 10-20 d, each plant has 5-8 long roots, the root length is 4-5 cm, and the rooting rate is 98%.
The standard of the clone rooted seedling of the witloof moss judged by the invention is the same as the requirement of the rooted seedling obtained by rooting culture of other plants in the prior art.
The witloof leaf is difficult to sterilize, and the browning degree of the disinfected and sterilized leaf is serious, so that the in vitro rapid propagation technology is not reported at present. The method selects tender and robust leaves of the witloof mosses as explant materials, carries out pretreatment by running water washing and low temperature before disinfection and sterilization, optimizes the disinfection steps of the witloof mosses explants, and successfully induces the explants to grow regenerated buds by adjusting the component concentration of a basic culture medium, adding an anti-browning agent, dark culture and other methods; and subculture multiplication culture is carried out on the regenerated buds, a large number of excellent seedlings can be obtained in a short time, and a foundation is laid for conservation, sustainable utilization and large-scale planting of the germplasm resources of the witloof moss.
The technical solutions of the present invention are further described in detail with reference to the following specific examples, which include but are not limited to the following examples.
Example 1
1) Pretreatment of explants: cutting young and tender leaves of witchgrass with good growth vigor and no plant diseases and insect pests, washing for 12h with running water, then sucking dry surface water with absorbent paper, wrapping with sterile absorbent paper, and placing in a refrigerator at 5 +/-2 ℃ for low-temperature treatment for 14h;
2) Explant disinfection: selecting pretreated leaves with fresh green color and no damage, soaking in 0.2% by weight of washing powder water for 10min, brushing and washing the surface of the leaves with running water, surface-disinfecting with 75% by volume of alcohol 45s 2 Sterilizing for 4min, rinsing with sterile water, removing water with sterile absorbent paper, and cutting into 1.5cm segments.
3) Primary induction: inoculating the leaf segments into a primary induction culture medium, culturing in dark for 8 days, culturing in light, wherein 60% of the leaf segments are bright green after 30 days, the leaves can induce and regenerate new seedlings, the obtained seedlings are bright green in color, thick and strong in stem, and good in growth, and the induction rate is about 52%; the formula of the primary induction culture medium is as follows: 1/2MS + 6-BA0.5mg. L -1 +IBA0.05mg·L -1 +Vc10.0mg·L -1 + 1.0 g.L of activated carbon -1 + sucrose 20 g.L -1 + agar 3.5 g. L -1 ，pH5.8。
4) Subculture proliferation: shearing the obtained sterile seedlings into stem segments with at least 1 axillary bud, transferring the stem segments into a subculture multiplication medium, and carrying out illumination culture for 40d to obtain clustered seedlings with the plant height of 5.5cm, wherein the multiplication coefficient is 5.8, and the leaves of the clustered buds are fresh green and grow strongly; the formula of the subculture multiplication medium is as follows: MS + 6-BA0.2mg.L -1 +IBA0.05mg·L -1 +10% banana + sucrose 30 g.L -1 + agar 3.5 g.L -1 ，pH5.8。
5) Rooting culture: selecting subculture seedlings with the height of 3cm and 4-6 leaves, transferring the subculture seedlings into a rooting culture medium, culturing 10 seedlings in each bottle in light for 15 days to obtain the rooted seedlings of witloof mosla, and obtaining rooted seedling plantsThe root system is thick and strong, and the rooting rate is 98% through statistics; the rooting medium comprises the following components in parts by weight: 1/2MS + NAA0.2mg.L -1 + sucrose 20 g.L -1 + agar 3.5 g.L -1 ，pH5.8。
6) And (4) hardening the obtained rooted seedlings according to a conventional method to obtain excellent clone seedlings of the witloof mosla.
Example 2
The same as the technical scheme of the embodiment 1, except that:
modifying the formula of the primary induction culture medium in the step 3) into: 1/2MS + 6-BA0.4mg.L -1 +IBA0.04mg·L -1 +Vc10.0mg·L -1 + activated carbon 1.0 g.L -1 + sucrose 20 g.L -1 + agar 3.5 g.L -1 pH5.8. 55% of the leaf segments after 30 days are bright green in color, new seedlings can be induced and regenerated by the leaves, the obtained sprouts are bright green in color, thick and strong in stems and good in growth, and the induction rate is about 47%; the survival rate of the small sections of the explant leaves and the induction rate of the regenerated seedlings are reduced compared with those of the example 1, and the reduction of the concentration of the used plant growth hormone is probably caused.
Example 3
The same technical scheme as that of the embodiment 1 is different:
modifying the formula of the subculture multiplication medium in the step 4) into: MS + 6-BA0.6mg.L -1 +IBA0.06mg·L -1 +10% banana + sucrose 30 g.L -1 + agar 3.5 g.L -1 pH5.8. After culturing for 40 days, obtaining cluster seedlings with the plant height of 4.2cm, wherein the multiplication coefficient of the cluster seedlings is 5.5, the leaves of the obtained cluster buds are fresh green, the callus grows out from the base part, and part of the stem sections are thin and weak; the proliferation coefficient is reduced compared with that of the example 1, the growth state of part of plants is poor, and probably the concentration of the used plant growth hormone is increased, the hormone proportion is increased, and the partial plants are slightly dedifferentiated and vitrified. Indicating that the higher the auxin concentration is not the better.
Example 4
The same technical scheme as that of the embodiment 2 is different from that:
the dark culture time in the step 3) is changed into: 10d; the time required for the leaf to induce and regenerate new seedlings is 35d, the color of small sections of the leaves is bright green, the obtained bud seedlings grow well, and the induction rate is about 42%; compared with the induction time of the new seedling in example 2, the induction rate is reduced, and the dark culture time is prolonged, so that the regeneration initiation of the cells is delayed, and the time required by the growth of the new bud seedling is increased.
Example 5
The same technical scheme as that of the embodiment 2 is different from that:
the formula of the rooting medium in the step 5) is modified as follows: 1/2MS + NAA0.1mg.L -1 + sucrose 20 g.L -1 + agar 3.5 g.L -1 pH5.8; performing illumination culture for 20 days to obtain rooted seedlings of the witloof moss, wherein the obtained rooted seedlings are thick and strong in plants, more and thick in root systems, and the rooting rate is 97% by statistics; the rooting time is increased, possibly with a decrease in NAA concentration, resulting in an increase in the time required for rooting.
Example 6
The same as the technical scheme of the embodiment 2, except that:
modifying the formula of the subculture multiplication medium in the step 4) into: MS + 6-BA1.0mg.L -1 +IBA0.02mg·L -1 +10% banana + sucrose 30 g.L -1 + agar 3.5 g.L -1 pH5.8; after 40 days of culture, obtaining cluster seedlings with the plant height of 3.2cm, wherein the obtained cluster seedlings are light green in leaves, slender in stems and the multiplication coefficient is 3.6. The obtained cluster buds are shorter and weaker than the cluster buds obtained in the embodiment 2, and the concentration of BA is increased, the concentration of IBA is reduced, the ratio of cytokinin to auxin is increased, partial plants are vitrified, and the result shows that the higher the concentration of cytokinin is, the better the concentration of cytokinin is.
Example 7
The same technical scheme as that of the embodiment 3 is adopted, except that:
modifying the formula of the subculture multiplication medium in the step 4) into: MS + 6-BA0.1mg.L -1 +IBA0.01mg·L -1 +10% banana + sucrose 30 g.L -1 + agar 3.5 g. L -1 pH5.8. After culturing for 40 days, obtaining cluster seedlings with the plant height of 6.0cm, wherein the obtained cluster seedlings are fresh green in leaves, thick and strong in stems, good in growth and 3.0 in multiplication coefficient. The decrease in the proliferation factor may be due to a decrease in the hormone concentration.
Example 8
The same technical scheme as that of the embodiment 3 is adopted, except that:
modifying the formula of the subculture multiplication medium in the step 4): MS + 6-BA0.2mg.L -1 +IBA0.01mg·L -1 +10% banana + sucrose 30 g.L -1 + agar 3.5 g.L -1 pH5.8. After culturing for 40 days, obtaining cluster seedlings with the plant height of 5.6cm, wherein the obtained cluster seedlings are fresh green in leaves, thick and strong in stems, good in growth and 4.2 in multiplication coefficient. The decrease in the proliferation coefficient may be due to a decrease in the concentration of hormones and a difference in the ratio of cytokinins to auxins.
Example 9
The same as the technical scheme of the embodiment 3, except that:
modifying the formula of the rooting culture medium in the step 5) into the formula: 1/2MS + NAA0.5mg. L -1 + sucrose 20 g.L -1 + agar 3.5 g.L -1 pH5.8. The light culture is carried out for 20 days, the rooting rate of the witloof mosses is 90%, the obtained rooted seedlings are thicker, new tillering buds grow out of part of the plants, and the root system is medium; the rooting time is increased, and the concentration of the auxin NAA is increased possibly, so that the regeneration of partial plants is promoted, the rooting is started slowly, and the rooting rate is reduced.
Example 10
The same as the technical scheme of the embodiment 1, except that:
the running water flushing time of tap water in the step 1) is changed into: 12h; the low-temperature treatment time in the refrigerator is modified as follows: and (5) 16h. After the leaves are cultured for 45 days by illumination, 46 percent of the leaf segments are bright green in color, the leaves can induce and regenerate new seedlings, the obtained sprouts are bright green in color, thick and strong in stems and good in growth, and the induction rate is about 38 percent. When the method is used, part of the leaves are browned and die, the induction rate of regenerated seedlings is reduced, and the pretreatment time is possibly prolonged, so that part of the leaves are damaged, the damage is aggravated after disinfection, and part of the leaves cannot be recovered and die; most of the leaves have reduced regeneration capability and reduced inductivity.
Example 11
The same technical scheme as that of the embodiment 1 is different:
the running water flushing time of tap water in the step 1) is changed into: 6h; the low-temperature treatment time in the refrigerator is modified as follows: and 6h. Illumination culture is carried out, after 30 days, 40% of leaf segments are bright green in color, new seedlings can be induced and regenerated by the leaves, the obtained sprouts are bright green in color, thick and strong in stems and good in growth, and the induction rate is about 40%; when part of the leaves are browned and dead, the induction rate of the regenerated seedlings is reduced, and the pretreatment time is possibly short, so that the phenolic substances in the leaves are excessively remained, the leaves are browned and dead, and the survival rate and the induction rate of the leaves are reduced.
Comparative example 1
The same as the technical scheme of the embodiment 1, except that:
the running water flushing time of tap water in the step 1) is changed into: and (5) 2h. After dark culture for 3d, the brown of partial leaf edges is observed, about 75% of leaves in 7d are completely browned, the leaves which are transferred to the illumination culture stage are about 100% browned in 7d, the leaf pollution rate is increased, no regeneration seedlings grow out after continuous culture for 50d, and the browning degree of the leaves is increased. It is likely that the washing time with running water is short and the leaves cannot be washed effectively from phenolic substances and microorganisms contained therein, resulting in browning and a serious degree of contamination.
Comparative example 2
The same technical scheme as that of the embodiment 1 is different:
in the step 1), the second step of operation is directly started after the exophytic plants are washed by running water of tap water for 12 hours. After dark culture for 5 days, observing that part of the leaf edges turn brown, transferring to a light culture stage for 7 days, completely turning brown on the leaf surfaces, and continuously culturing for 50 days, and then aggravating the death of the leaves. Probably, the leaves are not treated at low temperature, the activity of polyphenol oxidase in the leaves is higher, and enzymatic browning reaction occurs after disinfection, so that the browning degree of the leaves is deepened.
Comparative example 3
The same technical scheme as that of the embodiment 1 is different:
modifying the explant pretreatment in the step 1) into: and (4) cutting young and tender leaves of the witloof mosses which grow well and have no diseases and pests, washing the young and tender leaves for 2 hours by running water, and then directly starting the second step of operation. After dark culture for 2d, observing that part of the leaf edges are browned, completely browning about 80% of leaves after 7d, transferring to a light culture stage, browning 100% of leaves, increasing the pollution rate of the leaves, still not growing out the regenerated seedlings after 50d, and increasing the browning degree of the leaves. Probably, the washing time of running water is short, and phenolic substances and microorganisms contained in the leaves cannot be effectively washed away, so that browning occurs; it is also possible that the leaves are not treated at low temperature, the activity of polyphenol oxidase in the leaves is higher, and enzymatic browning reaction occurs after disinfection, resulting in browning of the leaves.
Comparative example 4
The same technical scheme as that of the embodiment 1 is different:
0.1% of HgCl in the method for disinfecting explants in step 2) 2 The disinfection time is modified as follows: and (5) 10min. After illumination culture, the leaves gradually become brown, after 15 days, 100 percent of the leaves become brown, after 50 days, no regeneration seedlings grow out, and the leaves are continuously cultured to die. Possibly HgCl 2 The disinfection time is too long, which causes irrecoverable damage to the leaves and finally causes browning and death of the leaves.
Comparative example 5
The same as the technical scheme of the embodiment 1, except that:
in the step 3), the basic culture medium of the primary induction culture medium is replaced by an MS culture medium. After 12 days of light culture, 100% of the leaf surface is browned, after 30 days of continuous culture, the part of the leaf is browned to deepen death, after 50 days of continuous culture, no regeneration seedling grows, and the leaf browning degree is increased. Probably, the concentration of inorganic salts in the basic culture medium is increased, the osmotic pressure of the culture medium is enhanced, and the phenol substances are promoted to overflow, so that the browning degree of the leaves is deepened.
Comparative example 6
The same technical scheme as that of the embodiment 1 is different:
in the step 3), vitamin C and activated carbon are not added into the primary induction culture medium. After the leaf surface is browned 100% after being cultivated for 10 days, most of the leaf is browned and dies after being cultivated for 30 days, no regrowth grows after being cultivated for 50 days, and the leaf is browned more heavily. The phenols are oxidized into quinones to form browning, and the vitamin C is an antioxidant and can reduce and inhibit oxidation reaction, so that the browning is reduced and inhibited; the activated carbon can adsorb polyphenol oxides and toxic substances, so that browning can be reduced or inhibited, and the browning degree of the leaves is deepened probably due to the lack of antioxidants and adsorbents.
Comparative example 7
The same as the technical scheme of the embodiment 1, except that:
in the step 4), the bananas are not added into the subculture multiplication medium. After 20 days of light culture, 100% of the leaf surface is browned, the brown degree of partial leaves is deepened and the leaves die after the continuous culture for 30 days, no regeneration seedlings grow out after the continuous culture for 50 days, and the brown degree of the leaves is increased. The banana has a pH buffering effect, and can also provide some necessary nutrient components, physiologically active substances, growth hormone and the like for the culture, and the culture environment is changed after the banana is not added, so that the damage to the leaves is aggravated, the phenol substances are overflowed, and the browning degree of the leaves is deepened.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (9)
1. A tissue culture method of witloof moss is characterized by comprising the following steps:
the method comprises the steps of taking tender leaves of witloof as explants to be pretreated, wherein the pretreatment method comprises the steps of washing the explants for 6-24 h with running water, and then placing the explants at 3-7 ℃ for low-temperature treatment for 6-24 h; sterilizing the pretreated explant, and sequentially performing primary induction culture, secondary multiplication culture and rooting culture;
the primary induction culture medium is 1/2MS basal culture medium plus 6-BA 0.1-1.0 mg.L -1 +IBA 0.01～0.1mg·L -1 + vitamin C5.0-20.0 mg.L -1 + 1.0-2.0 g.L of active carbon -1 + sucrose 20-30 g.L -1 + agar 3-4 g.L -1 ，pH5.0～5.8；
The subculture multiplication medium is MS as a basic medium plus 0.2-1.0 mg.L of 6-BA -1 +IBA 0.02～0.1mg·L -1 +5% -10% banana + cane sugar 20-30 g.L -1 + agar 3-4 g.L -1 ，pH 5.0～5.8；
The rooting culture medium is 1/2MS as a basic culture medium and 0.1-1.0 mg.L of NAA -1 + sucrose 20-30 g.L -1 + agar 3-4 g.L -1 ，pH 5.0～5.8；
The disinfection comprises soaking the leaves of the witloof moss in washing powder water for 5-10 min, scrubbing and washing the surface of the leaves with running water, disinfecting the surface with 75% alcohol by volume for 30-60s, 0.1% of HgCl 2 And (5) disinfecting for 3-8 min.
2. The method of claim 1, wherein the sterilized explants are cut into 1-1.5 cm pieces and inoculated into primary induction medium.
3. The method of claim 1, wherein the primary induction culture comprises a dark culture stage and a light culture stage, and the dark culture period is 7-14 days.
4. The method as claimed in claim 1, wherein the sterile seedlings obtained from the primary induction culture are cut into stem segments with 1 or 2 axillary buds, and transferred to a subculture multiplication medium for light culture.
5. The method as claimed in claim 1, wherein the plantlet is cultured in rooting medium for further culture.
6. The method according to claim 1, wherein the temperature of the primary induction culture, the secondary proliferation culture and the rooting culture is 25 to 31 ℃.
7. The method of claim 1, wherein the light conditions for the primary induction culture, the secondary proliferation culture and the rooting culture are: the illumination time is 10 to 15 h.d -1 The illumination intensity is 35 to 45 mu mol/m -2 ·s -1 。
8. The method of claim 1, wherein the primary induction culture time is 30-50 days, the secondary proliferation culture time is 30-60 days, and the rooting culture time is 10-20 days.
9. A culture medium for tissue culture of witloof moss is characterized by consisting of an initial generation induction culture medium, a secondary generation multiplication culture medium and a rooting culture medium;
the primary induction culture medium is 1/2MS basal culture medium plus 6-BA 0.1-1.0 mg.L -1 +IBA 0.01～0.1mg·L -1 + vitamin C5.0-20.0 mg.L -1 + 1.0-2.0 g.L of active carbon -1 + sucrose 20-30 g.L -1 + agar 3-4 g.L -1 ，pH5.0～5.8；
The subculture multiplication medium is MS as a basic medium plus 0.2-1.0 mg.L of 6-BA -1 +IBA 0.02～0.1mg·L -1 +5% -10% banana + cane sugar 20-30 g.L -1 + agar 3-4 g.L -1 ，pH 5.0～5.8；
The rooting culture medium is 1/2MS basal culture medium and 0.1-1.0 mg.L of NAA -1 + sucrose 20-30 g.L -1 + agar 3-4 g.L -1 ，pH 5.0～5.8。
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