Tissue culture breeding method for stem tips of passiflora edulis
By establishing a sterile system for Passiflora daphne through tissue culture technology, the problems of difficult explant disinfection and endophytic bacteria contamination were solved, rapid germination of axillary buds, rapid proliferation of young shoots and rich root system were achieved, the regeneration cycle was successfully shortened, and technical support for germplasm preservation and utilization was provided.
Patent Information
- Application Number
- CN202511013224.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-23
- Publication Date
- 2025-09-12
AI Technical Summary
In the existing technology, the tissue culture process of Passiflora plants often faces problems such as difficulty in explant disinfection, browning and endophyte contamination. The lack of systematic research makes it difficult to propagate Passiflora odorifera through tissue culture.
Tissue culture technology was used to establish a sterile system for Passiflora daphne through various experimental methods. Ordinary MS medium was used to induce axillary bud germination, and modified MS medium was used to promote cluster bud formation. Rejuvenated and regenerated plants were obtained through root induction medium, including explant pretreatment, stem tip axillary bud induction, bud subculture and rooting induction.
The rapid germination of axillary buds of the explants of Passiflora daphne was achieved, with a germination rate of over 81% and an average germination coefficient of over 3.0. The young shoots proliferated and elongated rapidly, the root system was abundant, and the regeneration cycle was shortened, providing technical support for germplasm preservation and utilization.
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Abstract
Description
Technical Field
[0001] The invention relates to the fields of plant propagation and cultivation and agricultural biotechnology, and in particular to a tissue culture propagation method for the stem tip of passionflower. Background Art
[0002] Passiflora Passifloraceae Juss.exRoussel), including the genus Passiflora ( Passiflora Linnaeu) and Capsules ( Adenia Forssk.). Passiflora is the most representative genus in the family, containing over 520 species, of which over 60 are edible and the rest are ornamental vines. Passiflora plants have become research targets in the fields of cosmetics, medicine, and food due to their ornamental flowers and edible fruits. There are 20 species of Passiflora distributed naturally in my country, of which 13 are wild species and the rest are introduced and cultivated species. Passiflora 'Soi Fah') is an ornamental herbaceous vine of the genus Passiflora introduced into my country. Passiflora incarnata × Passiflora laurifolia This hybrid of a plant native to India has a cylindrical, smooth, glabrous, purple-green main stem with purple tendrils. Its leaves are leathery, palmately three-lobed, dark green with serrated margins and two glands near the leaf base. It blooms year-round, with a predominantly purple-blue corolla and flowers 6 cm in diameter. The filaments are white at the tips and bases, the petals and calyx are mottled purple-blue, and the secondary corona is white and bluish-purple. The styles are three and face downward. Passiflora daphne has a rich aroma, strong resistance to stress, and vigorous growth. It has attracted significant attention abroad as an ornamental species for gardens, green spaces, and parks, but its research and application in my country has been limited.
[0003] Currently, there are few reports on systematic research on tissue culture propagation of Passiflora daphne. The wild species of Passiflora worldwide are facing environmental destruction and threats to their germplasm preservation. Tissue culture propagation technology is an efficient, rapid, and germplasm-preserving propagation method. If it can be successfully applied to Passiflora daphne, it will open up new avenues for its germplasm preservation and utilization. However, during the tissue culture process of Passiflora plants, problems such as difficulty in explant disinfection, browning, and endophyte contamination are often encountered. These problems may also exist in the tissue culture propagation of Passiflora daphne. Due to the lack of relevant systematic research, how to overcome these obstacles has become a problem that needs to be solved urgently. Summary of the Invention
[0004] The purpose of the present invention is to establish a sterile system for Passiflora daphne by utilizing tissue culture technology through a variety of experimental methods, successfully induce axillary bud germination in a common MS culture medium, promote cluster bud formation in a modified MS culture medium, and obtain regenerated plants with improved rejuvenation level through tissue culture rooting induction, thereby providing resource guarantee for Passiflora daphne seedling cultivation and germplasm innovation.
[0005] In view of the above-mentioned defects of the prior art, the present invention proposes a tissue culture propagation method of the stem tip of Passiflora daphne to solve the problems raised by the above-mentioned background technology.
[0006] In order to achieve the above object, the present invention provides the following technical solutions: A tissue culture propagation method for the stem tip of Passiflora daphne comprises the following steps: (1) Explant pretreatment: Cut healthy, vigorously growing shoots at the front end of 4 cm in length, cut off the tendrils, leaves and petioles, soak them in neutral detergent, place them on clean gauze and rinse them with running water until there is no foam, gently wipe the surface of the explant stem with a moist cotton swab, place them in a tissue culture bottle to keep them moist, and place them at 4℃ for 3 h~4 h. The explants have been pretreated and are ready for use. (2) Explant sterilization: Cut the pretreated explants obtained in step (1) to a length of 2 cm to 3 cm, soak them in 75% ethanol for 30 s, then rinse them once with sterile water; sterilize them with mercuric chloride solution, and finally rinse them five times with sterile water to obtain sterile explants; (3) Shoot tip axillary bud induction culture stage: Cut the sterile explants obtained in step (2) to a length of 5 mm to 10 mm, inoculate them upright according to the growth polarity, and after culturing for 10 to 15 days, take the new shoot tips and inoculate them into the stem tip axillary bud induction medium to obtain axillary buds; The shoot tip axillary bud induction medium is based on ordinary MS medium, and is supplemented with 1.0 mg / L to 2.0 mg / L 6-BA and 2000× to 3000× BEB ganoderma lucidum. (4) Bud subculture stage: The axillary buds obtained in step (3) are cultured in a bud subculture medium for 25 to 30 days to obtain secondary buds; The bud subculture medium is based on a modified MS medium and is supplemented with 1.5 mg / L to 2.5 mg / L 6-BA and 2300× to 2700× BEB ganoderma lucidum. (5) Rooting induction culture stage: the clustered buds obtained in step (4) are cut into multiple single buds, transferred to the rooting induction medium and cultured for 12 days to 25 days to obtain rooted seedlings of Passiflora odorifera; The root induction medium is based on a modified MS medium and is supplemented with 0.2 mg / L to 1.2 mg / L NAA and 0.5 mg / L to 1.5 mg / L IAA.
[0007] Preferably, the neutral detergent in step (1) is dishwashing liquid, and the soaking time is 15 min to 20 min.
[0008] Preferably, the mass concentration of the mercuric chloride aqueous solution in step (2) is 0.15% to 0.6%.
[0009] Preferably, the disinfection time in step (2) is 5 min to 11 min.
[0010] Preferably, the shoot tip axillary bud induction medium in step (3) is based on ordinary MS medium, supplemented with 2.0 mg / L 6-BA and 2000×BEB Ganoderma lucidum.
[0011] Preferably, the bud subculture culture medium in step (4) is based on a modified MS medium supplemented with 1.5 mg / L 6-BA and 2500×BEB Ganoderma lucidum.
[0012] Preferably, the concentrations of trace elements in the modified MS medium in step (4) and step (5) are MnSO4·4H2O44.6 mg / L, ZnSO4·7H2O 17.2 mg / L, CuSO4·5H2O 0.05 mg / L, H3BO3 12.4 mg / L, KI 1.66 mg / L, CoCl2·6H2O 0.05 mg / L and Na2MoO4·2H2O 0.5 mg / L; the concentrations of iron salts are FeSO4·7H2O 13.9 mg / L, Na2-EDTA 18.65 mg / L; the concentrations of organic matter are C2H5NO2 1.0 mg / L, C12H 17 ClN4OS·HCl 0.05 mg / L, C8H 12 ClNO30.25 mg / L, C6H5NO20.25 mg / L, C6H 12 O650 mg / L, and the concentrations of other nutrients remained unchanged.
[0013] Preferably, the concentrations of macroelements in the common MS medium are KNO3 1900 mg / L, NH4NO3 1650 mg / L, MgSO4·7H2O 370 mg / L, KH2PO4 170 mg / L, and CaCl2·2H2O 440 mg / L; the concentrations of trace elements are MnSO4·4H2O 22.3 mg / L, ZnSO4·7H2O 8.6 mg / L, CuSO4·5H2O 0.025 mg / L, H3BO3 6.2 mg / L, KI 0.83 mg / L, CoCl2·6H2O 0.025 mg / L, and Na2MoO4·2H2O 0.25 mg / L; the concentrations of iron salts are FeSO4·7H2O 27.8 mg / L and Na2-EDTA 37.3 mg / L; the concentration of organic matter is C2H5NO2 2.0 mg / L,C 12 H 17 ClN4OS·HCl (VB1) 0.1 mg / L, C8H 12 ClNO3 (VB6) 0.5 mg / L, C6H5NO2 (VB3) 0.5 mg / L, C6H 12 O6100 mg / L. 30 g / L sucrose and 6.5 g / L agar powder need to be added to adjust the pH to 6.0.
[0014] Preferably, the root induction medium in step (5) is a modified MS medium as the base medium, and is supplemented with 1.2 mg / L NAA + 0.5 mg / L IAA.
[0015] Preferably, the culture conditions in steps (3) to (5) are a temperature of 28±0.5°C, a light intensity of 2500 lx~3200 lx, and a photoperiod of 16 h / d~18 h / d.
[0016] Preferably, the length of the single bud in step (5) is ≥2.5 cm.
[0017] Compared with the prior art, the present invention has the following beneficial effects: under the conditions of a light intensity of 2500 lx-3200 lx, a photoperiod of 16 h / d, and a temperature of 28±0.5°C, by adding 1.0 mg / L-2.0 mg / L 6-BA and 2000×-3000× BEB ganoderma lucidum to a shoot tip axillary bud induction culture medium, the present invention achieves rapid germination of axillary buds of passionflower explants, a germination rate of more than 81%, and an average germination coefficient of more than 3.0. By increasing the concentration of trace elements in ordinary MS medium to 2 times the original concentration and reducing the concentration of iron salts and organic matter to 0.5 times the original concentration, a modified MS medium was prepared. After adding 1.5 mg / L-2.5 mg / L 6-BA and 2300×-2700×BEB Ganoderma lucidum extract, rapid sprout proliferation and seedling elongation were achieved in one step, with an average sprout proliferation multiple of 3.27 and an average height of 25.2 mm. The obtained single sprouts were inoculated into root induction medium. Using the above-mentioned modified medium as the basal medium, and supplemented with 0.2 mg / L-1.2 mg / L NAA and 0.5 mg / L-1.5 mg / L IAA, the tender shoots were induced to gradually form roots after 20 days, with the number of roots reaching 4.6. After 110 days of culture, the seedlings had abundant roots and maintained good growth potential. The above-mentioned rapid propagation technology only requires three culture stages to obtain tissue culture rooted seedlings, which effectively shortens the cycle of the passion fruit regeneration system and provides technical support for the large-scale preservation and production of passion fruit seedlings in a short period of time. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 The effects of different disinfection treatments on explants; Figure 2 The growth of axillary buds induced by different concentrations of 6-BA and dilution ratios of BEB ganoderma lucidum; Figure 3 Comparison of sprout development in modified MS medium (Figures A, C) and ordinary MS medium (Figures B, D) with different 6-BA concentrations and dilution ratios of BEB Ganoderma lucidum extract; Figure 4 The figure shows the effects of different basal culture media on rooting seedlings; A is ordinary MS medium and B is improved MS medium. DETAILED DESCRIPTION
[0019] In order to enable those skilled in the art to better understand the technical content of the present invention, the technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.
[0020] Example A tissue culture propagation method for the stem tip of Passiflora daphne comprises the following steps: (1) Explant pretreatment: Cut healthy, vigorously growing shoots at the front end of 4 cm in length, cut off the tendrils, leaves and petioles, soak them in detergent for 15 min to 20 min, then place them on clean gauze and rinse them with running water until there is no foam. Gently wipe the surface of the explant stem with a moist cotton swab, place them in a tissue culture bottle to keep them moist, and place them at 4 ° C for 3 h to 4 h. The explants have been pretreated and are ready for use. (2) Explant disinfection: Cut the pretreated explants obtained in step (1) to a length of 2 cm to 3 cm, soak them in 75% ethanol for 30 seconds, and then rinse them once with sterile water; then disinfect them with a 0.15% to 0.6% mercuric chloride solution for 5 minutes to 11 minutes, and finally rinse them five times with sterile water to obtain sterile explants; (3) Shoot tip axillary bud induction culture stage: Cut the sterile explants obtained in step (2) to a length of 5 mm to 10 mm, inoculate them upright according to the growth polarity, and after culturing for 10 to 15 days, take the new shoot tips and inoculate them into the stem tip axillary bud induction medium to obtain axillary buds; The shoot tip axillary bud induction medium is based on ordinary MS medium, and is supplemented with 1.0 mg / L to 2.0 mg / L 6-BA and 2000× to 3000× BEB ganoderma lucidum. (4) Bud subculture stage: The axillary buds obtained in step (3) are cultured in a bud subculture medium for 25 to 30 days to obtain secondary buds; The bud subculture medium is based on a modified MS medium and is supplemented with 1.5 mg / L to 2.5 mg / L 6-BA and 2300× to 2700× BEB ganoderma lucidum. (5) Rooting induction culture stage: The clustered buds obtained in step (4) are cut into multiple single buds with a length of more than 2.5 cm, and transferred to the rooting induction medium for culture for 12 days to 25 days to obtain the rooted seedlings of Passiflora odorifera; The root induction medium is based on a modified MS medium and is supplemented with 0.2 mg / L to 1.2 mg / L NAA and 0.5 mg / L to 1.5 mg / L IAA.
[0021] The concentrations of trace elements in the modified MS medium in steps (4) and (5) are MnSO4·4H2O 44.6 mg / L, ZnSO4·7H2O 17.2 mg / L, CuSO4·5H2O 0.05 mg / L, H3BO3 12.4 mg / L, KI 1.66 mg / L, CoCl2·6H2O 0.05 mg / L and Na2MoO4·2H2O 0.5 mg / L; the concentrations of iron salts are FeSO4·7H2O 13.9 mg / L, Na2-EDTA 18.65 mg / L; the concentrations of organic matter are C2H5NO2 1.0 mg / L, C12H 17 ClN4OS·HCl 0.05 mg / L, C8H 12 ClNO30.25 mg / L, C6H5NO20.25 mg / L, C6H 12 O650 mg / L, and the concentrations of other nutrients remained unchanged.
[0022] The culture conditions in steps (3) to (5) are as follows: a temperature of 28±0.5°C, a light intensity of 2500 lx~3200 lx, and a light cycle of 16 h / d~18 h / d.
[0023] Experimental Example 1 Different disinfection methods 1. Materials and Methods 1.1 Materials The experimental materials were obtained from the Passiflora Germplasm Resource Garden in Hainan Province (Danzhou) and the bases of the Chinese Academy of Tropical Agricultural Sciences. The new shoots of the current year's Passiflora odorifera were collected.
[0024] 1.2 Experimental methods 1.2.1 Explant pretreatment Cut new shoots with a length of 4 cm, cut off the tendrils, leaves and petioles, soak them in 0.1%~1.0% detergent solution for 15 minutes, place them on clean gauze and rinse with running water until there is no foam, gently wipe the surface of the tender stem of the explant with a moist cotton swab, and place them at 4℃ for 3 h~4 h.
[0025] 1.2.2 Explant disinfection Cut the pretreated new shoots 2 cm to 3 cm, soak them in 75% alcohol for 30 s, and rinse with sterile water; the concentrations of the designed disinfectant (HgCl2 solution) were 0.15%, 0.3%, and 0.6%, respectively, and the disinfection time was 5 min, 8 min, and 11 min. L9 (3 4 ) Orthogonal experimental design, as shown in Table 1, with rinsing with sterile water 5 times and sterile culture for 15 days.
[0026] Table 1 L9(34 ) Orthogonal experimental design
[0027] 1.3 Experimental Results The results are as follows Figure 1 As shown, as the concentration of the disinfectant HgCl₂ solution increased, the explant contamination rate exhibited a decrease-increase-decrease pattern, while the survival rate followed an increase-decrease-increase trend. The contamination rate was the same for both D3 and D4, at 7%, but the highest survival rate was achieved with D4, at 93%. The contamination rates for D6 and D7 were the same, both at 37%, the highest of all disinfection treatments. In summary, D4 demonstrated the best explant disinfection effect, with a concentration of 0.3% HgCl₂ and a disinfection time of 5 minutes being the optimal parameters for explant disinfection of Passiflora edulis.
[0028] Experimental Example 2 Screening of culture medium for induction of shoot apical axillary buds 2.1 Methods The sterilized explants were inoculated upright according to growth polarity. After culturing for 15 days, 5 mm to 10 mm new shoot tips were taken as explants and inoculated into a shoot tip axillary bud induction medium to obtain axillary buds. The culture conditions at this stage were as follows: temperature 28±0.5°C, light intensity 2500 lx to 3200 lx, and photoperiod 16 h / d to 18 h / d. The shoot tip axillary bud induction medium was based on ordinary MS medium supplemented with 1.0 mg / L to 2.0 mg / L 6-BA and 2000× to 3000× BEB ganoderin, where 2000× BEB ganoderin and 3000× BEB ganoderin indicated that the BEB ganoderin mother solution was diluted 2000 times and 3000 times with ultrapure water, respectively. An orthogonal experiment was designed, and culture media with only 2000×BEB ganoderin and 3000×BEB ganoderin added to ordinary MS culture medium were set as control culture media to observe the effects of different plant growth regulator concentration ratios on the induction of axillary buds in the stem tip of Passiflora odorifera. The effects of different culture media for axillary bud induction on the induction of axillary buds in the stem tip are shown in Table 2.
[0029] 2.2 Experimental Results As shown in Table 2 and Figure 2As shown, among nine axillary bud induction media and two control media, the germination rate of explants induced by media I2, I3, I5, and I8 exceeded 81%. Explants in these media exhibited rapid axillary bud germination and tender green leaves. Among them, medium I8 produced the greatest axillary bud length and germination coefficient, at 23.6 mm and 3.08, respectively. The main stems of axillary buds elongated significantly, with purple-green stems and green, open leaves, demonstrating vigorous growth. These values were higher than those in media I2, I3, and I5, but the axillary bud germination rate was lower than the other three. The results showed that different concentrations of 6-BA interacted with different multiples of BEB ganoderin to increase axillary bud germination rate and length. At the same 6-BA concentration, the addition of different multiples of BEB ganoderin to standard MS medium significantly increased axillary bud length (p < 0.05). At the same dilution of BEB ganoderin, the germination rate increased with decreasing 6-BA concentration (Table 2). This suggests that medium I8 is the optimal medium for inducing axillary bud germination in P. edulis explants. Axillary bud germination rate = number of buds / total number of inoculated plants.
[0030] Table 2 Effects of different shoot apex axillary bud induction media on shoot apex axillary bud induction
[0031] Note: Ordinary MS medium + 30 g / L sucrose + 6.5 g / L agar powder, pH 6.0 Experimental Example 3 Screening of bud subculture medium and the effect of improved MS medium on cluster bud growth 3.1 Experimental methods Screening of bud subculture media: 6-BA and BEB ganoderin were used as factors affecting axillary bud proliferation and elongation. Modified MS medium was used as the basal medium, supplemented with 6-BA at concentrations of 1.5 mg / L, 2.0 mg / L, and 2.5 mg / L, respectively. The BEB ganoderin stock solution was diluted with ultrapure water to different concentrations of 2300×, 2500×, and 2700×, respectively, to prepare different bud subculture media (S1-S9). Control media (S10-S11) were prepared by adding only 2000× and 3000× BEB ganoderin to the modified medium. An orthogonal experiment was designed, in which 50 buds were inoculated into each treatment, replicated three times. After 30 days of culture, the proliferation rate, average height, and clustered bud growth potential of the adventitious buds were counted and analyzed.
[0032] The effects of modified MS medium and standard MS medium on the growth of clustered shoots were compared. Standard MS medium was used as the control. Modified MS medium was obtained by halving the concentrations of FeSO₄·7H₂O, Na₂-EDTA, and organic matter in the standard MS medium and doubling the concentration of trace elements. 6-BA was added at concentrations of 1.5 mg / L, 2.0 mg / L, and 2.5 mg / L, respectively. BEB Ganoderma lucidum stock solution was diluted with ultrapure water to different concentrations of 2300×, 2500×, and 2700×, respectively, as treatments. Fifty axillary buds were inoculated in each treatment, replicated three times. Cultures were maintained under a light intensity of 2500–3200 lx for 16 h / d and a temperature of 28±0.5°C for 25 d. Bud growth patterns were then counted and analyzed.
[0033] 3.2 Experimental Results As shown in Table 3 and Figure 3 As shown in the figure, in the modified MS medium, the average bud proliferation times of S2-S4 medium were all above 3.0. Although the proliferation times in S4 medium was the highest, and the plants showed dark green shoots and slender leaves, there was no significant difference in the proliferation times between S2 medium and S4 medium, and the plants showed dark green shoots, expanded leaves, thick stems, and fast growth. With the increase of 6-BA concentration, when the concentration of BEB ganoderin was diluted by more than 2500×, the average bud proliferation times decreased, indicating that the combination of high concentration of 6-BA and low concentration of BEB ganoderin reduced the tillering ability of young shoots and the average bud proliferation times; without 6-BA, the proliferation times in S10 and S11 medium containing only BEB ganoderin were the lowest, and single buds grew and the leaves turned yellow ( Figure 3 A, C), indicating that BEB ganoderin promotes low tillering ability of young shoots. Low concentration of 6-BA interacts with high concentration of BEB ganoderin to promote proliferation and height increase. In ordinary MS medium, when 6-BA and BEB ganoderin concentration ratio are the same, most of the clustered shoots show slow plant growth, yellowing leaves, and even death ( Figure 3 B, D); This indicates that during the growth and development of Passiflora daphne, its demand for trace elements is higher than normal, while its demand for iron salts and organic matter is lower than normal.
[0034] Table 3 Effects of different bud subculture media on the proliferation of Passiflora odorifera buds
[0035] Experimental Example 4 Screening of root induction medium and effect of improved MS medium on root growth 4.1 Experimental methods Screening of rooting induction media: Nine rooting induction media were prepared using modified MS medium as the base medium and NAA (0.2 mg / L-1.2 mg / L) and IAA (0.5 mg / L-1.5 mg / L) as rooting induction factors. Subcultured shoots were cut into single shoots ≥2.5 cm in height and inoculated into the rooting induction media. Rooting was induced for 12-25 days under the following conditions: a temperature of 28±0.5°C, a light intensity of 2500-3200 lx, and a photoperiod of 16-18 h / d. Rooting induction media were then analyzed and analyzed to identify the optimal medium for rooting young shoots of Passiflora daphne. The following is a screening of different NAA and IAA concentrations in the rooting induction media.
[0036] The effects of modified MS medium and standard MS medium on the growth of clustered shoots were compared. Standard MS medium was used as the basal medium as a control. Modified MS medium was obtained by halving the concentrations of FeSO₄·7H₂O, Na₂-EDTA, and organic matter, while doubling the concentration of trace elements. NAA and IAA were added at concentrations of 1.2 mg / L and 1.5 mg / L, respectively. Fifty axillary buds were inoculated in each treatment, replicated three times. Bud growth patterns were counted and analyzed.
[0037] 4.2 Experimental Results As shown in Table 4, Table 5 and Figure 4 As shown in the results, among the nine rooting formulas, the R7 formula had the best effect in inducing root growth and development in the modified MS medium, with a rooting rate of 93.1% for the induced shoots, a root length of 41.9 cm, an average number of roots of 4.6 per plant, and the earliest rooting time of 12 days. The plant phenotype showed expanded, thick, dark green leaves and abundant root systems. After 110 days of culture without replacing the medium, the plants still showed strong growth and vigorous growth ( Figure 4 B). In ordinary MS medium, the base of the tender stem did not take root, the leaves of the seedlings were narrow, yellowed and fell off, the leaf yellowing rate was 66.7%, and the plant growth potential was low ( Figure 4 Therefore, modified MS medium supplemented with 1.2 mg / L NAA and 0.5 mg / L IAA was the optimal medium for inducing and cultivating rooting in sterile seedlings of Passiflora daphne. Rooting rate (%) = number of roots / total number of inoculated cells × 100%.
[0038] Table 4 Comparison of different rooting formulas on root induction of tender stems of Passiflora odorata
[0039] Table 5 Comparison of the growth of rooted seedlings between ordinary MS medium and improved MS medium
[0040] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.
Claims
1. A tissue culture breeding method for the stem tip of Passiflora daphne, characterized in that, The following steps are involved: (1) Explant pretreatment: Take healthy, vigorously growing shoots at the front end, soak them in neutral detergent, rinse them clean, wipe them, place them in a tissue culture bottle to keep them moist, and refrigerate them to obtain pretreated explants for later use; (2) Explant disinfection: The pretreated explant obtained in step (1) is first soaked in 75% by volume ethanol, then rinsed clean, and then disinfected with mercuric chloride solution to obtain a sterile explant; (3) Shoot tip axillary bud induction culture stage: The sterile explants obtained in step (2) are inoculated upright according to the growth polarity. After culturing for 10 to 15 days, the new shoot tips are inoculated into the stem tip axillary bud induction medium to obtain axillary buds; The shoot tip axillary bud induction medium is based on ordinary MS medium, and is supplemented with 1.0 mg / L to 2.0 mg / L 6-BA and BEB ganoderma lucidum diluted 2000 times to 3000 times; (4) Bud subculture stage: The axillary buds obtained in step (3) are cultured in a bud subculture medium for 25 to 30 days to obtain secondary buds; The bud subculture medium is based on a modified MS medium, supplemented with 1.5 mg / L to 2.5 mg / L 6-BA and 2300-2700-fold diluted BEB ganoderma lucidum. (5) Rooting induction culture stage: the clustered buds obtained in step (4) are cut into multiple single buds, transferred to the rooting induction medium and cultured for 12 days to 25 days to obtain rooted seedlings of Passiflora odorifera; The root induction medium is a modified MS medium as a base medium, and is supplemented with 0.2 mg / L-1.2 mg / L NAA and 0.5 mg / L-1.5 mg / L IAA.
2. The tissue culture breeding method according to claim 1, wherein The neutral detergent in step (1) is dishwashing liquid, and the soaking time is 15 min to 20 min.
3. The tissue culture breeding method according to claim 1, wherein The mass concentration of the mercuric chloride solution in step (2) is 0.15% to 0.6%.
4. The tissue culture breeding method according to claim 1, wherein The disinfection time of step (2) is 5 min~11 min.
5. The tissue culture breeding method according to claim 1, wherein The shoot tip axillary bud induction medium in step (3) is based on ordinary MS medium, and is supplemented with 2.0 mg / L 6-BA and 2000-fold diluted BEB Ganoderma lucidum.
6. The tissue culture breeding method according to claim 1, wherein The bud subculture culture medium in step (4) is based on the modified MS medium, and is supplemented with 1.5 mg / L 6-BA and 2500-fold diluted BEB Ganoderma lucidum.
7. The tissue culture breeding method according to claim 1, wherein The concentrations of trace elements in the modified MS medium in step (4) and step (5) are MnSO4·4H2O 44.6 mg / L, ZnSO4·7H2O 17.2 mg / L, CuSO4·5H2O 0.05 mg / L, H3BO3 12.4 mg / L, KI 1.66 mg / L, CoCl2·6H2O 0.05 mg / L and Na2MoO4·2H2O 0.5 mg / L; the iron salt concentration is FeSO4·7H2O 13.9 mg / L, Na2-EDTA 18.65 mg / L; the organic matter concentration is C2H5NO2 1.0 mg / L, C12H 17 ClN4OS·HCl 0.05 mg / L, C8H 12 ClNO30.25 mg / L, C6H5NO20.25 mg / L, C6H 12 O650 mg / L, and the concentrations of other nutrients remained unchanged.
8. The tissue culture breeding method according to claim 1, wherein The root induction medium in step (5) is based on a modified MS medium and is supplemented with 1.2 mg / L NAA and 0.5 mg / L IAA.
9. The tissue culture breeding method according to claim 1, wherein The culture conditions in steps (3) to (5) are as follows: a temperature of 28±0.5°C, a light intensity of 2500 lx to 3200 lx, and a photoperiod of 16 h / d to 18 h / d.
10. The tissue culture breeding method according to claim 1, wherein The length of the single bud in step (5) is ≥2.5 cm.