Tissue culture rapid propagation and adventitious bud regeneration method for cerasus yunnanensis

Abstract

According to the tissue culture and rapid propagation and adventitious bud regeneration method, a tissue culture and rapid propagation and organ regeneration system of the Cerasus serrulata is established for the first time, stems, leaves or seeds of nutritive organs of the Cerasus serrulata are used as explants, the primary induction rate reaches up to 74.4%, the propagation coefficient is about 3.9, the rooting rate reaches 83.5%, efficient and rapid propagation can be achieved, and the tissue culture and rapid propagation of the Cerasus serrulata can be realized. Strong buds without rooting can also be used as scions for grafting propagation. In the seed disinfection process, the plant tissue culture antibacterial agent PPM is applied to cerasus plant seed disinfection for the first time, the pollution rate is well controlled, the pollution rate is remarkably reduced, the reuse times of a mercury bichloride disinfectant are discussed for the first time, and it is found that the disinfection effect is reduced along with increase of the reuse times when the seeds are disinfected. It is found that the disinfection effect of mercury bichloride used for three or more times is remarkably reduced. In addition, the same culture medium is used for seed sterile explant obtaining, sterile seedling induction culture and multiplication culture, the operation process is simplified, and the production cost is reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of tissue culture, and particularly relates to a method for rapid propagation of cherry blossoms through tissue culture and adventitious bud regeneration. Background Art

[0002] Cherry blossoms (Prunus spp.) are a general term for plants in the subgenus Cerasus of the genus Prunus. The subgenus Cerasus is a famous woody ornamental plant since ancient times, and the genus includes more than 150 species.

[0003] Cherry trees have beautiful shapes, rich varieties, and diverse colors. When they bloom, the trees are full of flowers, like clouds and rosy clouds. After the flowers naturally wither, you can also enjoy the fruits and leaves. In garden applications, they can be planted in large quantities to form a "sea of ​​flowers", or they can be arranged with other garden plants for landscaping. They can also be used as hedges, street trees, or small bonsai. In recent years, cherry blossoms have been more widely used in modern garden landscaping as early spring flowering plants. They have great market application prospects and the demand for seedlings is increasing. In addition, as people's spiritual world becomes richer, higher requirements are put forward for the ornamental value and garden application level of cherry blossoms, which have prompted continuous innovation in production and reproduction methods.

[0004] At present, cutting or grafting propagation is widely used in production. However, according to the research of Peng Yufu et al. (2019), different varieties are prone to incompatibility between rootstocks and scions during grafting propagation, and are easily affected by factors such as time, climate, and environment, which is not conducive to large-scale promotion. It is more difficult for cuttings of cherry blossoms to take root. Compared with grafting propagation, the survival rate is lower, the growth is slower, the tree shape is weaker, and there are higher requirements for the cutting season, cutting environment, and cutting selection (Zou Na et al., 2007). According to the research of Duan Xiaomei et al. (2002), most of the seeds of this genus are deep dormant seeds, and the seedling rate of direct sowing is very low. Low temperature vernalization or gibberellin treatment is required to break dormancy. The research of Zhan Linxing (2018) shows that seedling propagation is cumbersome to prepare before sowing, and needs to go through temperature-changing cold storage, sand storage and other treatments. The sowing time is restricted by the season, and it takes a long time from seed development to mature plant. Plant tissue culture can culture isolated plant organs (such as roots, stems, leaves, etc.), tissues (cambial layer, anther tissue, endosperm, etc.), cells (somatic cells or reproductive cells) and protoplasts under artificially controlled conditions under sterile conditions, so that they can quickly grow into complete plants. After the proliferation stage, its reproduction coefficient is high, and a large number of seedlings can be obtained in a short period of time. It is not affected by seasons and climate, and can be produced throughout the year. It can also maintain the excellent traits of the mother plant and better meet market needs.

[0005] Campanulata is a small deciduous tree of the genus Prunus in the Rosaceae family, with an early flowering period. Campanulata 'Feihan' is an excellent cultivar of the tree, with large flowers, bright pink color, open petals, graceful posture, and a shape similar to a white butterfly. It has good cold and heat resistance, strong growth, strong resistance to diseases and pests, wide adaptability, and beautiful tree shape. The flowering period is between the early cherry varieties that bloom in early February and the mid-cherry varieties that bloom in March-April. It has high potential for landscaping applications and broad application prospects.

[0006] At present, the reports on Feihan cherry are concentrated on genetic diversity analysis (Zhou Chengcheng et al., 2020), garden application (Zhang Yanfang, 2016), cultivation management (Gao Zhaoliang et al., 2021), etc. In terms of reproduction methods, only seedling and grafting propagation have been studied (Kuang Hongling et al., 2020; Zhou Ling et al., 2022). The research on tissue culture rapid propagation of this variety is still in a blank stage.

[0007] References:

[0008] Peng Yufu, Peng Huohui, Chen Hualing, et al. Preliminary study on the budding propagation of five types of cherry blossoms[J]. Modern Horticulture, 2019, 42(23): 45-46.

[0010] Zou Na, Cao Guangqiu, Lin Sizu. Research progress on ornamental cherry propagation technology[J]. Journal of Southwest Forestry University, 2007, (06): 42-46.

[0012] Zhan Linxing. Experiment on sowing and seedling cultivation of mountain cherry in Fujian[J]. Modern Agricultural Science and Technology, 2018, (19): 164-165. Zhou Chengcheng, Yang Deming, Li Shikun, et al. ISSR analysis of the genetic relationship of 18 cherry materials[J]. Journal of Forestry and Environment, 2020, 40(1): 46-53.

[0013] Zhang Yanfang. Common cherry varieties and their garden applications[J]. Garden, 2016(4): 72-75.

[0014] Gao Zhaoliang, Zhu Jianjun, Jun Jiaxiang, et al. Effect of compound fertilizer combined with microbial agent on the growth of 'Feihanying' seedlings[J]. Shanghai Journal of Agriculture, 2021, 37(05): 116-120.

[0015] Zhou Ling. Effects of rootstock type and grafting method on the grafting effect of cherry blossom[J]. Green Science and Technology, 2022, 24(21): 142-145.

[0017] Kuang Hongling, Nie Chaoren, Xu Haiqin, et al. Research on sowing and seedling raising technology of 8 cherry varieties[J]. Hubei Forestry Science and Technology, 2020, 49(01): 25-26. Summary of the invention

[0018] The purpose of the present invention is to provide a method for rapid propagation and adventitious bud regeneration of cherry blossoms through tissue culture. The tissue culture method has high germination rate, good rooting rate, short culture period and high survival rate.

[0019] In order to achieve the above object, the technical solution of the present invention is as follows:

[0020] A method for rapid propagation and adventitious bud regeneration of cherry blossoms through tissue culture, characterized in that the method comprises the following steps:

[0021] (1) Selection and disinfection of explants: Select current year’s unlignified stem segments, leaves or seeds with buds and disinfect them respectively;

[0022] D. Disinfection of stem segments: First, disinfect with 75% alcohol for 30 seconds, rinse with sterile water 2-3 times, then disinfect with 1% sodium hypochlorite solution for 3-10 minutes or 0.1% mercuric chloride for 3-7 minutes, and rinse with sterile water 5-6 times;

[0023] E. Disinfection of stem segments: First, disinfect with 75% alcohol for 30 seconds, rinse with sterile water 2-3 times, then disinfect with 1% sodium hypochlorite solution for 1-5 minutes or 0.1% mercuric chloride for 3-7 minutes, and rinse with sterile water 5-6 times;

[0024] F. Seed disinfection: Cut the shell and crack the kernel, soak the embryo in detergent water for 20 minutes, rinse under tap water for 1 hour, soak in 0.1% carbendazim solution for 20 minutes, disinfect with 75% alcohol for 30 seconds, rinse with sterile water 3-4 times, then disinfect with 0.1% mercuric chloride solution for 10-15 minutes, rinse with sterile water for more than 5 times, and soak in 1-4% PPM for 1-4 hours;

[0025] (2) Stem bud induction and proliferation culture: The sterilized stem segments were inoculated into an induction medium. After 10-15 days, the axillary buds began to sprout and form small buds. The induction medium contained the following ingredients: MS or 1 / 2MS or WPM + 6-BA 0.5-2.0 mg / L + NAA 0.1-1-0.3 mg / L + sucrose 30 g / L + agar 7.0 g / L, pH = 5.8-6.0. When the sterile buds obtained by the primary culture grew to 2.5 cm, they were cut off and transferred into an axillary bud proliferation medium. After 30 days, clustered buds were formed. The proliferation medium contained the following ingredients: MS + ZT 0.3-1.0 mg / L + NAA 0.1-0.5 mg / L + GA3 0.5-1.5 mg / L + sucrose 30 g / L + agar 7.0 g / L, pH = 5.8-6.0.

[0026] (3) Seed priming and proliferation culture: After disinfection, the seed coats of the seeds were peeled off and the tips were inserted into the seed germination medium downwards. The components of the seed germination medium were: MS + 6-BA 1.0-3.0 mg / L + NAA 0.1-0.3 mg / L + GA3 1.0-2.0 mg / L + 0.3% PPM + sucrose 30 g / L + agar 7.0 g / L, pH = 5.8-6.0; the PPM is a plant tissue culture antibacterial agent;

[0027] (4) Leaf adventitious bud regeneration culture

[0028] The disinfected leaves are inoculated with callus induction medium. After about 1 week, the edges of the cuts curl and arch toward the center. After 10-15 days, small callus protrusions appear at the leaf veins and edge cuts of the leaflets. The callus induction medium comprises the following components: MS or 1 / 2MS or WPM+6-BA 0.5-2.0mg / L+2,4-D 1.0-2.0mg / L+sucrose 30g / L+agar 6.0-7.5g / L, pH=5.8-6.0. When the sterile buds obtained by the primary culture grow to 2.5cm, they are cut into squares and transferred to the differentiation medium. After 6-7 weeks, adventitious buds are formed. The differentiation medium comprises the following components: MS+6-BA 0.5-2.0mg / L+2,4-D 0.1-0.5mg / L+TDZ 0.5-2.0mg / L+200mg / L hydrolyzed casein+30g / L sucrose+7.0g / L agar, pH=5.8-6.0.

[0029] (5) Rooting culture

[0030] Select large seedlings with a height of more than 3 cm, cut off the callus tissue and ineffective proliferation buds at the base of the stem, inoculate them on a rooting medium, and culture them in the dark for 3-5 days; then culture them under light, and new roots begin to form after 15 days. The rooting medium composition is: 1 / 2MS + NAA 0.2-0.5mg / L + IBA 0.4-0.8mg / L + sucrose 20g / L + agar 6.0g / L, pH = 5.8-6.0;

[0031] (6) Hardening and transplanting

[0032] After 2-3 days of hardening, the Feihan cherry rooted seedlings are transplanted into a mixed medium and cultured for 3-4 weeks. The indoor humidity is controlled at 70-90% and the temperature is controlled at 22-30°C to obtain the cherry rooted seedlings.

[0033] Preferably, the specific method of selecting the explant in step (1) is as follows: selecting healthy and pest-free excellent plants, taking the current year's bud clusters or semi-lignified branches in February-April, cutting them into branches with 2-3 buds, inserting them into a matrix with a volume ratio of peat soil to perlite of 1:1, and soaking and disinfecting the matrix and cuttings with a 1000-fold solution of carbendazim before cutting; placing them in a growth chamber, spraying 1% gibberellin solution and 2% cyanamide solution; when the buds grow to 5-7 cm, cutting non-lignified single bud stem segments or terminal buds as explants; cutting the sprouted branches into 2-3 cm long stem segments with buds for pretreatment to ensure that each segment has at least one bud; collecting pest-free branches and relatively tender leaves in February-April for standby use; collecting relatively mature fruits in March-April, washing off excess pulp, drying the seeds, and placing them in a 4°C refrigerator for standby use.

[0034] In the present invention, in the disinfection treatment of stem segments, an optimal implementation method is to first disinfect with 75% alcohol for 30 seconds, rinse with sterile water 3 times, then disinfect with 0.1% mercuric chloride for 7 minutes, and rinse with sterile water 6 times. 11. The method according to claim 1 is characterized in that in the disinfection treatment of leaves, first disinfect with 75% alcohol for 30 seconds, rinse with sterile water 3 times, then disinfect with 0.1% mercuric chloride for 3 minutes, and rinse with sterile water 6 times.

[0035] In the seed disinfection treatment, an optimal implementation method is to first disinfect with 75% alcohol for 30 seconds, rinse with sterile water 4 times, then disinfect with 0.1% mercuric chloride solution for 15 minutes, rinse with sterile water for more than 5 times, and then soak in 4% PPM for 2 hours. The mercuric chloride solution is reused 3 times or more.

[0036] During the experiment, the conditions of various culture media were optimized, and it was concluded that an optimal implementation method was:

[0037] The induction medium in step (2) is: 1 / 2MS medium + 6-BA 2mg / L + NAA 0.2mg / L + sucrose 30g / L + agar 7.0g / L; the axillary bud proliferation medium composition is: MS + ZT 0.5mg / L + NAA 0.1mg / L + GA31mg / L + sucrose 30g / L + agar 7.0g / L.

[0038] The components of the seed germination medium in step (3) are: MS+6-BA 2mg / L+NAA 0.2mg / L+GA32.0mg / L+0.3%PPM+sucrose 30g / L+agar 7.0g / L.

[0039] The leaf callus induction medium in step (4) comprises: WPM+6-BA 0.5 mg / L+2,4-D 2.0 mg / L+sucrose 30 g / L+agar 7.0 g / L.

[0040] The composition of the rooting medium in step (5) is 1 / 2MS+0.2mg / L NAA+0.8mg / L IBA+20g / L sucrose+6.0g / L agar.

[0041] The mixed matrix in step (6) is peat soil, perlite and vermiculite, and the volume ratio of the peat soil, perlite and vermiculite is 2:1:1.

[0042] Advantages of the present invention:

[0043] (1) We took the lead in establishing a tissue culture rapid propagation and organ regeneration system for the cherry tree, using its vegetative organ stem segments, leaves or seeds as explants, achieving a primary induction rate of up to 74.4%, a proliferation coefficient of about 3.9, and a rooting rate of 83.5%. This can achieve efficient and rapid propagation, and is not restricted by factors such as season, climate and region. Unrooted strong buds can also be used as spikelets for grafting propagation. Its leaf regeneration system also provides infection materials for genetic transformation and gene editing.

[0044] (2) During the seed disinfection process, the plant tissue culture antimicrobial agent PPM was used for the first time to disinfect the seeds of the Sauerkraut subgenus. The contamination rate was well controlled and significantly reduced to a minimum of about 6.38%, which can provide a scientific reference for the tissue culture disinfection of the Sauerkraut subgenus.

[0045] (3) For the first time, the number of times mercuric chloride disinfectant can be reused was studied. It was found that the disinfection effect of mercuric chloride for seed disinfection decreased with the increase in the number of reuses. After experimental discussion, it was found that the disinfection effect of mercuric chloride was significantly reduced when used three times or more.

[0046] (4) The same culture medium is used for obtaining sterile seed explants, inducing and proliferating sterile seedlings, thereby simplifying the operation process and reducing production costs.

[0047] (5) During the proliferation culture process, it was found that the plants of the genus Sakura would quickly lignify after germination, which is consistent with the research results of Xu Chenjie et al. (2020). In the early stage of the experiment, the stem segments were retained for proliferation culture. It was found that the proliferation results after lignification were poor, and the culture was almost unchanged. In the later stage, the lignified stem segments gradually turned brown and died, showing a recalcitrant phenomenon. The recalcitrant phenomenon in plant in vitro culture refers to the phenomenon that cells, tissues and organs lack or lose response to culture operations under in vitro conditions. After that, 6-BA was replaced with ZT, which has a stronger effect, and subculture was carried out before the stem segments became lignified. During this period, the bottles were transferred several times. After the mother plant stem segments were completely lignified, the rooted seedlings and other proliferation seedlings obtained by tissue culture were used as explants to continue proliferation, and the proliferation effect was improved. In addition, the concentration of the regulator used in some treatments was relatively high, and the inoculated buds did not proliferate, and gradually turned yellow and died. This may be due to the accumulation of the regulator in the plant body and the toxicity. In the later stage, the concentration of the regulator was reduced and the thinner buds were placed in a blank culture medium for a period of time before proliferation culture was carried out. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 For the collected explants.

[0049] Figure 2 This is a photo of seeds contaminated by bacteria.

[0050] Figure 3 The germination status of the stem segments of Cherry Blossom in different treatments. In the figure, a: treatment 1; b: treatment 2; c: treatment 3; d: treatment 4; e: treatment 5; f: treatment 6; g: treatment 7; h: treatment 8; i: treatment 9.

[0051] Figure 4 This is a diagram of the germination status of the Feihan cherry seeds; in the figure, a: Feihan cherry seeds; bf: growth status of experimental treatment 5 (b: just after inoculation; c: 4-5 days after inoculation; d: 10 days after inoculation; ef: 25 days after inoculation).

[0052] Figure 5 The proliferation of axillary buds of Cherry Blossom under different treatments; in the figure, a: treatment 1; b: treatment 2; c: treatment 3; d: treatment 4; e: treatment 5; f: treatment 6; g: treatment 7; h: treatment 8; i: treatment 9.

[0053] Figure 6 The callus induction conditions of different treatments of the leaves of Cherry Blossoms in Taiwan; in the figure, a: treatment 1; b: treatment 2; c: treatment 3; d: treatment 4; e: treatment 5; f: treatment 6; g: treatment 7; h: treatment 8; i: treatment 9.

[0054] Figure 7The callus induction and differentiation of Prunus cerasifera leaves; ab: callus tissue transferred to differentiation medium; cg: the process of callus differentiation to form adventitious buds; gh: adventitious buds transferred to proliferation medium.

[0055] Figure 8 The rooting conditions of cherry blossoms under different treatments; in the figure, a: treatment 1; b: treatment 2; c: treatment 3.

[0056] Fig. 9 This is the situation of transplanting the domesticated seedlings of Feihan cherry. DETAILED DESCRIPTION

[0057] The present invention will be further described below in conjunction with specific examples, and the advantages and features of the present invention will become clearer as the description proceeds. However, the specific experimental methods involved in the following examples are all conventional methods or implemented under the conditions recommended by the manufacturer's instructions unless otherwise specified.

[0058] Unless otherwise specified, the technical means used in the examples are conventional means known to those skilled in the art. The test methods in the following examples are conventional methods unless otherwise specified. Unless otherwise specified, the reagents and materials used can all be purchased from the market.

[0059] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0060] 1. Test materials

[0061] Explants of Prunus cyrtonema include current year unlignified stem segments with buds and seeds ( Figure 1 ), the collection time was from late February to mid-May, and the collection location was located at the Cherry Blossom Trail in Guishan Park, Wuhan (30.55545°N, 114.28009°E).

[0062] Select strong, disease-free and pest-free plants, take the current year's cluster buds or semi-lignified branches, cut them and quickly bring them back to the laboratory for watering. Bring back the lignified branches with full dormant buds that have not yet fully germinated, cut them into branches with 2-3 buds, and insert them into a matrix with a volume ratio of peat soil and perlite of 1:1. Before cutting, the matrix and cuttings are soaked and disinfected with 1000 times carbendazim solution; place them in a growth room with an external environmental condition of 22-25℃, 12h light / 8h dark. At the same time, spray 1% gibberellin solution and 2% cyanamide solution; when the buds grow to about 5-7cm, cut the unlignified single bud stem segments or terminal buds as explants. Collect the semi-lignified branches that have sprouted and bring them back to cut them into 2-3cm long stem segments with buds for pretreatment, ensuring that each segment has at least one bud. Collect the leaves that are free of insect pests and are relatively young and tender for use. Collect relatively mature fruits, wash off excess pulp, dry them in the air and place them in a 4°C refrigerator for later use.

[0063] 2. Test methods

[0064] 2.1 Establishment of explant disinfection system

[0065] 2.1.1 Stem segment disinfection

[0066] Remove the leaves from the stems and branches with buds collected, retain some petioles, soak them in detergent and washing powder solution, place them on a shaker for 20 minutes, and then brush them to remove dust and oil on the surface. Rinse them with running water for 2-3 hours and move them to a clean bench. Disinfect with 75% alcohol (treatment time 30 seconds), rinse with sterile water 2-3 times, then use 1% sodium hypochlorite solution or 0.1% mercuric chloride to disinfect the stems of Feihan cherry for different times, then rinse with sterile water 5 times, each washing time is not less than 30 seconds, and then use filter paper to absorb the surface moisture, and inoculate the sterilized explants in MS blank culture medium. Each treatment was inoculated with 20 bottles, repeated 3 times, and the contamination rate of the stems was counted after 30 days.

[0067] The test results are shown in Table 1. There are significant differences in the disinfection effects of different disinfection treatments on the stem segments of the cherry tree. When the disinfection time of 75% alcohol remains unchanged, the contamination rate of the stem segments shows an overall downward trend as the disinfection time of another disinfectant is extended. Among them, the contamination rate of treatment 6 is the lowest.

[0068] Table 1 Comparison of disinfection of Feihan cherry stem segments

[0069]

[0070]

[0071] 2.1.2 Effects of different growth stages on stem contamination rate

[0072] As time went by, the contamination rate of explants gradually increased and the survival rate decreased. The main reason was that the contaminants gradually accumulated on the plants, making sterilization difficult. This is consistent with the research conclusions of Xu Chenjie (2020).

[0073] Table 2 Effects of collection at different growth stages on the contamination rate of P. campanulata 'Feihan' stem segments

[0074]

[0075] 2.1.3 Seed disinfection

[0076] Before the test, the shell was broken by mechanical methods, and the embryo was soaked in detergent water for about 20 minutes, then rinsed under tap water for 1-2 hours, and then soaked in 0.1% carbendazim solution for 30 minutes, and then placed in the clean bench. Disinfect with 75% alcohol for 30 seconds, rinse with sterile water 3-4 times, and then disinfect with 0.1% mercuric chloride solution for 10, 12, and 15 minutes, and then rinse with sterile water for more than 5 times. After treatment, it was placed in a sterile bottle filled with sterile water overnight, and then inoculated in a 3% (v / v) PPM culture medium. It was found that the seeds carried a large number of endophytes, so after conventional disinfection, plant tissue culture antibacterial agent plant preservative mixture (PPM) was used for further disinfection. PPM is a broad-spectrum antimicrobial preparation that can effectively inhibit microbial contamination in plant tissue culture, and will not affect seed germination, callus proliferation and regeneration at a certain concentration and time. The factors were mercuric chloride disinfection time, PPM immersion concentration, and PPM immersion time. Each factor had 3 levels, with a total of 9 treatments, 20 bottles in each treatment, and 3 replications.

[0077] As can be seen from Table 3, different combinations of sterilizing agents and sterilization time have a significant effect on the contamination rate of explants. Among the three different treatments of mercuric chloride disinfection time, the 0.1% mercuric chloride disinfection time of 15 minutes has the best effect. In addition, after disinfection with PPM immersion, the contamination rate is significantly lower than that of the treatment using only conventional disinfection steps, and the disinfection effect of 4% (v / v) concentration is the best. Among all treatments, the contamination rate of treatment 9 is the lowest.

[0078] Table 3 Disinfection of Feihan cherry embryos

[0079]

[0080]

[0081] Note: When the immersion time is 0h, only the conventional disinfection method of alcohol + mercuric chloride is used.

[0082] It can be seen from the variance analysis table 4 that there are extremely significant differences in the effects of mercuric chloride disinfection time, PPM immersion concentration and time on the contamination rate of Feihan cherry seeds, and the P values ​​are all less than 0.01.

[0083] Table 4 Analysis of variance of disinfection of cherry blossom seeds

[0084]

[0085] In the specific operation, it was found that some residues would remain after the repeated use of mercuric chloride after rinsing the seeds. It was speculated that its repeated use would affect the disinfection process of the Feihan cherry seeds, and an experiment was designed to explore it. The disinfection was carried out using a scheme of 75% alcohol for 30s + 0.1% mercuric chloride for 15min + 1% (v / v) PPM for 4h. The results showed that the use of mercuric chloride for disinfection that was reused three times or more had a significant effect on the final disinfection effect of the Feihan cherry seeds.

[0086] Secondly, during the specific operation of the experiment, it was found that the embryos were greatly affected by bacterial contamination. Figure 2 The results show the situation of seeds contaminated by bacteria. After preliminary experiments, it was speculated that the repeated use of mercuric chloride had a significant effect on the disinfection effect of seed embryos. An experiment was designed to explore the effect. The disinfection was carried out using a scheme of 75% alcohol for 30 seconds + 0.1% mercuric chloride for 15 minutes + 1% (v / v) PPM for 4 hours. As can be seen from Table 5, the use of mercuric chloride that was reused three times or more for disinfection had a significant effect on the final disinfection effect of Feihan cherry seeds.

[0087] Table 5 Effect of repeated times of mercuric chloride on seed disinfection effect

[0088]

[0089]

[0090] 2.1.4 Leaf disinfection

[0091] The leaves on the leafy branches collected were used as explants. The surface dust was brushed with a brush, soaked in detergent water and shaken for about 20 minutes, then rinsed under tap water for 1 hour, and then placed in a clean bench. Disinfected with 75% alcohol and 1% sodium hypochlorite solution (1, 3, 5 minutes) or 0.1% mercuric chloride (3, 5, 7 minutes) as disinfectants. The leaves were inoculated on MS blank culture medium, 20 plates were inoculated in each group of experiments, 4-5 leaf pieces were inoculated in each culture dish, and repeated 3 times. After 30 days, the contamination rate, browning rate and survival rate were statistically analyzed, and finally the optimal sterilization scheme was selected. The test results are shown in Table 6. There are significant differences in the disinfection effect of different disinfection treatments on the leaves of Feihan cherry. When the 75% alcohol disinfection time remains unchanged, the contamination rate of the stem segment shows an overall downward trend as the disinfection time of another disinfectant is extended. The disinfection effect of 0.1% mercuric chloride is better than that of 1% NaClO solution. Overall, treatment 5 has the best effect, with lower contamination rate and browning rate.

[0092] Table 6 Disinfection of leaves of cherry blossoms

[0093]

[0094] 2.2 Initiation of stem segment culture

[0095] The uncontaminated stem segments were transferred to the induction medium, and three different plant basal mediums were set up, namely WPM, MS, and 1 / 2MS basal medium. Two plant growth regulators were added to each medium. The first was cytokinin 6-BA, which was 0.5 mg / L, 1.0 mg / L, and 2.0 mg / L respectively; the second was auxin NAA, which was 0.1 mg / L, 0.2 mg / L, and 0.3 mg / L respectively. The bud induction and growth conditions were recorded and observed 30 days after inoculation.

[0096] The stem segments of Cherry Blossoms inoculated with different growth regulator combinations and different culture media can induce buds, but their quality and development speed vary with different growth regulator combinations. The results are as follows (Table 7, Figure 3 ), different exogenous hormone combinations had a significant effect on the induction rate of axillary buds, among which the bud induction rates of experimental treatments 9, 6, and 3 were higher, which were 74.44%, 62.22%, and 60.83%, respectively. When the concentration of NAA increased, the germination of axillary buds might be inhibited. Different basal culture media also had certain differences in the survival rate of bud induction and the number of buds induced. 1 / 2MS+2.0mg / L 6-BA+0.2mg / L NAA was the best start-up culture scheme for the stem segments of Cherry Blossom.

[0097] Table 7 Effects of different treatment combinations on germination of stem segments of Cherry Blossom

[0098]

[0099] Note: + indicates normal growth, ++ indicates good growth, and +++ indicates robust growth. Same below. Different lowercase letters in the same column indicate significant differences (P < 0.05), same below.

[0100] It can be seen from the variance analysis Table 8 that the basic culture medium and NAA concentration have no significant effect on the germination rate of the stem segments of Cherry Blossom, with P values ​​of 0.945 and 0.87, respectively, both greater than 0.05, while the effect of 6-BA on the germination rate is extremely significant, with P value less than 0.001.

[0101] Table 8 Analysis of variance on germination of stem segments of Cherry Blossom

[0102]

[0103] 2.3 Seed embryo initiation culture

[0104] The sterilized Feihan cherry embryos were transferred to the induction medium, with MS as the basic medium, and three plant growth regulators were added to the medium, including two concentration gradients of 6-BA, 2.0 mg / L and 3.0 mg / L; three concentration gradients of NAA, 0.1 mg / L, 0.2 mg / L, and 0.3 mg / L; and three concentration gradients of gibberellin GA3, 1.0 mg / L, 2.0 mg / L, and 3.0 mg / L. Thirty cells were inoculated for each treatment, repeated three times, and the induction rate was calculated after 30 days.

[0105] The results are shown in Table 9 and Figure 4 Different treatment combinations have a significant effect on the induction rate of embryos. The fastest 4-5 days will see some embryo cotyledons turn green and gradually germinate. Generally, 2-3 true leaves can grow after 10-20 days. The results of adding different concentrations of NAA were not significantly different, indicating that the embryo was not sensitive to the concentration of NAA. The results of adding different concentrations of 6-BA were significantly different. With the increase of 6-BA concentration, the induction rate showed an upward trend, and the seedlings grew better. This shows that the 6-BA concentration suitable for the induction of Feihan cherry embryos should be 2.0 mg / L or even higher. Too low a concentration of 6-BA cannot stimulate embryo germination well. Comprehensive analysis of existing data shows that the culture medium with the best seed induction effect is: MS+2.0 mg / L 6-BA+0.2 mg / L NAA+2.0 mg / L GA3. In addition, the experiment found that Feihan cherry embryos can be directly induced to form clusters on the germination medium.

[0106] Table 9 Effects of different treatment combinations on seed germination of Cherry Blossom

[0107]

[0108] It can be seen from the variance analysis table 10 that the effects of NAA concentration and GA3 concentration on the germination rate of Hikan cherry seeds were not significant, with P values ​​of 0.07 and 0.25, respectively, both greater than 0.05, while the effects of 6-BA on the germination rate were significantly different, with P values ​​less than 0.05.

[0109] Table 10 Analysis of variance on germination of cherry blossom seeds

[0110]

[0111]

[0112] 2.4 Proliferation culture

[0113] Stem segments were cut to induction medium and the robust shoots were inoculated into MS subculture medium supplemented with three regulators for proliferation culture. After 15 days of inoculation, a small amount of callus tissue grew at the base of some shoots and leaflets began to grow. Figure 5 It can be seen that the bud proliferation coefficient was the highest in treatment 4. In the proliferation culture, the proliferation coefficient first increased and then decreased with the increase of ZT concentration. The proliferation coefficient in the treatment with 0.5 mg / L ZT was generally greater than that in the treatments with 0.3 mg / L and 1.0 mg / L.

[0114] Table 11 Effects of different treatment combinations on the proliferation of axillary buds of Cherry Blossom

[0115]

[0116] 2.5 Leaf callus induction culture

[0117] About 1 week after the disinfected and uncontaminated Hikan cherry leaves were inoculated into the induction medium, the edges of the cut curled up toward the center. After about 10-15 days, small callus protrusions appeared at the leaflet veins and edge cuts. Most of the callus tissue was yellow-brown in color, loose in texture, and had rough granular protrusions on the surface; a small part was milky white with a firm structure, which was presumed to be embryonic callus. According to Table 12 and Figure 6 Only treatment 7 showed significant differences in callus induction rate, but the callus quality of different treatments was significantly different. Among them, the treatments with WPM as the basic medium all induced embryonic callus with good quality. The callus induced by the treatments with MS and 1 / 2MS as the basic medium were mostly non-embryonic callus, which could not be used for subsequent plant regeneration. According to the existing experimental results, WPM+0.5mg / L 6-BA+2mg / L 2,4-D is a suitable callus induction scheme for Feihan leaves.

[0118] Table 12 Callus induction culture scheme of cherry blossom leaves

[0119]

[0120] From the variance analysis table 13, it can be seen that the type of culture medium and the concentration of 6-BA have no significant effect on the induction rate of the leaves of Hikan cherry, with P values ​​of 0.496 and 0.175, respectively, both greater than 0.05. However, the concentration of 2,4-D has a significant effect on the induction rate, with a P value less than 0.05.

[0121] Table 13 Analysis of variance of callus induced by Prunus cerasifera

[0122]

[0123] 2.6 Callus differentiation culture

[0124] In the tissue culture of Sakura subgenus plants, it is relatively difficult to differentiate adventitious buds from callus, and its differentiation rate is restricted by many factors, among which hormones are one of the relatively important factors. The obtained callus tissue is cut into small pieces and inoculated on the differentiation medium. As the culture time goes by, the callus tissue gradually becomes compact and the growth slows down. Granular green callus tissue appears on some surfaces, and a small part of the green callus tissue differentiates into bud points ( Figure 7 ). Some treatments never differentiated adventitious buds. After about 6-7 weeks, the surface color gradually turned black or brown, and gradually turned brown or dried up and died as the culture time prolonged. As shown in Table 14, adventitious buds differentiated in treatments 1, 4, 5, and 8, among which treatment 5 had the highest differentiation rate of 24.44%; callus tissues in other control test groups did not effectively form adventitious buds. Through the analysis of the above test results, when a higher concentration of auxin regulators is used, the activity of related enzymes that synthesize endogenous cytokinins in cells may be reduced, which in turn directly affects the formation of buds and reduces the differentiation rate of adventitious buds; appropriate concentrations of 6-BA and TDZ help cells synthesize endogenous cytokinins, thereby inducing adventitious bud differentiation.

[0125] Table 14 Callus differentiation induction scheme of cherry blossom leaves

[0126]

[0127] From the variance analysis table 15, it can be seen that the effect of TDZ concentration on the callus differentiation rate of Feihan cherry leaves is not significant, with a P value of 0.781, greater than 0.05. However, the effects of 6-BA concentration and 2,4-D concentration on the differentiation rate are significant, with P values ​​less than 0.05, and the combined effect of 2,4-D and TDZ on the callus differentiation rate of Feihan cherry leaves is significant, with a P value less than 0.05.

[0128] Table 15 Analysis of variance of callus differentiation of Cherry Blossom

[0129]

[0130] 2.7 Rooting culture

[0131] Select the well-growing subcultured seedlings and inoculate them into the rooting medium. Generally, some seedlings will produce small white roots 20 days after inoculation. As can be seen from Table 16, the buds can all take root in the existing rooting medium. Treatment 3 has the best effect, with the highest rooting rate reaching 83.33%.

[0132] Table 16 Effects of different plant growth regulators on rooting culture of Cherry Blossom

[0133]

[0134] 2.8 Acclimation and transplanting

[0135] The rooted Feihan cherry seedlings were transplanted after 2-3 days of hardening. After about 30 days, two true leaves grew out and the transplant survived. The results are as follows ( Fig. 9 ). During the transplanting process, attention should be paid to shading and moisturizing. After 3-5 days of opening the bottle for seedling hardening, the seedlings were transplanted into a mixed matrix of peat soil: perlite: vermiculite = 2:1:1 (treatment 1), peat soil: vermiculite = 3:1 (treatment 2) and peat soil: perlite = 3:1 (treatment 3) (Table 17). Before transplanting the seedlings, the agar at the roots should be washed, and the transplanting matrix should also be disinfected with 1000 times carbendazim solution in advance. Within one month after transplanting, the seedlings should be covered with a seedling box to allow the seedlings to gradually adapt to the external environment. After 30 days, the transplanting survival rates of the three matrices were 91.11%, 60.15% and 67.78% respectively. The leaves of the seedlings after transplanting gradually turned green, the height gradually increased, and the growth was gradually good. From the comparison of survival rates, treatment 1 had the best effect.

[0136] Table 17 Comparison of different cultivation substrates

[0137]

[0138] The embodiments described above are only preferred embodiments of the present invention and are only used to explain the present invention, not to limit the scope of implementation of the present invention. For those skilled in the art, other implementation methods can certainly be easily made by replacement or modification based on the technical contents disclosed in this specification. Therefore, all changes and improvements made on the principles of the present invention should be included in the scope of the patent application of the present invention.

Claims

1. A method for rapid propagation and adventitious bud regeneration of cherry blossoms through tissue culture, characterized in that: The method comprises the following steps: (1) Selection and disinfection of explants: Select current year’s unlignified stem segments, leaves or seeds with buds and disinfect them respectively; A. Disinfection of stem segments: First, disinfect with 75% alcohol for 30 seconds, rinse with sterile water 2-3 times, then disinfect with 1% sodium hypochlorite solution for 3-10 minutes or 0.1% mercuric chloride for 3-7 minutes, and rinse with sterile water 5-6 times; B. Disinfection of stem segments: First, disinfect with 75% alcohol for 30 seconds, rinse with sterile water 2-3 times, then disinfect with 1% sodium hypochlorite solution for 1-5 minutes or 0.1% mercuric chloride for 3-7 minutes, and rinse with sterile water 5-6 times; C. Seed disinfection: Cut the shell and crack the kernel, soak the embryo in detergent water for 20 minutes, rinse under tap water for 1 hour, soak in 0.1% carbendazim solution for 20 minutes, disinfect with 75% alcohol for 30 seconds, rinse with sterile water 3-4 times, then disinfect with 0.1% mercuric chloride solution for 10-15 minutes, rinse with sterile water for more than 5 times, and soak in 1-4% PPM for 0-4 hours; (2) Stem bud induction and proliferation culture: The sterilized stem segments were inoculated into an induction medium. After 10-15 days, the axillary buds began to sprout and form small buds. The induction medium contained the following ingredients: MS or 1 / 2MS or WPM + 6-BA 0.5-2.0 mg / L + NAA 0.1-1-0.3 mg / L + sucrose 30 g / L + agar 7.0 g / L, pH = 5.8-6.

0. When the sterile buds obtained by the primary culture grew to 2.5 cm, they were cut off and transferred into an axillary bud proliferation medium. After 30 days, clustered buds were formed. The proliferation medium contained the following ingredients: MS + ZT 0.3-1.0 mg / L + NAA 0.1-0.5 mg / L + GA3 0.5-1.5 mg / L + sucrose 30 g / L + agar 7.0 g / L, pH = 5.8-6.

0. (3) Seed priming and proliferation culture: After disinfection, the seed coats of the seeds were peeled off and the tips were inserted into the seed germination medium downwards. The components of the seed germination medium were: MS + 6-BA 1.0-3.0 mg / L + NAA 0.1-0.3 mg / L + GA3 1.0-2.0 mg / L + 0.3% PPM + sucrose 30 g / L + agar 7.0 g / L, pH = 5.8-6.0; the PPM is a plant tissue culture antibacterial agent; (4) Leaf adventitious bud regeneration culture The disinfected leaves were inoculated with callus induction medium. After about 1 week, the edges of the cut curled and arched toward the center. After 10-15 days, small callus protrusions appeared at the leaf veins and edge cuts of the leaflets. The callus induction medium composition was: MS or 1 / 2MS or WPM + 6-BA 0.5-2.0mg / L + 2, 4-D 1.0-2.0mg / L + sucrose 30g / L + agar 6.0-7.5g / L, pH = 5.8-6.

0. When the sterile buds obtained by the primary culture grew to 2.5cm, they were cut into squares and transferred to the differentiation medium. After 6-7 weeks, adventitious buds were formed. The differentiation medium composition was: MS + 6-BA 0.5-2.0mg / L + 2, 4-D 0.1-0.5mg / L+TDZ0.5-2.0mg / L+200mg / L hydrolyzed casein+sucrose 30g / L+agar 7.0g / L, pH=5.8-6.

0. (5) Rooting culture Select large seedlings with a height of more than 3 cm, cut off the callus tissue and ineffective proliferation buds at the base of the stem, inoculate them on a rooting medium, and culture them in the dark for 3-5 days; then culture them under light, and new roots begin to form after 15 days. The rooting medium composition is: 1 / 2MS + NAA 0.2-0.5mg / L + IBA 0.4-0.8mg / L + sucrose 20g / L + agar 6.0g / L, pH = 5.8-6.0; (6) Hardening and transplanting After 2-3 days of hardening, the Feihan cherry rooted seedlings are transplanted into a mixed medium and cultured for 3-4 weeks. The indoor humidity is controlled at 70-90% and the temperature is controlled at 22-30°C to obtain the cherry rooted seedlings.

2. The method according to claim 1, characterized in that: The specific method of selecting the explant in step (1) is as follows: selecting strong and pest-free excellent plants, taking clustered buds or semi-lignified branches of the current year in February-April, cutting them into branches with 2-3 buds, inserting them into a matrix with a volume ratio of peat soil to perlite of 1:1, and soaking and disinfecting the matrix and cuttings with a 1000-fold solution of carbendazim before cutting; placing them in a growth chamber, spraying 1% gibberellin solution and 2% cyanamide solution; when the buds grow to 5-7 cm, cutting non-lignified single bud stem segments or terminal buds as explants; cutting the sprouted branches into 2-3 cm long stem segments with buds for pretreatment, ensuring that each segment has at least one bud; collecting branches without pests and relatively tender leaves in February-April for standby use; collecting relatively mature fruits in March-April, washing off excess pulp, and placing the seeds in a 4°C refrigerator for standby use after drying.

3. The method according to claim 1, characterized in that In the disinfection of stem segments, 75% alcohol was first used for disinfection for 30 seconds, and then rinsed with sterile water for 3 times. Then, 0.1% mercuric chloride was used for disinfection for 7 minutes, and then rinsed with sterile water for 6 times.

4. The method according to claim 1, characterized in that: In the leaf disinfection treatment, first use 75% alcohol to disinfect for 30 seconds, rinse with sterile water three times, then use 0.1% mercuric chloride to disinfect for 3 minutes, and rinse with sterile water six times.

5. The method according to claim 1, characterized in that During the seed disinfection treatment, first disinfect with 75% alcohol for 30 seconds, rinse with sterile water 4 times, then disinfect with 0.1% mercuric chloride solution for 15 minutes, rinse with sterile water for more than 5 times, and then soak in 4% PPM for 2 hours. The mercuric chloride solution is reused 3 times or more.

6. The method according to claim 1, characterized in that The induction medium in step (2) is: 1 / 2MS medium + 6-BA 2mg / L + NAA 0.2mg / L + sucrose 30g / L + agar 7.0g / L; the axillary bud proliferation medium composition is: MS + ZT0.5mg / L + NAA 0.1mg / L + GA31mg / L + sucrose 30g / L + agar 7.0g / L.

7. The method according to claim 1, characterized in that The components of the seed germination medium in step (3) are: MS+6-BA 2mg / L+NAA 0.2mg / L+GA32.0mg / L+0.3%PPM+sucrose 30g / L+agar 7.0g / L.

8. The method according to claim 1, characterized in that The leaf callus induction medium in step (4) comprises: WPM+6-BA 0.5 mg / L+2,4-D 2.0 mg / L+sucrose 30 g / L+agar 7.0 g / L.

9. The method according to claim 1, characterized in that: The composition of the rooting medium in step (5) is 1 / 2MS+0.2mg / L NAA+0.8mg / L IBA+20g / L sucrose+6.0g / L agar.

10. The method according to claim 1, characterized in that The mixed matrix in step (6) is peat soil, perlite and vermiculite, and the volume ratio of the peat soil, perlite and vermiculite is 2:1:1.

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