Tissue culture method of hemerocallis fulva scape

Abstract

The invention discloses a tissue culture method of a double-petal hemerocallis fulva scapes, which comprises the following steps: taking a double-petal hemerocallis fulva scapes, taking a growing point as a center, transversely cutting into long sections which are not less than 5cm, washing and disinfecting; putting the disinfected long segments into enzymatic hydrolysate for cell wall enzymolysis, taking out the long segments after enzymolysis is completed, and washing the long segments with sterile water; transversely cutting the long section subjected to enzymolysis into small sections of 1-2cm by taking the growing point as the center; and carrying out tissue culture by taking the small sections as explants. The method applies the double-petal hemerocallis fulva variety, and has the advantages that materials can be taken at any time in the flowering phase, the tissue culture efficiency is high, and the quality is good.

Description

Technical Field

[0001] The present invention relates to the field of tissue culture, and more specifically, to a tissue culture method for double-petal daylily scapes. Background Art

[0002] Daylily is one of the three major perennial flowers in the world. It has the advantages of large and beautiful flowers, various flower shapes, diverse colors, and long flowering period. It has become an important garden ornamental plant. In addition, daylily has a well-developed root system and has a strong ability to absorb water and consolidate soil, which can be used to reduce soil erosion; it has strong drought resistance, high temperature resistance, cold resistance, etc., and has a wide adaptability to soil, so it can be used for slope and road greening; it has strong vitality, can be extensively managed, and is easy to maintain, saving labor and effort, and low cost, and has broad prospects.

[0003] At present, the main method of propagating daylilies is to propagate by division, which has a slow propagation speed and each plant can only reproduce 3 to 5 plants per year. If large-scale propagation is required, on the one hand, a certain number of breeding plants and a large area of ​​production land are required. On the other hand, due to the low propagation coefficient and long production cycle, it is impossible to meet the needs of commercialization and large-scale production of excellent varieties, and it is difficult to meet the market, which leads to high prices, which seriously hinders its application and promotion speed. Especially some new and excellent double-petal varieties, such as the "cream roll" double-petal daylily, which has bright color, unique flower shape, plump flower shape, long green period, and improved overall ornamental value, but the difficulty of propagation has resulted in a small number of seedlings, which has hindered the process of large-scale promotion and application.

[0004] Tissue culture is an effective way to accelerate the breeding process. However, existing reports on tissue culture propagation of daylilies using scapes as explants are mainly for "Jianzhuang Lilan", "Datong Daylily", "Pink Beauty", "Golden Baby", etc., all of which are single-petal flower types. There are few reports on the successful establishment of tissue culture systems to propagate seedlings using scapes as explants in double-petal daylily varieties. In addition, the traditional tissue culture method using scapes as explants is to directly cut the scapes into small pieces for disinfection, which can easily cause mechanical damage to the tissue and damage to the growing point, affecting the subsequent induction of callus tissue and differentiation of adventitious buds.

[0005] Enzymatic hydrolysis is often used in protoplast isolation or cell wall research, but has not been reported in tissue culture. How to combine enzymatic hydrolysis with tissue culture to solve the problems in the existing tissue culture process needs further research. Summary of the invention

[0006] The object of the present invention is to provide a tissue culture method for double-petal daylily scapes to at least solve the above-mentioned problems.

[0007] In order to achieve the purpose and other advantages of the present invention, a tissue culture method of double-petal daylily scape is provided, comprising: taking a double-petal daylily scape, cutting it into long segments of not less than 5 cm with the growth point as the center, and washing and disinfecting it; placing the disinfected long segment into an enzymatic hydrolysis solution for cell wall enzymatic hydrolysis, taking it out after the enzymatic hydrolysis is completed, and washing it with sterile water; cutting the long segment after enzymatic hydrolysis into small segments of 1-2 cm with the growth point as the center; and carrying out tissue culture with the small segment as an explant; wherein the enzymatic hydrolysis solution is composed of 10 mg / mL-20 mg / mL of cellulase, 5 mg / mL-10 mg / mL of pectinase, 0.5 mg / mL-1 mg / mL of tea polyphenols, 73 mg / mL of mannitol, 20 mM of MES buffer, 1.1 mg / mL of calcium chloride and sterile water, and is filtered and sterilized through a 0.22 μm filter membrane; the enzymatic hydrolysis temperature is 25-30°C, and the enzymatic hydrolysis time is 30-90 min.

[0008] Preferably, the disinfection is to first disinfect the long section with 75% alcohol for 10 to 30 seconds and then disinfect with 2% sodium hypochlorite for 5 to 15 minutes.

[0009] Preferably, samples are taken at 30 min, 50 min and 70 min of enzymatic hydrolysis, and the cell status of the scape epidermis is observed under a microscope. If the cell wall is obviously thinned or partially degraded and the cell morphology is intact, the enzymatic hydrolysis is stopped; otherwise, the enzymatic hydrolysis is continued for 90 min.

[0010] Preferably, tissue culture is carried out with the small segment as an explant, comprising: step 1, inoculating the explant onto an induction medium, first culturing in the dark for 26 to 30 days to induce callus tissue, and then culturing under light for 26 to 30 days to induce callus tissue to differentiate into adventitious buds, wherein the induction medium comprises MS+6-BA 1.0 mg / L+NAA 0.2 mg / L+TDZ 0.01 mg / L to 0.1 mg / L; step 2, inoculating the adventitious buds as single plants or clustered buds onto a proliferation medium, and culturing under light for 26 to 30 days, wherein the proliferation medium comprises MS+6-BA1.0 mg / L+NAA 0.1 mg / L+TDZ 0.001 mg / L to 0.01 mg / L; step 3, dividing the adventitious buds into single plants, inoculating them into a rooting medium to induce rooting, and culturing under light for 26 to 30 days, wherein the rooting medium comprises 1 / 2MS+NAA 0mg / L~0.5mg / L; Step 4, after hardening the rooted seedlings, take them out from the rooting medium, clean the roots with a fungicide, and transplant them into a transplanting medium to obtain double-petal daylily plants.

[0011] Preferably, the induction medium comprises MS+6-BA 1.0 mg / L+NAA 0.2 mg / L+TDZ 0.05 mg / L.

[0012] Preferably, the proliferation medium comprises MS+6-BA 1.0 mg / L+NAA 0.1 mg / L+TDZ 0.005 mg / L.

[0013] Preferably, the rooting medium comprises 1 / 2MS+NAA 0.3 mg / L.

[0014] Preferably, the culture temperature of the dark culture is 20°C to 30°C, and the relative humidity of the air is 40% to 60%; the light intensity of the light culture is 1000lx to 2 000lx, the photoperiod is 12 hours of light / 12 hours of darkness, the culture temperature is 20°C to 30°C, and the relative humidity of the air is 40% to 60%.

[0015] Preferably, the double-petal daylily variety is one of "cream curl", "golden yellow" and "flesh pink".

[0016] The present invention has at least the following beneficial effects: First, the method of the present invention uses the scape with growth points as explants, which can indirectly induce the production of adventitious buds, increase the propagation method of double-petal daylily tissue culture, and can carry out large-scale propagation at any time during the flowering period, which is conducive to the large-scale production of factory-based seedling cultivation to meet the needs of the garden greening market; Second, the present invention cuts the scape into long segments with the growth point as the center, disinfects the long segment and then performs enzymolysis, and then cuts the long segment after enzymolysis into small segments with the growth point as the center. First, cutting into long segments and then disinfecting helps to better protect the growth point during the disinfection process and reduce the damage of the disinfectant to it. Second, the long segment is enzymolyzed by a limited enzymolysis solution, and the scape becomes soft after enzymolysis. After enzymolysis, it is cut into small segments, which causes little mechanical damage to the tissue, and is not easy to cause microbial contamination during subsequent operations. Moreover, the cells are more easily released after enzymolysis, which is more suitable for the growth and differentiation of double-petal daylily scape cells, and can more effectively induce callus tissue, adventitious buds, etc., thereby improving the efficiency and quality of double-petal daylily tissue culture; Third, the present invention adds NAA and 6-BA plant growth regulators in both the induction culture stage and the proliferation culture stage. In addition, TDZ is also used in combination. The three act synergistically to effectively improve the callus induction rate, adventitious bud differentiation rate and proliferation coefficient, which is very beneficial for the rapid and large-scale reproduction of double-petal daylily tissue culture seedlings.

[0017] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a schematic diagram of the material collection position of the scape with growth point of the present invention; Figure 2 It is a diagram showing the morphological changes of the scape during the adventitious bud induction culture process of the present invention; Figure 3 The growth conditions of the scape of the present invention in different induction culture media; Figure 4 It is the growth condition of the scape of the present invention in a defined induction medium under a defined explant treatment mode. DETAILED DESCRIPTION

[0019] The present invention is further described in detail below in conjunction with embodiments and drawings so that those skilled in the art can implement the invention with reference to the description.

[0020] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0021] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0022] Example 1: Tissue culture method of "cream roll" scape (1) Selection and processing of explants: The scapes of the double-petaled daylily variety “Butter Roll” with growth points (the growth points are defined as the attachment points of the bracts, the same below) were selected as explant materials (the sampling sites are as follows: Figure 1As shown in the figure, cut the stem into sections not less than 5 cm long with the growth point as the center. Soak the sections in soapy water for 15 min, rinse with running water for 1 h to clean the surface sand, rinse with sterile water twice under a clean bench, soak with 75% (v / v, the same below) ethanol for 30 s, rinse with sterile water twice, and finally soak with 2% (v / v, the same below) sodium hypochlorite for 5 min for disinfection. The disinfected long segments were placed in enzymatic hydrolysis solution for cell wall enzymatic hydrolysis. The enzymatic hydrolysis solution consisted of 15 mg / mL cellulase (manufacturer: Sigma-Aldrich, model: C2730, enzyme activity: ≥0.3 units / mg solid (with carboxymethyl cellulose as substrate), the same below), 7.5 mg / mL pectinase (manufacturer: Sigma-Aldrich, model: P4716, enzyme activity: ≥0.8 units / mg solid (with polygalacturonic acid as substrate), the same below), 0.75 mg / mL tea polyphenols, 73 mg / mL mannitol, 20 mM MES buffer, 1.1 mg / mL calcium chloride and sterile water, and was sterilized by filtration through a 0.22 μm filter membrane; the enzymatic hydrolysis temperature was 25°C and the enzymatic hydrolysis time was 60 min (at 50 min of enzymatic hydrolysis, sampling and observation showed that the cell wall was significantly thinned or partially degraded and the cell morphology was intact). The long segment after enzymatic hydrolysis was cut into small segments of about 1 cm with the growth point as the center. MS + 30 g / L sucrose + 7.0 g / L agar was used as the basic culture medium. Three explants were inoculated in each bottle, and 10 bottles were inoculated. After 28 days of dark culture in an incubator, the contamination rate was 3.03% and the start-up rate was 92.65%.

[0023] (2) Induction culture of adventitious buds: The small segments were inoculated onto the induction medium, cultured in the dark for 28 days to induce callus tissue, and then cultured in the light for 28 days to induce callus tissue to differentiate into adventitious buds. The induction medium included MS+6-BA 1.0mg / L+NAA0.2mg / L+TDZ 0.05mg / L+sucrose 30 g / L+agar 7.0g / L. Two explants were inoculated in each bottle, and 10 bottles were inoculated. The callus induction rate was 93.15%, and the adventitious bud differentiation rate was 88.92%.

[0024] During the adventitious bud induction culture, the morphological changes of the small segments are as follows Figure 2 Among them, after 7 days of dark culture, the lower incision of the morphology showed swelling, the color gradually became lighter, and a milky white swelling protrusion appeared at the growth point ( Figure 2 A); After 14 days of dark culture, loose callus appeared at the growth point, with a yellow-white irregular spherical shape. The upper end of the callus dried up and died, and the lower end swelled and then browned ( Figure 2 B); After 28 days of dark culture, callus rapidly proliferated ( Figure 2C). After 28 days of dark culture, the cells were switched to light culture. After 7 days of light culture, green granular protrusions gradually appeared in the yellow callus tissue, and a small number of adventitious buds appeared in some of them ( Figure 2 D); After 14 days of light culture, a large number of yellow-white adventitious buds appeared ( Figure 2 E); After 21 days of light culture, the number of adventitious buds further increased, and the color of the adventitious buds gradually turned into light green ( Figure 2 F); After 28 days of light culture, the adventitious buds grew to 1-3 cm and formed bud clusters ( Figure 2 G).

[0025] (3) Proliferation culture of adventitious buds: Inoculate the adventitious buds with strong growth, uniform growth, 2 cm ± in height, 2 to 3 leaves in the form of single plants or clustered buds on the proliferation medium and culture them under light for 28 days. The proliferation medium formula is: MS + 6-BA 1.0 mg / L + NAA 0.1 mg / L + TDZ 0.005 mg / L + sucrose 30 g / L + agar 7.0 g / L. Inoculate 2 plants in each bottle, and inoculate 10 bottles. The proliferation coefficient is 8.55, and the height reaches 2 cm in 84.88%.

[0026] (4) Rooting culture of adventitious buds: When the adventitious buds grow to 2-3 cm in height and 2-3 leaves, they are divided into individual plants and inoculated into rooting medium (activated carbon is added to the medium) to induce rooting. The culture is light-cultured for 28 days. The formula of the rooting medium is: 1 / 2MS + NAA 0.3 mg / L + sucrose 30 g / L + agar 7.0 g / L. Two plants are inoculated in each bottle, and 10 bottles are inoculated. The rooting rate is 86.67%, and the number of roots is 10.23.

[0027] (5) Hardening and transplanting: When the sterile seedlings grow 3 to 4 leaves and the new roots are 2 cm to 3 cm long, open the bottle cap and place them in the culture room for 3 days, and then in the greenhouse for 4 days. Then, take the plants out of the rooting medium, clean the roots with 0.1% carbendazim, and plant them in the sterilized transplanting medium (the ratio of the transplanting medium is: peat: perlite: vermiculite = 3:1:1). The transplanting survival rate is 96.67%.

[0028] (6) The culture conditions are as follows: the culture temperature for dark culture is 25°C, and the relative humidity of the air is controlled at 45%-55%; the light intensity for light culture is 1500 lx, the photoperiod is 12 hours light / 12 hours dark, the culture temperature is 25°C, and the relative humidity of the air is controlled at 45%-55%.

[0029] Comparative Example 1: Effects of different treatment methods on explant contamination and priming The "cream roll" double-petal daylily variety with consistent growth and growth point scapes were selected as explant materials. Treatment 1 used the small segments obtained by the method described in Example 1 as explants. Treatment 2 was based on Example 1, and the formula of the enzymatic hydrolyzate was changed to: 15 mg / mL cellulase, 7.5 mg / mL pectinase, and then the long segments after enzymatic hydrolysis were cut into small segments of about 1 cm with the growth point as the center as explants. Treatment 3 was a conventional method, in which the scapes were directly cut into small segments of about 1 cm with the growth point as the center, and then soaked in soapy water for 15 minutes, then rinsed with running water for 1 hour, the surface sand was washed off, rinsed with sterile water twice under a clean bench, soaked in 75% ethanol for 30 seconds, rinsed with sterile water twice, and finally soaked in 2% sodium hypochlorite for 10 minutes for disinfection to obtain explants. The explants obtained from treatments 1 to 3 were cultured in MS + 30 g / L sucrose + 7.0 g / L agar as the basic culture medium. Three explants were inoculated in each bottle, and 10 bottles were inoculated. After 28 days of dark culture in an incubator, the contamination rate and start-up rate were calculated. The results are shown in Table 1.

[0030] Table 1 Effects of different treatment methods on scape contamination rate and initiation rate Note: Lowercase letters indicate significant differences (P<0.05), the same below.

[0031] As shown in Table 1, different treatments have significant effects on the contamination and initiation of scapes. Treatment 1, i.e., first cross-cutting into long segments, disinfecting and then enzymolysis, and then cross-cutting into small segments, has the lowest contamination rate and the highest initiation rate, with a contamination rate of 3.03% and an initiation rate of 92.65%. This may be due to the disinfection of the long segments before enzymolysis, which effectively reduces the microbial contamination on the surface of the long segments. During disinfection, the growth point is far away from the end incision, which can better protect the growth point and reduce the damage of the disinfectant to it. After enzymolysis, the scape becomes soft and is cut into small segments after enzymolysis, which can reduce the mechanical damage to the tissue. It is not easy to cause microbial contamination during subsequent operations, and the cells are more easily released from the tissue after enzymolysis, which promotes the initiation of the explant. In addition, in this treatment method, the formula of the enzymolysis solution, the enzymolysis temperature, the enzymolysis time, and the post-enzymolysis treatment are all limited to ensure that the cell wall can be effectively enzymolyzed and the growth point can be protected to avoid excessive damage to the growth point. Compared with treatment 1, treatment 2 had the highest contamination rate and the lowest activation rate, with a contamination rate of 17.97% and an activation rate of 59.19%. This may be because although treatment 2 was also disinfected, the enzymatic hydrolyzate lacked tea polyphenols, mannitol, and MES buffer. The lack of tea polyphenols may cause a certain degree of damage to the cells during the enzymatic hydrolysis process, and the lack of mannitol and MES buffer may cause osmotic pressure and pH instability, affecting cell activity, thereby increasing the risk of contamination and reducing the activation ability of the cells. Treatment 3 used conventional treatment methods, and the contamination rate and activation rate were between treatment 1 and treatment 2, with a contamination rate of 8.33% and an activation rate of 70.45%.

[0032] Comparative Example 2: Effects of different induction medium compositions and ratios on callus differentiation of buds in scapes The scapes with growth points of the double-petaled daylily variety "Cream Roll" were selected as explant materials. After the explants were obtained by the conventional method of Treatment 3 in Comparative Example 1, they were inoculated onto an induction medium to induce callus differentiation into clustered buds. The composition, ratio and induction results of the induction medium are shown in Table 2.

[0033] Table 2 Effects of different plant hormone ratios on callus induction and adventitious bud differentiation of scape As shown in Table 2, the callus induction rate and adventitious bud differentiation rate were significantly different in the four schemes using 6-BA and NAA in combination. With the increase of NAA concentration, the induction rate and differentiation rate of scape showed an upward trend to a certain extent, but the induction rate and differentiation rate of the four schemes did not exceed 70%. After adding a lower concentration of TDZ, the induction rate and differentiation rate of scape were significantly improved, both reaching more than 70%, and with the increase of TDZ concentration, the callus induction rate showed an upward trend, and the adventitious bud differentiation rate showed a trend of first increasing and then decreasing. When the TDZ concentration was 0.05 mg / L, the induction rate and differentiation rate both reached 80%, and the adventitious buds grew vigorously ( Figure 3 A); Further increasing the TDZ concentration, there was no significant difference in callus induction rate and differentiation rate, and the induced adventitious buds grew poorly ( Figure 3 B). When the concentration reaches 0.2 mg / L, the differentiation rate will show a downward trend. Therefore, the best induction medium for scape is MS+6-BA1.0 mg / L+NAA 0.2 mg / L+TDZ 0.05 mg / L.

[0034] Further, the scape with growth point of the double-petal daylily variety "cream roll" was selected as the explant material, and the explant was obtained by the method of treatment 1 in comparative example 1, and then the explant was inoculated in the induction medium determined in comparative example 2 to induce callus and differentiate clustered buds. The callus induction rate reached 93.15%, the adventitious bud differentiation rate was 88.92%, and the adventitious buds grew vigorously ( Figure 4 ). This shows that the treatment method of first cross-cutting into long segments, disinfecting and then enzymatically hydrolyzing, and then cross-cutting into small segments in the present application not only has a significant impact on explant contamination and priming, but is also more suitable for the growth and differentiation of double-petal daylily scape cells, and can more effectively induce callus tissue, adventitious buds, etc., thereby improving the efficiency and quality of double-petal daylily tissue culture.

[0035] The number of equipment and processing scale described here are used to simplify the description of the present invention. The application, modification and variation of the tissue culture method of double-petaled daylily scape of the present invention will be obvious to those skilled in the art.

[0036] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. A method for tissue culture of double-petal daylily scapes, characterized in that: include: Take the scape of the double-petal daylily variety, cut it into sections no less than 5 cm long with the growth point as the center, and rinse and disinfect it; The sterilized long segment is placed in an enzymatic hydrolysis solution for cell wall enzymatic hydrolysis, and after the enzymatic hydrolysis is completed, it is taken out and rinsed with sterile water; Cut the long segment after enzymatic hydrolysis into small segments of 1 to 2 cm with the growth point as the center; The small segment is used as an explant for tissue culture; wherein, The enzymatic hydrolysis solution is composed of 10 mg / mL to 20 mg / mL of cellulase, 5 mg / mL to 10 mg / mL of pectinase, 0.5 mg / mL to 1 mg / mL of tea polyphenols, 73 mg / mL of mannitol, 20 mM of MES buffer, 1.1 mg / mL of calcium chloride and sterile water, and is sterilized by filtration through a 0.22 μm filter membrane; the enzymatic hydrolysis temperature is 25 to 30° C., and the enzymatic hydrolysis time is 30 to 90 min.

2. The tissue culture method of double-petal daylily scape according to claim 1, characterized in that: The disinfection is to first disinfect the long section with 75% alcohol for 10 to 30 seconds, and then disinfect with 2% sodium hypochlorite for 5 to 15 minutes.

3. The tissue culture method of double-petal daylily scape according to claim 1, characterized in that: Samples were taken at 30, 50 and 70 minutes of enzymatic hydrolysis, and the cell status of the scape epidermis was observed under a microscope. If the cell wall was obviously thinned or partially degraded and the cell morphology was intact, the enzymatic hydrolysis was stopped; otherwise, the enzymatic hydrolysis was continued for 90 minutes.

4. The tissue culture method of double-petal daylily scape according to claim 1, characterized in that: The small segment is used as an explant for tissue culture, comprising: Step 1, inoculating the explant onto an induction medium, first culturing in the dark for 26 to 30 days to induce callus tissue, and then culturing in the light for 26 to 30 days to induce the callus tissue to differentiate into adventitious buds, wherein the induction medium includes MS+6-BA 1.0 mg / L+NAA 0.2 mg / L+TDZ 0.01 mg / L to 0.1 mg / L; Step 2: Inoculate the adventitious buds in the form of single plants or clustered buds on a proliferation medium and culture them under light for 26 to 30 days. The proliferation medium includes MS+6-BA 1.0 mg / L+NAA 0.1 mg / L+TDZ 0.001 mg / L to 0.01 mg / L; Step 3: Divide the adventitious buds into individual plants, inoculate them in a rooting medium to induce rooting, and culture them under light for 26 to 30 days. The rooting medium includes 1 / 2MS+NAA 0mg / L to 0.5mg / L; Step 4: After hardening the rooted seedlings, take them out from the rooting medium, clean the roots with a fungicide, and transplant them into a transplanting medium to obtain double-petal daylily plants.

5. The tissue culture method of double-petal daylily scape according to claim 4, characterized in that: The induction medium includes MS+6-BA 1.0 mg / L+NAA 0.2 mg / L+TDZ 0.05 mg / L.

6. The tissue culture method of double-petal daylily scape according to claim 4, characterized in that: The proliferation medium includes MS+6-BA 1.0 mg / L+NAA 0.1 mg / L+TDZ 0.005 mg / L.

7. The tissue culture method of double-petal daylily scape according to claim 4, characterized in that: The rooting medium includes 1 / 2MS+NAA 0.3 mg / L.

8. The tissue culture method of double-petal daylily scape according to claim 4, characterized in that: The culture temperature of the dark culture is 20°C to 30°C, and the relative humidity of the air is 40% to 60%; the light intensity of the light culture is 1000lx to 2 000lx, the photoperiod is 12 hours of light / 12 hours of darkness, the culture temperature is 20°C to 30°C, and the relative humidity of the air is 40% to 60%.

9. The tissue culture method of double-petal daylily scape according to claim 1, characterized in that: The double-petal daylily variety is one of "cream roll", "golden yellow" and "flesh pink".

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