CN112640785A - Method for establishing asexual rapid propagation system of double-petal lily tundra - Google Patents
Method for establishing asexual rapid propagation system of double-petal lily tundra Download PDFInfo
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Abstract
The invention discloses a method for establishing a rapid asexual propagation system of lilium bicolor perianth in the technical field of tissue culture of lilium brownii plants, which comprises the following steps of S1, obtaining sterile explants, taking perianth of lilium bicolor 'Isabella' as the explants, picking unopened buds, cleaning and then sterilizing; s2, preparing a culture medium, and combining different types and concentrations of plant hormones by taking MS as a basic culture medium; s3, starting culture, inoculating the trapped perianth anth into a starting culture medium for culture, and inducing adventitious buds; s4, proliferation culture, namely inoculating the adventitious buds into a proliferation culture medium to induce the adventitious buds to proliferate; s5, rooting culture, screening adventitious buds, and placing the adventitious buds into a rooting culture medium to induce rooting, so as to obtain a complete sterile test-tube plantlet. Compared with the prior art, the technical scheme establishes a rapid asexual propagation technical system of the double-petal lily tundra, and lays a foundation for the industrialized production of high-quality test-tube seedlings.
Description
Technical Field
The invention belongs to the technical field of lily plant tissue culture, and particularly relates to a method for establishing a rapid asexual propagation system of double-petal lily perianth.
Background
Bulbus Lilii (Lilium spp.) is a perennial herb of the genus Lilium (Lilium) of the family Liliaceae, with an underground bulb[1]. As a flower with extremely high ornamental value, the flower is suitable for garden, flower bed cultivation and potted plant, is deeply loved by people with the meaning of 'conjugal felicity', and becomes one of the flowers which are very popular in the market; as one of the four fresh cut flowers, the sales volume of the seed balls and the cut flowers of the lily are always in the forefront of the world, and the lily is called as the king of bulbous flowers.
The traditional propagation of lily mainly adopts methods such as bulb separation, bulbil separation, scale cuttage and the like, but the propagation coefficient is low, and particularly after multi-generation propagation, virus accumulation is easily caused, the seed properties are degraded, and the yield and the quality of lily are influenced. The application of the plant tissue culture technology to rapidly propagate and produce the horticultural flower plants has the advantages of no limitation of climate, season and region, convenience for industrial production and the like, and the advantages make up for the defects of the traditional propagation.
The lilium bivalvate is a new horticultural variety of lilium brownie, has extremely high ornamental and economic values, and a great deal of research is carried out on tissue culture of lilium brownie by people before, but no report is found on tissue culture of lilium bivalvate at present. Firstly, the flower quilt sheet is adopted as the explant, MS culture medium is adopted to combine with plant hormone proportions of different types and concentrations, the tissue culture is carried out on the heavy lily 'Isabella' flower quilt sheet, researches are respectively carried out on the aspects of induced differentiation, proliferation seedling emergence, rooting culture and the like, a technical system for asexual rapid propagation of the heavy lily 'Isabella' is established, and a foundation is laid for the industrialized production of high-quality test tube seedlings of the heavy lily.
Disclosure of Invention
In order to solve the problems, the invention aims to establish an asexual rapid propagation technical system by taking the lilium bicolor 'ixabella' perianth tablet as an explant and lay a foundation for producing high-quality lilium bicolor test-tube seedlings.
In order to achieve the purpose, the technical scheme of the invention is as follows:
s1, obtaining sterile explants, taking perianth sheets of the lilium bicolor 'Isabella' as the explants, picking unopened buds, cleaning and then disinfecting;
s2, preparing the culture medium by taking MS as a basic culture medium and mixing 0.5-2.0mg/L of phytohormone 6-BA and 0.1-1.0mg/L of NAA and 0.5-1mg/L of 2,4-D in different types and concentrations;
s3, starting culture, inoculating the trapped perianth anth into a starting culture medium for culture, and inducing and differentiating adventitious buds;
s4, proliferation culture, namely inoculating the adventitious buds into a proliferation culture medium to induce the adventitious buds to proliferate;
s5, rooting culture, screening adventitious buds, and placing the adventitious buds into a rooting culture medium to induce rooting, so as to obtain a complete sterile test-tube plantlet.
Further, the method for preparing the propagation medium in S4 includes a), b), c), d), e) or f)
a) Mixing 6-BA1.0mg/L and NAA0.1mg/L by taking MS as a basic culture medium;
b) mixing 6-BA0.5mg/L and NAA0.2mg/L with MS as a basic culture medium;
c) mixing 6-BA1.0mg/L and NAA0.2mg/L with MS as a basic culture medium;
d) mixing MS as basic culture medium with 6-BA0.5mg/L and NAA0.3mg/L;
e) mixing MS as basic culture medium with 6-BA1.0mg/L and NAA0.3mg/L;
f) simple MS medium.
Further, the method for preparing the rooting medium in the step S5 includes g), h), i), k), l), f), i.e., g)1/2MS + NAA0.1, h)1/2MS + NAA0.5, i)1/2MS, k) MS + NAA0.1, l) MS + NAA0.5, f) MS, and the statistical rooting rate is observed with reference to the following rooting rate (number of rooted explants/number of inoculated explants) × 100%.
Further, in the disinfection treatment process, firstly, unopened buds are placed under running water and washed for 20-30 min, an operator wipes the buds dry by using absorbent paper, then the buds are placed in a sterilized glass bottle, a superclean workbench in the glass bottle is soaked in 70-75% alcohol for 20-30 s, then the explants are washed for 3-4 times by using sterile water, then the explants are disinfected for 3-4 min by using 10-time diluted chlorine dioxide disinfectant, and finally the explants are washed for 4-5 times by using the sterile water.
Further, in the step S2, 5-7 g/L of agar and 27-32 g/L of sucrose are added until the pH value is 5.8-6.0, and then the culture medium is placed in an autoclave at 120-125 ℃ for sterilization for 25-30 min.
Further, the temperature of the illumination incubator is adjusted to 25 +/-1 ℃ in the day, 18 +/-1 ℃ in the night, the illumination intensity is 2000-3000 lx, the illumination time is 16h/d, and the dark culture is 8h/d, so that the composite regulation and control of ecological factors under the simulated natural environment are carried out.
Further, in step S1, the double-petal lily is sliced into 5X 5mm small pieces.
Further, step S3 includes stirring the culture medium with a stirring rod, where the bottom of the stirring rod is in a spiral multi-head shape, and through holes are formed at two radial sides of the stirring rod, and a notch is formed at one side of each through hole.
After the scheme is adopted, the following beneficial effects are realized:
1. the technical scheme establishes a rapid asexual propagation technical system for the lilium bivalvate, lays a foundation for producing high-quality test-tube seedlings and lays an important foundation for the lilium bivalvate genetic transformation.
2. According to the technical scheme, various proliferation culture media are debugged, and the influence of different culture media on adventitious bud induction is observed, so that an optimal culture scheme is established.
3. Compared with the prior art of utilizing mercuric chloride to disinfect in the prior art, utilize novel environmental protection disinfectant chlorine dioxide to disinfect among this technical scheme, promoted the feature of environmental protection.
4. Compared with the prior art of bulb culture, the technical scheme utilizes the floral quilt sheet for culture, creates a new culture system and protects the stock plant body.
5. Compared with the prior art for protecting the stock plant body, the technical scheme initiates the cultivation of the lilium bivalvata and fills the blank in the prior art.
6. Compared with other pioneering culture technologies, the technical scheme utilizes the illumination incubator to perform composite regulation and control on conditions such as temperature, illumination, humidity and the like, and simulates the culture condition in a natural state.
7. Compared with other prior arts of exogenous hormone addition, the optimal proportion is obtained by orthogonal experiments by using different levels of plant hormones.
8. Compared with the prior art adopting orthogonal experiments, the multi-head stirring rod is adopted to stir the disturbed flow of the culture medium in the technical scheme, because the hormone and the agar added in the culture medium realize layering due to different densities, the culture medium is mixed by stirring operation, and the disturbed flow and convection effect are realized by the double-helix structure.
9. Compared with the prior art of stirring layering, the through-hole that the stirring rod was seted up among this technical scheme is convenient for stir convection in-process flowers and is passed the through-hole, and the breach introduces the culture medium on every side to smear the culture medium to flowers and quilt piece surface at the vortex in-process.
Drawings
FIG. 1 is a flow chart of an embodiment of the present invention;
FIG. 2 is a graph showing the effect of different hormone combinations on adventitious bud induction of double-petal lily buds;
FIG. 3 shows the effect of different hormone combinations on the multiplication culture of envelope pieces of lilium bivalvia flowers;
FIG. 4 shows the effect of different culture media and hormone concentrations on the rooting culture of the peridium of lily;
FIG. 5 is a schematic view showing the construction of a stirring rod in the second embodiment.
Detailed Description
The following is further detailed by way of specific embodiments:
the reference signs are: stirring rod 1, through-hole 2.
Example one
Referring to fig. 1, the specific implementation process is as follows: a method for establishing a asexual rapid propagation system of lilium bicolor duvet slices comprises the steps of taking the lilium bicolor duvet slices as explants, picking unopened buds which are about 6cm long, washing the buds for 30min under running water, wiping the buds with absorbent paper, putting the buds into sterilized glass bottles, soaking the buds in 70% alcohol for 30s in an ultraclean workbench, washing the buds with sterile water for 3 times, preparing the buds into a ratio of 1: 10 by adopting a novel environment-friendly disinfectant, disinfecting the explants for 3-4 min, and finally washing the explants with the sterile water for 4-5 times for later use.
MS is used as a basic culture medium, 6g/L of agar, 30g/L of cane sugar and 5.8-6.0 of PH value are added. The medium was sterilized in an autoclave at 121 ℃ for 30 min. The culture adopts variable temperature culture, the day temperature is 25 +/-1 ℃, the night temperature is 18 +/-1 ℃, the illumination intensity is 2000-3000 lx, and the illumination time is 16 h/d.
Starting culture
The sterilized double-petal lily quilt slices are cut into small pieces of 5 multiplied by 5mm, and the small pieces are respectively inoculated on each group of start culture medium, and the hormone types and the hormone concentrations adopt three-factor three-level orthogonal design, which is shown in table 1.
TABLE 1 three-factor three-level orthogonal design
Proliferation culture
Selecting materials with good growth conditions for starting culture, and inoculating the materials to 6 proliferation culture media: a) MS +6-BA1.0mg/L + NAA0.1mg/L (the same units are shown below), b) MS +6-BA0.5+ NAA0.2, c) MS +6-BA1.0+ NAA0.2, d) MS +6-BA0.5+ NAA0.3, e) MS +6-BA1.0+ NAA0.3, f) MS (control group), and 3 pieces were inoculated in each culture dish. Differentiation rate (number of budding explants/number of inoculated explants) × 100%; the growth coefficient is the number of sprouts after subculture/the number of sprouts before subculture.
Rooting culture
Selecting plantlets which grow to 3-4cm and have good growth conditions in the proliferation culture, transferring the plantlets into rooting culture media containing plant hormones with different concentrations for rooting culture, and setting 1/2MS without adding hormones, wherein the MS culture media are used as controls. g)1/2MS + NAA0.1, h)1/2MS + NAA0.5, i)1/2MS, k) MS + NAA0.1, l) MS + NAA0.5, f) MS, and the rooting rate was observed and counted. The rooting rate is (number of rooted explants/number of inoculated explants) x 100%
Results and analysis of the experiments
Effect of different hormone combinations on adventitious bud induction of floral quilt sheet
Please refer to fig. 2, wherein a: hormone combination 1B: hormone combination 2C: hormone combination 3D: hormone combination 4E: hormone combination 5, F: hormone combination 6G: hormone combination 7H: hormone combination 8I: hormone combination 9, (start culture)
The perianth lobes begin to thicken and expand after 7 days of inoculation, only a very small amount of bud germination can be seen after 45 days of observation, after subculture, light yellow tender buds appear at the cut of about 25 days of perianth lobes after 6-BA2.0+ NAA0.5 and 6-BA1.0+ NAA0.1+2 and 4-D0.5 of hormone combination, and the statistical result of 45 days is shown in table 2.
TABLE 2 orthogonal test protocol and result analysis for double-petal lily quilt sheet
Note: kj1Is the sum of differentiation rates corresponding to 1 level of factor j, Kj2Is the sum of differentiation rates corresponding to 2 levels of factor j, Kj13Is the sum of differentiation rates corresponding to 3 levels of factor j, which represents factor A, B or C; k is a radical ofj1Mean differentiation rate, k, corresponding to level 1 of factor jj2Mean differentiation rate, k, corresponding to 2 levels of factor jj3The mean differentiation rate corresponding to the 3 levels of factor j.
As can be seen from table 2, the induced differentiation rates of the phytohormones are different according to different plant hormone ratios, and the range analysis shows that the range corresponding to 6-BA (factor a) is the largest (R ═ 0.344), which indicates that the effect of factor a is the most obvious, and NAA is the next.
As can be seen from the orthogonal test Table 2, the average values of the differentiation rates at 3 levels of 6-BA (factor A) were 0.122, 0.389 and 0.476, respectively, indicating that the water content of 6-BAThe flat variation has an effect on the test according to kA1，kA2，kA3In the order of magnitude (k)A3>kA2>kA1)，A3The optimal level for factor A, 6-BA at 2.0mg/L, is the most suitable concentration for differentiation in this study; also NAA according to kB1，kB2，kB3In the order of magnitude (k)B2>kB3>kB1)，B2Is the optimal level for factor B, i.e., NAA at a level of 0.5mg/L is the most suitable concentration for differentiation in this study; likewise 2,4-D according to kC1，kC2，kC3In the order of magnitude (k)C1>kC3>kC2)，C1The optimal level for factor C, 2,4-D at 0mg/L, is the most suitable concentration for differentiation in this study; thus, the results of the orthogonal experiments showed that the optimal phytohormone combination was A3B2C1Namely MS +6-BA2.0mg/L + NAA0.5mg/L is a suitable culture medium for inducing the differentiation of the double-petal lily perianth.
Proliferation culture
Please refer to fig. 3, wherein J: hormone combination 1K: hormone combination 2, L: hormone combination 3, M: hormone combination 4, N: hormone combination 5, O: a combination of hormones 6 which is selected from the group consisting of,
the appearance of buds is observed after 10 days of inoculation, green seedlings are obviously increased after 20 days, the seedlings grow to 4-5cm after 40 days, and the statistical result is shown in table 3 after 40 days.
Table 3 shows that: the adventitious buds of the double-petal lily perianth tablet can be proliferated within the concentration ranges of 0.5-1.0 mg/L of 6-BA and 0.1-0.3 mg/L of NAA, and variance analysis shows that the 6 groups of hormone combinations have obvious difference among groups. The hormone combination with good proliferation effect is MS +6-BA1.0mg/L + NAA0.1mg/L and MS +6-BA0.5 mg/L + NAA0.3mg/L, the proliferation coefficients are respectively 6.951 and 6.801 which are obviously superior to those of a control group, but the number of seedlings growing on a culture medium with the hormone combination of MS +6-BA1.0mg/L + NAA0.1mg/L is more than that of seedlings growing on a culture medium with the hormone combination of MS +6-BA0.5 mg/L + NAA0.3mg/L, and the color is dark, so that MS +6-BA1.0mg/L + NAA0.1mg/L is a culture medium suitable for bud proliferation.
TABLE 3 Effect of different hormone combinations on the propagation culture of Lilium polyphylla
Root induction
Referring to fig. 4, P: hormone combination 1, Q: hormone combination 2, R: hormone combination 3, S: hormone combination 4, T: hormone combination 5, U: a combination of hormones 6 which is selected from the group consisting of,
selecting plantlets which grow to 3-4cm and have good growth conditions in the enrichment culture, transferring the plantlets into a rooting culture medium for rooting culture, and setting a control group. Rooting is started after 15 days, the number of roots, the average number of roots and the like are counted after 30 days, and the statistical results are shown in a table 4.
TABLE 4 Effect of different media, hormone concentrations on the rooting culture of adventitious buds of Lilium graveolens
Table 4 shows that: the experimental group 1/2MS + NAA 0.1-0.5 mg/L and MS + NAA 0.1-0.5 mg/L culture medium and the control group 1/2MS and MS two basic culture media can induce rooting, but the rooting rate of the experimental group is obviously higher than that of the control group. Wherein when the hormone combination is MS + NAA0.1mg/L, the rooting rate is the highest and can reach 90.0%, and the average rooting rate is 9.75, and is the most, which is superior to 1/2MS + NAA0.1mg/L; and in the case of the same basic culture medium, the NAA0.1mg/L is better than the NAA0.5mg/L in a proper NAA concentration range.
Therefore, the suitable rooting medium of the double-petal lily perianth is MS + NAA0.1mg/L.
Conclusion
The suitable culture medium for induction culture (start culture) of the heavy-petal lily perianth is MS +6-BA2.0mg/L + NAA0.5mg/L, the suitable culture medium for propagation culture is MS +6-BA1.0mg/L + NAA0.1mg/L, and the suitable culture medium for rooting culture is MS + NAA0.1mg/L.
Example two
The technical scheme is different from the scheme in that the step S3 further comprises the step of stirring the stirred culture medium by using a stirring rod 1, the bottom of the stirring rod 1 is in a spiral multi-head shape, through holes 2 are formed in two radial sides of the stirring rod 1, and a notch is formed in one side of each through hole 2.
Adopt bull puddler 1 to carry out the vortex stirring to the culture medium among this technical scheme, thereby because hormone and the agar that adds realize the layering because density difference in the culture medium, thereby need the stirring operation to mix the culture medium, moreover double helix structure realizes vortex and convection action, through-hole 2 that puddler 1 was seted up is convenient for stir convection in-process flower quilt piece and passes through-hole 2, and the breach introduces the culture medium on every side, thereby paints the culture medium to flower quilt piece surface at the vortex in-process.
The foregoing is merely an example of the present invention, and common general knowledge in the field of known specific structures and characteristics is not described herein in any greater extent than that known in the art at the filing date or prior to the priority date of the application, so that those skilled in the art can now appreciate that all of the above-described techniques in this field and have the ability to apply routine experimentation before this date can be combined with one or more of the present teachings to complete and implement the present invention, and that certain typical known structures or known methods do not pose any impediments to the implementation of the present invention by those skilled in the art. It should be noted that, for those skilled in the art, without departing from the structure of the present invention, several changes and modifications can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicability of the patent. The scope of the claims of the present application shall be determined by the contents of the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.
Claims (8)
1. The method for establishing the asexual rapid propagation system of the double-petal lily tundra is characterized by comprising the following steps of: comprises the following steps of (a) carrying out,
s1, obtaining sterile explants, taking perianth sheets of the lilium bicolor 'Isabella' as the explants, picking unopened buds, cleaning and then disinfecting;
s2, preparing a culture medium, taking MS as a basic culture medium, and adding plant hormones with different types and concentrations;
s3, starting culture, inoculating the trapped perianth anth into a starting culture medium for culture, and inducing adventitious buds;
s4, proliferation culture, namely inoculating the adventitious buds into a proliferation culture medium to induce the adventitious buds to proliferate;
s5, rooting culture, screening adventitious buds, and placing the adventitious buds into a rooting culture medium to induce rooting, so as to obtain a complete sterile test-tube plantlet.
2. The method for establishing an asexual rapid propagation system of the double-petal lily angiospermum according to claim 1, which is characterized in that: the method for preparing the propagation medium in S4 includes a), b), c), d), e), f)
a) Mixing 6-BA1.0mg/L and NAA0.1mg/L by taking MS as a basic culture medium;
b) mixing 6-BA0.5mg/L and NAA0.2mg/L with MS as a basic culture medium;
c) mixing 6-BA1.0mg/L and NAA0.2mg/L with MS as a basic culture medium;
d) mixing MS as basic culture medium with 6-BA0.5mg/L and NAA0.3mg/L;
e) mixing MS as basic culture medium with 6-BA1.0mg/L and NAA0.3mg/L;
f) simple MS medium.
3. The method for establishing an asexual rapid propagation system of the double-petal lily angiospermum according to claim 2, which is characterized in that: the method for preparing the rooting medium in the step S5 comprises g), h), i), k), l) and f): g)1/2MS + NAA0.1, h)1/2MS + NAA0.5, i)1/2MS, k) MS + NAA0.1, l) MS + NAA0.5, f) MS, and observed statistical rooting rate, which is (number of rooted explants/number of inoculated explants) × 100% with reference to the following.
4. The method for establishing an asexual rapid propagation system of the double-petal lily angiospermum according to claim 1, which is characterized in that: in the disinfection treatment process, firstly, unopened buds are placed under running water and washed for 20-30 min, an operator wipes the buds dry by using absorbent paper, then the buds are placed in a sterilized glass bottle, a superclean workbench in the glass bottle is soaked in 70% -75% alcohol for 20-30 s, then the explants are disinfected for 3-4 min by using sterile water, then the explants are disinfected for 3-4 min by using 10-time diluted chlorine dioxide disinfectant, and finally the explants are washed for 4-5 times by using sterile water.
5. The method for establishing an asexual rapid propagation system of the double-petal lily angiospermum according to claim 4, which is characterized in that: in the step S2, 5-7 g/L agar and 27-32 g/L sucrose are added until the pH value is 5.8-6.0, and then the culture medium is placed in an autoclave at 125 ℃ for sterilization for 25-30 min.
6. The method for establishing an asexual rapid propagation system of the double-petal lily angiospermum according to claim 5, which is characterized in that: the temperature of the incubator is regulated to 25 +/-1 ℃ in the day, 18 +/-1 ℃ in the night, 2000-3000 lx in illumination intensity, 16h/d in illumination time and 8h/d in dark culture, and the ecological factors under the simulated natural environment are subjected to composite regulation.
7. The method for establishing an asexual rapid propagation system of the double-petal lily angiospermum according to claim 6, which is characterized in that: in step S1, the double-petal lily is sliced into small pieces of 5X 5 mm.
8. The method for establishing an asexual rapid propagation system of the double-petal lily angiospermum according to claim 1, which is characterized in that: and step S3, stirring the culture medium by using a stirring rod, wherein the bottom of the stirring rod is in a spiral multi-head shape, through holes are formed in the radial two sides of the stirring rod, and a notch is formed in one side of each through hole.
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