Method for tissue culture of double peony flower peduncle

Abstract

This invention discloses a method for tissue culture of double-flowered daylily scapes, comprising: taking the scape of a double-flowered daylily variety, transversely cutting it into segments no less than 5 cm long with the growing point as the center, and rinsing and disinfecting the segments; placing the disinfected segments into an enzymatic hydrolysis solution for cell wall enzymatic hydrolysis, removing the segments after hydrolysis, and rinsing them with sterile water; transversely cutting the hydrolyzed segments into 1-2 cm segments with the growing point as the center; and using the segments as explants for tissue culture. This method, using double-flowered daylily varieties, has the advantages of allowing for the collection of materials at any time during the flowering period, resulting in high tissue culture efficiency and good quality.

Description

Technical Field

[0001] This invention relates to the field of tissue culture. More specifically, this invention relates to a method for tissue culture of double-flowered daylily scapes. Background Technology

[0002] Daylilies are one of the world's three major perennial flowers, prized for their large, beautiful blooms, diverse shapes, varied colors, and long flowering period, making them an important ornamental plant in gardens. Furthermore, daylilies have a well-developed root system with strong water absorption and soil-binding capabilities, helping to reduce soil erosion; they also exhibit strong drought resistance, high temperature tolerance, and cold resistance, making them adaptable to a wide range of soil types and suitable for slope and roadside greening; their vigorous growth allows for extensive management and simple maintenance, saving labor, effort, and costs, thus offering broad prospects for future development.

[0003] Currently, daylilies are mainly propagated through division, which is slow, with each plant only producing 3-5 new plants per year. Large-scale propagation requires a certain number of seedlings and a large area of ​​land. Furthermore, the low propagation coefficient and long production cycle make it difficult to meet the demands of commercial, large-scale production of superior varieties, leading to high prices and hindering their application and promotion. This is especially true for some new and superior double-flowered varieties, such as the "Creamy Curl" double-flowered daylily, which boasts bright colors, unique flower shapes, full blooms, long green periods, and enhanced overall ornamental value. However, the difficulty in propagation results in a limited number of seedlings, further hindering large-scale application.

[0004] Tissue culture is an effective way to accelerate the breeding process. However, existing reports on the propagation of daylilies by tissue culture using flower stalks as explants mainly focus on varieties such as "Jianzhuanglilan", "Datong Huanghuacai", "Fenmeiren", and "Jinwawa", all of which are single-petaled. There are few reports on the successful establishment of tissue culture systems for propagating seedlings of double-petaled daylily varieties using flower stalks as explants. Furthermore, the traditional tissue culture method using flower stalks as explants involves directly cutting the flower stalk into small segments and sterilizing them, which can easily cause mechanical damage to the tissue and damage to the growth point, affecting the subsequent induction of callus tissue and the differentiation of adventitious buds.

[0005] Enzymatic digestion techniques are commonly used in protoplast isolation or cell wall-related studies, but their application in tissue culture has not yet been reported. Further research is needed to explore how to combine enzymatic digestion techniques with tissue culture to address existing problems in the tissue culture process. Summary of the Invention

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

[0007] To achieve the objectives and other advantages of this invention, a method for tissue culture of double-flowered daylily scapes is provided, comprising: taking the scape of a double-flowered daylily variety, transversely cutting it into segments no less than 5 cm long with the growth point as the center, and rinsing and disinfecting the segments; placing the disinfected segments into an enzymatic hydrolysis solution for cell wall enzymatic hydrolysis, removing the segments after enzymatic hydrolysis, and rinsing them with sterile water; transversely cutting the hydrolyzed segments into 1-2 cm segments with the growth point as the center; and using the segments as explants for tissue culture; wherein the enzymatic hydrolysis solution consists of cellulase 10 mg / mL-20 mg / mL, pectinase 5 mg / mL-10 mg / mL, tea polyphenols 0.5 mg / mL-1 mg / mL, mannitol 73 mg / mL, MES buffer 20 mM, calcium chloride 1.1 mg / mL, and sterile water, and is filtered through a 0.22 μm filter membrane for sterilization; the enzymatic hydrolysis temperature is 25-30℃, and the enzymatic hydrolysis time is 30-90 min.

[0008] Preferably, the disinfection involves first disinfecting the long section with 75% alcohol for 10-30 seconds, and then disinfecting it with 2% sodium hypochlorite for 5-15 minutes.

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

[0010] Preferably, tissue culture using the aforementioned explant segment includes: Step 1: Inoculating the explant onto an induction medium, first culturing in the dark for 26–30 days to induce callus formation, then culturing under light for 26–30 days to induce callus differentiation into adventitious shoots. The induction medium comprises MS + 6-BA 1.0 mg / L + NAA 0.2 mg / L + TDZ 0.01 mg / L–0.1 mg / L; Step 2: Inoculating the adventitious shoots, either as single plants or in clusters, onto a proliferation medium and culturing under light for 26–30 days. The proliferation medium comprises MS + 6-BA 1.0 mg / L + NAA 0.1 mg / L + TDZ 0.001 mg / L–0.01 mg / L; Step 3: Dividing the adventitious shoots into single plants, inoculating them into a rooting medium to induce rooting, and culturing under light for 26–30 days. The rooting medium comprises 1 / 2 MS + NAA. 0 mg / L~0.5 mg / L; Step 4: After the rooted seedlings have hardened off, remove them from the rooting medium, wash the roots with a fungicide, and then transplant them into the transplanting substrate to obtain double-flowered 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 / 2 MS + NAA 0.3 mg / L.

[0014] Preferably, the dark culture temperature is 20°C to 30°C and the relative humidity is 40% to 60%; the light culture intensity is 1000 lx to 2000 lx, the photoperiod is 12 hours of light / 12 hours of darkness, the culture temperature is 20°C to 30°C, and the relative humidity is 40% to 60%.

[0015] Preferably, the double-flowered daylily variety is one of "Creamy Roll", "Golden Yellow", or "Flesh Pink".

[0016] The present invention has at least the following beneficial effects:

[0017] First, the method of the present invention uses the flower stalk with growth point as explant, which can indirectly induce the production of adventitious buds, increases the propagation method of tissue culture of double daylily, and can carry out large-scale propagation at any time during the flowering period, which is conducive to realizing the large-scale production of factory seedlings to meet the needs of the landscaping market.

[0018] Secondly, this invention involves transversely cutting the flower stalk into long segments centered on the growth point, disinfecting the long segments, enzymatically hydrolyzing them, and then transversely cutting the hydrolyzed long segments into smaller segments centered on the growth point. Firstly, disinfecting after transverse cutting helps to better protect the growth point during disinfection, reducing damage from the disinfectant. Secondly, enzymatic hydrolysis of the long segments using a specific enzymatic hydrolysate softens the flower stalk, and cutting it into smaller segments after hydrolysis causes less mechanical damage to the tissue, reducing the risk of microbial contamination during subsequent operations. Furthermore, the cells are more easily released after hydrolysis, making it more suitable for the growth and differentiation of double-flowered daylily flower stalk cells, and more effectively inducing callus tissue and adventitious buds, thereby improving the efficiency and quality of double-flowered daylily tissue culture.

[0019] Third, this invention incorporates 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 conjunction with these substances. The synergistic effect of these three substances effectively improves the callus induction rate, adventitious bud differentiation rate, and proliferation coefficient, which is very beneficial for the rapid and large-scale propagation of double-flowered daylily tissue culture seedlings.

[0020] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the material taken from the flower stalk with growth point according to the present invention;

[0022] Figure 2 This is a diagram showing the morphological changes of the flower stalk during the adventitious bud induction culture process of the present invention;

[0023] Figure 3 This describes the growth of the flower stalk of the present invention in different induction culture media;

[0024] Figure 4 This describes the growth of the flower stalk of the present invention in a defined induction medium under a defined explant treatment method. Detailed Implementation

[0025] The present invention will now be described in further detail with reference to the embodiments and accompanying drawings, so that those skilled in the art can implement it based on the description.

[0026] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0027] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0028] Example 1: Tissue Culture Method of "Cream Roll" Flower Stalk

[0029] (1) Selection and treatment of explants: Flower stalks with growth points (defined as the attachment point of bracts, the same below) of the "Creamy Roll" double-flowered daylily variety were selected as explant materials (the parts taken are as follows). Figure 1As shown in the figure, with the growth point as the center, cut it into segments no less than 5 cm long. Soak the segments in soapy water for 15 minutes, then rinse them with running water for 1 hour to remove surface dirt. Under a clean bench, rinse them twice with sterile water, soak them in 75% (v / v, the same below) ethanol for 30 seconds, rinse them twice with sterile water, and finally soak them in 2% (v / v, the same below) sodium hypochlorite for 5 minutes for disinfection. The sterilized long segments were placed in an enzymatic hydrolysis solution for cell wall hydrolysis. The 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 filtered through a 0.22 μm filter membrane for sterilization. The hydrolysis temperature was 25℃ and the hydrolysis time was 60 min (after 50 min of hydrolysis, samples were taken for observation, and the cell walls were significantly thinned or partially degraded while the cell morphology remained intact). The long segments after enzymatic hydrolysis were transversely cut into small segments of about 1 cm with the growth point as the center. Using MS + sucrose 30 g / L + agar 7.0 g / L as the basic culture medium, 3 explants were inoculated in each bottle, and 10 bottles were inoculated. After 28 days of dark incubation in an incubator, the contamination rate was 3.03% and the initiation rate was 92.65%.

[0030] (2) Induction and culture of adventitious shoots: Small explants were inoculated onto induction medium and cultured in the dark for 28 days to induce callus formation, followed by 28 days of light culture to induce callus differentiation into adventitious shoots. The induction medium consisted of MS + 6-BA 1.0 mg / L + NAA 0.2 mg / L + TDZ 0.05 mg / L + sucrose 30 g / L + agar 7.0 g / L. Two explants were inoculated per bottle, and 10 bottles were inoculated. The callus induction rate was 93.15%, and the adventitious shoot differentiation rate was 88.92%.

[0031] During the adventitious shoot induction culture, the morphological changes of small segments are as follows: Figure 2 As shown. In the dark culture for 7 days, morphological swelling appeared at the lower cut end, the color gradually lightened, and milky-white swollen protrusions appeared at the growth point. Figure 2 A); After 14 days of dark culture, loose callus tissue was observed at the growth point, appearing as irregular yellowish-white spheres. The upper morphological end was dried up and dead, while the lower morphological end swelled and turned brown. Figure 2 B); after 28 days of dark culture, callus tissue proliferated rapidly ( Figure 2C). After 28 days of dark culture, the tissue was switched to light culture. After 7 days of light culture, green granular protrusions gradually appeared on the yellow callus tissue, and some of the tissue also showed a small number of adventitious buds. Figure 2 D); After 14 days of light cultivation, a large number of yellowish-white adventitious buds appeared ( Figure 2 E); After 21 days of light cultivation, the number of adventitious buds further increased, and the color of the adventitious buds gradually turned light green (E). Figure 2 F); after 28 days of light cultivation, adventitious buds grow to 1-3 cm and form bud clusters ( Figure 2 G).

[0032] (3) Proliferation culture of adventitious buds: Healthy, uniformly growing adventitious buds with a height of 2cm± and 2-3 leaves were inoculated onto proliferation medium, either individually or in clusters, and cultured under light for 28 days. The proliferation medium formula was: MS + 6-BA 1.0mg / L + NAA 0.1mg / L + TDZ 0.005mg / L + sucrose 30g / L + agar 7.0 g / L. Two buds were inoculated per bottle, and 10 bottles were inoculated. The proliferation coefficient was 8.55, and 84.88% of the buds reached a height of 2cm.

[0033] (4) Rooting culture of adventitious buds: When the adventitious buds grow to a height of 2-3 cm and have 2-3 leaves, they are divided into individual plants and inoculated into rooting medium (with activated carbon added) to induce rooting. The plants are cultured under light for 28 days. The rooting medium formula is: 1 / 2 MS + NAA 0.3 mg / L + sucrose 30 g / L + agar 7.0 g / L. Two plants are inoculated per bottle, for a total of 10 bottles. The rooting rate is 86.67%, and the number of roots is 10.23.

[0034] (5) Hardening off and transplanting: When the sterile seedlings have grown 3-4 leaves and the new roots are 2-3 cm long, open the bottle cap and place them in the culture room for 3 days, then place them in the greenhouse for 4 days. Then, take the plants out of the rooting medium, wash the roots with 0.1% carbendazim, and plant them in sterilized transplanting substrate (transplanting substrate ratio: peat moss: perlite: vermiculite = 3:1:1). The transplant survival rate is 96.67%.

[0035] (6) The cultivation conditions are as follows: the cultivation temperature for dark cultivation is 25°C and the relative humidity of the air is controlled at 45% to 55%; the light intensity for light cultivation is 1500 lx, the photoperiod is 12 hours of light / 12 hours of darkness, the cultivation temperature is 25°C, and the relative humidity of the air is controlled at 45% to 55%.

[0036] Comparative Example 1: Effects of different treatments on explant contamination and initiation

[0037] Flower stalks with growth points of the double-flowered daylily variety "Creamy Roll" with uniform growth 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, but the formulation of the enzymatic hydrolysate was changed to: cellulase 15 mg / mL and pectinase 7.5 mg / mL. Then, the long segments after enzymatic hydrolysis were transversely cut into small segments of about 1 cm with the growth point as the center as explants. Treatment 3 used the conventional method, directly cutting the flower stalk into small segments of about 1 cm with the growth point as the center. Then, the segments were first soaked in soapy water for 15 min, then rinsed with running water for 1 h to wash off the surface dirt. Under a clean bench, the segments were rinsed twice with sterile water, soaked in 75% ethanol for 30 s, rinsed twice with sterile water, and finally soaked in 2% sodium hypochlorite for 10 min for disinfection to obtain explants. The explants obtained from treatments 1-3 were cultured on MS medium containing 30 g / L sucrose and 7.0 g / L agar. Three explants were inoculated per bottle, and 10 bottles were inoculated. After 28 days of dark incubation, the contamination rate and initiation rate were calculated. The results are shown in Table 1.

[0038] Table 1. Effects of different treatment methods on the pollution rate and initiation rate of flower stalks.

[0039]

[0040] Note: Lowercase letters indicate significant differences (P<0.05), and the same applies below.

[0041] Table 1 shows that different treatment methods significantly affected the contamination and initiation of the flower stalk. Treatment 1, which involved first cutting the stalk into long segments, then disinfecting and enzymatically hydrolyzing it, and finally cutting it into smaller segments, had the lowest contamination rate and the highest initiation rate, with a contamination rate of 3.03% and an initiation rate of 92.65%. This is likely because disinfecting the long segments before enzymatic hydrolysis effectively reduced microbial contamination on the surface of the long segments. During disinfection, the growth point was further away from the end cut, which better protected the growth point and reduced damage from the disinfectant. After enzymatic hydrolysis, the flower stalk softened, and cutting it into smaller segments after enzymatic hydrolysis reduced mechanical damage to the tissue. This made it less likely for microbial contamination to occur during subsequent operations, and the cells were more easily released from the tissue after enzymatic hydrolysis, promoting the initiation of the explant. In addition, this treatment method limited the formulation of the enzymatic hydrolysate, the enzymatic hydrolysis temperature, the enzymatic hydrolysis time, and the post-enzymatic hydrolysis treatment to ensure that the cell wall was effectively hydrolyzed while protecting the growth point and avoiding excessive damage to the growth point. Compared to Treatment 1, Treatment 2 had the highest contamination rate and the lowest initiation rate, with a contamination rate of 17.97% and an initiation rate of 59.19%. This may be because although Treatment 2 also underwent sterilization, the enzymatic hydrolysate lacked tea polyphenols, mannitol, and MES buffer. The lack of tea polyphenols may have caused some damage to the cells during enzymatic hydrolysis, while the lack of mannitol and MES buffer may have led to unstable osmotic pressure and pH, affecting cell viability and thus increasing the risk of contamination and reducing the cells' initiation ability. Treatment 3 used conventional treatment methods, and its contamination rate and initiation rate were between those of Treatment 1 and Treatment 2, with a contamination rate of 8.33% and an initiation rate of 70.45%.

[0042] Comparative Example 2: Effects of different induction medium compositions and ratios on the differentiation of callus tissue into shoot clusters in flower scapes

[0043] The flower stalk with growth point of the double-flowered daylily variety "Creamy Roll" was selected as the explant material. After obtaining the explants using the conventional method of treatment 3 in Comparative Example 1, they were inoculated onto the induction medium to induce callus differentiation and shoot formation. The composition, ratio and induction results of the induction medium are shown in Table 2.

[0044] Table 2 Effects of different plant hormone ratios on callus induction and adventitious bud differentiation in flower stalks.

[0045]

[0046] Table 2 shows that among the four schemes using 6-BA and NAA combinations, the callus induction rate and adventitious bud differentiation rate differed significantly. With increasing NAA concentration, the induction and differentiation rates of flower stalks showed an upward trend to some extent, but none of the four schemes exceeded 70%. Adding a lower concentration of TDZ significantly improved the induction and differentiation rates of flower stalks, both reaching over 70%. Furthermore, with increasing TDZ concentration, the callus induction rate showed an upward trend, while the adventitious bud differentiation rate showed a trend of first increasing and then decreasing. At a TDZ concentration of 0.05 mg / L, both the induction and differentiation rates reached 80%, and the adventitious buds exhibited vigorous growth. Figure 3 A); Further increasing the TDZ concentration did not significantly affect the callus induction rate and differentiation rate, and the induced adventitious shoots showed poor growth. Figure 3 B). When the concentration reaches 0.2 mg / L, the differentiation rate will show a decreasing trend. Therefore, the optimal induction medium for flower stalks is MS + 1.0 mg / L 6-BA + 0.2 mg / L NAA + 0.05 mg / L TDZ.

[0047] Furthermore, the scape with a growth point of the double-flowered daylily cultivar "Creamy Roll" was selected as explant material. After obtaining explants using the method of treatment 1 in Comparative Example 1, the explants were inoculated in the induction medium determined in Comparative Example 2 to induce callus tissue and differentiate into clustered shoots. The callus induction rate reached 93.15%, and the adventitious shoot differentiation rate was 88.92%, with vigorous growth of adventitious shoots. Figure 4 This indicates that the treatment method described in this application—first cutting the tissue into long segments, then disinfecting and enzymatically digesting it, and finally cutting it into smaller segments—not only has a significant impact on explant contamination and initiation, but is also more suitable for the growth and differentiation of double-flowered daylily scape cells, and can more effectively induce callus tissue, adventitious buds, etc., thereby improving the efficiency and quality of double-flowered daylily tissue culture.

[0048] The number of devices and processing scale described herein are for the purpose of simplifying the description of the invention. Applications, modifications, and variations of the tissue culture method for double-flowered daylily scapes of the present invention will be readily apparent to those skilled in the art.

[0049] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A tissue culture method for double-flowered daylily scapes, characterized in that, include: Take the flower stalk of the double-flowered daylily variety, cut it horizontally into sections no less than 5cm long with the growing point as the center, and then rinse and disinfect it. The sterilized long segment was placed in an enzymatic hydrolysis solution for cell wall enzymatic hydrolysis. After the enzymatic hydrolysis was completed, it was removed and rinsed with sterile water. After enzymatic hydrolysis, the long segment is cut into 1-2 cm segments with the growth point as the center. The aforementioned segment was used as an explant for tissue culture; wherein, The enzymatic hydrolysate consists of 10 mg / mL to 20 mg / mL cellulase, 5 mg / mL to 10 mg / mL pectinase, 0.5 mg / mL to 1 mg / mL tea polyphenols, 73 mg / mL mannitol, 20 mM MES buffer, 1.1 mg / mL 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. The disinfection process involves first disinfecting the long section with 75% alcohol for 10-30 seconds, and then disinfecting it with 2% sodium hypochlorite for 5-15 minutes. Tissue culture using the aforementioned segment as an explant includes: Step 1: Inoculate the explants onto the induction medium, first culture in the dark for 26-30 days to induce callus tissue, then culture under light for 26-30 days to induce callus tissue to differentiate into adventitious shoots. The induction medium is MS + 6-BA 1.0 mg / L + NAA 0.2 mg / L + TDZ 0.01 mg / L-0.1 mg / L + sucrose 30 g / L + agar 7.0 g / L; Step 2: Inoculate the adventitious buds onto the proliferation medium as single plants or clusters of buds, and culture under light for 26-30 days. The proliferation medium 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. Step 3: Divide the adventitious buds into individual plants, inoculate them in rooting medium to induce rooting, and culture under light for 26-30 days. The rooting medium is 1 / 2 MS + NAA 0.3 mg / L + sucrose 30 g / L + agar 7.0 g / L. Step 4: After the rooted seedlings have hardened off, remove them from the rooting medium, clean the roots with a fungicide, and then transplant them into the transplanting substrate to obtain double-flowered daylily plants.

2. The tissue culture method for double-flowered daylily scape as described in claim 1, characterized in that, Samples were taken at 30 min, 50 min and 70 min of enzymatic hydrolysis, and the cell state of the flower stalk epidermis was observed under a microscope. If the cell wall was significantly thinned or partially degraded and the cell morphology was intact, the enzymatic hydrolysis was stopped; otherwise, the enzymatic hydrolysis was continued until 90 min.

3. The tissue culture method for double-flowered daylily scape as described in claim 1, characterized in that, The induction medium consists of MS + 6-BA 1.0 mg / L + NAA 0.2 mg / L + TDZ 0.05 mg / L.

4. The tissue culture method for double-flowered daylily scape as described in claim 1, characterized in that, The dark culture temperature is 20°C to 30°C, and the relative humidity is 40% to 60%. The light culture has a light intensity of 1000 lx to 2000 lx, a photoperiod of 12 hours of light / 12 hours of darkness, a culture temperature of 20°C to 30°C, and a relative humidity of 40% to 60%.

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