CN112931217A - Tissue culture regeneration method of Denmark shrubalthea - Google Patents
Tissue culture regeneration method of Denmark shrubalthea Download PDFInfo
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Abstract
The invention discloses a tissue culture regeneration method of hibiscus daniella, relating to the technical field of hibiscus daniella, and comprising the following steps: taking the annual tender stem with axillary buds of the excellent strain of the Denmark hibiscus syriacus as an explant, and performing induction and multiplication culture of adventitious buds after surface disinfection; and when the adventitious bud extends to 2-3 cm and is accompanied with 2-4 complete leaves, performing in-bottle or out-bottle adventitious root induction to finally obtain a complete regeneration plant of the hibiscus danense. The invention has the beneficial effects that: the invention has important significance for the large-scale propagation and variety improvement research of the excellent strain of the hibiscus danense.
Description
Technical Field
The invention relates to the technical field of hibiscus denudata, in particular to a tissue culture regeneration method of hibiscus denudata.
Background
The Denmark hibiscus syriacus is a perennial plant of the genus hibiscus of the family Malvaceae, is an improved new species of hibiscus syriacus, and has the advantages of dark green leaf color, rich flower color and long flowering period (annual flowering can be realized only by ensuring sufficient nutritional conditions after flowering); and the adaptability is strong, the cultivation can be carried out indoors and outdoors, and the ornamental value and the economic value are extremely high.
In recent years, with the improvement of living standard of people and the increase of market demand for the hibiscus denudata, the development of industrial cultivation of hibiscus denudata faces unprecedented opportunities. But lack of high-efficiency high-quality seedling breeding technology, and become a bottleneck restricting the industrial development of the seedlings.
The patent with publication number CN 107006326A discloses a method for efficiently breeding a good strain of hibiscus daniella, which is used for efficiently breeding the good strain of hibiscus daniella, but in the production, the breeding of hibiscus daniella mainly comprises introducing seedlings from abroad, so that the cost is high, and the large-scale breeding, popularization and later-stage variety improvement of hibiscus daniella are seriously limited. Due to the plant tissue culture and rapid propagation technology, a large number of excellent seedlings with consistent genetic background can be obtained in a short time, and on one hand, the demand of large-scale planting on the high-quality seedlings of the hibiscus denudata can be met to a certain extent; on the other hand, on the basis of the technology, the genetic improvement work of the good variety of the hibiscus denudata can be realized in the later period. However, the research on breeding of hibiscus danense by using plant tissue culture technology is still blank at present.
Therefore, in view of the problems of the breeding of hibiscus syriacus, there is a need to develop a method for efficient tissue culture and regeneration of hibiscus syriacus to meet the demand for rapid breeding and variety improvement of hibiscus syriacus elite lines.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for the efficient tissue culture and regeneration of the hibiscus denudata by applying a plant tissue culture technology.
The invention solves the technical problems through the following technical means:
a tissue culture regeneration method of the hibiscus danense comprises the following steps:
(1) taking the annual tender stem with axillary buds of the hibiscus danense as an explant, and disinfecting the surface of the explant;
(2) cutting the disinfected stem into sections with proper size, inoculating the sections into an adventitious bud induction culture medium, and inducing adventitious buds;
(3) after 3-4 weeks of illumination culture, transferring the stem segments inducing the adventitious bud clusters into an elongation culture medium for elongation culture of the adventitious buds;
(4) after 2-3 weeks of illumination culture, when the buds grow to 2-3 cm and 2-3 leaves are accompanied, in-bottle or out-bottle rooting induction is carried out to obtain a regeneration plant of the hibiscus daniellii with a robust root system; the ex-bottle rooting induction is to place the morphological lower end of the adventitious bud in rooting promoting liquid added with 10-25 mM melatonin and 100-500 mg/L IAA for 20-60s, and then fix the morphological lower end in a seedling culture plate for ex-bottle rooting induction.
Has the advantages that: in the breeding process, stem segments are adopted to directly induce adventitious buds, and after the adventitious buds are elongated, in one aspect, in-bottle rooting is adopted, so that a foundation is laid for later molecular genetic improvement; on the other hand, the ex-vitro rooting can be directly carried out, so that the breeding coefficient is improved, meanwhile, the in-vitro rooting and the later domestication and transplantation of the tissue culture seedlings are avoided, the breeding complexity is greatly reduced, and the breeding period is shortened.
The regeneration efficiency is high, the induction rate of the cluster buds can reach 98.4 percent, 8.5 adventitious buds can be generated in each explant on average, and the rooting rate outside the adventitious bud bottle can reach 98.6 percent; and the stem section of the obtained aseptic seedling can be further expanded and propagated, and an important technical support is provided for the rapid propagation and genetic improvement of the excellent hibiscus danense strain.
When the melatonin and the IAA are combined for use, the melatonin and the IAA have a synergistic effect, and the induction effect of adventitious roots can be greatly improved; and when the treatment time of the rooting promoting liquid exceeds 60s, the induction effect of the adventitious roots is gradually weakened, and the induction rate of the adventitious roots is reduced, the number of the adventitious roots is gradually reduced, and the length of the adventitious roots is gradually shortened.
The stem section is obtained from the current-year young and tender branch of the Denmark hibiscus line, and on one hand, the germplasm characteristic of a female parent plant can be maintained; on the other hand, the materials are convenient to obtain and rich; can realize the rapid propagation of the good single plant of the hibiscus denudata.
Preferably, the concentration of the melatonin is 25mM, and the concentration of the IAA is 100-250 mg/L.
Has the advantages that: under the concentration, the rooting rate outside the elongation bud bottle of the hibiscus denudata reaches more than 90 percent.
Preferably, the concentration of the melatonin is 25mM, the concentration of the IAA is 250mg/L, and the treatment time is 60 s.
Has the advantages that: under the concentration, the rooting rate outside the elongation bud bottle of the hibiscus denudata is as high as 98.6%.
Preferably, the adventitious bud induction culture medium comprises 0.2-3.0 mg/L TDZ and 2.0-10.0mg/L AgNO32.0-3.0% (w/v) sucrose and 0.6-0.8% (w/v) agar.
Has the advantages that: when the concentration of TDZ in the adventitious bud induction culture medium is 2.0mg/L, AgNO3When the concentration is 10mg/L, the induction rate of the stem adventitious bud of the hibiscus danense is as high as 98.4 percent, and each explant generates 4.3 adventitious buds on average.
Preferably, the elongation culture medium comprises 0.5-1.0 mg/L TDZ + 0.25-1.0 mg/L GA3+0.05-0.3mg/L KT + 2.0% -3.0% (w/v) sucrose + 0.6% -0.8% (w/v) agar DKW culture medium.
Has the advantages that: the combination of low-concentration TDZ and KT in the culture medium is favorable for the proliferation of adventitious buds when the TDZ and KT are combined with GA with lower concentration3When used in combination, the extract can promote the elongation of the adventitious bud of Hibiscus Densiflora.
Preferably, the step (1) of sterilizing the surface of the stem section is to cut the tender stem section of the hibiscus danense in the current year into 7-10 cm-sized cut sections after leaf removal, wash the cut sections under running water for 5-10 min, and add 0.5-2 ml of liquid soap 1-3 times in the period; then wiping the stem segments with 70-75% of absolute ethyl alcohol; then placing the stem segments in a sterile operation table, washing the stem segments with sterile water for 4-6 times, then sequentially using 70-75% of absolute ethyl alcohol to perform surface disinfection for 30-45 s, using 0.1% (w/v) of mercuric chloride solution to perform disinfection for 2-5 min, and finally washing the stem segments with sterile water for 6-8 times; sucking water on the surface of the stem segments, and cutting into stem segments with proper size and with an axillary bud for later use.
Preferably, the in-bottle rooting induction in the step (4) is to perform adventitious root induction culture on the elongated adventitious bud graft in a rooting medium, wherein the rooting medium comprises 1/2DKW medium added with 10-100 MuM melatonin, 0.05-1.0 mg/L IAA, 1.5-2.0% (w/v) sucrose and 0.6-0.8% (w/v) agar.
Has the advantages that: compared with a control, the induction rate of the adventitious roots of the elongation buds can be improved by independently adding melatonin and IAA with proper concentrations. However, when both are used in combination, the effect of inducing adventitious roots can be further improved.
Preferably, in the step (4), the morphological lower end is planted in a seedling culture tray filled with a nutrient soil and vermiculite mixed matrix (1:1), a plastic cover is covered, and ex-bottle rooting induction is carried out in a greenhouse.
Preferably, the stem adventitious bud induction, proliferation and elongation, and in-vitro rooting culture conditions in the step (2), the step (3) and the step (4) are as follows: the temperature is 25 +/-2 ℃, the illumination intensity is 2500-3000 lx, and the illumination/dark period is 14/10 h.
The invention has the advantages that: in the breeding process, stem segments are adopted to directly induce adventitious buds, and after the adventitious buds are elongated, in one aspect, in-bottle rooting is adopted, so that a foundation is laid for later molecular genetic improvement; on the other hand, the ex-vitro rooting is directly carried out, so that the breeding coefficient is improved, meanwhile, the in-vitro rooting and the later domestication and transplantation of the tissue culture seedlings are avoided, the breeding complexity is greatly reduced, and the breeding period is shortened. Thirdly, the regeneration efficiency is high, the induction rate of the cluster buds can reach 98.4 percent, each explant can generate 8.5 adventitious buds at most on average, and the rooting rate outside the adventitious bud bottle can reach 98.6 percent at most; and the stem section of the obtained aseptic seedling can be further expanded and propagated, and an important technical support is provided for the rapid propagation and genetic improvement of the excellent hibiscus danense strain.
The stem section is obtained from the current-year young and tender branch of the Denmark hibiscus line, and on one hand, the germplasm characteristic of a female parent plant can be maintained; on the other hand, the materials are convenient to obtain and rich; can realize the rapid propagation of the good single plant of the hibiscus denudata.
Drawings
FIG. 1 is a diagram of a sterile stem section of Hibiscus denudensis planted in adventitious bud induction medium in example 2 of the present invention;
FIG. 2 is a drawing showing the induction of adventitious buds of stem segments of Hibiscus danicus in example 2 of the present invention;
FIG. 3 is a graph showing the proliferation and elongation of adventitious buds of a stem segment in example 2 of the present invention;
FIG. 4 shows the in-vitro rooting of tissue-cultured and extended buds of Hibiscus danicus in example 2 of the present invention;
FIG. 5 is a graph showing the ex vitro rooting of the extended bud of Hibiscus danicus in example 4 of the present invention; in the figure, a is the tissue culture extended bud of the hibiscus denudati fixedly planted in the mixed matrix disc; b is the complete regeneration plant of the hibiscus danense obtained after the extracorporal rooting.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments. 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.
Test materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The specific techniques or conditions not specified in the examples can be performed according to the techniques or conditions described in the literature in the field or according to the product specification.
Example 1
A tissue culture regeneration method of Denmark shrubalthea comprises the following specific operations:
(1) selecting a good growing strain of the hibiscus danense, and cutting branches which are not lignified in the current year as explants; cutting into 7-10cm pieces, loading into 1000ml beaker, washing with flowing water for 5min, and adding 2ml liquid soap 1 time for washing; then wiping the stem segments with 75% absolute ethyl alcohol; then placing the stem segments in a sterile operating platform, washing with sterile water for 4 times, sequentially sterilizing the surface with 75% anhydrous ethanol for 30s, sterilizing with 0.1% (w/v) mercuric chloride solution for 2min, and finally washing with sterile water for 6 times; sucking water on the surface of the stem section, and cutting the stem section into a stem section with an axillary bud for later use.
(2) Vertically inoculating the stem segments in the step (1) into an adventitious bud induction culture medium, and inducing adventitious buds in a constant-temperature culture room with the temperature of 25 +/-2 ℃, the illumination intensity of 2500lx and the illumination time of 14 h. After 3 weeks of illumination culture, adventitious buds are induced at the cut of the stem segment, the induction rate of the adventitious buds is 61.3 percent, and each explant averagely generates 3.4 adventitious buds. Wherein the adventitious bud induction culture medium is added with 0.2mg/L TDZ and 2.0mg/L AgNO3DKW medium of 2.0% (w/v) sucrose, 0.6% (w/v) agar, pH 5.8.
(3) And (3) transferring the stem segments induced with the adventitious buds in the step (2) into an elongation culture medium, and performing proliferation and elongation culture on the adventitious buds in a constant-temperature culture room with the temperature of 25 +/-2 ℃, the illumination intensity of 2500lx and the illumination time of 14 h. After 4 weeks of illumination culture, the obtained product has 2-3 complete leaves, the average adventitious number is 4.8, and the average adventitious bud length is 2.4 cm. Wherein the adventitious bud elongation culture medium comprises: adding 0.5mg/L TDZ and 0.25mg/L GA3DKW medium of 0.05mg/L KT, 2.0% (w/v) sucrose, 0.6% (w/v) agar, pH 5.8.
(4) And (4) inoculating the adventitious bud elongated in the step (3) into a rooting culture medium, and performing induction culture in an adventitious root bottle in a constant-temperature culture room with the temperature of 25 +/-2 ℃, the illumination intensity of 2500-3000 lx and the illumination time of 14 h. After 4 weeks of light culture, the induction rate of adventitious roots was 73.9%, 2.1 adventitious roots were produced per explant on average, and the length of the adventitious roots was 3.2cm on average. Wherein the rooting medium is 1/2DKW medium supplemented with 10 μ M melatonin, 0.05mg/L IAA, 1.5% (w/v) sucrose, and 0.6% agar, and has pH of 5.8.
Example 2
A tissue culture regeneration method of Denmark shrubalthea comprises the following specific operations:
(1) selecting a good growing strain of the hibiscus danense, and cutting a semi-lignified branch of the current year as an explant; cutting the leaves into 8 cm-sized sections after removing the leaves, subpackaging the sections into 1000ml beakers, washing for 10min under running water, and adding 0.5-1 ml of liquid soap for washing 2 times; then wiping the stem segments with 75% absolute ethyl alcohol; then placing the stem segments in a sterile operating platform, washing with sterile water for 6 times, sequentially sterilizing the surface with 75% anhydrous ethanol for 40s, sterilizing with 0.1% (w/v) mercuric chloride solution for 3min, and finally washing with sterile water for 8 times; sucking water on the surface of the stem section, and cutting the stem section into a stem section with an axillary bud for later use.
(2) As shown in FIG. 1, the stem segments obtained in step (1) were vertically inoculated into an adventitious bud induction medium, and adventitious bud induction was carried out in a constant temperature culture room at a temperature of 25. + -. 2 ℃ under illumination for 3000lx for 14 hours. After 3 weeks of illumination culture, adventitious bud clusters are induced at the cut of the stem segment, the induction rate of the adventitious buds is 98.4%, and on average, each explant generates 4.3 adventitious buds as shown in FIG. 2. Wherein the adventitious bud induction culture medium is supplemented with 2.0mg/L TDZ and 5.0mg/L AgNO3DKW medium of 3.0% (w/v) sucrose, 0.7% (w/v) agar, pH 5.8.
(3) And (3) transferring the stem segments induced with the adventitious buds in the step (2) into a proliferation culture medium, and performing proliferation and elongation culture on the adventitious buds in a constant-temperature culture room with the temperature of 25 +/-2 ℃, the illumination intensity of 3000lx and the illumination time of 14 h. After 4 weeks of light culture, adventitious shoots with 2-3 intact leaves and an average length of 2.8cm were obtained, yielding an average of 8.5 adventitious shoots per explant, as shown in FIG. 3. Wherein the adventitious bud proliferation culture medium is supplemented with 0.75mg/L TDZ and 0.5mg/L GA3DKW of 0.2mg/L KT, 3.0% (w/v) sucrose, 0.7% (w/v) agar, pH 5.8.
(4) Inoculating the adventitious bud elongated in the step (3) into a rooting culture medium, and performing induction culture in an adventitious root bottle in a constant-temperature culture room with the temperature of 25 +/-2 ℃, the illumination intensity of 3000lx and the illumination time of 14 h. After 4 weeks of light culture, the induction rate of adventitious roots was 89.6%, and on average 3.4 adventitious roots were produced per explant, with an average adventitious root length of 4.2cm, as shown in FIG. 4. Wherein the rooting culture medium is 1/2DKW culture medium added with 50 μ M melatonin, 0.5mg/L IAA, 2.0% (w/v) sucrose, and 0.7% agar, and has pH of 5.8;
example 3
A tissue culture regeneration method of Denmark shrubalthea comprises the following specific operations:
(1) selecting a good-growing Denmark hibiscus syriacus strain, and cutting a lignified branch of the current year as an explant; cutting into 10cm pieces after removing leaves, subpackaging in 1000ml beaker, washing under flowing water for 10min, adding 0.5ml liquid soap for washing 3 times; then wiping the stem segments with 75% absolute ethyl alcohol; then placing the stem segments in a sterile operating platform, washing with sterile water for 6 times, sequentially sterilizing the surface with 75% anhydrous ethanol for 45s, sterilizing with 0.1% (w/v) mercuric chloride solution for 5min, and finally washing with sterile water for 8 times; sucking water on the surface of the stem section, and cutting the stem section into a stem section with an axillary bud for later use.
(2) Vertically inoculating the stem segments in the step (1) into an adventitious bud induction culture medium, and inducing adventitious buds in a constant-temperature culture room with the temperature of 25 +/-2 ℃, the illumination intensity of 2800lx and the illumination time of 14 h. After 4 weeks of illumination culture, adventitious buds are induced at the cut of the stem segment, the induction rate of the adventitious buds is 42.4 percent, and each explant averagely generates 1.9 adventitious buds. Wherein the adventitious bud induction culture medium is supplemented with 2.0mg/L TDZ and 10.0mg/L AgNO3DKW medium of 3.0% (w/v) sucrose, 0.8% (w/v) agar, pH 5.8.
(3) And (3) transferring the stem segments induced with the adventitious buds in the step (2) into a proliferation culture medium, and performing proliferation and elongation culture on the adventitious buds in a constant-temperature culture room with the temperature of 25 +/-2 ℃, the illumination intensity of 2800lx and the illumination time of 14 h. After 4 weeks of light culture, adventitious buds with 2-3 intact leaves and an average length of 1.8cm were obtained, yielding an average of 5.9 adventitious buds per explant. Wherein the adventitious bud proliferation culture medium is supplemented with 1.0mg/L TDZ and 1.0mg/L GA3DKW medium of 0.3mg/L KT, 3.0% (w/v) sucrose, 0.6% (w/v) agar, pH 5.8.
(4) And (4) inoculating the adventitious bud elongated in the step (3) into a rooting culture medium, and performing induction culture in an adventitious root bottle in a constant-temperature culture room with the temperature of 25 +/-2 ℃, the illumination intensity of 2500-3000 lx and the illumination time of 14 h. After 4 weeks of light culture, the induction rate of adventitious roots was 76.9%, and on average 4.1 adventitious roots were produced per explant, with an average adventitious root length of 2.5 cm. Wherein the rooting culture medium is 1/2DKW added with 100 μ M melatonin, 1.0mg/L IAA, 2.0% (w/v) sucrose, and 0.8% agar, and has pH of 5.8;
example 4
This example tested different concentrations of TDZ and AgNO3The method has the following specific steps of influencing the induction of the stem adventitious bud of the hibiscus danense:
(1) selecting a good growing strain of the hibiscus danense, and cutting a semi-lignified branch of the current year as an explant; cutting the leaves into 8 cm-sized sections after removing the leaves, subpackaging the sections into 1000ml beakers, washing for 10min under running water, and adding 0.5-1 ml of liquid soap for washing 2 times; then wiping the stem segments with 75% absolute ethyl alcohol; then placing the stem segments in a sterile operating platform, washing with sterile water for 6 times, sequentially sterilizing the surface with 75% anhydrous ethanol for 40s, sterilizing with 0.1% (w/v) mercuric chloride solution for 3min, and finally washing with sterile water for 8 times; sucking water on the surface of the stem section, and cutting the stem section into a stem section with an axillary bud for later use.
(2) Vertically inoculating the cut stem segments obtained in the step (1) into a culture medium added with different TDZ concentrations (0.2-2.0mg/L) and different AgNO3Culturing adventitious bud in adventitious bud induction culture medium with concentration of 2.0-10.0mg/L at 25 + -2 deg.C under illumination intensity of 2500lx for 14 h. Wherein the adventitious bud induction culture medium is supplemented with 2.0mg/L TDZ and 5.0mg/L AgNO3DKW medium of 3.0% (w/v) sucrose, 0.7% (w/v) agar, pH 5.8. After 3 weeks of illumination culture, the induction rate of adventitious buds of the stem segments and the average number of adventitious buds generated by each explant are counted. The results of the study are shown in Table 1.
TABLE 1 different concentrations of TDZ and AgNO3Influence on the induction of adventitious buds of stem segments of hibiscus danense
| TDZ(mg/L) | AgNO3(mg/L) | Adventitious bud induction ratio (%) | Mean adventitious bud number/explant |
| 0.0 | 0.0 | 0.0 | 0.0 |
| 0.2 | 2.0 | 62.1 | 3.5 |
| 0.2 | 10.0 | 73.5 | 3.1 |
| 0.5 | 2.0 | 77.4 | 3.8 |
| 0.5 | 5.0 | 82.3 | 4.1 |
| 1.0 | 5.0 | 84.7 | 3.2 |
| 1.0 | 10 | 67.2 | 2.6 |
| 2.0 | 5.0 | 98.4 | 4.3 |
| 2.0 | 10.0 | 42.4 | 1.9 |
Note: data are mean values, each treatment contained 120 explants, and each treatment was replicated three times.
The study in table 1 shows that: different concentrations of TDZ and AgNO3Plays an important role in the induction process of the adventitious bud of the stem segment of the hibiscus danense. When the concentration of TDZ in the adventitious bud induction culture medium is 2.0mg/L, AgNO3When the concentration is 10mg/L, the induction rate of the stem adventitious bud of the hibiscus danense is as high as 98.4 percent, and each explant generates 4.3 adventitious buds on average.
Example 5
In this example, the effect of different concentrations and species of plant growth regulators on the proliferation and elongation of the adventitious bud of the stem segment of hibiscus denudata was tested, and the specific steps were as follows:
(1) selecting a good growing strain of the hibiscus danense, and cutting a semi-lignified branch of the current year as an explant; cutting the leaves into 8 cm-sized sections after removing the leaves, subpackaging the sections into 1000ml beakers, washing for 10min under running water, and adding 0.5-1 ml of liquid soap for washing 2 times; then wiping the stem segments with 75% absolute ethyl alcohol; then placing the stem segments in a sterile operating platform, washing with sterile water for 6 times, sequentially sterilizing the surface with 75% anhydrous ethanol for 40s, sterilizing with 0.1% (w/v) mercuric chloride solution for 3min, and finally washing with sterile water for 8 times; sucking water on the surface of the stem section, and cutting the stem section into a stem section with an axillary bud for later use.
(2) Vertically inoculating the stem segments in the step (1) into an adventitious bud induction culture medium, and inducing adventitious buds in a constant-temperature culture room with the temperature of 25 +/-2 ℃, the illumination intensity of 2500lx and the illumination time of 14 h. Wherein adventitious bud inductionThe culture medium is supplemented with 2.0mg/L TDZ and 5.0mg/L AgNO3DKW medium of 3.0% (w/v) sucrose, 0.7% (w/v) agar, pH 5.8.
(3) And (3) transferring the stem sections induced with the adventitious buds in the step (2) into proliferation culture media added with plant growth regulators of different types and concentrations, and performing proliferation and elongation culture on the adventitious buds in a constant-temperature culture room with the temperature of 25 +/-2 ℃, the illumination intensity of 2500lx and the illumination time of 14 h. After 4 weeks of light culture, the number of adventitious buds generated per explant and the height of the average adventitious buds were counted. The results of the study are shown in Table 2.
TABLE 2 Effect of different concentrations and species of plant growth regulators on the proliferation and elongation of adventitious buds of the stem segments of Hibiscus dandeli
| TDZ(mg/L) | GA3(mg/L) | KT(mg/L) | Number of adventitious buds | Average indefinite bud length (cm) |
| 0.0 | 0.0 | 0.0 | 4.3 | 1.4 |
| 0.5 | 0.25 | 0.05 | 4.8 | 2.4 |
| 0.5 | 0.5 | 0.1 | 5.3 | 2.6 |
| 0.5 | 1.0 | 0.2 | 5.6 | 1.9 |
| 0.75 | 0.25 | 0.3 | 7.2 | 2.2 |
| 0.75 | 0.5 | 0.2 | 8.5 | 2.8 |
| 1.0 | 0.25 | 0.1 | 5.7 | 2.3 |
| 1.0 | 0.5 | 0.05 | 5.4 | 2.5 |
| 1.0 | 1.0 | 0.3 | 5.9 | 1.8 |
Note: data are mean values, each treatment contained 120 explants, and each treatment was replicated three times.
The study in table 2 shows that: different concentrations of TDZ, KT and GA3The use of (2) can promote the proliferation and elongation of the adventitious bud of the stem segment of hibiscus danicus. The combination of low-concentration TDZ and KT in the culture medium is favorable for the proliferation of adventitious buds when the TDZ and KT are combined with GA with lower concentration3When used in combination, the extract can promote the elongation of the adventitious bud of Hibiscus Densiflora. When the concentration of TDZ in the culture medium is 0.75mg/L, GA3At a concentration of 0.5mg/L and a KT concentration of 0.2mg/L, on average 8.5 adventitious buds were produced per hibiscus danense stem segment and the average length of the adventitious buds was 2.8 cm.
Example 6
The present example tests the influence of exogenous melatonin and IAA concentrations on rooting in a hibiscus syriacus growing bud bottle, and the specific steps are as follows:
(1) selecting a good growing strain of the hibiscus danense, and cutting a semi-lignified branch of the current year as an explant; cutting the leaves into 8 cm-sized sections after removing the leaves, subpackaging the sections into 1000ml beakers, washing for 10min under running water, and adding 0.5-1 ml of liquid soap for washing 2 times; then wiping the stem segments with 75% absolute ethyl alcohol; then placing the stem segments in a sterile operating platform, washing with sterile water for 6 times, sequentially sterilizing the surface with 75% anhydrous ethanol for 40s, sterilizing with 0.1% (w/v) mercuric chloride solution for 3min, and finally washing with sterile water for 8 times; sucking water on the surface of the stem section, and cutting the stem section into a stem section with an axillary bud for later use.
(2) As shown in FIG. 1, the stem segments obtained in step (1) were vertically inoculated into an adventitious bud induction medium, and adventitious bud induction was carried out in a constant temperature culture room at a temperature of 25. + -. 2 ℃ under illumination for 3000lx for 14 hours. Wherein the adventitious bud induction culture medium is added2.0mg/L TDZ and 5.0mg/L AgNO are added3DKW medium of 3.0% (w/v) sucrose, 0.7% (w/v) agar, pH 5.8.
(3) And (3) transferring the stem segments induced with the adventitious buds in the step (2) into a proliferation culture medium, and performing proliferation and elongation culture on the adventitious buds in a constant-temperature culture room with the temperature of 25 +/-2 ℃, the illumination intensity of 3000lx and the illumination time of 14 h. Wherein the adventitious bud proliferation culture medium is supplemented with 0.75mg/L TDZ and 0.5mg/L GA3DKW of 0.2mg/L KT, 3.0% (w/v) sucrose, 0.7% (w/v) agar, pH 5.8.
(4) And (4) inoculating the adventitious buds elongated in the step (3) into rooting culture media added with melatonin and IAA with different concentrations, and performing induction culture in adventitious root bottles in a constant-temperature culture room with the temperature of 25 +/-2 ℃, the illumination intensity of 3000lx and the illumination time of 14 h. After 4 weeks of illumination culture, the induction rate of the adventitious roots is counted, and the number of the adventitious roots generated by each explant and the average length of the adventitious roots are averaged. The results of the study are shown in Table 3. Research results show that the concentrations of melatonin and IAA in the culture medium have an important promotion effect on rooting in the extended bud bottle of the hibiscus denudata. Compared with a control, the induction rate of the adventitious roots of the elongation buds can be improved by independently adding melatonin and IAA with proper concentrations. When both are used in combination, the effect of inducing adventitious roots can be improved. Wherein when the concentration of melatonin is 50 μ M and the IAA concentration is 0.5mg/L, the induction rate of the adventitious roots in the bottle of the hibiscus denudata is up to 89.6%, 3.4 adventitious roots are generated on average per explant, and the average root length is 4.2 cm.
TABLE 3 influence of melatonin and IAA concentrations on rooting in extended bud bottles of Denmark Hibiscus syriacus
Note: data are mean values, each treatment contained 120 explants, and each treatment was replicated three times.
Example 7
In this example, the influence of exogenous melatonin and IAA concentrations on the external rooting of the extended bud of hibiscus denudata was tested, and the specific steps were as follows:
(1) selecting a good growing strain of the hibiscus danense, and cutting a semi-lignified branch of the current year as an explant; cutting the leaves into 8 cm-sized sections after removing the leaves, subpackaging the sections into 1000ml beakers, washing for 10min under running water, and adding 0.5-1 ml of liquid soap for washing 2 times; then wiping the stem segments with 75% absolute ethyl alcohol; then placing the stem segments in a sterile operating platform, washing with sterile water for 6 times, sequentially sterilizing the surface with 75% anhydrous ethanol for 40s, sterilizing with 0.1% (w/v) mercuric chloride solution for 3min, and finally washing with sterile water for 8 times; sucking water on the surface of the stem section, and cutting the stem section into a stem section with an axillary bud for later use.
(2) Vertically inoculating the stem segments in the step (1) into an adventitious bud induction culture medium, and inducing adventitious buds in a constant-temperature culture room with the temperature of 25 +/-2 ℃, the illumination intensity of 2500lx and the illumination time of 14 h. Wherein the adventitious bud induction culture medium is supplemented with 2.0mg/L TDZ and 5.0mg/L AgNO3DKW medium of 3.0% (w/v) sucrose, 0.7% (w/v) agar, pH 5.8.
(3) And (3) transferring the stem segments induced with the adventitious buds in the step (2) into a proliferation culture medium, and performing proliferation and elongation culture on the adventitious buds in a constant-temperature culture room with the temperature of 25 +/-2 ℃, the illumination intensity of 2500lx and the illumination time of 14 h. Wherein the adventitious bud proliferation culture medium is supplemented with 0.75mg/L TDZ and 0.5mg/L GA3DKW medium of 0.2mg/L KT, 3.0% (w/v) sucrose, 0.7% (w/v) agar, pH 5.8.
(4) And (4) after the adventitious bud in the step (3) is elongated to about 2.5cm, separating the elongated adventitious bud from the tissue culture bottle, washing the adventitious bud with running water, absorbing the surface moisture, and placing the morphological lower end of the adventitious bud in rooting promoting liquid containing melatonin and IAA with different concentrations for 30 s. Then, morphological lower ends of the treated adventitious buds are planted in a seedling culture tray filled with a mixed matrix (nutrient soil: vermiculite: 1), a plastic cover is covered, and adventitious root induction outside a bottle is carried out in a greenhouse, as shown in fig. 5. After 4 weeks of culture in the greenhouse, the effect of exogenous melatonin and IAA concentrations on the external rooting of the extended buds of the hibiscus danense was counted, and the results of the study are shown in Table 4.
TABLE 4 Effect of exogenous melatonin and IAA concentrations on vitro rooting of extended buds of Hibiscus danaeus
| Melatonin (mM) | IAA(mg/L) | Rooting percentage (%) | Average number of roots | Average root length (cm) |
| 0 | 0 | 7.5 | 1.1 | 3.6 |
| 10 | 0.0 | 45.6 | 2.2 | 4.3 |
| 25 | 0.0 | 68.2 | 4.6 | 5.2 |
| 50 | 0.0 | 47.1 | 3.7 | 5.8 |
| 0.0 | 100 | 53.5 | 2.1 | 6.1 |
| 0.0 | 250 | 77.3 | 3.4 | 5.5 |
| 0.0 | 500 | 59.4 | 4.2 | 4.9 |
| 25 | 100 | 90.2 | 5.5 | 6.3 |
| 25 | 250 | 96.5 | 6.3 | 5.9 |
| 25 | 500 | 87.8 | 5.2 | 5.4 |
Note: data are mean values, each treatment contained 120 explants, and each treatment was replicated three times.
The study in table 4 shows that: exogenous melatonin and IAA concentrations have important effects on the external rooting of the extended buds of the hibiscus danense. Compared with a control, the exogenous melatonin and the IAA which are independently used can promote the formation of the adventitious roots of plants and can promote the increase of the length and the number of the adventitious roots. In addition, further analysis finds that the induction effect of melatonin on adventitious roots is slightly weak. When the two are used in combination, the induction effect of the adventitious roots can be greatly improved. Wherein when the concentration of the melatonin is 25mM and the concentration of the IAA is 250mg/L, the induction rate of the hibiscus denudata adventitious root is up to 96.5 percent, each explant averagely generates 6.3 adventitious roots, and the average root length is 5.9 cm.
Example 8
This example tests the effect of treatment time of the mixture of exogenous melatonin and IAA on the external rooting of extended buds of Hibiscus dannamei. The inventors treated the morphological lower ends of the obtained elongated tissue-cultured adventitious buds of Hibiscus danesensis in a solution of 25mM melatonin and 250mg/L IAA for different periods of time (10s, 20s, 30s, 60s and 90s) with only changing the treatment time of the mixture of exogenous melatonin and IAA. The rest of the procedure was the same as in example 4. After 4 weeks of culture in the greenhouse, the effect of treatment time on rooting outside the extended bud of hibiscus denudate was counted and the results of the study are shown in table 5. Research results show that the treatment time of the mixed solution of the exogenous melatonin and the IAA has an important influence on the external rooting of the extended buds of the hibiscus danense. Within a certain range, the induction rate of the adventitious roots, the number of the adventitious roots and the average length of the adventitious roots gradually increase with the increase of the treatment time, and the treatment time reaches the optimum value at 60 s. Then, as the treatment time was prolonged, the effect of induction of adventitious roots was gradually reduced, and this indicated that the induction rate of adventitious roots was decreased, the number of adventitious roots was gradually decreased, and the length of adventitious roots was gradually shortened. Presumably, the reason for this is that the callus formation was accompanied at the base of the stem segment with the increase in the treatment time, and the formation of adventitious roots was suppressed to some extent.
TABLE 5 Effect of treatment time of mixture of exogenous melatonin and IAA on rooting outside the extended bud of Hibiscus danaensis
Note: data are mean values, each treatment contained 120 explants, and each treatment was replicated three times.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (10)
1. A tissue culture regeneration method of the hibiscus danense is characterized by comprising the following steps: the method comprises the following steps:
(1) taking the annual tender stem with axillary buds of the hibiscus danense as an explant, and disinfecting the surface of the explant;
(2) cutting the disinfected stem into sections with proper size, inoculating the sections into an adventitious bud induction culture medium, and inducing adventitious buds;
(3) after 3-4 weeks of illumination culture, transferring the stem segments inducing the adventitious bud clusters into an elongation culture medium for elongation culture of the adventitious buds;
(4) after 2-3 weeks of illumination culture, when the buds grow to 2-3 cm and 2-3 leaves are accompanied, in-bottle or out-bottle rooting induction is carried out to obtain a regeneration plant of the hibiscus daniellii with a robust root system; the ex-bottle rooting induction is to place the morphological lower end of the adventitious bud in rooting promoting liquid added with 10-25 mM melatonin and 100-500 mg/L IAA for 20-60s, and then fix the morphological lower end in a seedling culture plate for ex-bottle rooting induction.
2. The tissue culture regeneration method of hibiscus denudata according to claim 1, wherein: the concentration of the melatonin is 25mM, and the concentration of the IAA is 100-250 mg/L.
3. The tissue culture regeneration method of hibiscus denudata according to claim 1, wherein: the concentration of the melatonin is 25mM, the concentration of the IAA is 250mg/L, and the treatment time is 60 s.
4. The tissue culture regeneration method of hibiscus denudata according to claim 1, wherein: the adventitious bud induction culture medium comprises 0.2-3.0 mg/L TDZ and 2.0-10.0mg/L AgNO3A DKW culture medium containing 2.0-3.0% of cane sugar and 0.6-0.8% of agar.
5. The tissue culture regeneration method of hibiscus denudata as claimed in claim 4, wherein: the adventitious bud induction culture medium comprises 2.0mg/L TDZ and 10.0mg/L AgNO3A DKW culture medium containing 2.0-3.0% of cane sugar and 0.6-0.8% of agar.
6. The tissue culture regeneration method of hibiscus denudata according to claim 1, wherein: the elongation culture medium comprises 0.5-1.0 mg/L TDZ + 0.25-1.0 mg/L GA3+0.05-0.3mg/L KT + 2.0% -3.0% sucrose + 0.6% -0.8% agar DKW culture medium.
7. The tissue culture regeneration method of hibiscus denudata according to claim 1, wherein: the step (1) of sterilizing the surface of the stem section is to cut the tender stem section of the current-year hibiscus danense into 7-10 cm-sized cut sections after removing leaves, wash the cut sections under running water for 5-10 min, and add 0.5-2 ml of liquid soap for 1-3 times in the period; then wiping the stem segments with 70-75% of absolute ethyl alcohol; then placing the stem segments in a sterile operation table, washing the stem segments with sterile water for 4-6 times, then sequentially using 70-75% of absolute ethyl alcohol to perform surface disinfection for 30-45 s, using 0.1% (w/v) of mercuric chloride solution to perform disinfection for 2-5 min, and finally washing the stem segments with sterile water for 6-8 times; sucking water on the surface of the stem segments, and cutting into stem segments with proper size and with an axillary bud for later use.
8. The tissue culture regeneration method of hibiscus denudata according to claim 1, wherein: and (4) performing adventitious root induction culture on the elongated adventitious bud in a rooting culture medium in the in-bottle rooting induction step, wherein the rooting culture medium comprises 1/2DKW culture medium added with 10-100 mu M of melatonin, 0.05-1.0 mg/L of IAA, 1.5-2.0% of cane sugar and 0.6-0.8% of agar.
9. The tissue culture regeneration method of hibiscus denudata according to claim 1, wherein: and (4) planting the morphological lower end in a seedling culture tray filled with a nutrient soil and vermiculite mixed matrix, covering a plastic cover, and performing ex-vitro rooting induction in a greenhouse.
10. The tissue culture regeneration method of hibiscus denudata according to claim 1, wherein: the stem adventitious bud induction, proliferation and elongation, and in-bottle and out-bottle rooting culture conditions in the step (2), the step (3) and the step (4) are as follows: the temperature is 25 +/-2 ℃, the illumination intensity is 2500-3000 lx, and the illumination/dark period is 14/10 h.
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