Tissue culture and rapid propagation method of'purple lotus' Chinese rose

Abstract

The invention discloses a tissue culture and rapid propagation method of'purple lotus' Chinese roses, and belongs to the technical field of plant tissue culture. Comprising the following steps: (1) explant detoxification: selecting a stem segment with one bud as an explant, sterilizing, inoculating to a primary culture medium, and carrying out primary culture to induce axillary bud germination; (2) subculture: cutting the axillary buds, and inoculating the axillary buds to a subculture medium for subculture to obtain tissue culture seedlings; (3) rooting culture: transferring the tissue culture seedlings growing to 3-5cm into a rooting culture medium for rooting culture; (4) domestication and transplanting: taking out the tissue culture seedlings, and transplanting the tissue culture seedlings in a domestication and transplanting medium for seedling recovery. According to the method disclosed by the invention, the explant with the bud stem section of the Chinese ancient Chinese rose purple lotus is used as a test material, so that a rapid tissue propagation technical system of the Chinese ancient Chinese rose purple lotus is established, and a scientific basis is provided for perfecting a tissue culture rapid propagation technology and large-scale production of the Chinese ancient Chinese rose.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, in particular to a tissue culture and rapid propagation method for "Zihe" rose. Background Art

[0002] Chinese rose (Rosa hybrida) is an important cut flower species in the world. It is widely used in cut flowers, potted flowers and landscaping, and has extremely high ornamental value, cultural value and economic value. As the distribution center of the world's Rosa plants, my country has a long history of cultivation and a wide variety of species. Among them, ancient Chinese roses such as 'Oldblush', 'Crimson China', 'Hume's BlushTea-scented China' and 'Park's Yellow Tea-scented China' are deeply loved by people for their elegance, fragrance and multi-season flowering characteristics. It is precisely because of the characteristics of ancient Chinese roses that modern roses have the excellent traits of continuous blooming in all seasons, special flower colors and fragrances, and certain disease resistance and stress resistance, which provide important germplasm resources for the cultivation of new rose varieties. At the same time, ancient Chinese roses are also precious national heritage and an important manifestation of my country's ancient horticultural history. The "Rose Catalogue" of the Qing Dynasty recorded 131 varieties. However, in the past hundred years, a large number of ancient Chinese rose varieties that have been preserved have been lost overseas, and some varieties have even been lost. There are only more than 50 varieties left.

[0003] In order to preserve the excellent genes of ancient roses, the preservation of germplasm resources is particularly important. As an important means of asexual reproduction of plants, plant tissue culture has the advantages of being unaffected by seasons, short growth cycle, and high reproduction coefficient, which makes up for the defects of traditional reproduction. With the development of rose tissue culture technology, research on rose tissue culture technology has emerged in an endless stream in recent years. Studies have shown that the main factors affecting the rapid reproduction of roses are the selection and sterilization of explants, the selection of culture media and culture conditions in each stage of tissue culture, etc. Zong Bo et al. used the axillary buds of the ancient Chinese rose "Alluring" as explants, and screened out the best induction culture medium as MS+0.5mg / L 6-BA through comparative experiments; the best proliferation culture medium was MS+2.0mg / L 6-BA+0.01mg / L NAA; the best rooting culture medium was 1 / 2MS+0.25mg / LIBA; and the best transplanting matrix was vermiculite. However, there are currently few reports on the research on the tissue culture technology of ancient Chinese roses, so it is very necessary to study its suitable reproduction method for its application and promotion.

[0004] The ancient Chinese rose "Purple Lotus" has bright purple flowers that look like a lotus when fully opened. The plant is strong and tall, with smooth branches and no thorns. It grows quickly and has a strong rose fragrance. It is the "ancestor" of many fragrant roses. After cultivation and observation in Shenzhen, it blooms all year round, with a large amount of flowers, strong growth, strong disease resistance, and strong adaptability. However, since the existing number of "Purple Lotus" is very small, asexual reproduction techniques such as tissue culture can effectively protect the genetic resources of this endangered ancient rose variety and prevent its extinction. Summary of the invention

[0005] The purpose of the present invention is to provide a tissue culture and rapid propagation method for "Zihe" rose to solve the problems existing in the above-mentioned prior art.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] One of the technical solutions of the present invention is: 1. A tissue culture and rapid propagation method for "Zihe" rose, comprising the following steps:

[0008] (1) Explant detoxification: A stem segment with one bud was selected as an explant, and after disinfection, it was inoculated into a primary culture medium for primary culture to induce axillary bud germination;

[0009] (2) Subculture: excise the axillary buds, inoculate them into a subculture medium for subculture to obtain tissue culture seedlings; the subculture medium comprises: MS + 1.0-2.0 mg / L 6-BA + 0.5 mg / L NAA + 30 g / L sucrose + 7.8-8 g / L agar, with a pH of 5.8-5.9;

[0010] (3) Rooting culture: The tissue culture seedlings grown to 3-5 cm were transferred to a rooting medium for rooting culture; the composition of the rooting medium was: 1 / 2MS + 0.25-0.5 mg / L IBA + 30 g / L sucrose + 7.8-8 g / L agar, pH 5.8-5.9;

[0011] (4) Acclimation and transplanting: Take out the tissue culture seedlings and plant them in the acclimation and transplanting medium to accelerate the growth of the seedlings.

[0012] Based on the above technical solution, the present invention has the following technical effects:

[0013] 1. When the rose explants are detoxified and enter the primary culture stage, in different disinfection time gradients: the best disinfection time for the ancient Chinese rose "Zihe" is 75% alcohol washing for 30 seconds and 6% sodium hypochlorite disinfection for 3 minutes, the stem segment induction rate is 90%, the mortality rate is 4%, and the contamination rate is 6%.

[0014] 2. Induced sterile axillary buds of roses to enter the subculture proliferation stage, in subculture proliferation culture medium with different hormone concentrations: the best subculture culture medium for the ancient Chinese rose "Purple Lotus" was MS+1.0-2.0 mg / L 6-BA+0.5 mg / L NAA+agar 7.8-8 g / L, the plant heights were 6.32 cm and 5.49 cm, the crown widths were 5.69 cm and 5.81 cm, and the proliferation coefficients were all 3.78.

[0015] 3. In the rooting culture stage of rose inducing adventitious buds, in the rooting medium with different hormone concentrations: the rooting performance of the ancient Chinese rose "Zihe" in the medium containing IBA (0.25mg / L, 0.5mg / L) is similar. When the rooting medium is 1 / 2MS+0.25mg / L IBA+sucrose 30g / L+agar 7.8-8g / L, the rooting rate reaches 40% in 15 days, the average root length is 0.45cm, and the average number of roots is 8.80. The rooting rate reaches 100% in 30 days, the average root length is 0.73cm, and the average number of roots is 12.10; when the rooting medium is 1 / 2MS+0.5mg / L When IBA+sucrose 30g / L+agar 7.8-8g / L was used, the rooting rate reached 27.5% after 15 days, the average root length was 0.25cm, and the average number of roots was 3.60. The rooting rate reached 99% after 30 days, the average root length was 0.59cm, and the average number of roots was 16.0.

[0016] 4. Gradually open the bottle cap of the rooted rose sterile seedlings to adapt to the external environment. After 3 days, take out the agar gel and wash the roots, and enter the seedling hardening and transplanting stage. The transplanting matrix is ​​V vermiculite: V perlite: V nutrient soil = 1:1:3. After transplanting, sprinkle enough rooting water and cover with film to maintain a moist environment. About 1 week after transplanting, the heart leaves begin to grow and the leaves begin to spit beads, and the transplanting is successfully completed.

[0017] The present invention uses the bud stem segment explants of the ancient Chinese rose "Zihe" as test materials and uses plant tissue culture technology to study the main influencing factors of each stage of explant disinfection time, subculture proliferation, rooting culture, seedling hardening and transplanting, in order to establish a tissue rapid propagation technology system of the ancient Chinese rose "Zihe", and provide a scientific basis for improving the tissue culture rapid propagation technology and large-scale production of the ancient Chinese rose. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1It is the form of purple lotus flower, leaf, stem and plant. Among them, a is the purple lotus flower; b is the purple lotus leaf; c is the purple lotus stem segment; d is the red dragon pearl plant.

[0020] Figure 2 This is the growth status of the ancient Chinese rose "Zihe" under different disinfection times.

[0021] Figure 3 This shows the growth of the ancient Chinese rose "Zihe" under different proliferation and subculture culture media.

[0022] Figure 4 The effect of different root growth medium on the rooting of the ancient Chinese rose "Zihe" (0d). Among them, a is the initial state of the whole plant of the ancient Chinese rose "Zihe" inoculated with different root growth mediums, and b is the initial state of the base incision of the ancient Chinese rose "Zihe" inoculated with different root growth mediums. Z1: 1 / 2MS + 0.1mg / LNAA; Z2: 1 / 2MS + 0.5mg / LNAA; Z3: 1 / 2MS + 0.25mg / L IBA; Z4: 1 / 2MS + 0.5mg / L IBA, the same below.

[0023] Figure 5 The effect of different root growth medium on the rooting of the ancient Chinese rose "Zihe" (15d). Among them, a is the state of the whole plant of the ancient Chinese rose "Zihe" inoculated with different root growth medium on the 15th day, and b is the state of the rooting at the base of the ancient Chinese rose "Zihe" inoculated with different root growth medium on the 15th day.

[0024] Figure 6 The effect of different root growth medium on the rooting of the ancient Chinese rose "Zihe" (30d). Among them, a is the state of the whole plant of the ancient Chinese rose "Zihe" inoculated with different root growth medium on the 30th day, and b is the state of the rooting at the base of the ancient Chinese rose "Zihe" inoculated with different root growth medium on the 30th day.

[0025] Figure 7 The pictures are of the domestication and transplantation of "Chilong Hanzhu" and "Zihe" outside the bottle. Among them, a is the transplantation soil culture picture of "Zihe"; b is the rooting situation of the transplanted tissue culture seedlings of "Zihe". DETAILED DESCRIPTION

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0028] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0029] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0031] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0032] The embodiment of the present invention provides 1. a tissue culture and rapid propagation method of "Zihe" rose, comprising the following steps:

[0033] (1) Explant detoxification: A stem segment with one bud was selected as an explant, and after disinfection, it was inoculated into a primary culture medium for primary culture to induce axillary bud germination;

[0034] (2) Subculture: excise the axillary buds, inoculate them into a subculture medium for subculture to obtain tissue culture seedlings; the subculture medium comprises: MS + 1.0-2.0 mg / L 6-BA + 0.5 mg / L NAA + 30 g / L sucrose + 7.8-8 g / L agar, with a pH of 5.8-5.9;

[0035] (3) Rooting culture: The tissue culture seedlings grown to 3-5 cm were transferred to a rooting medium for rooting culture; the composition of the rooting medium was: 1 / 2MS + 0.25-0.5 mg / L IBA + 30 g / L sucrose + 7.8-8 g / L agar, pH 5.8-5.9;

[0036] (4) Acclimation and transplanting: Take out the tissue culture seedlings and plant them in the acclimation and transplanting medium to accelerate the growth of the seedlings.

[0037] In some specific implementation schemes, the disinfection method is: first disinfect with 75% alcohol for 30 seconds, and then disinfect with 6% sodium hypochlorite for 3 minutes.

[0038] In some specific embodiments, the components of the primary culture medium are: MS+1.0 mg / L 6-BA+0.1 mg / L NAA+30 g / L sucrose+7.8-8 g / L agar, and the pH is 5.8-5.9.

[0039] In some specific embodiments, the culture medium for acclimation and transplanting is vermiculite:perlite:nutrient soil in a volume ratio of 1:1:3.

[0040] In some specific embodiments, the method for slowing down the growth of seedlings is: after watering thoroughly, cover with a film to keep moisture for 1 week, and gradually open the doors until they are fully opened.

[0041] In some specific embodiments, the culture temperature for slow seedling growth is 25±2°C, the light intensity is 2000-30001x, and the light time is 14 hours per day.

[0042] The present invention uses the stem segments of the ancient Chinese rose "Zihe" as explants, and studies the effects of different sterilization times, different proliferation and subculture culture media and different rooting culture media, in order to establish a complete, efficient and practical culture and rapid propagation system for the ancient Chinese rose, to provide a scientific basis for the large-scale propagation and production of seedlings in the future, and also to lay a foundation for the germplasm preservation, molecular breeding and garden application of ancient rose varieties.

[0043] Example 1

[0044] 1. Materials and Methods

[0045] 1.1 Plant materials

[0046] The ancient Chinese rose variety "Purple Lotus" (such as Figure 1), an upright shrub with 2-3 flower buds on a single branch and a plant height of 80-100cm; the flowers are rose-red, with a diameter of about 7-10cm, 30-35 petals, double-petaled, with a strong rose scent, and the petals are obovate with curled edges; the sepal edges are moderately extended, the pedicel is 10-12cm long, with glandular hairs; the leaves have 3-7 leaflets, 10-12cm in length, with clear veins, green, and moderate gloss on the leaf surface; the top leaflets are oblong, 4-5cm in diameter, with no obvious contraction at the tip, single serrations on the leaf margins, reddish-brown young branches, and reddish-brown young leaves; the stems are green, some of which are slightly brown-red, and the plant stems are thornless. The plant has good growth potential and good disease resistance, and can be used as a garden, potted plant, or ground cover.

[0047] 1.2 Methods

[0048] (a) Explant detoxification

[0049] Select healthy branches that have grown in the current year, have full buds, and are free of pests and diseases. After removing the young leaves and petioles on the branches, cut them into stem segments with one bud as explants. Try to choose the same stem segments according to their positions on the branches. Put the processed stem segments into water with detergent and scrub them carefully with a brush. Soak and shake them constantly for 15 minutes, then rinse them with running water for 30-60 minutes, transfer them to a clean bench, and disinfect them with 75% alcohol for 30 seconds.

[0050] Rinse with sterile water for 3 times, disinfect with 6% sodium hypochlorite for 5, 10, and 15 min at 3 time gradients. The number of stem segments for each treatment is 50-60. Rinse with sterile water for 5 times, wipe dry, and inoculate into primary culture medium. Observe and count the contamination and axillary bud induction every day for 30 consecutive days.

[0051] (b) Subculture

[0052] The axillary buds sprouting from the stem segments were cut off and inoculated into culture media containing different types and concentrations of plant growth regulators for subculture. The growth was observed every week for 120 consecutive days.

[0053] (c) Rooting culture

[0054] The tissue culture seedlings that grew to 3-5 cm were transferred to the rooting medium and the rooting conditions were observed for 30 consecutive days.

[0055] (d) Transplantation outside the bottle

[0056] Open the cover of the tissue culture bottle for rooted seedlings gradually, take out the tissue culture seedlings, wash the root culture medium, plant them in a matrix of vermiculite: perlite: nutrient soil (1:1:3), water them thoroughly, cover them with a film to keep them moist for 1 week, and gradually open them until they are fully opened. The culture temperature is 25±2℃, the light intensity is 2000~30001x, and the light time is 14 hours a day.

[0057] 1.3 Data calculation standards

[0058] Microsoft Excel and SPSS Statistics 27.0 were used for data statistical analysis.

[0059] Contamination rate = number of contaminated explants / total number of inoculated explants × 100%.

[0060] Mortality rate = number of dead uncontaminated explants / total number of inoculated explants × 100%.

[0061] Stem segment induction rate = number of stem segments that induced adventitious buds / total number of inoculated stem segments × 100%.

[0062] Proliferation coefficient = current number of plants / original number of plants.

[0063] Rooting rate = number of rooted seedlings / number of inoculated seedlings × 100%.

[0064] Average number of roots = total number of roots of rooted seedlings / number of rooted seedlings.

[0065] Average root length = total root length / total number of roots.

[0066] 1.4 Culture medium formula table

[0067] (a) Primary culture medium: MS + 1.0 mg / L 6-BA + 0.1 mg / L NAA + 30 g / L sucrose + 7.8-8 g / L agar, pH adjusted to 5.8-5.9.

[0068] (b) Subculture medium: MS + 1.0-3.0 mg / L 6-BA + 0.02-0.5 mg / L NAA + 30 g / L sucrose + 7.8-8 g / L agar, pH adjusted to 5.8-5.9.

[0069] It is divided into three gradients: 1.0mg / L 6-BA, 2.0mg / L 6-BA, and 3.0mg / L 6-BA.

[0070] It is divided into three gradients: 0.02mg / L NAA, 0.1mg / L NAA, and 0.5mg / L NAA.

[0071] The subculture medium with different gradients is shown in Table 1.

[0072] Table 1 Numbers of different gradient subculture medium

[0073]

[0074] (c) Rooting medium: 1 / 2MS + 0.1-0.5 mg / L NAA or 0.25-0.5 mg / L IBA + 30 g / L sucrose + 7.8-8 g / L agar, with pH adjusted to 5.8-5.9.

[0075] It is divided into two gradients: 0.1mg / LNAA and 0.5mg / LNAA.

[0076] There are two gradients: 0.25mg / L IBA and 0.5mg / L IBA.

[0077] The formulas of rooting medium with different gradients are shown in Table 2.

[0078] Table 2 Different gradient rooting medium numbers

[0079]

[0080] 2. Results and Analysis

[0081] 2.1 Effects of different disinfection times on explants

[0082] This experiment used 75% alcohol disinfection for 30 seconds and 6% sodium hypochlorite disinfection at different time gradients to disinfect the explants. The effects of different disinfection times on the ancient Chinese rose "Zihe" are as follows. Figure 2 As shown in Table 3, as the disinfection time of 6% sodium hypochlorite was prolonged, the contamination rate of the stem segments of "Purple Lotus" showed a trend of first increasing and then decreasing, while the mortality rate showed a trend of gradually increasing. When disinfected for 3 minutes, the contamination rate and mortality rate of the stem segments were the lowest, and the induction rate of the stem segments was the highest, which was significantly better than other disinfection times. In summary, 75% alcohol disinfection for 30 seconds and 6% sodium hypochlorite disinfection for 3 minutes were the optimal treatment times for detoxification of "Purple Lotus" explants.

[0083] Table 3 Effects of different disinfection times on the explants of the ancient Chinese rose “Zihe”

[0084]

[0085] 2.2 Effects of different hormone ratios on bud proliferation

[0086] There were significant differences in the lateral bud height, crown width, lateral bud proliferation rate and growth potential of the different concentration combinations of 6-BA and NAA. Figure 3It can be seen that during the proliferation culture process, at the same 6-BA concentration, with the increase of NAA concentration, the lateral bud height, crown width, and lateral bud value-added rate showed a gradual increasing trend; at the same NAA concentration, with the increase of 6-BA concentration, the lateral bud height, crown width, and lateral bud value-added rate showed a gradual decreasing trend. "Zihe" had the highest average height of 6.32±0.54a, an average crown width of 5.69±0.59a, and a maximum proliferation coefficient of 3.78±0.67a when M6 (2.0mg / L6-BA+0.5mg / L NAA), and a good growth. Comprehensive analysis showed that 1.0~2.0mg / L 6-BA+0.5mg / L NAA was the best hormone combination to induce lateral bud proliferation.

[0087] Table 4 Effects of different gradients of subculture medium on rose height and crown width

[0088]

[0089] Table 5 Effects of different gradients of subculture medium on the proliferation coefficient and growth potential of "Zihe"

[0090]

[0091] 2.3 Effects of different hormone ratios on rooting of tissue culture seedlings

[0092] Select sterile seedlings with strong and uniform growth and inoculate them into 1 / 2MS medium containing IBA (0.25mg / L, 0.5mg / L) and NAA (0.1mg / L, 0.5mg / L). Figure 4 , 5 As shown in Table 6, the root growth rate of the medium with IBA added is faster, while the growth rate of the medium with NAA added is slower. Among them, Z3: 1 / 2MS + 0.25mg / L IBA has a rooting rate of 40%, an average number of roots of 8.8 and an average root length of 0.45cm, with the best growth.

[0093] Table 6 Effects of different auxins on rooting of the ancient Chinese rose “Zihe” (15d)

[0094]

[0095] After 30 days of culture, Figure 6As shown in Table 7, it was found that the rooting medium with added IBA hormone had better rooting effect than the rooting medium with added NAA hormone. Among them, Z3: 1 / 2MS + IBA 0.25mg / L and Z4: 1 / 2MS + IBA 0.5mg / L IBA two culture media have their own advantages in rooting effect. Z3: 1 / 2MS + IBA 0.25 mg / L has a rooting rate of 100%, an average number of roots of 12.1 and an average root length of 0.73cm, a long root system, and good growth; Z4: 1 / 2MS + IBA 0.5mg / L has a rooting rate of 99%, an average number of roots of 16.0 and an average root length of 0.59cm, many roots, and good growth. Z3 is mainly reflected in the long average root length, and Z4 is mainly reflected in the large average number of roots.

[0096] Table 7 Effects of different auxins on rooting of the ancient Chinese rose “Zihe” (30 days)

[0097]

[0098] 2.4 Acclimation and transplanting outside the bottle

[0099] Open the cover of the rooting seedling tissue culture bottle gradually, take out the rooted sterile rose tissue culture seedlings, remove the agar gel from the cleaned roots after 3 days, plant them in a matrix with a ratio of vermiculite: perlite: nutrient soil (1:1:3), cover with film to maintain a moist environment, about 1 week after planting, the heart leaves grow, the leaves begin to spit beads, and the transplanting is successfully completed ( Figure 7 ).

[0100] In summary, the present invention explores the formula for rapid tissue culture of ancient Chinese roses from the aspects of the disinfection method of exogenous plants and the mass concentration of growth hormones. Disinfection and aseptic operation of explants are particularly important in tissue culture experiments. The results of this experimental study showed that for "Purple Lotus", under the conditions of 75% alcohol disinfection for 30s and 6% sodium hypochlorite disinfection of explants with different time gradients, the disinfection time of 3min was the best, which could achieve a lower contamination rate and a high stem segment induction rate.

[0101] Exogenous plant hormones play a key role in bud proliferation, and different concentrations of 6-BA and NAA combinations show a significant effect on the growth of lateral buds. Low concentrations of 6-BA are most suitable for promoting the proliferation of axillary buds, while high concentrations will inhibit the growth and development of axillary buds; the concentration of NAA will affect the rate of bud proliferation. In the experiment, at the same 6-BA concentration, with the increase of NAA concentration, the lateral bud height, crown width and lateral bud proliferation rate showed a gradual increasing trend. At the same NAA concentration, with the increase of 6-BA concentration, the lateral bud height, crown width and lateral bud proliferation rate showed a gradual decreasing trend. Specifically, "Zihe" performed best under the conditions of M3 and M6 (6-BA 1.0~2.0 mg / L+NAA0.5 mg / L), which is the best hormone combination to promote lateral bud proliferation.

[0102] The appropriate addition of IBA and NAA to the culture medium is conducive to the rooting of rose varieties. "Zihe" showed significant differences in different rooting culture media. The rooting-promoting effect of IBA in "Zihe" was significantly better than that of NAA. "Zihe" had its own advantages in growth under Z3 (1 / 2MS+IBA0.25mg / L) and Z4 (1 / 2MS+IBA0.5 mg / L IBA) conditions. The roots grew fast and well on Z3 culture medium, and the number of roots was large on Z4 culture medium.

[0103] In conclusion, this study provides a certain theoretical basis for the reproduction of ancient Chinese roses, but it still needs further improvement and in-depth research to provide more comprehensive and in-depth theoretical support and practical guidance for the cultivation and production of ancient Chinese roses.

[0104] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A tissue culture and rapid propagation method for "Zihe" rose, characterized in that: The following steps are involved: (1) Explant detoxification: A stem segment with one bud was selected as an explant, and after disinfection, it was inoculated into a primary culture medium for primary culture to induce axillary bud germination; (2) Subculture: excise the axillary buds, inoculate them into a subculture medium for subculture to obtain tissue culture seedlings; the subculture medium comprises: MS + 1.0-2.0 mg / L 6-BA + 0.5 mg / L NAA + 30 g / L sucrose + 7.8-8 g / L agar, with a pH of 5.8-5.9; (3) Rooting culture: The tissue culture seedlings grown to 3-5 cm were transferred to a rooting medium for rooting culture; the composition of the rooting medium was: 1 / 2MS + 0.25-0.5 mg / L IBA + 30 g / L sucrose + 7.8-8 g / L agar, pH 5.8-5.9; (4) Acclimation and transplanting: Take out the tissue culture seedlings and plant them in the acclimation and transplanting medium to accelerate the growth of the seedlings.

2. The tissue culture rapid propagation method according to claim 1, characterized in that: The disinfection method is: first disinfect with 75% alcohol for 30 seconds, and then disinfect with 6% sodium hypochlorite for 3 minutes.

3. The tissue culture rapid propagation method according to claim 1, characterized in that The components of the primary culture medium are: MS+1.0 mg / L 6-BA+0.1 mg / L NAA+30 g / L sucrose+7.8-8 g / L agar, and the pH is 5.8-5.

9.

4. The tissue culture rapid propagation method according to claim 1, characterized in that: The culture medium for acclimation and transplanting is a culture medium in which the volume ratio of vermiculite:perlite:nutrient soil is 1:1:

3.

5. The tissue culture rapid propagation method according to claim 1, characterized in that: The method for slowing down the seedlings is: after watering thoroughly, cover with a film to keep moisture for 1 week, and gradually open the seedlings until they are fully opened.

6. The tissue culture rapid propagation method according to claim 5, characterized in that: The culture temperature of the seedling acclimatization is 25±2° C., the light intensity is 2000-30001x, and the light time is 14 hours per day.

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