Method for rapidly establishing phallus impudicus variety Donglin violet tissue culture system through underground buds

Abstract

The invention provides a method for rapidly establishing a phallus impudicus variety Donglin violet tissue culture system through underground buds. According to the method, underground buds of 'Donglin purple' are used as explants, clean and tender underground buds ingeniously slip out through the methods of base cutting, fixing and light clamping, cluster buds are directly induced after the propagation coefficient is increased through longitudinal cutting, then roots are induced to form in-vitro plants, the sterile rate in 5 days is 38.48%, the fungal rate is 0.21%, the sterile rate in 25 days of primary subculture is 74.79%, and the rooting rate in 50 days is 87.12%. According to the method, the callus induction stage is not needed, the time for obtaining the high-quality sterile Donglin purple in-vitro plant is shortened, the technology is easy to master, the cost is low, the efficiency is high, and a foundation is laid for establishing a high-efficiency improved variety propagation system through further subculture of cluster buds or induction of callus redifferentiation subsequently. The invention provides a solid guarantee for improved variety breeding, stem apex detoxification, ploidy breeding and genetic engineering genetic improvement research of iris varieties, especially phallus impudicus' Donglin purple '.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture, and in particular relates to a method for quickly establishing a tissue culture system of the cultivar 'Donglinzi' of the cultivar Iris strychni through underground buds. Background Art

[0002] Iris sanguinea Donn ex Hornem. is a famous perennial ornamental flower of the genus Iris. It is native to Northeast my country and has strong cold resistance and high ornamental value. It is widely used in garden landscaping and cut flowers. In recent years, with the deepening of research on Iris sanguinea, hybridization and breeding of Iris sanguinea varieties with various flower colors have been carried out, such as 'Donglin Zi' (Yang, J. et al. (2022). 'Dong Lin Zi': A New Iris sanguinea Cultivar. HortScience, 57(2), 197-199.), 'Donglin Yixia' (Shi, G. et al. (2024). Donglin Yixia: A New Iris sanguinea Cultivar. HortScience, 59(10), 1475-1476.) and 'Yise Ziqun' (Xu, N. et al. (2024). 'Yise Ziqun': A New Iris sanguinea Cultivar. HortScience, 59(7), 972-973.) etc. 'Donglinzi' is a new variety of Iris sanguinea bred by the College of Landscape Architecture of Northeast Forestry University through hybrid breeding. Its flowers are bright purple-red, with half-drooping petals and half-erect standard petals. The flowering period is from May to June, the fruiting period is from August to September, and the green period is relatively long. It is a rare perennial ornamental flower in cold regions.

[0003] The varieties of iris spatholobi rely heavily on asexual reproduction to maintain their excellent traits. Common ramet propagation has the limitations of heavy workload, long time consumption, and variety degeneration. Tissue culture is one of the important ways to breed asexual varieties and preserve genetic resources, and can achieve good varieties in a short period of time. The efficient production and breeding of new varieties is the guarantee for its promotion and application. Domestic and foreign scholars have made certain progress in the establishment of tissue culture systems for iris spatholobi. The explants are mainly selected from seeds, young leaves, new rhizomes, flower stem nodes, root tips and other parts. Li Li (Li Li et al. Research on in vitro rapid propagation technology of iris spatholobi [J]. Land and Natural Resources Research, 2017, (05): 90-91.), Tang Biao (Tang Biao. Establishment of iris spatholobi regeneration system and research on transformation of GUS gene [D]. Northeast Forestry University, 2018.) and others have obtained a relatively efficient in vitro regeneration system for iris spatholobi, but the tissue culture system established with seeds as explants cannot meet the needs of asexual reproduction of varieties. The regeneration ability of Iridaceae is lower than that of Amaryllidaceae, Araceae and Liliaceae (Wei Xiaoyu et al. Research Progress on Tissue Culture and Rapid Propagation Technology of Iris Plants [J]. Molecular Plant Breeding, 2024, 22(19): 6500-6513.). Although the tissue culture system established by the young leaves, flower stem nodes and other parts of Iris can be used for the propagation of asexual lines of varieties, it requires callus induction to form in vitro plants, which takes a long time. The callus induction rate of the root tip is low and the contamination rate of the new rhizomes is high. Therefore, it has not been used to further establish the tissue culture system of Iris. There are still many problems to be solved in the establishment of tissue culture system of Xisan varieties, such as massive contamination by fungi and bacteria, browning and vitrification after sterile treatment, difficulty in inducing callus tissue and differentiating it into plants, and difficulty in rooting clustered buds. Currently, there are few research reports on methods for establishing tissue culture system of Xisan varieties. Therefore, establishing a fast and efficient tissue culture system for Xisan varieties is crucial for the breeding and promotion of new Xisan varieties. Summary of the invention

[0004] The purpose of the present invention is to provide a method for quickly establishing an in vitro plant tissue culture system of the cultivar 'Donglin Zi' of the cultivar 'Donglin Zi' by using underground buds as explants, through the steps of explant disinfection, cluster bud induction, rooting culture, etc., without the need for a callus approach. The technology is easy to master, can effectively reduce the infection rate of the primary culture, shorten the time to obtain high-quality sterile 'Donglin Zi' in vitro plants, and provide a new way to establish an efficient tissue culture and rapid propagation system of the cultivar 'Donglin Zi' of the cultivar 'Donglin Zi'.

[0005] In order to realize the above technology, the technical solution adopted by the present invention is: a method for quickly establishing a tissue culture system of the cultivar 'Donglinzi' of the cultivar of Ilex strychnifolia through underground buds, comprising the following steps:

[0006] Step 1: Obtaining explants, select underground buds of the cultivar 'Donglinzi' as explant materials. After the underground buds are formed in autumn or before the underground buds sprout and grow in spring, dig out the mature 'Donglinzi' seedlings from the field, trim the leaves and roots, and obtain clean underground buds.

[0007] Step 2: Disinfection of explants. Soak the clean underground buds treated in step 1 in 50% carbendazim wettable powder diluted 1500 times with tap water for 5 minutes. Rinse with tap water, shake and clean the underground buds with detergent water for 1 minute, and then rinse with tap water for 20 minutes to wash thoroughly. Then, shake and clean with 75% ethanol solution for 30 seconds in a sterile table, rinse with sterile water 3 times, 1 minute each time, and then put in 5% NaClO solution containing 0.1% Tween20 and shake and disinfect for 15 minutes, rinse with sterile water 5 times, 1 minute each time, place the disinfected underground buds on clean filter paper, and then quickly cut off the base of the outermost leaf of the underground bud, use the side of the blade to press the top of the underground bud, use tweezers to gently clamp the base of the underground bud, squeeze and slide the young underground buds in the center of the underground buds, and quickly use newly sterilized clean tweezers to clamp the young underground buds and place them on clean filter paper.

[0008] Step 3: Induction of clustered buds, cut the young underground buds obtained in step 2 into 2-3 pieces longitudinally, each piece needs to retain the base of the underground buds and young leaves, and press the cut side down into the clustered bud culture medium horizontally. The culture dish uses a 90mm specification, and 6-10 cut pieces are placed in each culture dish. After the culture dish is sealed, it is cultured in a culture room at a temperature of 25±1℃, a light intensity of 2000-3000lx, and a photoperiod of 12h light and 12h dark. After 5 days, select a culture dish without fungal contamination, select the underground bud cuts without bacteria and re-inoculate them into a new clustered bud culture medium. After 25 days, continue to select the robust underground bud cuts without bacteria from the culture dish without fungal contamination, and re-inoculate them into a new clustered bud culture medium with the morphological lower end facing down, and continue to induce clustered buds. The culture medium is replaced every 25 days until clustered buds are produced.

[0009] Step 4: Rooting culture, take out the completely sterile clustered buds obtained in step 3, insert their bases into the rooting medium, induce rooting under the culture conditions described in step 3, and finally obtain high-quality sterile in vitro plants of 'Donglin Purple'.

[0010] The clustered bud culture medium described in step 3 is MS+0.5mg / L 6-BA+1.0mg / L KT+0.2mg / L NAA+30g / L sucrose+7.8g / L agar, with a pH of 5.8-6.0.

[0011] The rooting medium described in step 4 is MS+0.5mg / L NAA+30g / L sucrose+7.0g / L agar, with a pH of 5.8-6.0.

[0012] Advantages of the present invention: The present invention uses the underground buds of the cultivar 'Donglin Zi' of the cultivar as explants, and directly induces clustered adventitious buds by cutting off the base, fixing, and lightly clamping the young underground buds that are not in contact with the outside world, and then induces roots to form in vitro plants. The technology is easy to master, can greatly reduce the infection rate of the primary culture, does not need to go through the callus induction stage, shortens the time to obtain high-quality sterile 'Donglin Zi' in vitro plants, and establishes an efficient tissue culture system of the cultivar 'Donglin Zi'. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The present invention will be described in detail below with reference to the accompanying drawings and in combination with embodiments, and the advantages and implementation modes of the present invention will become more obvious. The contents shown in the accompanying drawings are only used to explain the present invention and do not constitute any limitation to the present invention in any sense. In the accompanying drawings:

[0014] Figure 1 Schematic diagram of the initial processing of explants.

[0015] Figure 2 To obtain underground buds.

[0016] Figure 3 It is the process of sliding out young underground buds that have no contact with the outside world by cutting off the base, fixing, and gently clamping.

[0017] Figure 4 Schematic diagram of underground bud excisions in a culture dish and cultured on cluster medium.

[0018] Figure 5 Schematic diagram of the culture of sterile underground shoots in rooting medium.

[0019] Figure 6 This is a tissue culture system of the Ilex strychnifolia variety 'Donglinzi' that is established quickly through underground buds. DETAILED DESCRIPTION

[0020] The present invention is further described in detail below in conjunction with embodiments and drawings to help understand the content of the present invention.

[0021] Unless otherwise specified, the experimental methods used in the examples are all conventional methods, and researchers in the field can achieve the results by following conventional tissue culture methods; the materials, reagents, etc. used in the examples, unless otherwise specified, can be obtained from commercial channels.

[0022] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a method for quickly establishing a tissue culture system of the cultivar 'Donglinzi'.

[0023] The relevant culture medium preparation method in the embodiment of the present invention is as follows:

[0024] Cluster bud culture medium: MS+0.5mg / L 6-BA+1.0mg / L KT+0.2mg / L NAA+30g / L sucrose+7.8g / L agar, pH 5.8-6.0.

[0025] Rooting medium: MS + 0.5 mg / L NAA + 30 g / L sucrose + 7.0 g / L agar, pH 5.8-6.0.

[0026] The calculation methods of sterility rate, fungal rate and rooting rate in the embodiment are as follows:

[0027] Sterility rate = number of underground bud cuts without fungi or bacteria growing / total number of underground bud cuts × 100%.

[0028] Fungal rate = number of underground bud cuttings with fungi / total number of underground bud cuttings × 100%.

[0029] Rooting rate = number of underground bud cuttings placed on the rooting medium / total number of underground bud cuttings × 100%.

[0030] A method for quickly establishing a tissue culture system of the cultivar 'Donglinzi' of the cultivar of Ilex strychnifolia through underground buds provided in an embodiment of the present invention comprises the following steps:

[0031] (1) Obtaining explants

[0032] Select the cultivar 'Donglinzi' of the brook spathiphyllum, which is healthy, free of pests and diseases, and more than three years old, as the mother plant. After the underground buds are formed in autumn or before the underground buds sprout and grow in spring, dig them out from the field, use pruning shears to cut the withered leaves, shake off the excess soil, and divide them into small pieces for later use. Figure 1 This is the state of the explant after preliminary treatment. Further, remove the excess roots and use sharp pruning shears to cut the strong and plump underground buds about 3-4 cm long from the rhizome. Figure 2 To obtain the state of underground buds, spread filter paper on a clean table, use a sharp scalpel to cut off 1-2 layers of outer leaves of the underground buds, expose the relatively clean underground buds, and place them on the clean filter paper for later use.

[0033] (2) Disinfection of explants

[0034] Use 50% carbendazim wettable powder diluted 1500 times with tap water to soak the clean underground buds of 'Donglin Purple' for 5 minutes. Rinse with tap water and shake the explants with clean water for 1 minute. Then place the explants in a clean conical bottle, with about a quarter of the volume of the explant in each conical bottle. Seal the bottle with a layer of clean gauze, place it in running tap water and rinse for 20 minutes to wash it thoroughly. Then, in the sterile table, use tissue culture bottles as containers, place about half of the volume of explants in each culture bottle, use 75% ethanol solution that has submerged the explants to shake and clean for 30 seconds, rinse with sterile water 3 times, 1 minute each time, then put the explants in 5% NaClO solution containing 0.1% Tween20, cover the tissue culture bottle cap and shake and disinfect for 15 minutes, rinse with sterile water 5 times, 1 minute each time, place the disinfected explants on clean filter paper to absorb water, and then quickly cut off the base of the outermost leaves of the underground buds to expose the stem tip. For larger underground buds, it is necessary to cut cross-sections at about 8mm from the top of the leaves. Use the side of the blade to press the top of the underground bud, use tweezers to gently clamp the base of the underground bud, squeeze and slide the young underground buds in the center of the underground buds, and quickly use clean tweezers that have been burned again and completely cooled to clamp the sterile underground buds and place them on clean filter paper. The young underground buds need to have stem tips and young leaves, and as many stem structures as possible should be retained to facilitate rooting. Figure 3 It is the process of sliding out young underground buds that have no contact with the outside world by cutting off the base, fixing, and gently clamping.

[0035] (3) Induction of clustered shoots

[0036] Cut the young underground buds into 2-3 pieces longitudinally, retaining the stem tip and young leaves at the base of the underground buds in each piece. Place the cut side down and press horizontally into the cluster bud culture medium. Use a 90mm specification culture dish, and place 6-10 cut pieces in each culture dish. After the culture dish is sealed, culture it in a culture room at 25±1℃, with a light intensity of 2000-3000lx and a photoperiod of 12h light and 12h darkness. Figure 4 This is a photo taken from the bottom of the culture dish of an underground bud cut into pieces cultured on cluster bud culture medium.

[0037] After 5 days of induction, select the culture dishes without fungal contamination, select the underground buds without bacteria from them and re-inoculate them into new cluster culture medium. After 25 days, continue to select the robust underground buds without bacteria from the culture dishes without fungal contamination, re-inoculate them into new cluster bud culture medium with their morphological lower end facing downward, continue to induce cluster buds, and change the culture medium every 25 days until cluster buds are produced. The sterility rate and fungal rate at 5 days and 25 days were calculated respectively.

[0038] According to statistics, the sterility rate at 5 days was 38.48%, the fungal rate was 0.21%, and the sterility rate at 25 days was 74.79%, the fungal rate was 0.27%.

[0039] (4) Rooting culture

[0040] The well-growing clustered buds were transferred to the rooting medium and cultured in a culture room at a temperature of 25±1°C, a light intensity of 2000-3000lx, and a photoperiod of 12h light and 12h darkness. Figure 5 The schematic diagram of the aseptic underground buds cultured in the rooting medium. After 50 days of cultivation, the rooting rate was 87.12%. Finally, the high-quality aseptic in vitro plants of 'Donglinzi' were obtained. Figure 6 This is a tissue culture system of the Ilex strychnifolia variety 'Donglinzi' that is established quickly through underground buds.

[0041] The method for establishing the tissue culture system of the 'Donglin Purple' variety of the iris cultivar provided in the embodiment of the present invention uses underground buds as explants, and skilfully slides out the tender underground buds that are not in contact with the outside world by cutting the base, fixing, and lightly clamping. After the longitudinal section improves the reproduction coefficient, the clustered adventitious buds are directly induced, and then the roots are induced to form in vitro plants, which can greatly reduce the infection rate of the primary culture. Through the four-step culture of bud taking, bud cutting, bud promotion, and rooting, there is no need to go through the callus induction stage, shortening the time to obtain high-quality sterile 'Donglin Purple' in vitro plants, and establishing an efficient 'Donglin Purple' tissue culture system of the iris cultivar, which is easy to master, low in cost, and high in efficiency, and lays the foundation for the subsequent further subculture of clustered buds or inducing callus redifferentiation to establish an efficient improved variety expansion system. The present invention provides a solid guarantee for the research on improved variety breeding, stem tip detoxification, ploidy breeding, and genetic engineering genetic improvement of iris cultivars, especially the 'Donglin Purple' variety of the iris cultivar.

[0042] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A method for quickly establishing a tissue culture system of the cultivar 'Donglinzi' of the Iris strychnifolia through underground buds, characterized in that: The following steps are involved: Step 1: Acquisition of explants: Select underground buds of the cultivar 'Donglinzi' of the Iris strychnifolia as explant materials; Step 2: Disinfection of explants: Disinfect underground buds; Step 3: Induction of clustered buds: Cut the sterilized underground buds into clustered bud medium MS + 0.5 mg / L 6-BA + 1.0 mg / L KT + 0.2 mg / L NAA + 30 g / L sucrose + 7.8 g / L agar; Step 4: Rooting culture: Inoculate the completely sterile clustered buds into the rooting medium MS + 0.5 mg / L NAA + 30 g / L sucrose + 7.0 g / L agar; The pH values ​​of all the above culture media were adjusted to 5.8-6.0, the temperature of the culture room was 25±1°C, the illumination time was 12h / d, and the illumination intensity was 2000-3000lx.

2. A method for rapidly establishing a tissue culture system of the cultivar 'Donglinzi' of the Xisun cultivar through underground buds according to claim 1, characterized in that The underground buds of the cultivar 'Donglinzi' described in step 1 refer to the underground buds obtained after the 'Donglinzi' seedlings are dug out from the field and the leaves and roots are trimmed after the underground buds are formed in autumn or before the underground buds sprout and grow in spring.

3. A method for rapidly establishing a tissue culture system of the cultivar 'Donglinzi' of the cultivar of Cnidium monnieri through underground buds according to claim 1, characterized in that The disinfection treatment described in step 2 refers to soaking the underground buds in claim 2 in 50% carbendazim wettable powder diluted 1500 times with tap water for 5 minutes, disinfecting with 75% ethanol solution for 30 seconds, and then disinfecting with 5% NaClO solution containing 0.1% Tween20 for 15 minutes, quickly cutting off the base of the outermost leaves of the underground buds, using the side of a blade to press the top of the underground buds, using tweezers to lightly clamp the base of the underground buds, and squeezing out the young and tender underground buds in the center of the underground buds.

4. A method for rapidly establishing a tissue culture system of the cultivar 'Donglinzi' of the Xisun cultivar through underground buds according to claim 1, characterized in that The induction of clustered buds described in step 3 refers to cutting the young underground buds in claim 3 into 2-3 pieces longitudinally, retaining the base of the underground buds and the young leaves as much as possible in each piece, and pressing the incision side down horizontally into the clustered bud culture medium. After 25 days, select the robust underground buds without bacteria, and insert them into the clustered bud culture medium with their morphological lower ends facing down to induce clustered buds.

5. A method for rapidly establishing a tissue culture system of the cultivar 'Donglinzi' of the Xisun cultivar through underground buds according to claim 1, characterized in that The rooting culture described in step 4 refers to inserting the base of the sterile clustered buds produced in claim 4 into a rooting medium to induce rooting.

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