Tissue culture method of root stocks of hemerocallis fulva

Abstract

The invention discloses a tissue culture method of root stocks of hemerocallis fulva, which comprises the following steps: step 1, selecting a robust root stock of a hemerocallis fulva variety as an explant, transversely cutting the root stock into small sections, and cleaning and disinfecting the small sections; 2, the disinfected explant is longitudinally cut under the sterile condition and then rapidly transferred to an MS solution containing 0.4-0.6 mg / L of 6-BA and 0.08-0.12 mg / L of NAA to be soaked for 30 min, the temperature of the solution is controlled to be about 20 DEG C, and then the explant is inoculated to an induction culture medium in an upright arrangement mode to induce adventitious buds; step 3, inoculating the adventitious buds on a proliferation culture medium in a single plant or cluster bud mode for proliferation; 4, dividing the adventitious buds into single plants, and inoculating the single plants into a rooting culture medium to induce rooting; 5, after the rooting seedlings are hardened, transplanting is conducted, and hemerocallis fulva plants are obtained. The method applies the double-petal hemerocallis fulva variety, and has the advantages of being capable of obtaining materials all year round, high in propagation coefficient and the like.

Description

Technical Field

[0001] The present invention relates to the field of tissue culture, and more particularly to a tissue culture method for double-petal daylily rhizomes. 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, the existing reports on tissue culture propagation of daylilies using rhizomes as explants are all based on single-petal daylily varieties such as "Daylily", "Datong Daylily" and "Golden Baby". There are differences in genetic characteristics and physiological structure between double-petal daylilies and single-petal daylilies, which means that the tissue culture of double-petal daylilies cannot directly refer to the tissue propagation method of single-petal daylily varieties. So far, there have been no reports on tissue culture propagation of double-petal daylilies using rhizomes as explants. Summary of the invention

[0005] The object of the present invention is to provide a tissue culture method for double-petal daylily rhizomes to at least solve the above-mentioned problems.

[0006] In order to achieve the purpose and other advantages of the present invention, a tissue culture method for double-petal daylily rhizomes is provided, comprising: step 1, selecting a robust rhizome of a double-petal daylily variety as an explant, cutting it into small sections, and washing and disinfecting it; step 2, cutting the disinfected explant longitudinally under sterile conditions and quickly transferring it to an MS solution containing 0.4-0.6 mg / L 6-BA and 0.08-0.12 mg / L NAA, soaking it for 30 minutes, with the solution temperature controlled at about 20°C, and then inoculating it upright on an induction culture medium to induce adventitious buds, and culturing it under light for 26-30 days, wherein the induction culture medium comprises MS+6-BA 2.0 mg / L+NAA 0.2 mg / L+TDZ 0.1 mg / L-0.2 mg / L; step 3, inoculating the adventitious buds in the form of single plants or clustered buds on a proliferation culture medium, and culturing it under light for 26-30 days, wherein the proliferation culture medium comprises MS+6-BA1.0 mg / L+NAA 0.1mg / L+TDZ 0.001mg / L~0.01mg / L; Step 4, divide the adventitious buds into individual plants, inoculate them in a rooting medium to induce rooting, and culture them under light for 26~30 days, wherein the rooting medium includes 1 / 2MS+NAA 0mg / L~0.5mg / L; Step 5, after hardening the rooted seedlings, take them out from the rooting medium and transplant them into a transplanting medium to obtain double-petal daylily plants.

[0007] Preferably, the disinfection is to disinfect the cleaned explant with 75% alcohol for 10s to 30s, and then disinfect with 2% sodium hypochlorite for 5min to 15min.

[0008] Preferably, the induction medium comprises MS+6-BA 2.0 mg / L+NAA 0.2 mg / L+TDZ 0.1 mg / L+glycine 100 mg / L+vitamin C 50 mg / L+inositol 20 mg / L.

[0009] Preferably, the proliferation medium comprises MS+6-BA 1.0 mg / L+NAA 0.1 mg / L+TDZ 0.005 mg / L+proline 80 mg / L+niacin 30 mg / L.

[0010] Preferably, the rooting medium comprises 1 / 2MS+NAA 0.3 mg / L.

[0011] Preferably, in step 2, the first illumination culture condition is used for illumination culture, wherein the first illumination culture condition is: on the 1st to 6th day, the light intensity is 1000 lx, the photoperiod is 12 hours of light / 12 hours of darkness, the culture temperature is 22°C, and the relative humidity of the air is 70% to 80%; on the 7th to 14th day, the light intensity is 1000 lx, the photoperiod is 13 hours of light / 11 hours of darkness, the temperature is raised to 25°C during the 4th to 10th hour of illumination, and the culture temperature is maintained at 22°C for the rest of the time. , the relative humidity of the air was 70% ~ 80%; on the 15th to 21st day, the light intensity was 1500lx, the photoperiod was 14 hours light / 10 hours dark, the culture temperature was 24℃ in light, 20℃ in darkness, and the relative humidity of the air was 60% ~ 70%; on the 22nd to 28th day, the light intensity was 2000lx, the photoperiod was 14 hours light / 10 hours dark, the culture temperature was 25℃ in light, 20℃ in darkness, and the relative humidity of the air was 60% ~ 70%.

[0012] Preferably, in step three, a second illumination culture condition is used for illumination culture, and the second illumination culture condition is: on the 1st to 6th day, the light intensity is 2500lx, the photoperiod is 16 hours of light / 8 hours of darkness, the culture temperature is 27°C during illumination, the culture temperature is 22°C during darkness, and the relative humidity of the air is 60% to 70%; on the 7th to 28th day, the light intensity is 2500lx, the photoperiod is 16 hours of light / 8 hours of darkness, the culture temperature is 27°C during illumination and the temperature is raised to 28°C two hours before the end of the illumination each day, the culture temperature is 22°C during darkness, and the relative humidity of the air is 60% to 70%.

[0013] Preferably, in step 4, a third illumination culture condition is adopted for illumination culture, and the third illumination culture condition is: on the 1st to 14th day, the light intensity is 1500lx, the photoperiod is 14 hours light / 10 hours dark, the culture temperature is 24°C during illumination, the culture temperature is 20°C during darkness, and the relative humidity of the air is 50% to 60%; on the 15th to 28th day, the photoperiod is 14 hours light / 10 hours dark, in the first 6 hours and the last 6 hours of illumination each day, the light intensity is 1500lx, the culture temperature is 26°C, the light intensity for the middle 2 hours is 1000lx, the culture temperature is 24°C, the culture temperature is 20°C in darkness, and the relative humidity of the air is 50% to 60%.

[0014] Preferably, the sterilized explant is inoculated into the induction medium in a manner of cross-sectioning and then half longitudinally sectioning and placing the explant upright to induce adventitious buds.

[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 rhizomes of the double-petal daylily variety as explants, which can directly induce the production of adventitious buds and accelerate the reproduction speed of the double-petal daylily; the rhizomes can be obtained all year round, breaking the limitations of seasonal restrictions, and large-scale mass reproduction can be carried out at any time, which is easy to realize factory-based seedling production to meet the needs of the landscaping market as soon as possible.

[0017] Second, 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 adventitious bud induction rate, the number of adventitious buds and the proliferation coefficient, which is very beneficial for the rapid and large-scale reproduction of double-petal daylily tissue culture seedlings.

[0018] Third, the present invention sterilizes the explant by cross-sectioning, cuts it into 1 / 2 longitudinal section under sterile conditions and quickly transfers it to a MS solution containing 0.4-0.6 mg / L 6-BA and 0.08-0.12 mg / L NAA and soaks it for 30 minutes, controls the solution temperature at about 20°C, and then inoculates it upright on an induction medium to induce adventitious buds, which can effectively improve the induction rate and number of adventitious buds, and the adventitious buds grow well.

[0019] Fourth, the present invention can effectively improve the adventitious bud induction rate, the number of adventitious buds, the proliferation coefficient, the rooting rate and the number of roots by improving the lighting culture conditions during the culture process, and the obtained plants have good growth potential and high survival rate.

[0020] 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

[0021] Figure 1 It is a schematic diagram of the material collection position of the rhizome of the present invention; Figure 2 The growth conditions of adventitious buds of the rhizome of the present invention in different induction culture media; Figure 3 The growth conditions of adventitious buds of the rhizome of the present invention under different treatment modes; Figure 4 The invention discloses the growth conditions of the adventitious buds of the rhizome in different proliferation culture media. DETAILED DESCRIPTION

[0022] 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.

[0023] 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.

[0024] 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.

[0025] Example 1: Tissue culture method of "cream roll" rhizome (1) Selection and disinfection of explants: Select the robust rhizomes of the double-petaled daylily variety "Cream Roll" as explant materials (the material extraction site is as follows: Figure 1 As shown, the same below), cut it into 1cm± small sections, soak it in soapy water for 15min, then rinse it with running water for 1h to clean the surface sand, rinse it with sterile water twice under the clean bench, soak it in 75% (v / v, the same below) ethanol for 30s, rinse it with sterile water twice, and finally soak it in 2% (v / v, the same below) sodium hypochlorite for 10min for disinfection. MS+sucrose 30g / L+agar 7.0 g / L was used as the basic culture medium, 3 explants were inoculated in each bottle, 10 bottles were inoculated, and statistics after 28 days of light culture showed that the contamination rate was 13.54% and the start-up rate was 80.74%.

[0026] (2) Induction culture of adventitious buds: After sterilization, the healthy rhizomes were cut into 1 / 2 longitudinal sections under sterile conditions and quickly transferred to MS solution containing 0.4~0.6mg / L 6-BA and 0.08~0.12mg / L NAA and soaked for 30 minutes. The solution temperature was controlled at about 20℃, and then inoculated on the induction medium in an upright manner to induce adventitious buds. The induction medium formula was: MS+6-BA 2.0 mg / L+NAA 0.2 mg / L+TDZ 0.1mg / L+sucrose 30 g / L+agar 7.0 g / L. Two explants were inoculated in each bottle, and 10 bottles were inoculated. After 28 days of light culture, the adventitious bud induction rate was 83.33%, and the number of adventitious buds was 4.62.

[0027] (3) Proliferation culture of adventitious buds: The adventitious buds with strong growth, relatively uniform growth, plant height of 2 cm ±, and 2 to 3 leaves were inoculated in the form of single plants or clustered buds on the proliferation medium. The formula of 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 7.0g / L. Two plants were inoculated in each bottle, and 10 bottles were inoculated. After 28 days of light culture, the proliferation coefficient was 6.8, and 81.23% of the heights reached 2 cm.

[0028] (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 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. After 28 days of light culture, the rooting rate is 80% and the number of roots is 10.16.

[0029] (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 91.67%.

[0030] (6) The lighting culture conditions are as follows: light intensity of 1500 lx, photoperiod of 12 h light / 12 h dark, culture temperature of 25 °C, and relative air humidity of 50% to 60%.

[0031] Example 2: Tissue culture method of "golden yellow" rhizomes (1) Selection and disinfection of explants: The robust rhizomes of the "golden yellow" double-petal daylily variety were selected as explant materials, cut into 1 cm ± small segments, first soaked in soapy water for 15 minutes, then rinsed with running water for 1 hour to clean the surface mud and sand, rinsed twice with sterile water under a clean bench, soaked in 75% ethanol for 30 seconds, rinsed twice with sterile water, and finally soaked in 2% sodium hypochlorite for 10 minutes for disinfection. MS + sucrose 30 g / L + agar 7.0 g / L was used as the basic culture medium, 3 explants were inoculated in each bottle, and 10 bottles were inoculated. After 28 days of light culture, the contamination rate was 10.42% and the activation rate was 90.32%.

[0032] (2) Induction culture of adventitious buds: After disinfection, the healthy rhizomes were cut into 1 / 2 longitudinal sections under sterile conditions and quickly transferred to MS solution containing 0.4~0.6 mg / L 6-BA and 0.08~0.12 mg / L NAA for 30 minutes. The solution temperature was controlled at around 20°C, and then inoculated upright onto the induction medium to induce adventitious buds. The induction medium formula was: MS+6-BA 2.0 mg / L+NAA 0.2 mg / L+TDZ 0.1 mg / L+glycine 100 mg / L+vitamin C 50 mg / L+inositol 20 mg / L+sucrose 30 g / L+agar 7.0 g / L. Two explants were inoculated in each bottle, and 10 bottles were inoculated. The culture was counted after 28 days of light culture. The light culture conditions were as follows: on the 1st to 6th day, the light intensity was 1000 lx, the photoperiod was 12 hours of light / 12 hours of darkness, the culture temperature was 22°C, and the relative humidity of the air was 70% to 80%; on the 7th to 14th day, the light intensity was 1000 lx, the photoperiod was 13 hours of light / 11 hours of darkness, the temperature was raised to 25°C during the 4th to 10th hour of light, and the culture temperature was maintained at 22°C for the rest of the time, and the relative humidity was 70% to 80%. The relative humidity was 70%-80%; on the 15th to 21st day, the light intensity was 1500 lx, the photoperiod was 14 hours light / 10 hours dark, the culture temperature was 24°C during light, 20°C during dark, and the relative humidity was 60%-70%; on the 22nd to 28th day, the light intensity was 2000 lx, the photoperiod was 14 hours light / 10 hours dark, the culture temperature was 25°C during light, 20°C during dark, and the relative humidity was 60%-70%. The induction rate of adventitious buds was 96.24% and the number of adventitious buds was 5.81.

[0033] (3) Proliferation culture of adventitious buds: Inoculate the adventitious buds with strong growth, relatively uniform growth, plant height of 2 cm ±, and 2 to 3 leaves in the form of single plants or clusters on the proliferation medium. The formula of the proliferation medium is: MS + 6-BA 1.0 mg / L + NAA 0.1 mg / L + TDZ 0.005 mg / L + proline 80 mg / L + niacin 30 mg / L + sucrose 30 g / L + agar 7.0 g / L. Two strains were inoculated in each bottle, and 10 bottles were inoculated. The data were counted after 28 days of light culture. The light culture conditions were as follows: on the 1st to 6th day, the light intensity was 2500lx, the photoperiod was 16 hours of light / 8 hours of darkness, the culture temperature was 27℃ during light, 22℃ during darkness, and the relative humidity was 60% to 70%; on the 7th to 28th day, the light intensity was 2500lx, the photoperiod was 16 hours of light / 8 hours of darkness, the culture temperature was 27℃ during light and the temperature was raised to 28℃ two hours before the end of light each day, the culture temperature was 22℃ during darkness, and the relative humidity was 60% to 70%. The proliferation coefficient was 8.68, and 93.13% of the heights reached 2 cm.

[0034] (4) Rooting culture of adventitious buds: When the adventitious buds grow to a height of 2 cm to 3 cm and have 2 to 3 leaves, they are divided into individual explants and inoculated into rooting medium (with activated carbon added to the medium) to induce rooting. 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 were inoculated in each bottle, and 10 bottles were inoculated. After 28 days of light culture, the data were counted. The light culture conditions were as follows: on the 1st to 14th day, the light intensity was 1500 lx, the photoperiod was 14 hours of light / 10 hours of darkness, the culture temperature was 24°C during light, 20°C during darkness, and the relative humidity was 50% to 60%; on the 15th to 28th day, the photoperiod was 14 hours of light / 10 hours of darkness, the light intensity was 1500 lx, the culture temperature was 26°C in the first 6 hours and the last 6 hours of light each day, the light intensity was 1000 lx, the culture temperature was 24°C in the middle 2 hours, the culture temperature was 20°C in darkness, and the relative humidity was 50% to 60%. The rooting rate was 96.67%, and the number of roots was 15.23.

[0035] (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 5 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 97.33%.

[0036] (6) The lighting culture conditions are as follows: light intensity is 1500 lx~2000 lx, the photoperiod is 12 hours light / 12 hours dark, the culture temperature is (25±2)°C, and the relative humidity of the air is 40%~60%.

[0037] Comparative Example 1: Effects of different disinfection times on explant contamination and priming The robust rhizomes of the double-petaled daylily variety "Cream Roll" were selected as explant materials, cut into 1 cm ± small segments, and disinfected with 75% alcohol and 2% sodium hypochlorite for different time combinations. MS + sucrose 30 g / L + agar 7.0 g / L was used as the basic culture medium, and 3 explants were inoculated in each bottle for culture. The results are shown in Table 1.

[0038] Table 1 Effects of different disinfection treatments on rhizome contamination rate and initiation rate Note: Lowercase letters indicate significant differences (P<0.05), the same below.

[0039] As shown in Table 1, different disinfection times have significant effects on the contamination and activation of explants. With the increase of sterilization time, the contamination rate and activation rate of explants show a downward trend, and the decrease is greater with the increase of alcohol disinfection time, indicating that the combination of the two disinfectants can effectively remove the surface bacteria of explants. The disinfection effect of 75% alcohol is significantly stronger than that of 2% sodium hypochlorite, but its damage to explants is also more serious. For rhizomes, when alcohol disinfection is 30s and sodium hypochlorite disinfection is 10min, the contamination rate of rhizomes is low, which is 13.54%, and the activation rate reaches about 80%. Further increasing the time of sodium hypochlorite, although the contamination rate reaches 10.42%, the activation rate drops to 67.43%. Therefore, the disinfection effect of rhizomes is best in treatment 8, that is, after disinfection with 75% alcohol for 30s, it is disinfected with 2% sodium hypochlorite for 10min.

[0040] Comparative Example 2: Effects of different induction medium compositions and ratios on adventitious bud induction The robust rhizomes of the double-petaled daylily variety "Cream Roll" were selected as explant materials, cut into 1 cm ± small sections, and disinfected by the disinfection method determined in Comparative Example 1. The disinfected robust rhizomes were cut into 1 / 2 longitudinal sections under sterile conditions and quickly transferred to an MS solution containing 0.4~0.6 mg / L 6-BA and 0.08~0.12 mg / L NAA and immersed for 30 minutes. The solution temperature was controlled at about 20°C, and then inoculated in an upright manner on an induction medium to induce adventitious buds. The cells were cultured under light for 28 days. The composition, ratio and induction results of the induction medium are shown in Table 2.

[0041] Table 2 Effects of different combinations and ratios of plant growth regulators on adventitious bud induction from rhizomes As can be seen from Table 2, there is no significant difference in the number of adventitious buds in the four schemes of combined use of 6-BA and NAA, the induction rate of adventitious buds is all below 70%, and the number of adventitious buds is difficult to exceed 2. There is no obvious regularity in the effect of different concentrations on the induction rate and number of adventitious buds. When the ratio of the two is 10:1, the induction rate and the number of adventitious buds are relatively high. After adding a lower concentration of TDZ, the induction rate and number of adventitious buds are significantly improved, and show an upward trend with the increase of TDZ concentration. When the TDZ concentration is 0.1 mg / L, the induction rate is relatively high, which is 83.3%, and the number of adventitious buds is also large, which is 4.62. The leaf color is dark green and the height reaches 3 cm ( Figure 2 A); Further increasing the TDZ concentration, although there was no significant difference in the induction rate and number of adventitious buds, it was more likely to produce adventitious buds through callus differentiation, and the growth was relatively poor, with a height of only about 1 cm ( Figure 2B). Therefore, the optimal medium for rhizome induction is MS+6-BA 2.0 mg / L+NAA 0.2 mg / L+TDZ 0.1 mg / L.

[0042] Comparative Example 3: Effects of different treatments on adventitious bud induction The robust rhizomes of the double-petal daylily variety "Cream Roll" were selected as explant materials, cut into 1 cm ± small sections, disinfected by the disinfection method determined in Comparative Example 1, and then inoculated into the induction medium determined in Comparative Example 2 in different treatment methods for light culture for 28 days. The different treatment methods and induction results are shown in Table 3; wherein, the cross-section upright position is to directly inoculate the disinfected rhizomes in an upright manner into the induction medium; the 1 / 2 longitudinal section upright position A after cross-section is to directly inoculate the disinfected rhizomes in an upright manner after 1 / 2 longitudinal section under sterile conditions; the 1 / 2 longitudinal section upright position B after cross-section is to quickly transfer the disinfected rhizomes to the MS containing 0.4~0.6 mg / L 6-BA and 0.08~0.12 mg / L NAA after 1 / 2 longitudinal section under sterile conditions. Soak in the solution for 30 minutes, control the solution temperature at about 20℃, and then inoculate it into the induction culture medium in an upright manner; after cross-cutting, cut 1 / 4 vertically and place it upright 2. Cut the disinfected rhizome 1 / 4 vertically under sterile conditions and quickly transfer it to MS solution containing 0.4~0.6mg / L 6-BA and 0.08~0.12mg / L NAA and soak it for 30 minutes, control the solution temperature at about 20℃, and then inoculate it into the induction culture medium in an upright manner.

[0043] Table 3 Effects of different treatments on adventitious bud induction from rhizomes As shown in Table 3, different treatments had significant effects on the induction rate and number of adventitious buds of rhizomes. With the increase of the number of longitudinal cuts, the induction rate of adventitious buds decreased, while the number of adventitious buds increased. When the cross-section was placed upright, the induction rate of adventitious buds was significantly higher than that of the other three treatments, reaching more than 90%. The leaves were dark green and thick, but a rootless seedling was directly formed through tissue repair, and the number of adventitious buds was significantly lower than that of the other three treatments ( Figure 3 A). When the 1 / 2 longitudinal section after cross-section was placed upright A, the induction rate and number of adventitious buds were significantly lower than those when the 1 / 2 longitudinal section after cross-section was placed upright B, and the adventitious buds obtained had weaker growth potential than those when the cross-section was placed upright ( Figure 3 B). There was a significant difference in the induction rate of adventitious buds between the two treatments of 1 / 2 vertical section after cross section B and 1 / 4 vertical section after cross section B, but no significant difference in the number of adventitious buds. When 1 / 2 vertical section after cross section B was placed vertically, the adventitious buds formed had dark green leaves and thick leaves ( Figure 3C), the number of adventitious buds reached 4.62; after cross-section, the number of adventitious buds reached 4.68 when the 1 / 4 longitudinal section was placed upright, but the leaves of the adventitious buds formed were slender and the growth was weak ( Figure 3 D). Therefore, the best way to handle the rhizome is to cut it horizontally and then cut 1 / 2 vertically and place it upright at B.

[0044] Comparative Example 4: Effects of different proliferation medium compositions and ratios on adventitious bud proliferation The robust rhizomes of the double-petaled daylily variety "Cream Roll" were selected as explant materials. After being disinfected by the disinfection method determined in Comparative Example 1, they were inoculated into the induction medium determined in Comparative Example 2 in the form of cross-section and 1 / 2 longitudinal section placed upright B. After culturing for 28 days, they were transferred to proliferation medium of different compositions and proportions in the form of single buds or clustered buds for proliferation culture. The composition, proportion and proliferation results of the proliferation medium are shown in Table 4.

[0045] Table 4 Effects of different combinations and ratios of plant growth regulators on adventitious bud proliferation As shown in Table 4, adventitious buds were transferred to the proliferation medium in the form of single buds or clustered buds and gradually produced new axillary buds after one week of proliferation culture. The proliferation coefficient was between 1.23 and 8.55, and the proportion of plant heights above 2 cm was between 54.43% and 81.23%. In the four schemes of combining 6-BA and NAA, the proliferation coefficient was difficult to exceed 2.0, and the proportion of plant heights above 2 cm was below 70%. After adding a certain concentration of TDZ, the proliferation coefficient and plant height were significantly improved. When the TDZ concentration was 0.005 mg / L, the proliferation coefficient could reach 6.80, the percentage of plant heights above 2 cm exceeded 80%, the adventitious buds developed normally, the leaves were dark green, and the growth was vigorous ( Figure 4 A); Continuing to increase the TDZ concentration, the proliferation coefficient increased significantly, the percentage of plants with a height higher than 2 cm decreased significantly, and callus tissue was easily formed at the base, and clustered buds continued to differentiate on the callus tissue, which was not conducive to subsequent cutting operations ( Figure 4 B), therefore, the optimal proliferation medium is MS+6-BA 1.0 mg / L+NAA 0.1 mg / L+TDZ 0.005 mg / L.

[0046] Comparative Example 5: Effects of different light conditions on adventitious bud induction The robust rhizomes of the "golden yellow" double-petal daylily variety were selected as explant materials. Based on Example 2, the effects of different light culture conditions on adventitious bud induction in step (2) were explored. Among them, light condition 1: light intensity of 1500lx, photoperiod of 12 hours light / 12 hours dark, culture temperature of 25°C, relative air humidity of 50%-60%, culture for 28 days; light condition 2: from 1st to 14th day, light intensity of 1000lx, photoperiod of 12 hours light / 12 hours dark, culture temperature of 22°C, relative air humidity of 70%-80%; from 15th to 28th day, light intensity of 1500lx, photoperiod of 14 hours light / 10 hours dark, culture temperature of 24°C during light, 20°C during dark, relative air humidity of 60%-70%; light condition 3: from 1st to 6th day, light intensity of 1000lx, photoperiod of 12 hours light / 12 hours dark, culture temperature of 22℃, relative humidity of air 70%~80%; on the 7th to 14th day, the light intensity was 1000lx, the photoperiod was 13 hours light / 11 hours dark, the temperature was raised to 25℃ during the 4th to 10th hour of light, and the culture temperature was kept at 22℃ for the rest of the time, and the relative humidity of air was 70%~80%; on the 15th to 21st day, the light intensity was 1500lx, the photoperiod was 14 hours light / 10 hours dark, the culture temperature was 24℃ during light, the culture temperature was 20℃ during darkness, and the relative humidity of air was 60%~70%; on the 22nd to 28th day, the light intensity was 2000lx, the photoperiod was 14 hours light / 10 hours dark, the culture temperature was 25℃ during light, the culture temperature was 20℃ during darkness, and the relative humidity of air was 60%~70%. The different light conditions and induction results are shown in Table 5.

[0047] Table 5 Effects of different light conditions on adventitious bud induction from rhizomes As shown in Table 5, different light conditions have significant effects on the induction rate and number of adventitious buds of rhizomes. Under light condition 3, the induction rate and number of adventitious buds are the highest. This may be because on the 1st to 6th day of light condition 3, the light intensity of 1000lx, the photoperiod of 12 hours light / 12 hours dark, the culture temperature of 22℃ and the relative humidity of 70%~80% provided a relatively mild and stable initial environment for the explants, which helped the explants to adapt smoothly to the in vitro culture conditions and reduce the stress response caused by changes in the external environment, so that the cells could carry out physiological activities in an orderly manner and prepare for subsequent differentiation and induction; on the 7th to 14th day, the photoperiod was adjusted to 13 hours light / 11 hours dark, and the temperature was raised to 25℃ during the 4th to 10th hour of light, and maintained at 22℃ for the rest of the time. This short-term increase in temperature can simulate the day and night temperature difference in the natural environment, stimulate the metabolic activity of rhizome cells, enhance their physiological activity, and promote the initiation of rhizome cells and the formation of tissue repair; on the 15th to 21st day, the light intensity was increased to 1500lx and the photoperiod was changed to 14 hours light / 11 hours dark. 10 hours of darkness, 24℃ in light and 20℃ in darkness. Higher light intensity and appropriate temperature difference enhance the efficiency of photosynthesis and make the metabolic activities in cells more active. A large amount of nutrients are synthesized and accumulated, providing sufficient energy and material support for the formation of adventitious buds. On the 22nd to 28th day, the light intensity is further increased to 2000lx, the temperature is stabilized at 25℃ in light and 20℃ in darkness. High-intensity light greatly promotes photosynthesis in the later stage of culture, so that a large amount of photosynthetic products accumulate in the cells. These rich substances provide a solid guarantee for the induction of a large number of adventitious buds, making the induction rate and number of adventitious buds under light condition 3 significantly higher than those under other conditions.

[0048] Comparative Example 6: Effects of different light conditions on adventitious bud proliferation The robust rhizomes of the "golden" double-petal daylily variety were selected as explant materials. Based on Example 2, the effects of different light culture conditions on the proliferation of adventitious buds in step (3) were explored. Among them, light condition 1: light intensity of 1500lx, photoperiod of 12 hours light / 12 hours dark, culture temperature of 25°C, relative air humidity of 50%-60%, culture for 28 days; light condition 4: light intensity of 2500lx, photoperiod of 16 hours light / 8 hours dark, culture temperature of 27°C during light and 22°C during dark, relative air humidity of 60%-70%, culture for 28 days; light condition 5: on the 1st to 6th day, The light intensity was 2500 lx, the photoperiod was 16 hours of light / 8 hours of darkness, the culture temperature was 27°C during light, 22°C during darkness, and the relative humidity was 60% to 70%; on the 7th to 28th day, the light intensity was 2500 lx, the photoperiod was 16 hours of light / 8 hours of darkness, the culture temperature was 27°C during light and the temperature was raised to 28°C two hours before the end of light each day, the culture temperature was 22°C during darkness, and the relative humidity was 60% to 70%. The different light conditions and proliferation results are shown in Table 6.

[0049] Table 6 Effects of different light conditions on adventitious bud proliferation As shown in Table 6, different light conditions have a significant effect on the proliferation of adventitious buds. Under light condition 5, the proliferation coefficient of adventitious buds and the percentage of plant height above 2 cm are the highest. This may be because on the 1st to 6th day of light condition 5, the high-intensity light of 2500lx, combined with the temperature of 27℃ in light, 22℃ in darkness and 60%~70% relative humidity of the air, created an active physiological environment for the adventitious buds, enhanced the photosynthesis and respiration of the cells, promoted the rapid division and differentiation of cells, and reserved sufficient energy and material basis for subsequent proliferation; on the 7th to 28th day, while maintaining high-intensity light and a specific light cycle, the temperature was raised from 27℃ to 28℃ two hours before the end of the light every day. The additional temperature change can further stimulate the physiological activity of the cells, enhance the activity of enzymes involved in photosynthesis and material synthesis in the cells, accelerate the accumulation of photosynthesis products, and enable the adventitious buds to obtain more sufficient nutrient supply during the proliferation process, thereby significantly improving the proliferation coefficient. In addition, the accumulation of more nutrients also contributes to the elongation growth of the adventitious buds, which greatly increases the percentage of plants with a height of more than 2cm, indicating that light condition 5 can effectively promote the proliferation and strong growth of adventitious buds.

[0050] Comparative Example 7: Effects of different light conditions on adventitious bud proliferation The robust rhizomes of the "golden" double-petal daylily variety were selected as explant materials. Based on Example 2, the effects of different light culture conditions on the rooting of adventitious buds in step (4) were explored. Among them, light condition 1: light intensity of 1500lx, photoperiod of 12 hours light / 12 hours dark, culture temperature of 25°C, relative air humidity of 50%-60%, and culture for 28 days; light condition 6: light intensity of 1500lx, photoperiod of 14 hours light / 10 hours dark, culture temperature of 24°C during light and 20°C during dark, relative air humidity of 50%-60%, and culture for 28 days; light condition 7: on the 1st to 14th day, light intensity of 1500lx, light per day of 12 hours dark, culture temperature of 25°C, relative air humidity of 50%-60%, and culture for 28 days. The cycle was 14 hours of light / 10 hours of darkness, the culture temperature was 24°C during light, 20°C during darkness, and the relative humidity was 50% to 60%; from 15 to 28 days, the photoperiod was 14 hours of light / 10 hours of darkness, the light intensity was 1500 lx and the culture temperature was 26°C in the first 6 hours and last 6 hours of light each day, the light intensity was 1000 lx and the culture temperature was 24°C in the middle 2 hours, the culture temperature was 20°C in darkness, and the relative humidity was 50% to 60%. The different light conditions and rooting results are shown in Table 7.

[0051] Table 7 Effects of different light conditions on rooting of adventitious buds As shown in Table 7, different light conditions have a significant effect on the rooting of adventitious buds. Under light condition 7, the rooting rate and number of adventitious buds are the highest. This may be because on the 1st to 14th day of light condition 7, the light intensity of 1500lx, the photoperiod of 14 hours of light / 10 hours of darkness, and the temperature environment of 24℃ in light and 20℃ in darkness provide a relatively stable and suitable growth environment for adventitious buds. The 14-hour light duration ensures that the adventitious buds have sufficient time to photosynthesize and synthesize the organic matter and energy required for rooting. The appropriate temperature difference helps to maintain the efficient progress of a series of physiological and biochemical reactions in the cells, promotes the expression of root-related genes and cell differentiation, and lays the foundation for the formation of the root system; on the 15th to 28th day, the light intensity of 1500lx and the culture temperature of 26℃ in the first 6 hours and the last 6 hours of light each day. The cultivation temperature enhances the efficiency of photosynthesis, allowing the adventitious buds to accumulate more photosynthetic products. The higher temperature also increases the activity of intracellular enzymes, accelerates material metabolism and energy conversion, and provides sufficient material and energy supply for the rooting process. The light intensity of 1000lx and the culture temperature of 24℃ in the middle 2 hours, the relatively mild environment help the adventitious buds balance their own physiological state and avoid damage to the cells caused by long-term high-intensity light and high temperature. This phased and variable light and temperature fluctuation can simulate the dynamic changes of light and temperature in the natural environment, effectively stimulate the growth of the root system, make it more robust and developed, thereby significantly increasing the rooting rate and number of roots under light conditions of 7.

[0052] The number of equipment and processing scales described here are used to simplify the description of the present invention. Applications, modifications and variations of the tissue culture method of the double-petaled daylily rhizome of the present invention will be obvious to those skilled in the art.

[0053] 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 tissue culture method for double-petal daylily rhizomes, characterized in that: include: Step 1: Select a robust rhizome of a double-petaled daylily variety as an explant, cut it into small sections, and clean and disinfect it; Step 2: After the explants are sterilized, they are cut longitudinally under sterile conditions and quickly transferred to a MS solution containing 0.4-0.6 mg / L 6-BA and 0.08-0.12 mg / L NAA, and then immersed for 30 minutes. The temperature of the solution is controlled at about 20°C, and then inoculated upright on an induction medium to induce adventitious buds, and cultured under light for 26-30 days. The induction medium includes MS+6-BA 2.0 mg / L+NAA 0.2 mg / L+TDZ 0.1 mg / L-0.2 mg / L; Step 3, inoculating the adventitious buds in the form of single plants or clustered buds on a proliferation medium, and culturing under light for 26 to 30 days, wherein 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 4: 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 5: After hardening the rooted seedlings, take them out from the rooting medium and transplant them into a transplanting medium to obtain double-petal daylily plants.

2. The tissue culture method of double-petal daylily rhizomes according to claim 1, characterized in that: The disinfection is to disinfect the cleaned explants with 75% alcohol for 10s to 30s, and then disinfect with 2% sodium hypochlorite for 5min to 15min.

3. The tissue culture method of double-petal daylily rhizomes according to claim 2, characterized in that: The induction medium includes MS+6-BA 2.0 mg / L+NAA 0.2 mg / L+TDZ 0.1 mg / L+glycine 100 mg / L+vitamin C 50 mg / L+inositol 20 mg / L.

4. The tissue culture method of double-petal daylily rhizomes according to claim 3, characterized in that: The proliferation medium includes MS+6-BA 1.0 mg / L+NAA 0.1 mg / L+TDZ 0.005 mg / L+proline 80 mg / L+niacin 30 mg / L.

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

6. The tissue culture method of double-petal daylily rhizomes according to claim 5, characterized in that: In step 2, the first illumination culture condition is used for illumination culture, wherein the first illumination culture condition is: on the 1st to 6th day, the light intensity is 1000 lx, the photoperiod is 12 hours of light / 12 hours of darkness, the culture temperature is 22°C, and the relative humidity of the air is 70% to 80%; on the 7th to 14th day, the light intensity is 1000 lx, the photoperiod is 13 hours of light / 11 hours of darkness, the temperature is raised to 25°C during the 4th to 10th hour of illumination, and the culture temperature is maintained at 22°C for the rest of the time, and the air relative humidity is 70% to 80%. The relative humidity was 70%-80%; on the 15th-21st day, the light intensity was 1500lx, the photoperiod was 14 hours light / 10 hours dark, the culture temperature was 24℃ in light, 20℃ in dark, and the relative humidity of the air was 60%-70%; on the 22nd-28th day, the light intensity was 2000lx, the photoperiod was 14 hours light / 10 hours dark, the culture temperature was 25℃ in light, 20℃ in dark, and the relative humidity of the air was 60%-70%.

7. The tissue culture method of double-petal daylily rhizomes according to claim 5, characterized in that: In step three, the second illumination culture condition is adopted for illumination culture, and the second illumination culture condition is: on the 1st to 6th day, the light intensity is 2500lx, the photoperiod is 16 hours of light / 8 hours of darkness, the culture temperature is 27°C during illumination, the culture temperature is 22°C during darkness, and the relative humidity of the air is 60% to 70%; on the 7th to 28th day, the light intensity is 2500lx, the photoperiod is 16 hours of light / 8 hours of darkness, the culture temperature is 27°C during illumination and the temperature is raised to 28°C two hours before the end of the light each day, the culture temperature is 22°C in darkness, and the relative humidity of the air is 60% to 70%.

8. The tissue culture method of double-petal daylily rhizomes according to claim 5, characterized in that: In step 4, the third illumination culture condition is adopted for illumination culture, and the third illumination culture condition is: on the 1st to 14th day, the light intensity is 1500lx, the photoperiod is 14 hours light / 10 hours dark, the culture temperature is 24°C during illumination, the culture temperature is 20°C during darkness, and the relative humidity of the air is 50% to 60%; on the 15th to 28th day, the photoperiod is 14 hours light / 10 hours dark, in the first 6 hours and the last 6 hours of illumination each day, the light intensity is 1500lx, the culture temperature is 26°C, the light intensity is 1000lx for the middle 2 hours, the culture temperature is 24°C, the culture temperature is 20°C in darkness, and the relative humidity of the air is 50% to 60%.

9. The tissue culture method of double-petal daylily rhizomes according to claim 1, characterized in that: The sterilized explant is inoculated into the induction medium in a manner of cross-cutting and half-cutting vertically and then being placed upright to induce adventitious buds.

10. The tissue culture method of double-petal daylily rhizomes 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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