Culture medium for tissue culture of endangered aquatic plant cauliflower and tissue culture method

Abstract

The invention provides a culture medium and a tissue culture method for tissue culture of endangered aquatic plant cauliflower, and the culture medium for tissue culture of endangered aquatic plant cauliflower comprises a cauliflower regeneration bud proliferation culture medium; the propagation culture medium for the regenerated buds of the water cauliflower comprises MS (Murashige and Skoog), 1.0 mg / L 6-BA, 0.2 mg / L IBA, 30g / L white sugar and 6g / L carrageenan; the method disclosed by the invention has the beneficial effects that the water cauliflower can be rapidly propagated by utilizing a tissue culture technology, a large number of excellent seedlings can be obtained in a short time, and a new technical approach is provided for the plant number of the water cauliflower and the realization of field regression.

Description

Technical Field

[0001] The invention relates to the technical field of tissue culture, and in particular to a culture medium and a tissue culture method for endangered aquatic plant water cabbage. Background Art

[0002] Ottelia cordata belongs to the genus Hydrocharitaceae and is an annual or perennial submerged herb[1] with well-developed fibrous roots and very short stems. The leaves are basal and heteromorphic; the submerged leaves are oblong, lanceolate or ribbon-shaped. The flowers are unisexual and dioecious, with white petals and pale yellow stamens. In 1999, Ottelia cordata was included in the "National Key Protected Wild Plant List (First Batch)" as a Class II protected species; in 2004, it was included in the "China Red List of Species". Ottelia cordata is mainly distributed in freshwater ditches and ponds in China, Thailand, Myanmar and Cambodia. In my country, it is only sporadically distributed in volcanic lava wetlands in Haikou, Wenchang and Chengmai in Hainan. Water rape likes shade and lives in ditches or ponds with clean flowing water. It is extremely sensitive to environmental changes and is one of the key indicator species for testing wetland environment and climate change. Due to the influence of human activities, environmental destruction and global warming, the environment suitable for the growth of water rape is becoming less and less. The survival status of water rape is not optimistic and it is listed as a second-level endangered plant.

[0003] Water rapeseed is propagated by seeds, and its germination rate is extremely low. The seeds of water rapeseed are small and will be washed away by running water after maturity. The seeds of water rapeseed are loved by snails and birds. The seeds are eaten by snails and birds before they are mature. In addition, the extremely low seed germination rate and environmental pollution have affected the survival of water rapeseed. Water rapeseed is becoming less and less and is extremely endangered. In order to protect water rapeseed, research on tissue culture rapid propagation technology has been carried out. Breeding a large number of seedlings is an important method to protect the survival of water rapeseed.

[0004] Currently, there is very little research on water rapeseed. Wu Tingtian et al. discussed the potential habitat selection and spatial pattern prediction of water rapeseed populations; Wang Luanfeng et al. studied the pollination biology of water rapeseed; Jin Quan et al. studied the chlorophyll fluorescence characteristics and HCO3- utilization ability of the heteromorphic leaves of water rapeseed; Liang Huiting et al. studied the community characteristics and plant diversity of water rapeseed in Hainan. However, there has been no report on the research on tissue culture technology of water rapeseed seedlings. Summary of the invention

[0005] The purpose of the present invention is to solve at least one of the above-mentioned deficiencies in the prior art. For example, one of the purposes of the present invention is to provide a tissue culture method for the endangered aquatic plant water cabbage; the method uses the tissue culture rapid propagation technology for water cabbage seedlings, and the method specifically provides a disinfection method suitable for water cabbage seeds, and provides an induction culture medium, proliferation and seedling rooting culture medium, which can obtain a large number of high-quality seedlings in a short period of time, and provide technical support for the protection and development and utilization of water cabbage germplasm resources.

[0006] In order to achieve the above-mentioned purpose, the present invention provides a tissue culture medium for the endangered aquatic plant water cabbage, including a water cabbage regeneration bud proliferation medium; the water cabbage regeneration bud proliferation medium comprises: MS, 1.0 mg / L 6-BA, 0.2 mg / L IBA, 30 g / L white sugar and 6 g / L carrageenan.

[0007] In a preferred embodiment of the present scheme, the culture medium for tissue culture of the endangered aquatic plant water cabbage also includes a water cabbage seed seedling induction medium; the water cabbage seed seedling induction medium includes: MS, 1.0 mg / L6-BA, 0.1 mg / LIBA, 30 g / L sugar and 7 g / L carrageenan.

[0008] In a preferred embodiment of the present scheme, the culture medium for tissue culture of the endangered aquatic plant water cabbage also includes a water cabbage tissue culture seedling rooting medium; the water cabbage tissue culture seedling rooting medium includes: MS, 0.5 mg / L NAA, 30 g / L sugar and 7 g / L carrageenan.

[0009] On the other hand, the present invention also provides a method for tissue culture of the endangered aquatic plant water rape, which utilizes the above endangered aquatic plant water rape tissue culture medium to carry out water rape tissue culture.

[0010] In a preferred embodiment of this scheme, the tissue culture method of the endangered aquatic plant water cabbage comprises the following steps:

[0011] Cut the regenerated buds of water cabbage into single clusters and inoculate them into the regenerated bud proliferation medium of water cabbage. Add sterile water so that the water level exceeds the explants. Then place them in a culture room at 26-28℃ and culture them in the dark for 5-7 days. Finally, place them in a culture room under the conditions of light intensity of 2000lx and photoperiod of 12-16h / d for regenerated bud proliferation culture.

[0012] In a preferred embodiment of the present scheme, the tissue culture method of the endangered aquatic plant water cabbage comprises the following steps:

[0013] S1. Obtain sterile seedlings

[0014] The water cauliflower seeds were sterilized and then inoculated into MS culture medium, and sterile seedlings were obtained after 20 to 25 days.

[0015] S2. Induction culture of water cabbage seedlings

[0016] Cut off 1 / 2 to 2 / 3 of the leaves of the sterile seedlings and inoculate them into a water cabbage seedling induction medium for water cabbage seedling induction culture to obtain water cabbage regeneration buds;

[0017] S3. Proliferation and culture of regenerated shoots of water cauliflower

[0018] Cutting the water cabbage flower regeneration buds into single clumps and inoculating them into a water cabbage flower regeneration bud proliferation medium for water cabbage flower regeneration bud proliferation culture to obtain regeneration bud seedlings;

[0019] S4. Rooting culture of water cauliflower seedlings

[0020] Cutting the water cauliflower tissue culture regeneration buds into single plants or small clusters of uniform size and inoculating them into the water cauliflower tissue culture seedling rooting medium for rooting culture to obtain rooted tissue culture seedlings;

[0021] S5. Hardening of water cauliflower tissue culture seedlings.

[0022] In a preferred embodiment of the present scheme, step S2 is specifically as follows: cut off 1 / 2 to 2 / 3 of the leaves of the sterile seed seedlings and inoculate them into the water cauliflower seed seedling induction culture medium, and pour in sterile water, the sterile water covering the induced explants, and then place them in a 26-28°C culture room for dark culture for 5-7 days, and then place them in a culture room under a light intensity of 2000lx and a light period of 12-16h / d for 30 days to obtain water cauliflower regeneration shoots.

[0023] In a preferred embodiment of the present scheme, step S3 is specifically as follows: cutting the water cabbage regeneration buds into single clusters and inoculating them into the water cabbage regeneration bud proliferation culture medium, adding sterile water, the sterile water covering the explants, and then placing them in a 26-28°C culture room for dark culture for 5-7 days, and then placing them under a light intensity of 2000lx and a light period of 12-16h / d for 30 days to obtain regenerated seedlings.

[0024] In a preferred embodiment of the present scheme, step S4 is specifically as follows: the water cabbage tissue culture regenerated seedlings are cut into single plants or small clusters of uniform size and inoculated into the water cabbage tissue culture seedling rooting medium. After inoculation, they are placed in a culture room at 26-28°C for dark culture for 5-7 days, and then placed in a culture room under light intensity of 3000lx and a light period of 12-16h / d for culture to obtain rooted tissue culture seedlings.

[0025] In a preferred embodiment of the present scheme, the step S5 specifically comprises: moving the rooted tissue culture seedlings of water rape in a vase to a refraction place for 6 to 8 days, taking out the tissue culture seedlings, washing the root culture medium and transplanting them into planting soil, watering them to cover the plants, moving them to a 60% shade net to harden the seedlings for 12 to 15 days, and then culturing them under natural light to obtain transplanted water rape seedlings; wherein the planting soil is a mixture of fine river sand and red soil, and the ratio of fine river sand to red soil is 2:1.

[0026] In a preferred embodiment of the present solution, the step S1 further includes step S0:

[0027] S0. Obtaining seeds

[0028] Pick the ripe cauliflower pods, rinse them under the tap, then dry them in an oven at 35°C, remove the peel, collect the seeds and save them for later use.

[0029] Compared with the prior art, the beneficial effects of the present invention include at least one of the following:

[0030] (1) This scheme provides a tissue culture method for the endangered aquatic plant water cabbage, which provides a disinfection method suitable for water cabbage seeds, and provides an induction culture medium, a proliferation medium and a seedling rooting medium. Therefore, this scheme can use tissue culture technology to propagate water cabbage, and a large number of high-quality seedlings can be obtained in a short period of time, providing technical support for the protection and development and utilization of water cabbage germplasm resources;

[0031] (2) The present invention provides a culture medium for tissue culture of the endangered aquatic plant water cabbage, which includes a water cabbage regeneration bud proliferation medium (MS+1.0mg / L 6-BA+0.2mg / LIBA+30g / L sugar+6g / L carrageenan). The water cabbage regeneration bud proliferation culture is used for regeneration bud proliferation culture, and the average proliferation coefficient is 8.2 times, and the sprouts grow robustly; and the proliferation coefficient of the water cabbage regeneration bud proliferation medium is much higher than that of the regeneration bud proliferation medium with other ratios of IBA and 6-BA, that is, in the present invention, the ratio of IBA to 6-BA is 1:5, which produces synergistic effect. The water cabbage regeneration bud proliferation medium may achieve the best cell division activity and bud proliferation efficiency in the target plant by precisely regulating the synergistic effect of auxin and cytokinin. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The above and other objects and / or features of the present invention will become more apparent through the following description in conjunction with the accompanying drawings, in which:

[0033] Figure 1 A comparison chart of the disinfection contamination rate and germination rate of water cauliflower seeds of an exemplary embodiment of a culture medium and tissue culture method for water cauliflower, an endangered aquatic plant, of the present invention is shown.

[0034] Figure 2 A comparison chart showing the induction of water cabbage seedlings using different induction culture media according to an exemplary embodiment of a culture medium for tissue culture of the endangered aquatic plant water cabbage and a tissue culture method of the present invention.

[0035] Figure 3 A comparison chart showing the effect of different proliferation culture media on the proliferation of water cauliflower tissue culture seedlings of an exemplary embodiment of a culture medium and tissue culture method for the endangered aquatic plant water cauliflower of the present invention.

[0036] Figure 4 A comparison chart showing the rooting of water cabbage tissue culture seedlings using different rooting culture media according to an exemplary embodiment of a culture medium and tissue culture method for the endangered aquatic plant water cabbage of the present invention. DETAILED DESCRIPTION

[0037] Hereinafter, a culture medium and a tissue culture method for the endangered aquatic plant water cabbage of the present invention will be described in detail in conjunction with exemplary embodiments.

[0038] Example 1

[0039] A tissue culture method for an endangered aquatic plant, water cabbage, comprises the following steps:

[0040] Cut the regenerated buds of water cabbage into single clusters and inoculate them into water cabbage regeneration bud proliferation medium (MS, 1.0 mg / L6-BA, 0.2 mg / LIBA, 30 g / L white sugar and 6 g / L carrageenan), add sterile water, the water level exceeds the explant, and then place it in a culture room at 26-28°C for dark culture for 5-7 days, and finally place it in a culture room under light intensity of 2000 lx and photoperiod of 12-16 h / d for regeneration bud proliferation culture.

[0041] Example 2

[0042] A tissue culture method for an endangered aquatic plant, water cabbage, specifically comprises the following steps:

[0043] S0. Obtaining seeds

[0044] Pick the ripe pods of water cauliflower, rinse them under the tap, and then dry them in an oven at 35℃, remove the peels, and collect the seeds for later use;

[0045] S1. Obtain sterile seedlings

[0046] The water cauliflower seeds were sterilized and then inoculated into MS culture medium, and sterile seedlings were obtained after 20 to 25 days.

[0047] S2. Induction culture of water cabbage seedlings

[0048] Cut off 1 / 2 to 2 / 3 of the leaves of the sterile seedlings and inoculate them into water cauliflower seedling induction medium (MS, 1.0 mg / L 6-BA, 0.1 mg / L 1 BA, 30 g / L sugar and 7 g / L carrageenan) to carry out water cauliflower seedling induction culture to obtain water cauliflower regeneration buds;

[0049] S3. Proliferation and culture of regenerated shoots of water cauliflower

[0050] Cut the water cauliflower regeneration buds into single clumps and inoculate them into water cauliflower regeneration bud proliferation medium (MS, 1.0 mg 6-BA, 0.2 mg / L 1 BA, 30 g / L white sugar and 6 g / L carrageenan) to perform water cauliflower regeneration bud proliferation culture to obtain regeneration bud seedlings;

[0051] S4. Rooting culture of water cauliflower seedlings

[0052] Cut the water cauliflower tissue culture regeneration seedlings into single plants or small clusters of uniform size and inoculate them into water cauliflower tissue culture seedling rooting medium (MS, 0.5 mg / L NAA, 30 g / L sugar and 7 g / L carrageenan) for rooting culture to obtain rooted tissue culture seedlings;

[0053] S5. Hardening of water cauliflower tissue culture seedlings.

[0054] In this exemplary embodiment, step S2 is specifically as follows: cut off 1 / 2 to 2 / 3 of the leaves of the sterile seed seedlings and inoculate them into the water cauliflower seed seedling induction culture medium, and pour in sterile water, the sterile water covering the induced explants, place them in a culture room at 26 to 28°C, first culture them in the dark for 5 to 7 days, and then culture them under the conditions of a light intensity of 2000lx and a light period of 12 to 16h / d for 30 days to obtain water cauliflower regeneration shoots.

[0055] In this exemplary embodiment, step S3 is specifically as follows: cutting the water cauliflower regeneration buds into single clusters and inoculating them into the water cauliflower regeneration bud proliferation culture medium, adding sterile water, the sterile water covering the explants, and then placing them in a 26-28°C culture room for dark culture for 5-7 days, and then placing them in a culture room under a light intensity of 2000lx and a light period of 12-16h / d for 30 days to obtain regenerated seedlings.

[0056] In this exemplary embodiment, step S4 is specifically as follows: the water cabbage tissue culture regenerated seedlings are cut into single plants or small clusters of uniform size and inoculated into the water cabbage tissue culture seedling rooting medium. After inoculation, they are placed in a culture room at 26-28°C for dark culture for 5-7 days, and then placed under a light intensity of 3000lx and a photoperiod of 12-16h / d to obtain rooted tissue culture seedlings.

[0057] In this exemplary embodiment, step S5 is specifically as follows: moving the rooted tissue culture seedlings in the water lettuce vase to a place with refraction for 6 to 8 days, and then gradually moving them to a place with better light to harden the seedlings for 6 to 8 days to obtain tissue culture seedlings; taking out the tissue culture seedlings, washing the root culture medium, and transplanting them into a flowerpot, watering them to cover the plants, and then culturing them under natural light.

[0058] Furthermore, in this exemplary embodiment, the step S5 is specifically as follows: moving the rooted tissue culture seedlings in the water cabbage vase to a refraction place for 6 to 8 days, taking out the tissue culture seedlings, washing the root culture medium and transplanting them into the planting soil, watering them to cover the plants, moving them to a 60% shade net to harden the seedlings for 12 to 15 days, and then culturing them under natural light; wherein the planting soil is a mixture of fine river sand and red soil, and the ratio of fine river sand to red soil is 2:1.

[0059] In order to better understand the above exemplary embodiments, they are further described below with reference to specific examples.

[0060] Example

[0061] Using water rape seeds, after disinfection, sterile seedlings were obtained to carry out experimental research on tissue culture rapid propagation technology, explore the disinfection method of water rape seeds, induction culture medium, proliferation and seedling rooting culture medium, obtain tissue culture rapid propagation method, and use tissue culture technology to reproduce water rape.

[0062] This experimental study specifically includes the following steps:

[0063] 1. Obtaining seeds

[0064] Pick the mature water cauliflower seeds (the water cauliflower seeds used in this experiment are from the Aquatic Plant Resource Garden of the Institute of Tropical Crop Variety Resources, Chinese Academy of Tropical Agricultural Sciences) and bring them back to the laboratory for later use. Rinse the pods under the tap, dry them in an oven at 35°C, remove the peel and save the seeds for later use.

[0065] 2. Seed disinfection

[0066] The seeds were placed in 100 ml Erlenmeyer flasks and disinfected using different methods (see Table 1). The disinfected seeds were inoculated into MS culture medium and cultured in a dark room at 28°C for 7 days to calculate the contamination rate.

[0067] Contamination rate = number of contaminated seeds / number of inoculated seeds*100%; germination rate = number of germinated uncontaminated seeds / number of uncontaminated seeds.

[0068] Table 1 Different disinfection methods for water cauliflower seeds

[0069]

[0070]

[0071] 3. Screening of induction medium for water cauliflower seedlings

[0072] The seeds disinfected by method A3 in step 2 were inoculated into MS medium, and sterile seedlings were obtained after 25 days. Then, MS medium was used as the basal medium, and different plant growth regulators (see Table 2) were added, sucrose 30 g / L, carrageenan 7 g / L were added, and the pH value was 5.8. 1 / 2 to 2 / 3 of the leaves of the sterile seedlings were cut off and inoculated into various induction mediums, and sterile water was poured in to just cover the induced explants. There was one control and three treatments, 2 explants were inoculated in each bottle, 10 bottles were inoculated in each treatment, and 3 replicates were placed in a 28°C culture room for dark culture for 7 days, and then cultured under the conditions of a light intensity of 2000 lx and a photoperiod of 12 h / d. The induction rate was calculated after 30 days.

[0073] Table 2 Addition of different plant growth regulators to induce culture medium

[0074] Culture medium number Induction medium B1 MS+30g / L sugar+7g / L carrageenan B2 MS+1.0mg / L6-BA+30g / L sugar+7g / L carrageenan B3 MS+1.0mg / L6-BA+0.1mg / LNAA+30g / L sugar+7g / L carrageenan B4 (Example) MS+1.0mg / L6-BA+0.1mg / LIBA+30g / L sugar+7g / L carrageenan B5 MS+1.0mg / L6-BA+0.1mg / LIAA+30g / L sugar+7g / L carrageenan

[0075] 4. Effect of adding different concentrations of 6-BA and IBA culture medium on the proliferation of water cauliflower regeneration shoots

[0076] Cut the regenerated buds of water cauliflower obtained in step 3 B4 into single clumps and inoculate them into 9 different proliferation culture media (see Table 3) to simulate the growth environment of water cauliflower. Each explant was inoculated into a solid culture medium, and then sterile water was added to exceed the explant. Ten bottles were inoculated for each treatment, with 2 explants per bottle. The buds were placed in a dark culture room at 28°C for 7 days, and then placed in a culture room with a light intensity of 2000lx and a photoperiod of 12h / d for culture. The results were observed and recorded after 30 days of culture.

[0077] Table 3 MS medium supplemented with different concentrations of 6-BA and IBA

[0078]

[0079]

[0080] 5. Effects of different rooting media on the rooting of water cauliflower tissue culture seedlings

[0081] The water cauliflower tissue culture regeneration seedlings obtained in step 4 C5 were cut into single plants or small clusters of uniform size and inoculated into different rooting mediums (see Table 4), with MS medium as the basic medium, 30 g / L sugar and 7 g / L carrageenan added. After inoculation, they were placed in a 28°C culture room for dark culture for 7 days, and then placed in a culture condition with a light intensity of 3000 lx and a photoperiod of 12 h / d for culture. The results were observed and recorded after 30 days of culture.

[0082] Table 4 Addition of different hormones and different concentrations of rooting medium

[0083] Culture medium number Different rooting media D1 MS+30g / L sugar+7g / L carrageenan D2 MS+0.1mg / LNAA+30g / L sugar+7g / L carrageenan D3 (Example) MS+0.5mg / LNAA+30g / L sugar+7g / L carrageenan D4 MS+1.0mg / LNAA+30g / L sugar+7g / L carrageenan D5 MS+0.1mg / LIBA+30g / L sugar+7g / L carrageenan D6 MS+0.5mg / LIBA+30g / L sugar+7g / L carrageenan D7 MS+1.0mg / LIBA+30g / L sugar+7g / L carrageenan

[0084] 6. Effects of different substrates on the hardening of water cauliflower tissue culture seedlings

[0085] The rooted tissue culture seedlings of water cabbage obtained in D3 in step 5 were moved to a refraction place for 7 days, the tissue culture seedlings were taken out and the root culture medium was washed, and then transplanted into the planting soil, and placed in 4 non-empty plastic flower pots with different seedling hardening formulas. The planting soils of the 4 methods were E1: water, E2: sand, E3 was red soil, and E4: sand: red soil = 1:1. Watering just covered the plants, and the water should not be too deep, otherwise the seedlings would float. Ten pots were planted for each treatment, with 1 plant in each pot, and repeated 3 times. The seedlings were moved to a 60% shade net for 12 days of hardening, and then cultured under natural light. The survival rate was counted after 30 days.

[0086] Results and Analysis

[0087] 1. Effects of different disinfection methods on the disinfection of water cauliflower seeds

[0088] As shown in Table 5, the contamination rate of disinfection method control A1 is the highest, reaching 92%, and the germination rate of uncontaminated seeds is 72%; the contamination rate of disinfection method A2 is 50%, and the germination rate of uncontaminated seeds is 69%; the contamination rate of disinfection method A3 is 10%, and the germination rate of uncontaminated seeds is 67.9%; the contamination rate of A4 is 7%, and the germination rate of uncontaminated seeds is 65.2%; the contamination rate of disinfection method A5 reaches 85%, and the germination rate of uncontaminated seeds is 48.1%; the contamination rate of disinfection method A6 is 76%, and the germination rate is 45.2%; the contamination rate of disinfection method A7 is 65%, and the germination rate of uncontaminated seeds is 38.5%. From the research results, it can be seen that sodium hypochlorite is more suitable as a disinfectant for water cabbage seeds than fungicide S206, with a low contamination rate and a relatively high uncontaminated germination rate. Since the germination rate of water cabbage seeds themselves is low, the germination rate after disinfection is affected.

[0089] In summary, A3 is the best disinfection method for water cauliflower, with a low contamination rate and a high germination rate for uncontaminated seeds. The best disinfection method for water cauliflower seeds as explants is A3: wash with sterile water 5 times → disinfect with 75% alcohol for 1 min → wash with sterile water 5 times → disinfect with 20% sodium hypochlorite for 20 min and inoculate into MS culture medium → wash with sterile water 5 times → inoculate into MS culture medium.

[0090] Table 5 Comparison of the results of different disinfection methods for water cauliflower seeds

[0091] Disinfection methods Contamination rate Uncontaminated seed germination rate A1 92 72 A2 50 69 A3 (Example) 10 67.5 A4 7 65.2 A5 85 48.1 A6 76 45.2 A7 65 38.5

[0092] 2. Effects of adding different plant growth regulators to culture medium on the induction of water cauliflower seedlings

[0093] From Table 6 and Figure 2It can be seen that the effects of adding different plant growth regulators to the induction of water cauliflower seed seedlings are very different. In the MS medium B1 without any plant growth regulator, only buds grew, the induction rate of induced regenerated buds was 0, there was no callus, and the seedlings were weak; in the MS medium B2 with the plant growth regulator 6-BA, a small number of 2 clusters of regenerated buds were found, the number of clustered buds was small, there was no callus, the seedlings were good, and the induction rate was only 23%; in the MS medium B3 with the plant growth regulator 6-BA and NAA, 1-3 clusters of regenerated seedlings, no callus, yellowish seedlings, a bit watery, not conducive to the subculture of clusters, 2 clusters of buds induction rate is only 35%; 91% of the MS medium B4 with plant growth regulators 6-BA and IBA can induce 3-5 clusters of regenerated seedlings, the seedlings are green, strong, growing well, with a few roots, but not affecting differentiation; 72% of the MS medium B5 with plant growth regulators 6-BA and IAA can induce 2-3 clusters of regenerated seedlings, but the seedlings are yellowish, affecting the differentiation of proliferation culture.

[0094] Therefore, the most suitable plant growth regulator for inducing water cauliflower seedlings is the culture medium supplemented with a combination of 6-BA and IBA.

[0095] Table 6 Effects of different plant growth regulator culture media on the induction of water cauliflower seedlings

[0096] Culture medium number Induction rate (%) Bud growth B1 0 Single sprout, no callus, no clustered sprouts, and the sprouts are weak. B2 23 There are 1-2 clusters of regenerated seedlings, with more seedlings in 1 cluster and a few seedlings in 2 clusters. There is no callus and the seedlings are in good condition. B3 35 There are 1-3 clusters of regenerated seedlings, without callus, the seedlings are yellowish and a bit watery. B4 (Example) 91 3-5 clusters of regenerated seedlings, no callus, the seedlings are green and strong, with a few roots, which does not affect the differentiation of cluster seedlings. B5 72 2-3 clusters of regenerated seedlings, no callus, and yellowish seedlings.

[0097] 3. Effects of different proliferation media on the proliferation of water cauliflower tissue culture seedlings

[0098] From Table 7 and Figure 3 It can be seen that the proliferation coefficient of the regenerated buds of water cauliflower in C1 medium is only 1.7 times, the regenerated buds are green, and some of the old leaves on the edge are yellow. Cutting off the old leaves does not affect the subculture; the proliferation coefficient of the regenerated buds of water cauliflower in C2 medium is 3.7 times, the regenerated buds are strong and growing well; the proliferation coefficient in C3 medium is 2.1, the regenerated buds are weak, and yellow leaves appear, which affects the subculture of the regenerated buds; the proliferation coefficient in C4 medium is 2.6 times, most of the regenerated buds are strong, a few have yellow leaves, and cutting off the yellow leaves does not affect the subculture. It does not affect subculture; in C5 culture medium, the proliferation coefficient is as high as 8.2 times, the regenerated seedlings are strong, and a few have yellow leaves. Removing the yellow leaves does not affect subculture; in C6 culture medium, the proliferation coefficient is 3.0 times, the regenerated seedlings are yellowish, which is not conducive to subculture; in C7 culture medium, the proliferation coefficient is 2.2, the regenerated seedlings are green, and the growth is weak; in C8 culture medium, the proliferation coefficient is 3.9 times, the regenerated seedlings are green and strong; in C9 culture medium, the proliferation coefficient is only 2.1 times, the leaves of the regenerated seedlings are yellow and die.

[0099] From the comparison of the experimental results of C1, C4, and C7, the comparison of the experimental results of C2, C5, and C8, and the comparison of the experimental results of C3, C6, and C9, it can be seen that the IBA concentration of 0.2 is the optimal IBA concentration for the cultivation of regenerated buds.

[0100] From the comparison of the experimental results of C1, C2, and C3, the comparison of the experimental results of C4, C5, and C6, and the comparison of the experimental results of C7, C8, and C9, it can be seen that the proliferation coefficient of the regenerated buds of water cauliflower increases with the increase of the hormone 6-BA concentration, and decreases after reaching a certain value, affecting the proliferation of the regenerated buds. When the hormone 6-BA is 1.0 mg / L, the proliferation coefficient of the regenerated buds of water cauliflower is the largest, and the regenerated buds grow robustly; the hormone IBA has a great effect on the proliferation of the regenerated buds of water cauliflower. When the concentration of IBA is 0.1 mg / L, the average proliferation coefficient does not exceed 2.6. When the IBA concentration is 0.2mg / L, the average proliferation coefficient exceeds 3.9, and the regenerated buds are strong and growing well; when the IBA concentration is 0.4mg / L, the regenerated buds grow weakly; the proliferation medium combined with hormones 1.0mg / L6-BA and 0.2mg / LIBA is more suitable for the proliferation of water cauliflower regeneration buds, with an average proliferation coefficient of 8.2 times, and the buds grow strong. Therefore, the best proliferation medium is C5: MS+1.0mg / L6-BA+0.2mg / LIBA+30g / L sugar+6g / L carrageenan. It is worth noting that when the hormones 1.0mg / L6-BA and 0.2mg / LIBA are combined (that is, when the ratio of 6-BA to IBA is 5:1), the proliferation coefficient is much higher than the other ratios of the formula combination, which shows that 6-BA and IBA in a specific ratio have a synergistic effect.

[0101] Table 7 Effects of different proliferation media on the proliferation of water cauliflower tissue culture seedlings

[0102] Culture medium number Proliferation coefficient Proliferation seedlings C1 1.3 The proliferation coefficient is 1.7 times, the regenerated buds are green, and the edges of the old leaves are partially yellow. C2 3.7 The proliferation coefficient is 3.7 times, the regenerated sprouts are strong and green C3 2.1 The proliferation coefficient is 2.1, the regenerated shoots are weak, and some leaves are yellowing. C4 2.6 The proliferation coefficient is 2.6. Most of the regenerated shoots are strong, and a few have yellow leaves. C5 (Example) 8.2 The proliferation coefficient is 8.2, the regenerated seedlings are strong, and a few of them have yellow leaves. C6 3.0 The proliferation coefficient is 3.0, and the regenerated sprouts are yellowish, which is not conducive to subculture. C7 2.2 The proliferation coefficient is 2.2, the regenerated seedlings are green and the growth is weak C8 3.9 The proliferation coefficient is 3.9, the regenerated sprouts are green and strong C9 2.1 The proliferation coefficient is 2.1, and the leaves of the regenerated seedlings are yellow.

[0103] 4. Effects of different rooting media on the rooting of water cauliflower tissue culture seedlings

[0104] As shown in Table 8, the effects of adding different concentrations of NAA and IBA rooting medium on the rooting of water cauliflower tissue culture seedlings were significantly different. In the control D1 medium without adding any hormones, the water cauliflower tissue culture seedlings had not taken root at 30 days; after adding NAA and IBA to the medium, the water cauliflower tissue culture seedlings all took root, and the rooting effect of adding NAA was better than that of IBA. When the NAA concentration was 0.5 mg / L, roots began to grow on the 15th day, the earliest rooting, and the average rooting rate was 85%. The growth condition of roots and seedlings was the best, the growth was good, and the number of roots was significantly higher than that of other treatment groups, reaching 3 to 8; when the NAA concentration was 0.1 mg / L, roots began to grow on the 17th day, the root state was better, there were 1 to 4 roots, and the rooting rate was 53%; when the NAA concentration was 1.0 mg / L, roots began to grow on the 20th day, the root state was better, there were 2 to 5 roots, and the rooting rate was 48.67%; It can be seen that the rooting rate of water cauliflower tissue culture seedlings increased with the increase of NAA concentration, but after adding NAA to a certain concentration, the rooting rate decreased with the increase of NAA concentration. When the IBA concentration was 0.1 mg / L, roots began to grow on the 21st day, the root state was better, there were 1 to 4 roots, and the rooting rate was 47.67%; when the IBA concentration was 0.5 mg / L, roots began to grow on the 23rd day, the root state was average, there were 1 to 3 roots, and the rooting rate was 35.33%; when the IBA concentration was 1.0 mg / L, roots began to grow on the 24th day, the root state was average, there were 1 to 2 roots, and the rooting rate was 23.33%. It can be seen that the best rooting medium for water cauliflower is D3: MS + 0.5 mg / L NAA + 30 g / L sugar + 7 g / L carrageenan.

[0105] Table 8 Effects of different rooting media on the rooting of water lettuce

[0106]

[0107]

[0108] 5. Effects of different cultivation media on transplanting of water cauliflower tissue culture seedlings

[0109] As can be seen from Table 9, the survival rate of water cabbage seedlings hardened in E1 water is low, the water is insufficient in nutrients, and after a long time, the leaves of the water cabbage seedlings become water-soaked, the leaves become thinner and yellower, affecting the photosynthesis of the leaves and causing the death of the plants; the average survival rate of the tissue culture seedlings hardened in E2 sand is 66.7%, the leaves are green in the early stage, the sand is insufficient in nutrients, and after a long time, the seedlings slowly turn yellow, affecting the growth of the seedlings; the average survival rate of the tissue culture seedlings hardened in E3 red soil is 46.7%, because water cabbage likes to grow in clean water, the water in the red soil is turbid, which affects the growth of water cabbage, the leaves are yellowish, and the survival rate is not high; the average survival rate of the tissue culture seedlings hardened in E4 fine river sand: red soil = 2:1 is 86.7%, the leaves are green, the water cabbage seedlings are strong, and the survival rate is high, which is suitable as a method for hardening water cabbage seedlings.

[0110] Table 9 Effects of different cultivation media on transplanting of water cauliflower tissue culture seedlings

[0111] Culture medium number Average survival rate (%) Seedling growth E1 20 The survival rate of water cauliflower tissue culture seedlings in water is low, and the leaves become water-soaked, thin, and die. E2 66.7 The leaves are green at first, but slowly turn yellow over time, affecting the growth of the seedlings E3 46.7 The leaves are yellowish and the soil is dirty, which affects the growth of water cauliflower seedlings E4 (Example) 86.7 The leaves are green, the survival rate is high, and the plants are strong.

[0112] in conclusion:

[0113] Water cabbage is a national second-level wild protected aquatic plant. It is extremely sensitive to environmental changes and its survival is not optimistic. In my country, it is only scattered in the volcanic lava wetland area in northern Hainan, and the number is very small. However, the relevant research progress is very slow, and there are no reports on tissue culture technology research. Water cabbage is affected by the habitat and is not easy to survive in artificial cultivation. It requires a clean flowing water environment. This experiment established a tissue culture rapid propagation system for water cabbage, providing a new technical approach for the number of water cabbage plants and the realization of wild return.

[0114] In this experiment, water cauliflower seedlings were used as explants. The selection of disinfectants for explants and the length of disinfection time not only affect the later contamination rate, but also the germination rate of the seeds. This experiment used 20% sodium hypochlorite and fungicide 2% S206 as comparative disinfectants, and the disinfection time was 10min, 20min and 30min respectively. The results showed that sodium hypochlorite was more suitable as a disinfectant for water cauliflower seeds. The average contamination rate of 20% sodium hypochlorite disinfection for 20min was 10%, and the germination rate was 69%. Although the contamination rate decreased with the increase of sodium hypochlorite disinfection time, the germination rate gradually decreased. This is because as the disinfection time increases, the disinfectant will have a certain toxic effect on the plant. In this experiment, the best disinfection method for water cauliflower seeds is: sterile water washing 5 times → 75% alcohol disinfection 1min → sterile water washing 5 times → 20% sodium hypochlorite disinfection 20min inoculated into MS culture medium → sterile water washing 5 times → inoculated into MS culture medium

[0115] Plant growth regulators play a major role in tissue culture and rapid propagation. Different plants use different types and concentrations of hormones at different stages of tissue culture. In tissue culture experiments, cell cytokinins can induce the formation of adventitious buds, and auxins can promote the rooting of plants. In the process of inducing water cabbage seedlings, the combination of 6-BA and IBA has the best induction rate. 91% of the explants can induce 3-5 regenerated buds, and the buds are strong. The experiment also found that 6-BA has a significant effect on the proliferation of water cabbage regeneration buds. When the concentration of IBA is constant, the proliferation coefficient of water cabbage regeneration buds will increase first and then decrease with the increase of 6-BA concentration; when the concentration of 6-BA is constant, IBA also has a great effect on the proliferation rate of water cabbage regeneration buds. With the increase of IBA concentration, the proliferation coefficient of water cabbage regeneration buds will increase first and then decrease. The test results show that the best proliferation medium for water cabbage regeneration buds is C5: MS+1.0mg / L6-BA+0.2mg / LIBA+30g / L sugar+6g / L carrageenan.

[0116] Plant growth regulators have an important influence on the rooting of tissue culture seedlings. NAA, IBA, and IAA are commonly used hormones for tissue culture rooting. Using one or more of them can promote rooting. In the experiment, it was found that the rooting effect of adding NAA in the rooting stage of water cauliflower was better than that of IBA, with a high rooting rate, the highest average rooting rate of 85%, early rooting, a large number of roots, and good root and seedling growth; while the rooting effect of adding IBA was average, with a highest average rooting rate of 47.67%, and slow rooting. Therefore, NAA is suitable as a rooting hormone for water cauliflower. Transplant test tube seedlings with fine river sand, red soil, and a mixture of fine river sand and red soil as the matrix. The survival rate of water cauliflower tissue culture seedlings with fine river sand: red soil = 2:1 was the highest, with an average survival rate of 86.7%, green leaves, and strong water cauliflower tissue culture seedlings.

[0117] The results of this experiment show that, with water cauliflower seedlings as explants, the average contamination rate was 10% when disinfected with 75% alcohol for 1 minute and 20% sodium hypochlorite for 20 minutes, but the average germination rate of non-contaminated seeds was as high as 67.5%; the suitable induction medium was B4: MS + 1.0mg / L6-BA + 0.1mg / LIBA + 30g / L sugar + 7g / L carrageenan, with an induction rate of 91%; the best proliferation medium was C5: MS + 1.0mg / L6-BA + 0.2mg / LIBA + 30g / L white sugar + 6g / L carrageenan, with a proliferation coefficient of 8.2; the best rooting medium was D3: MS + 0.5mg / LNAA + 30g / L sugar + 7g / L carrageenan, with an average rooting rate of 85%; the most suitable transplanting method was E4: fine river sand: red soil = 2:1, and the survival rate of water cauliflower tissue culture seedlings was the highest, with an average survival rate of 86.7%.

[0118] Although the present invention has been described above in conjunction with the exemplary embodiments and the accompanying drawings, it should be apparent to those skilled in the art that various modifications may be made to the above-described embodiments without departing from the spirit and scope of the claims.

Claims

1. A culture medium for tissue culture of the endangered aquatic plant water cabbage, characterized in that: It includes a water cauliflower regeneration bud proliferation culture medium; The water cauliflower regeneration bud proliferation culture medium comprises: MS, 1.0 mg / L 6-BA, 0.2 mg / L IBA, 30 g / L white sugar and 6 g / L carrageenan.

2. The culture medium for tissue culture of the endangered aquatic plant water cabbage according to claim 1, characterized in that: Also included is a water cauliflower seed seedling induction medium; The water cauliflower seed seedling induction culture medium comprises: MS, 1.0 mg / L 6-BA, 0.1 mg / L I BA, 30 g / L sugar and 7 g / L carrageenan.

3. The culture medium for tissue culture of the endangered aquatic plant water cabbage according to claim 2, characterized in that: Also included is a rooting medium for tissue culture seedlings of water cauliflower; The water cauliflower tissue culture seedling rooting medium comprises: MS, 0.5 mg / L NAA, 30 g / L sugar and 7 g / L carrageenan.

4. A tissue culture method for the endangered aquatic plant water cabbage, characterized in that: The endangered aquatic plant water cabbage tissue culture medium as claimed in any one of claims 1 to 3 is used to carry out water cabbage tissue culture.

5. The tissue culture method of the endangered aquatic plant water cabbage according to claim 4, characterized in that: The following steps are involved: Cut the regenerated buds of water cabbage into single clusters and inoculate them into the regenerated bud proliferation medium of water cabbage. Add sterile water so that the water level exceeds the explants. Then place them in a culture room at 26-28℃ and culture them in the dark for 5-7 days. Finally, place them in a culture room under the conditions of light intensity of 2000lx and photoperiod of 12-16h / d for regenerated bud proliferation culture.

6. The tissue culture method of the endangered aquatic plant water cabbage according to claim 4, characterized in that: The specific steps include: S1. Obtain sterile seedlings The water cauliflower seeds were sterilized and then inoculated into MS culture medium, and sterile seedlings were obtained after 20 to 25 days. S2. Induction culture of water cabbage seedlings Cut off 1 / 2 to 2 / 3 of the leaves of the sterile seedlings and inoculate them into a water cabbage seedling induction medium for water cabbage seedling induction culture to obtain water cabbage regeneration buds; S3. Proliferation culture of regenerated shoots of water cauliflower Cutting the water cabbage flower regeneration buds into single clumps and inoculating them into a water cabbage flower regeneration bud proliferation medium for water cabbage flower regeneration bud proliferation culture to obtain regeneration bud seedlings; S4. Rooting culture of water cauliflower seedlings Cutting the water cauliflower tissue culture regeneration buds into single plants or small clusters of uniform size and inoculating them into the water cauliflower tissue culture seedling rooting medium for rooting culture to obtain rooted tissue culture seedlings; S5. Hardening of water cauliflower tissue culture seedlings.

7. The tissue culture method of the endangered aquatic plant water cabbage according to claim 6, characterized in that: The step S2 is specifically as follows: Cut off 1 / 2 to 2 / 3 of the leaves of the sterile seed seedlings and inoculate them into the water cauliflower seed seedling induction culture medium, and pour in sterile water to cover the induced explants. Then place them in a culture room at 26 to 28°C and culture them in the dark for 5 to 7 days, and then culture them under the conditions of a light intensity of 2000lx and a photoperiod of 12 to 16h / d for 30 days to obtain the regenerated shoots of water cauliflower.

8. The tissue culture method of the endangered aquatic plant water cabbage according to claim 6, characterized in that: The step S3 is specifically as follows: Cut the regenerated buds of water cabbage into single clusters and inoculate them into the proliferation medium of the regenerated buds of water cabbage. Add sterile water to cover the explants, then place them in a culture room at 26-28°C and culture them in the dark for 5-7 days, then place them in a culture room with a light intensity of 2000lx and a photoperiod of 12-16h / d for 30 days to obtain regenerated seedlings.

9. The tissue culture method of the endangered aquatic plant water cabbage according to claim 6, characterized in that: The step S4 is specifically as follows: The water rape tissue culture regenerated seedlings are cut into single plants or small clusters of uniform size and inoculated into the water rape tissue culture seedling rooting medium. After inoculation, they are placed in a 26-28°C culture room for dark culture for 5-7 days, and then placed under a light intensity of 3000lx and a photoperiod of 12-16h / d to obtain rooted tissue culture seedlings.

10. The tissue culture method of the endangered aquatic plant water cabbage according to claim 6, characterized in that: The step S5 is specifically as follows: Move the rooted tissue culture seedlings in the vase to a place with refraction for 6 to 8 days, take out the tissue culture seedlings, wash the root culture medium, and transplant them into the planting soil, water them to cover the plants, move them to a 60% shade net to harden the seedlings for 12 to 15 days, and then place them under natural light for cultivation; The planting soil is a mixture of fine river sand and red soil, and the ratio of fine river sand to red soil is 2:1.

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