CN114208679A - Tissue culture rapid propagation method of oasis 1 juncao - Google Patents
Tissue culture rapid propagation method of oasis 1 juncao Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/002—Culture media for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
- A01G24/10—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material
- A01G24/12—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material containing soil minerals
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
- A01G24/10—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material
- A01G24/12—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material containing soil minerals
- A01G24/15—Calcined rock, e.g. perlite, vermiculite or clay aggregates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
- A01G24/20—Growth substrates; Culture media; Apparatus or methods therefor based on or containing natural organic material
- A01G24/28—Growth substrates; Culture media; Apparatus or methods therefor based on or containing natural organic material containing peat, moss or sphagnum
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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Abstract
The invention discloses a tissue culture and rapid propagation method of oasis 1 junci grass, which comprises the steps of culture material selection, explant treatment and disinfection, starting culture, proliferation culture, rooting culture and domestication and transplantation. The invention belongs to the technical field of junci grass production, and particularly relates to a tissue culture rapid propagation method of junci grass 1, which optimizes the schemes of explant sterilization, stem axillary bud induction and proliferation, rooting and seedling hardening transplantation of the junci grass 1, and provides technical support for rapid propagation and large-scale production of the junci grass 1.
Description
Technical Field
The invention belongs to the technical field of juncao production, and particularly relates to a tissue culture rapid propagation method of oasis 1 juncao.
Background
Medinilla oasca No.1 (Arundo donax cv. lvzhuu No.1) is a perennial herbaceous plant belonging to the genus Arundo (Arundo) of the family Poaceae. Oasis No.1 was cultivated by the institute for fungal and grass, university of fujian agriculture and forestry, and was originally used for cultivating edible and medicinal fungi. In recent years, the grass and the fungus are concerned and researched by a plurality of scholars at home and abroad due to special biological properties and rich values. The oasis 1 grasses have high crude protein content, good palatability, fast growth and high yield, have extremely high value in the feed aspect, and are a new favorite for livestock breeding green fodder and storage. The strain No.1 oasis is drought-tolerant and waterlogging-barren, has strong coverage and tillering regeneration capacity, and can be used as a biological sand barrier and a water and soil conservation plant variety to be planted in loess hilly areas. Currently, oasis No.1 is used as a forage concentrated feed or ecological plants, and has a huge domestic and international market. However, the traditional breeding approaches of asexual propagation plants of oasis number 1, namely planting seed buds, layering propagation or cuttage have the defects of low seedling raising efficiency, insufficient seedling supply, uneven seedling quality and the like, and obviously, standardized seedlings cannot be provided for large-scale planting of oasis number 1. In recent years, popularization of oasis No.1 in northern areas of China also faces problems of seedling external adjustment, high transportation and storage cost and the like which need to be solved urgently.
At present, the regeneration path of tissue culture seedlings in oasis No.1 is only reported in public. The plant tissue culture is a common method for population propagation because the genetic characteristic of a parent can be maintained, is a unique technology for massively propagating complete plants in a short time by using less plant materials, and has the advantages of seedling detoxification, high propagation coefficient, no limitation of seasons and environmental conditions and the like. Therefore, the tissue culture technology is applied to the propagation of oasis No.1, and the industrialization of the seedlings is made to be more important to solve the problem of crop shortage and to be popularized and utilized in a large scale.
Disclosure of Invention
In order to solve the problems, the invention provides a tissue culture rapid propagation method of the oasis 1 junci grass, which optimizes the schemes of explant sterilization, stem axillary bud induction and proliferation, rooting and seedling hardening transplantation of the oasis 1 junci grass and provides technical support for rapid propagation and large-scale production of the oasis 1 junci grass.
In order to realize the functions, the technical scheme adopted by the invention is as follows: a tissue culture and rapid propagation method of Juncao Hainanensis No.1 comprises the following steps:
(5) selecting culture materials:
selecting a No.1 excellent strain of junci grass, and collecting a stem section with a node (the diameter is 0.4-0.6 cm) at the front end of a twig of a part with vigorous growth as a culture material;
(6) explant treatment and disinfection:
peeling off the culture material, removing leaves, washing with tap water for 10min to remove surface stains, cutting into stem sections with nodes of 2-3 cm by using a scalpel, transferring to an ultra-clean workbench, carrying out oscillation disinfection in 2% NaClO (sodium hypochlorite solution) for 4-12min, rinsing with sterile water for 5 times, each time for 30s, then, drying moisture on sterile filter paper, cutting off 0.3-0.5 cm at two ends by using the scalpel, carrying out spiral cutting at the swelling part of the stem node to enable a new wound surface to appear, and immediately inoculating with a forceps;
(7) starting culture:
preparing starting culture medium by adding 6-BA5.0mg/L +0.2mg/L,4-D + NAA0.2mg/L of induced axillary bud hormone in the basic culture medium, and inoculating the sterilized explant in the starting culture medium for axillary bud induction;
(8) and (3) proliferation culture:
transferring the sterile single buds (with a small amount of basal tissues during cutting) which grow robustly and uniformly in the starting culture into a proliferation culture medium which takes MS as a minimal medium and contains hormone;
(5) rooting culture:
selecting strong cluster buds (3-5 cm) in multiplication culture, transferring the cluster buds to an MS rooting culture medium added with additives for culture, adding 30g/L of sucrose and 6.5g/L of agar powder into the culture medium, adjusting the pH value to 6.0, adding hormones, preparing and subpackaging, sterilizing by high-pressure steam at 121 ℃ for 20min, inoculating, and placing in an incubator at the temperature of (25 +/-3) ° C, the illumination intensity of 2500-3000 lx, the light period of 12h/d and the relative air humidity of 50-60%;
(6) domestication and transplantation:
unscrewing a bottle cap of a tissue culture seedling of oasis No.1 with the plant height of about 10cm, the root length of 1-4 cm and strong growth in an incubator for 1-2 days, then completely opening the cover, moving the seedling to natural illumination for ventilation adaptation for about 5 days, spraying water for 3 times per day for moisture preservation, and washing off the residual culture medium at the root by using normal-temperature water (20-22 ℃) before transplanting. Soaking the roots of the tissue culture seedlings in 10% carbendazim solution for 10min, washing again, planting the tissue culture seedlings in a matrix fully sterilized by 0.1% potassium permanganate, transplanting the tissue culture seedlings, placing the tissue culture seedlings in a small arch shed for maintenance, spraying water by an automatic spraying device to keep the air humidity to be 60-80%, and meanwhile properly ventilating.
Further, at least 1cm is reserved above and below the stem node in the step 2.
Further, the start culture in the step 3 comprises 1/2MS, WPM, one of White 4 minimal mediums.
Further, in the step 4, the hormone is 6-benzylaminopurine (6-BA), and one of 2, 4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA) or Naphthalene Acetic Acid (NAA) is added.
Further, the additive in the step 5 is one of NAA, IBA + NAA or potato filtrate.
Further, the potato filtrate is prepared by selecting commercially available potatoes which are free of virus invasion, complete in surface and free of sprouting, slicing, cooking, and filtering with gauze, wherein the filtrate is ready for use.
Further, in the step 6, the matrix is one of the following components: 1:1 of river sand and vermiculite; ② peat soil, vermiculite 1: 1; (iii) vermiculite: perlite: 1; fourthly, river sand, turfy soil and perlite are mixed in a ratio of 2:1: 1; fifthly, the river sand is vermiculite and perlite are 2:1: 1.
The invention adopts the structure to obtain the following beneficial effects: the tissue culture rapid propagation method of the oasis 1 junci grass provided by the invention is simple to operate, compact in structure and reasonable in design, takes the oasis 1 junci stem section as a material, improves the schemes of explant disinfection, axillary bud induction, subculture, seedling hardening and transplanting, establishes a set of complete scheme from oasis 1 production to seedling hardening and transplanting, and provides technical support for industrialization and high-quality seedling culture of the oasis 1 junci grass.
Drawings
FIG. 1 is a graph of the effect of 2% NaClO disinfection time on the periphyton contamination rate and germination rate on oasis number 1 (30 d);
FIG. 2 shows the induction effect of four auxins on adventitious roots in oasis number 1 (30 d);
FIG. 3 shows the effect of different substrates on the survival rate and plant height of transplanted seedlings (20 d).
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
This example is an example of a research procedure, in particular
The test material is obtained by selecting a No.1 excellent strain of Juncao oasis in 3-5 late decades of 2021, and collecting a stem section with a node (the diameter is 0.4-0.6 cm) at the front end of a twig of a part with vigorous growth as a test material. The test material is from greenhouse of Indonesian agriculture university Indonesia venture science and technology hatching park.
The test method comprises the following steps:
1. explant treatment and disinfection
Peeling off the epidermis of a newly picked twig, removing leaves, washing with tap water for 10min to remove surface stains, cutting into stem sections with nodes of 2-3 cm (at least 1cm is reserved above and below the stem node) by using a scalpel, transferring to an ultra-clean workbench, carrying out oscillation disinfection in 2% NaClO (sodium hypochlorite solution) for 4, 6, 8, 10 and 12min, rinsing for 5 times by using sterile water for 30s each time, then sucking water on the sterile scalpel, cutting off 0.3-0.5 cm at two ends by using the scalpel, carrying out spiral cutting at the swelling position of the stem node to enable a new wound surface to appear, and immediately inoculating to a start culture medium by using a forceps. 4 explants were inoculated in 250ml Erlenmeyer flasks, 15 flasks per treatment, 3 replicates. And observing the growth state of the No.1 oasis explants every 10 days, and counting the contamination rate and the germination rate after 30 days of inoculation.
2. Starting culture
The sterilized explants were inoculated in a start medium for axillary bud induction to establish sterile oasis No.1 system. 1/2MS, WPM and White 4 minimal mediums are selected for starting culture, and the optimal hormone combination for inducing axillary buds screened by a plurality of preliminary tests is added in each minimal medium: 6-BA5.0mg/L +0.2mg/L2,4-D + NAA0.2mg/L. 60 explants were inoculated per treatment and repeated 3 times. The growth state (leaf color, growth vigor) is recorded every 10 days after inoculation, and the axillary bud induction rate, the proliferation coefficient and the height of the new bud are calculated 30 days after inoculation.
3. Proliferation culture
Sterile single shoots (with small amounts of basal tissue when cut) that grow well and consistently in the priming culture are transferred to multiplication media containing different hormone combinations. In this experiment, MS was used as a minimal medium, and 6-benzylaminopurine (6-BA) was added at concentrations of 4, 6, and 8mg/L, 2, 4-dichlorophenoxyacetic acid (2,4-D) at concentrations of 0, 0.1, and 0.2mg/L, and L9(34) was orthogonally combined with indole-3-acetic acid (IAA) at concentrations of 1.0, 1.5, and 2.0mg/L, and Naphthalene Acetic Acid (NAA) at concentrations of 0, 0.1, and 0.2mg/L (see Table 5). There were 9 treatments, each 20 flasks and 3 axillary buds inoculated per flask, repeated 3 times. The growth state (sprouting speed, leaf color, growth vigor and the like) is recorded every 10 days after inoculation, and the number of cluster buds with the length of more than 5mm is counted after 30 days of culture, so that the proliferation coefficient is calculated.
TABLE 1L9(34) Level of orthogonality test factor
4. Rooting culture
Selecting strong cluster buds (3-5 cm) in enrichment culture, transferring the cluster buds to MS rooting culture media respectively added with 4 additives (each provided with 3 concentration gradients) such as NAA, IBA + NAA, potato filtrate and the like, and culturing for 12 treatments, wherein 60 subculture buds are inoculated in each treatment. And after 30 days of inoculation, counting the rooting rate, the rooting quantity and the root length index. The preparation method of the potato filtrate comprises the following steps: selecting potato without virus invasion, intact surface and no germination, slicing, cooking, filtering with gauze, and collecting filtrate.
Adding sucrose 30g/L and agar powder 6.5g/L into all the above culture media, adjusting pH to 6.0, adding hormone required in different treatments, packaging, and sterilizing with high pressure steam at 121 deg.C for 20 min. After inoculation, the mixture is placed in an incubator, the temperature is 25 +/-3 ℃, the illumination intensity is 2500-3000 lx, the photoperiod is 12h/d, and the relative air humidity is 50-60%.
5. Domesticated transplantation
And (3) unscrewing the bottle cap of the oasis No.1 tissue culture seedling which is about 10cm in plant height, 1-4 cm in root length and strong in growth in an incubator for 1-2 days, then completely opening the cover, moving the cover to natural illumination for ventilation adaptation for about 5 days, and spraying water for 3 times per day for moisturizing. And (3) washing residual culture medium at roots with normal-temperature water (20-22 ℃) before transplanting. Soaking the roots of the tissue culture seedlings in 10% carbendazim solution for 10min, washing again, and then respectively planting the tissue culture seedlings in a substrate fully disinfected by 0.1% potassium permanganate (five kinds of treatment Y1-Y5 are arranged in total): 1:1 of river sand and vermiculite; ② peat soil, vermiculite 1: 1; (iii) vermiculite: perlite: 1; fourthly, river sand, turfy soil and perlite are mixed in a ratio of 2:1: 1; fifthly, repeating the steps of treating and transplanting 50 tissue culture seedlings for 3 times, wherein the river sand is vermiculite and the perlite is 2:1: 1. And (4) placing the transplanted seedlings in a small arched shed for maintenance, spraying water by using an automatic spraying device (NS2L01, Guangdong) to keep the air humidity at 60-80%, and meanwhile properly ventilating. Observing the growth condition of the plants, counting the survival rate and the plant height after transplanting for 20d, and analyzing.
6. Data analysis
The graphs were plotted using Microsoft Excel 2010 software, statistical analyses such as one-way variance (ANOVA), range analysis, and multiple comparisons (Duncan test) were performed on the data using SPSS 25.0 software, and the significance level was set to 0.05.
7. Results
7.1 Effect of different Disinfection times on Disinfection of Stem segments of oasis No.1
The effect of different disinfection time on the disinfection of oasis No.1 stem segments is shown in fig. 1, as shown in fig. 1, the explant contamination rate continuously decreases with the increase of 2% NaClO treatment time, the germination rate increases first and then decreases, and the disinfection effect has a significant difference. When the sterilization time is 4min, the pollution rate is 35.6%, and the germination rate is at a lower level due to the influence of the pollution on the explants; when the sterilization time reaches 6min, the explant contamination rate is remarkably reduced, the germination rate is increased from 41.7% to 87.8%, the germination rate is increased by more than 2 times and reaches the highest value, and at the moment, the explant of oasis number 1 has a higher germination speed and a good growth condition; when the sterilization time of the explant is increased to 8 or 10min, although the pollution rate keeps a descending trend, the germination rate of axillary buds of the explant is remarkably reduced, and the effect is not ideal. When the sterilization is carried out for 12min, a large number of explants die gradually, and the pollution rate and the germination rate are both the lowest.
7.2 Effect of basic Medium on Aroasis No.1 Stem axillary bud Induction
Sterile segmented stem segments were inoculated into the start-up medium, and the difference in the induction capacity of 4 different minimal media for axillary buds of stem segment No.1 of oasis was significant (table 2). As shown in table 2, treatment with MS as the minimal medium, C2, was most significant, with an induction rate of 92.22%; c1, C3 induction rates were intermediate; the induction effect of C4 is poor, and the induction rate is only 36.25%. The 4 minimal mediums are ranked as MS > WPM >1/2MS > White according to the multiplication coefficient of adventitious buds from high to low, the multiplication coefficient of the MS medium reaches 3.32, and the multiplication coefficient of the White medium is 0.8 and is only 1/4 of the MS medium. In terms of growth conditions, the height of the adventitious bud treated by C2 is obvious, the average bud height is 7.7cm, and the leaf is unfolded and bright green; c1 treatment; shoots were short in C3 and C4 and were relatively less robust.
TABLE 2 minimal Medium screening (30d)
A, hormone combinations added into the culture media are 6-BA5.0mg/L + IBA1.5mg/L + NAA0.2mg/L +2, 4-D0.2mg/L;
note b that the data in the table are mean ± standard deviation; different letters in the same column indicate that the difference is statistically significant at a level of P < 0.05.
7.3 Effect of different combinations of plant growth regulators on subculture proliferation
The MS is taken as a basic culture medium, different additional plant growth regulator combinations exist, and the proliferation coefficient of the adventitious bud of oasis 1 is remarkably different (p is less than 0.05) (see table 3). The proliferation coefficient of the explants treated with J6 was 7.29, which is significantly higher than that of the other treatments. And (3) analyzing the range difference of the orthogonal test results, wherein the range difference of each factor is respectively 1.9, 0.81, 0.6 and 0.6, the primary and secondary ranking order of the influence of each factor is 6-BA >2,4-D > IAA-NAA, 6-BA is the factor which has the largest influence on the adventitious bud proliferation, and 2,4-D are the factors. In the test of the effect between the subjects (Table 4), the F values of the 4 hormones are 33.689(6-BA), 1.407(2,4-D), 1.086(NAA) and 0.856(IAA) in turn from large to small, and only 6-BA influences the proliferation coefficient to a very significant level (P < 0.01). The number of adventitious buds increases with the increase of the concentration of 6-BA, but the influence of 2,4-D, IAA and NAA on the proliferation coefficient is not obvious.
TABLE 3 orthogonal experimental design L9 (3)4) Sum and pole difference analysis
Note a that the minimal medium is MS.
Note b that the data in the table are mean ± standard deviation; different letters in the same column indicate that the difference is statistically significant at a level of P < 0.05.
TABLE 4 examination of the Effect between subjects
5. Effect of different auxins on the Effect of rooting
As can be seen from table 5, the 12 treatments with MS as the minimal medium, and with different concentrations of NAA, IBA + NAA, and potato filtrate all promoted rooting of the adventitious bud of oasis No.1, with a rooting rate of 72.76% to 97.22%, but with large differences in root morphology and mass (fig. 2). The influence of different culture medium formulas on the rooting rate, the root number and the root length of the adventitious bud of oasis No.1 is obviously different (P is less than 0.05). In the S9 process, each evaluation index of the adventitious root is significantly higher than that in the other processes. In a rooting culture medium with auxin NAA added alone, the rooting rate of the adventitious bud No.1 of oasis is improved along with the increase of the concentration of the NAA, the number and the root length are increased gradually, the rooting rate of a single bud and the number of roots are not obviously different in the treatment of S12 and S3, the root length index of S12 is obviously superior to that of S1, S2 and S3, and the organic potato filtrate has an obvious promotion effect on inducing the adventitious bud No.1 of oasis. In the culture medium added with IBA with different concentrations, each rooting index tends to rise first and then fall along with the increase of the IBA concentration, and each index processed by S5 is better; the rooting effect is found to be more remarkable by adding NAA with different mass concentrations into the S5 culture medium. In a comprehensive view, MS + IBA5.0mg/L + NAA0.9mg/L can be used as a rooting culture medium of the tissue culture seedling of oasis No.1, each index of the treated lower root is obvious, and the tissue culture seedling has good growth vigor and is beneficial to domestication and transplantation in a later period.
TABLE 5 Effect of different hormone ratios on adventitious bud rooting
Note that the data in the table are mean values. + -. standard deviations; different letters in the same column indicate that the difference is statistically significant at a P <0.05 level
6. Influence of transplanting matrix ratio on survival rate and plant height of tissue culture seedlings
The matrix suitable for oasis No.1 transplanting needs to have certain fertility and is breathable and moisture-retaining. The test results show that the transplanting growth of the tissue culture seedlings is influenced by the matrix significantly, and the influence of different matrix formulations on the survival rate and the plant height of the tissue culture seedlings of oasis No.1 is shown in fig. 3 (20 d). The survival rate of the tissue culture seedlings in the medium containing river sand is higher, particularly in the Y4 medium (river sand: turfy soil: perlite: 2:1:1), the transplanting survival rate reaches 96.67 percent and is obviously higher than that of other 4 groups of treatments. The height of the tissue culture seedlings is sequentially Y4, Y5, Y1, Y3 and Y2 from high to low, the height of the tissue culture seedlings in Y4 is 24.27cm, the tissue culture seedlings grow well, the plants are strong and fresh green, the leaves are extended, and the transplanting of tissue culture seedlings in oasis No.1 into the matrix is reasonable.
Example 2:
sterilizing stem segments of No.1 Juncao in oasis with 2% NaClO for 6min, wherein the germination rate is 87.8%; the most suitable starting culture medium is MS +6-BA5.0mg/L + IBA1.5mg/L +2,4-D0.2mg/L + NAA0.2mg/L, the induction rate is 92.22 percent, and the multiplication coefficient is 3.32; culturing in MS +6-BA 6mg/L +2,4-D0.2mg/L + IAA 1.0mg/L + NAA 0.1mg/L at proliferation stage, wherein proliferation coefficient reaches 7.29; the rooting medium MS + IBA5.0mg/L + NAA0.9mg/L has the most obvious effect, and the rooting rate is 97.22%; transplanting the domesticated tissue culture seedlings into a medium of river sand, turfy soil and perlite (2:1:1) (V: V), wherein the survival rate reaches 96.67%.
The present invention and its embodiments have been described above, and the description is not intended to be limiting, and the drawings are only one embodiment of the present invention, and the actual structure is not limited thereto. In summary, those skilled in the art should appreciate that they can readily use the disclosed conception and specific embodiments as a basis for designing or modifying other structures for carrying out the same purposes of the present invention without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (6)
1. A tissue culture rapid propagation method of oasis 1 juncao is characterized by comprising the following steps:
(1) selecting culture materials:
selecting a No.1 excellent strain of Juncao oasis, and collecting a stem section with a node at the front end of a twig of a part with vigorous growth as a culture material;
(2) explant treatment and disinfection:
peeling off the culture material, removing leaves, washing with tap water for 10min to remove surface stains, cutting into stem sections with nodes of 2-3 cm by using a scalpel, transferring to an ultra-clean workbench, carrying out oscillation disinfection in 2% NaClO for 4-12min, rinsing with sterile water for 5 times for 30s each time, then sucking water on sterile filter paper, cutting off 0.3-0.5 cm at two ends by using the scalpel, carrying out spiral cutting at the expansion part of the stem node to enable a new wound surface to appear, and immediately inoculating by using a forceps;
(3) starting culture:
preparing starting culture medium by adding 6-BA5.0mg/L +0.2mg/L2,4-D + NAA0.2mg/L induced axillary bud hormone in the basic culture medium, inoculating sterilized explant in the starting culture medium to induce axillary bud;
(4) and (3) proliferation culture:
transferring the sterile single buds which grow vigorously and have consistent growth vigor in the starting culture into a multiplication culture medium which takes MS as a basic culture medium and contains hormones;
(5) rooting culture:
selecting 3-5 cm of strong cluster buds in multiplication culture, transferring the cluster buds into an MS rooting culture medium added with additives for culture, adding 30g/L of sucrose and 6.5g/L of agar powder into the culture medium, adjusting the pH value to 6.0, adding hormones, preparing and packaging, sterilizing for 20min by high-pressure steam at 121 ℃, placing in an incubator after inoculation, and controlling the temperature: 22-28 ℃, light intensity: 2500-3000 lx, the photoperiod is 12h/d, and the relative air humidity is 50% -60%;
(6) domestication and transplantation:
unscrewing a bottle cap of a tissue culture seedling of oasis No.1 with the plant height of about 10cm, the root length of 1-4 cm and strong growth in an incubator for 1-2 days, then completely opening the cover, moving the seedling to natural illumination for ventilation adaptation for about 5 days, spraying water for 3 times per day for moisturizing, and washing off residual culture medium at the root part by using water at the temperature of 20-22 ℃ before transplanting. Soaking the roots of the tissue culture seedlings in 10% carbendazim solution for 10min, washing again, planting the tissue culture seedlings in a matrix fully sterilized by 0.1% potassium permanganate, transplanting the tissue culture seedlings, placing the tissue culture seedlings in a small arch shed for maintenance, spraying water by an automatic spraying device to keep the air humidity to be 60-80%, and meanwhile properly ventilating.
2. The tissue culture and rapid propagation method of Juncao, Haizhou No.1, according to claim 1, is characterized in that: in the step 2, the stem nodes are reserved for more than 1cm above and below.
3. The tissue culture and rapid propagation method of Juncao, Haizhou No.1, according to claim 2, characterized in that: in the step 4, the hormone is 6-benzylaminopurine, and one of 2, 4-dichlorophenoxyacetic acid, indole-3-acetic acid or naphthylacetic acid is added.
4. The tissue culture and rapid propagation method of Juncao, Haizhou No.1, according to claim 3, wherein: the additive in the step 5 is one of NAA, IBA + NAA or potato filtrate.
5. The tissue culture and rapid propagation method of Juncao, Haizhou No.1, according to claim 4, wherein: the potato filtrate is prepared by selecting commercially available potatoes which are free of virus invasion, complete in surface and free of sprouting, slicing, cooking, and filtering with gauze to obtain filtrate for later use.
6. The tissue culture and rapid propagation method of Juncao, Haizhou No.1, according to claim 5, is characterized in that: the matrix in the step 6 is one of the following components: river sand: 1, vermiculite: 1; turfy soil: 1, vermiculite: 1; vermiculite: perlite is 1: 1; river sand: turfy soil: perlite is 2:1: 1; river sand: vermiculite: perlite is 2:1: 1.
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