In vitro embryo culture method of paeonia hybrid seed

Abstract

The application provides an in-vitro seed embryo culture method of peony hybrid seeds, which comprises the following steps: 1) taking Chuan-shao No.1 or Chuan-shao No.2 as the female parent, Bozhou peony as the male parent to cross, collecting hybrid seeds, cleaning and disinfecting, taking embryos to obtain seed embryos; 2) taking the seed embryos, connecting into seed embryo starting seedling culture medium to culture, after the F1 generation seed embryo sprouts, first low-temperature treatment and then constant-temperature culture to obtain tissue culture seedlings; 3) taking the tissue culture seedlings to harden and transplant, which can be used. Under the culture of the specific culture medium, the double dormancy problem of the peony hybrid seeds is solved, and the low germination rate, high rot rate and embryo abortion of the peony hybrid seeds are overcome, so that the difficulty of the hybrid peony seedling quantity and quality is solved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of plant cultivation, and particularly relates to an in-vitro seed embryo culture of Paeonia hybrid seeds. BACKGROUND

[0002] Paeonia lactiflora Pall. belongs to Paeonia L. of Paeoniaceae and is a perennial herbaceous plant. It is a famous ornamental flower and a common medicinal material in China, and its root is used as medicine. As an ornamental flower, many ornamental Paeonia varieties have been bred through various breeding methods. However, in terms of medicine, the medicinal Paeonia varieties in China still have the shortcomings of single source and single gene, and it is necessary to improve the properties of medicinal Paeonia and breed new varieties of medicinal Paeonia through hybridization breeding and other means.

[0003] The white Paeonia produced in Sichuan Zhongjiang has excellent quality and high content of paeoniflorin, and is known as "silver core white Paeonia". However, the stamens of Paeonia in Sichuan Zhongjiang are petalized, and only flowers are produced without seeds, which leads to abnormal pollination and limits the seed propagation, thereby negatively affecting the cultivation and yield of white Paeonia and directly affecting the accumulation of germplasm resources and the breeding of new varieties of white Paeonia. The stamens of Paeonia in Anhui Bozhou are less petalized, and can be propagated by seeds. At present, the annual yield accounts for more than 70% of the total yield in China. The hybridization of white Paeonia in Sichuan Zhongjiang and Paeonia in Anhui Bozhou is beneficial to obtain new varieties of medicinal Paeonia which inherit the high quality of white Paeonia in Sichuan Zhongjiang and the high yield characteristics of Paeonia in Anhui Bozhou. However, there are significant differences in the compatibility between different varieties, and the hybrid seeds of Paeonia germinate slowly and have low germination rate under natural conditions, which seriously restricts the breeding of Paeonia and the cultivation of new varieties.

[0004] At present, there is no report on the successful hybridization of white Paeonia in Sichuan Zhongjiang and Paeonia in Anhui Bozhou to obtain hybrid Paeonia plants. SUMMARY

[0005] The present application aims to provide an in-vitro seed embryo culture method of hybrid seeds obtained by hybridization of white Paeonia in Sichuan Zhongjiang "Chuan-shao No. 1" (CBS-B) and "Chuan-shao No. 2" (CBS-H) as female parents and "Bozhou Paeonia" (BZSY) as male parent, which solves the double dormancy of hybrid seeds of Paeonia, and the problems of low germination rate, high corruption rate and poor quality of seedlings.

[0006] The present application provides an in-vitro seed embryo culture method of Paeonia hybrid seeds, which comprises the following steps:

[0007] 1) Hybridization of Chuan-shao No. 1 or Chuan-shao No. 2 as female parent and Bozhou Paeonia as male parent, collection of hybrid seeds, cleaning and disinfection, embryo extraction, and obtaining of seed embryos;

[0008] 2) take the seed embryo, connect into the seed embryo starting seedling culture medium culture, after the F1 generation seed embryo germination, first low temperature treatment then constant temperature culture get tissue culture seedling;

[0009] 3) the obtained tissue culture seedling acclimatization, transplant, can;

[0010] When taking Chuan Shao 1 as the female parent, the composition of the seed embryo starting seedling culture medium is: WPM culture medium+3.0mg / L 6-benzyl purine+3.0mg / L gibberellin+1.0g / L activated carbon+30g / L sucrose+8.0g / L agar;

[0011] When taking Chuan Shao 2 as the female parent, the composition of the seed embryo starting seedling culture medium is: 1 / 2MS culture medium+2.0mg / L 6-benzyl purine+2.0mg / L gibberellin+1.0g / L activated carbon+30g / L sucrose+8.0g / L agar;

[0012] Further, the pH of the seed embryo starting seedling culture medium is 5.5-6.0.

[0013] Further, the condition for culturing the seed embryo in the seed embryo starting seedling culture medium in step 2) is: temperature 25±2℃, humidity 40-50%, light intensity 2500-3 000lx, light cycle 14h light / 10h dark.

[0014] Further, the seed embryo is subjected to low temperature treatment when the seed embryo germination is to seedling length 1-2cm.

[0015] Further, the method for the low temperature treatment is: the seedling is cultured in the condition of temperature 4℃, humidity 30-40%, light intensity 2500-3000lx, light cycle 14h(light) / 10h(dark); when taking Chuan Shao 1 as the female parent, the culture time is 45d, and when taking Chuan Shao 2 as the female parent, the culture time is 30d.

[0016] Further, the condition for constant temperature culture in step 2) is: temperature 25±2℃, humidity 40-50%, light intensity 2500-3 000lx, light cycle 14h light / 10h dark.

[0017] Further, the length of the tissue culture seedling in step 3) is 4-5cm.

[0018] Further, the method for acclimatization in step 3) is: the tissue culture seedling is cultured in the condition of temperature 10℃, humidity 30-40%, light intensity 2500-3 000lx, light cycle 14h(light) / 10h(dark) for 72h.

[0019] Further, the transplanting is that the tissue culture seedling is washed with carbendazim solution to remove the root culture medium, then is transplanted into the substrate composed of nutrient soil, vermiculite and perlite, and is cultivated in a clean and ventilated room.

[0020] Further, the concentration of the carbendazim solution is 500 mg / L.

[0021] Further, the water content of the substrate is 25%-35%, the volume ratio of the nutrient soil, the vermiculite and the perlite is 1:1:1, the particle size of the perlite is 3-5mm, and the particle size of the vermiculite is 1-3mm.

[0022] The present application takes two new varieties of Paeonia lactiflora Pall. in Zhongjiang, Sichuan, "Chuan-shao No.1" (CBS-B) and "Chuan-shao No.2" (CBS-H) as the female parent, and "Bozhou Paeonia lactiflora" (BZSY) as the male parent, and the in-vitro seed embryo of the hybrid F1 generation seed is cultured in a specific medium, so that the double dormancy problem of the hybrid Paeonia lactiflora seed is solved, the low germination rate and high rotting rate of the hybrid Paeonia lactiflora seed and the seed embryo abortion are overcome, and the difficulty of the hybrid Paeonia lactiflora seedling quantity and quality is solved. Through the in-vitro seed embryo culture method of the hybrid Paeonia lactiflora seed, a hybrid Paeonia lactiflora seed embryo culture system of Paeonia lactiflora in Zhongjiang, Sichuan and Paeonia lactiflora in Bozhou, Anhui is constructed, new materials are provided for the breeding of new medicinal Paeonia lactiflora varieties, and technical support is provided for the factory production, large-scale production and industrialization production of the Paeonia lactiflora tissue culture seedlings.

[0023] Obviously, according to the above content of the present application, according to the ordinary technical knowledge and common means in the art, other various forms of modifications, replacements or changes can be made without departing from the above technical idea of the present application.

[0024] The above content of the present application will be further explained in detail through the following embodiment. However, it should not be understood that the above subject matter of the present application is limited to the following examples. Any technology achieved based on the above content of the present application belongs to the scope of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 Appearance of Paeonia lactiflora seed;

[0026] Figure 2 Structure of Paeonia lactiflora seed;

[0027] Figure 3 Embryo structure of Paeonia lactiflora seed;

[0028] Figure 4 Different concentrations of activated carbon on the germination of CBS-B x BZSY seed embryo; A. CBS-B x BZSY seed embryo; B. 1.0 g / L activated carbon on the start culture of CBS-B x BZSY seed embryo for 10 days; C. 1.0 g / L activated carbon on the start culture of CBS-B x BZSY seed embryo for 30 days; D. 0.0 g / L activated carbon on the start culture of CBS-B x BZSY seed embryo for 30 days;

[0029] Figure 5 Effects of different concentrations of activated carbon on the germination of CBS-H×BZSY embryos; A. CBS-H×BZSY embryos; B. CBS-H×BZSY embryos initiated with 1.0 g / L activated carbon for 10 days; C. CBS-H×BZSY embryos initiated with 1.0 g / L activated carbon for 30 days; D. CBS-H×BZSY embryos initiated with 0.0 g / L activated carbon for 30 days.

[0030] Figure 6 The effects of different durations of low-temperature treatment on the growth of CBS-B×BZSY embryos after emergence culture; A. CBS-B×BZSY embryos treated with low temperature for 0 days; B. CBS-B×BZSY embryos treated with low temperature for 15 days; C. CBS-B×BZSY embryos treated with low temperature for 30 days; D. CBS-B×BZSY embryos treated with low temperature for 45 days;

[0031] Figure 7 The effects of different durations of low-temperature treatment on the growth of CBS-H×BZSY embryos after emergence culture; A. CBS-H×BZSY embryos treated with low temperature for 0 days; B. CBS-H×BZSY embryos treated with low temperature for 15 days; C. CBS-H×BZSY embryos treated with low temperature for 30 days; D. CBS-H×BZSY embryos treated with low temperature for 45 days;

[0032] Figure 8 CBS-B×BZSY embryo initiation culture and growth status;

[0033] Figure 9 CBS-H×BZSY embryo initiation culture and growth status;

[0034] Figure 10 Growth status of CBS-B×BZSY seed embryos into seedlings;

[0035] Figure 11 Growth of CBS-H×BZSY seed embryos into seedlings;

[0036] Figure 12 Growth of peony CBS-B×BZSY hybrid tissue culture seedlings after hardening and transplanting;

[0037] Figure 13 Growth of peony CBS-H×BZSY hybrid tissue culture seedlings after hardening and transplanting;

[0038] Figure 14 Growth of peony CBS-B×BZSY hybrid tissue culture seedlings after 60 days of hardening-off;

[0039] Figure 15 Growth of peony CBS-H×BZSY hybrid tissue culture seedlings after 60 days of hardening; Detailed Implementation

[0040] The raw materials, reagents and equipment used in the present application are all commercially available. Among them, 1 / 2MS medium (without agar and sucrose) is provided by Jinan Geait Instrument and Equipment Co., Ltd., origin: Zhongtian Horticulture, product number: ZT-1 / 2MS; MS medium (without agar and sucrose) and B5 medium are provided by Beijing Solarbio Science & Technology Co., Ltd., product numbers are Cat#M8521 and Cat#B8831 respectively; WPM medium (without agar and sucrose) is provided by BASF, product number: Lot#EK160504.

[0041] One bag of perlite 70 liters, particle size 3-5mm, one bag of vermiculite 50 liters, particle size 1-3mm.

[0042] “Chuan Shao 1 (Paeonia lactiflora Pall.)” is the first recognized new variety of Paeonia lactiflora in Sichuan Province, with an approval (registration, recognition) number of Chuan Recognition Medicine 2021002, and a popularization and application area of more than 10,000 mu; “Chuan Shao 2 (Paeonia lactiflora Pall.)” is a recognized new variety of Paeonia lactiflora selected from the traditional cultivation of Paeonia lactiflora in Zhongjiang, Sichuan, by systematic breeding method, with an approval (registration, recognition) number of Chuan Recognition Medicine 2021003, and a popularization and application area of more than 5,000 mu; “Bozhou Paeonia lactiflora (Paeonia lactiflora Pall.)” is a specialty of Bozhou City, Anhui Province, and also a national geographical indication product; the three varieties of Paeonia lactiflora are introduced to Zhongjiang County, Sichuan, for in vitro embryo culture.

[0043] Example 1 In vitro embryo culture of hybrid seeds of “Chuan Shao 1” and “Chuan Shao 2” and “Bozhou Paeonia lactiflora”

[0044] I. Screening of embryo germination and seedling culture medium

[0045] In the early research process, it was found that when the hybrid seeds of “Chuan Shao 1” or “Chuan Shao 2” and “Bozhou Paeonia lactiflora” were used as explants for tissue culture, the medium for embryo germination not only affected the embryo germination of hybrid Paeonia lactiflora, but also had a significant impact on the rooting of subsequent tissue culture seedlings, which was directly related to the final quality of hybrid Paeonia lactiflora seedlings. The following further shows the influence of component nutrition on the growth of hybrid Paeonia lactiflora during the embryo germination period by adjusting the composition and dosage of the embryo germination and seedling culture medium.

[0046] 1. Method

[0047] 1) Material selection and treatment

[0048] “Chuan Shao 1” and “Chuan Shao 2” are new varieties of Paeonia lactiflora selected and recognized from Paeonia lactiflora in Zhongjiang, Sichuan.

[0049] (CBS-B) and "Chuan Shao No. 2" (CBS-H) as the female parent, "Bozhou Saoyao" (BZSY) as the male parent, specifically, the pollen of the male parent BZSY was collected from 8-10 am every day from April 12, 2022 to April 15, 2022. The flower buds at the stage of loose anthers were collected, the anthers were removed with tweezers and placed in a dry and ventilated place indoors where the sunlight could not directly shine, and the pollen was allowed to naturally disperse. After the pollen was dispersed, it was collected in a centrifuge tube for standby use. The cross-pollination was carried out on a sunny and windless day. When pollinating, the tip of a hairbrush was dipped in the pollen and gently touched on the stigma. After pollination, the cross-pollination bag was immediately covered, and a paper clip was used to tightly close the bag opening. When closing, the flower stem should not be damaged to prevent insects from pollinating. The cross-pollination bag was removed 7-10 days after pollination. When removing the bag, the action should be light to avoid damaging the hybrid capsules. After 90 days, the mature hybrid seeds with full grains were collected before the fruit skin cracked and the seeds were scattered. The seeds were washed with clean water to remove surface impurities, and the full-grain seeds were selected and stored in a 4°C refrigerator for standby use;

[0050] 2) Material disinfection

[0051] The stored hybrid seeds were soaked in sterile water for 24 h, and the seed coat was removed after soaking and washed with clean water for 0.5 h. In the clean bench, the seed coat was first sterilized with 75% alcohol for 45 s, washed with sterile water for 3 times, then sterilized with 0.1% mercuric chloride for 8 min, and washed with sterile water for 5-6 times until the water became clear. The endosperm was removed to obtain the hybrid seed embryo of Paeonia lactiflora, which was used for standby use;

[0052] 3) Paeonia lactiflora hybrid seed embryo germination and seedling culture

[0053] The sterilized Paeonia lactiflora hybrid seed embryo was selected as a sterile explant under an electron microscope, and was inoculated into the seed embryo initiation and seedling culture medium, and was placed in a culture room with a temperature of 25±2°C, a humidity of 40-50%, a light intensity of 2 500-3 000 lx, and a light cycle of 14 h (light) / 10 h (dark).

[0054] 1.1 Effect of different concentrations of activated carbon on seed embryo germination and seedling

[0055] MS+1.0 mg / L 6-BA+0.5 mg / L GA3+8 g / L agar+30 g / L sucrose was used as the basic medium for seed embryo initiation, different concentrations of activated carbon were added, and the seed embryos of CBS-B×BZSY and CBS-H×BZSY hybrid seeds were inoculated into the medium. After 30 days of culture, the germination rate and rot rate of the seed embryos, and the rooting rate were counted.

[0056] 1.2 Effect of different basic media on seed embryo germination and seedling

[0057] The sterilized CBS-B x BZSY and CBS-H x BZSY hybrid embryos were inoculated into the medium of (1 / 2MS, MS, WPM, B5) + 1.0 mg / L 6-BA + 0.5 mg / L GA3 + 8 g / L agar + 1.0 g / L activated carbon + 30 g / L sucrose, and the germination rate, rot rate, and rooting rate of the embryos were counted after 30 days of culture.

[0058] 1.3 Effects of different concentrations of 6-BA and GA3 on the germination and seedling growth of the embryos

[0059] The WPM + 1.0 mg / L activated carbon + 30 g / L sucrose + 8.0 g / L agar medium was used as the basic medium for the germination and seedling growth of the CBS-B x BZSY hybrid embryos, and different concentrations of 6-BA and GA3 were added to the medium. The 1 / 2MS + 1.0 g / L activated carbon + 30 g / L sucrose + 8.0 g / L agar medium was used as the basic medium for the germination and seedling growth of the CBS-H x BZSY hybrid embryos, and different concentrations of 6-BA and GA3 were added to the medium. The effects of different concentrations of 6-BA and GA3 on the germination and seedling growth of the embryos were investigated, and the germination rate, average number of true leaves, average seedling length, rooting rate, and average root length were counted after 60 days of culture.

[0060] Germination rate = number of germinated embryos in the medium / number of inoculated embryos;

[0061] Contamination rate = number of contaminated embryos in the medium / number of inoculated embryos;

[0062] Germination rate = number of germinated embryos in the medium / number of inoculated embryos;

[0063] Rooting rate = number of rooted embryos in the medium / number of inoculated embryos;

[0064] 2. Results

[0065] 2.1 Culture of hybrid seed embryos in the medium containing different concentrations of activated carbon for germination and seedling growth

[0066] Table 1 Effects of different concentrations of activated carbon on the germination and seedling growth of the embryos

[0067]

[0068] From Table 1 and Figures 4-5The germination rate of CBS-B x BZSY seed embryo in 0.0 g / L activated carbon treatment group was 81.11%, and the contamination rate was 12.22%. The germination rate of CBS-B x BZSY seed embryo in 1.0 g / L activated carbon treatment group was 66.67%, and the contamination rate was 7.78%. The germination rate of CBS-B x BZSY seed embryo in 0.0 g / L activated carbon treatment group was significantly different from that in 1.0 g / L activated carbon treatment group (P<0.05), and the contamination rate was not significantly different (P>0.05). The rooting rate of CBS-B x BZSY was 65.56% in 1.0 g / L activated carbon treatment group after 30 days of culture.

[0069] For CBS-H x BZSY 0.0 g / L hybrid combination, when the concentration of activated carbon was 0.0 g / L, the germination rate of seed embryo was 82.22%, and the contamination rate was 15.56%. When the concentration of activated carbon was 1.0 g / L, the germination rate was significantly different from that of 0.0 g / L treatment group (P<0.05), and the germination rate was lower than that of 0.0 g / L treatment group, which was 65.55%. The contamination rate was also significantly different from that of 0.0 g / L treatment group (P>0.05), and the contamination rate was lower than that of 0.0 g / L treatment group, which was 8.89%. The rooting rate of CBS-H x BZSY was 64.45% in 1.0 g / L activated carbon treatment group after 30 days of culture.

[0070] Therefore, activated carbon has a promoting effect on the development of radicle of CBS-H x BZSY and CBS-B x BZSY seed embryo.

[0071] 2.2 Culture of hybrid seed embryo in seed embryo germination and seedling culture medium prepared from different basic media

[0072] Table 2 Effect of different basic media on seed embryo germination and seedling

[0073]

[0074] Note: In the table, lowercase letters indicate the significance of differences at the 0.05 level.

[0075] The effects of 1 / 2MS, MS, B5 and WPM on the germination and seedling growth of CBS-B×BZSY and CBS-H×BZSY were studied. The results showed that the different basic medium had significant difference on the germination rate of CBS-B×BZSY (P<0.05). The germination rate was the highest in WPM medium, which was 92.22%, and the lowest in B5 medium, which was 58.89%. The infection rate of B5 treatment group had significant difference with MS and WPM treatment groups (P<0.05), and had no significant difference with 1 / 2MS treatment group (P>0.05). The infection rate was the lowest in WPM medium, which was 4.44%, and the highest in B5 medium, which was 17.78%. The rooting rate of 1 / 2MS had significant difference with MS and B5 treatment groups (P<0.05), and had no significant difference with WPM treatment group (P>0.05). The rooting rate was the highest in WPM medium, which was 86.67%, and the lowest in B5 medium, which was 55.56%.

[0076] The different basic medium had significant difference on the germination rate of CBS-H×BZSY (P<0.05), and had no significant difference on the infection rate (P>0.05). The germination rate was the highest in 1 / 2MS medium, which was 88.89%, and the lowest in B5 medium, which was 42.22%. The infection rate was 4.46% in 1 / 2MS medium, and 12.22% in B5 medium. The rooting rate of B5 and WPM treatment groups had no significant difference (P>0.05), and had significant difference with 1 / 2MS and MS treatment groups (P<0.05). The rooting rate was the highest in 1 / 2MS medium, which was 78.87%, and the lowest in B5 medium, which was 40.00%.

[0077] The results showed that the optimal basic medium for the germination of CBS-B×BZSY was WPM medium, and the optimal basic medium for the germination of CBS-H×BZSY was 1 / 2MS medium.

[0078] 2.3 The effects of different concentrations of 6-BA and GA3 on the germination and seedling growth of hybrid seeds

[0079] Table 3 The effects of different concentrations of 6-BA and GA3 on the germination and seedling growth of hybrid seeds

[0080]

[0081]

[0082] Note: The lower case letters in the table represent the significant difference at the 0.05 level.

[0083] The effects of different concentrations of 6-BA and GA3 on the seedling of CBS-H x BZSY and CBS-B x BZSY were investigated. As shown in Table 3, the results of seedling of CBS-B x BZSY were as follows: when the concentration of 6-BA was 3.0 mg / L, the average seedling length of 1.0 mg / L, 2.0 mg / L and 3.0 mg / L GA3 had significant difference (P<0.05); when the concentration of GA3 was 1.0 mg / L and 2.0 mg / L, the seedling rate, average leaf number, rooting rate and average root length had no significant difference (P>0.05), and all had significant difference with the seedling rate of 3.0 mg / L GA3, but had no significant difference in rooting rate and average root length (P>0.05); and when the concentration of GA3 was 3.0 mg / L, the seedling rate and average seedling length were the highest, which were 65.56% and 5.27 cm, respectively. When the concentration of GA3 was 1.0 mg / L, the seedling rate, average leaf number, average seedling length and average root length of 2.0 mg / L and 3.0 mg / L 6-BA had no significant difference (P>0.05), but had significant difference with 1.0 mg / L, 4.0 mg / L and 5.0 mg / L 6-BA (P<0.05); and when the concentration of 6-BA was 4.0 mg / L, the seedling rate and average leaf number were the highest, which were 54.44% and 2.94, respectively. Therefore, the best hormone ratio for seedling of CBS-B x BZSY was 3.0 mg / L 6-BA + 3.0 mg / L GA3.

[0084] The results of seedling of CBS-H x BZSY are as follows: when the concentration of 6-BA is 2.0 mg / L, the seedling rate, average number of true leaves, and average seedling length increase significantly first and then decrease significantly (P<0.05) with the increase of the concentration of GA3, and reach the maximum when the concentration of GA3 is 2.0 mg / L, the seedling rate is 71.11%, the average number of true leaves is 2.90, the average seedling length is 6.69 cm, and the average root length is 6.78 cm; the minimum is obtained when the concentration of GA3 is 1.0 mg / L, the seedling rate is 12.22%, the average number of true leaves is 1.50, the average seedling length is 2.16 cm, and the average root length is 5.42 cm; the rooting rate has no significant difference (P>0.05) and is between 88.89% and 94.44%; when the concentration of 6-BA is 3.0 mg / L, the seedling rate, average number of true leaves, average seedling length, and rooting rate have no significant difference (P>0.05) when the concentration of GA3 is 1.0 mg / L and 2.0 mg / L, but the average root length has significant difference (P<0.05); the maximum is obtained when the concentration of GA3 is 3.0 mg / L, the seedling rate is 67.78%, the average number of true leaves is 1.95, the average seedling length is 4.90 cm, and the average root length is 3.98 cm; the minimum is obtained when the concentration of GA3 is 1.0 mg / L, the seedling rate is 24.44%, the average number of true leaves is 1.66, the average seedling length is 3.12 cm, and the average root length is 6.51 cm. Therefore, the optimal hormone ratio for seedling of CBS-H x BZSY is 2.0 mg / L 6-BA + 2.0 mg / L GA3.

[0085] The results of screening of seedling culture medium of the seedlings show that the optimal seedling culture medium for CBS-B x BZSY is WPM medium + 3.0 mg / L 6-benzylaminopurine (6-BA) + 3.0 mg / L gibberellin (GA3) + 1.0 g / L activated carbon + 30 g / L sucrose + 8.0 g / L agar, and the pH value is 5.9.

[0086] The optimal seedling culture medium for CBS-H x BZSY is 1 / 2MS medium + 2.0 mg / L 6-benzylaminopurine (6-BA) + 2.0 mg / L gibberellin (GA3) + 1.0 g / L activated carbon + 30 g / L sucrose + 8.0 g / L agar, and the pH value is 5.9.

[0087] The hybrid seedlings of peony are cultured in the optimal seedling culture medium, and one-step seedling is achieved, and the quality of seedling is good.

[0088] II. Screening of low-temperature treatment time

[0089] Appropriate low temperature stress can improve the seedling index and dry matter accumulation of plants, thus promoting the growth and development of plants. However, excessive low temperature stress can cause cold injury, frost damage and other adverse effects on plants, resulting in slowed growth, blocked physiological and biochemical processes, and changes in cell structure and function. Therefore, when conducting low temperature treatment of embryo seedlings, the temperature and time must be controlled to maximize the benefits and avoid potential negative effects. The effects of different time low temperature treatment on hybrid peony growth were explored.

[0090] 1. Method

[0091] When the hybrid seeds of CBS-B x BZSY and CBS-H x BZSY germinated to 1-2 cm in length, they were placed in a low temperature environment of 4°C, with humidity of 40-50%, light intensity of 2500-3000 lx, and light cycle of 14 h (light) / 10 h (dark). They were cultured for 0, 15, 30, and 45 days, respectively, and then placed in a culture room at 25±2°C, with humidity of 40-50%, light intensity of 2500-3000 lx, and light cycle of 14 h (light) / 10 h (dark). After 30 days of culture, the average number of true leaves, average seedling length, rooting rate, and average root length were counted.

[0092] Rooting rate = number of seedlings with roots / total number of germinated seeds

[0093] 3. Results

[0094] Table 4 Effects of different time low temperature treatment on seedling growth

[0095]

[0096]

[0097] Note: The lowercase letters in the table indicate the significance of the difference at the 0.05 level

[0098] The effects of different time low temperature treatment on seedling growth of CBS-B x BZSY and CBS-H x BZSY were explored, as shown in Table 4 and Figures 6-7 When the hybrid seeds of CBS-B x BZSY were treated with low temperature, the cotyledon and radicle continued to grow for 0 and 15 days. The average number of true leaves showed significant difference for 30 and 45 days of low temperature treatment, while the average seedling length, rooting rate, and average root length showed no significant difference. The average number of true leaves was highest for 45 days of low temperature treatment, with an average of 2.38 true leaves.

[0099] The average true leaf number and average root length of CBS-H x BZSY embryo treated with low temperature for 15 days had no significant difference (P>0.05) compared with that of the untreated group, but had significant difference (P<0.05) compared with that of the groups treated for 30 days and 45 days. The average seedling length of the four groups had significant difference, but the rooting rate of the four groups had no significant difference. When the low temperature treatment time was 45 days, the maximum seedling length was 7.17 cm; when the low temperature treatment time was 30 days, the maximum seedling length was 5.81 cm, which was shorter than that of the group treated for 45 days, but the growth rate had a slowing trend, so the environment can be adjusted after the low temperature treatment for 30 days to promote further growth. Therefore, the optimum low temperature treatment time for CBS-B x BZSY embryo to grow into seedlings is 45 days, and the optimum low temperature treatment time for CBS-H x BZSY embryo to grow into seedlings is 30 days.

[0100] After obtaining the tissue culture seedlings, acclimatization treatment is needed to improve their adaptability to the external adverse environment, so as to prepare for subsequent field cultivation. The suitable acclimatization and transplanting method for CBS-B x BZSY and CBS-H x BZSY tissue culture seedlings is:

[0101] When the length of the tissue culture seedlings is 4-5 cm, they are placed in a constant temperature incubator at 10°C for 72 h, the humidity is 30-40%, the light intensity is 2500-3000 lx, and the light cycle is 14 h (light) / 10 h (dark). Then, after washing the root medium with 500 mg / L carbendazim solution, the tissue culture seedlings are transplanted into a mixed substrate composed of nutrient soil, vermiculite and perlite at a volume ratio of 1:1:1, and the water content is 25%-35%. The indoor environment should be kept clean and well ventilated to achieve the transplanting of the tissue culture seedlings.

[0102] The following Examples 2-3 are specific embodiments of the preferred method of in vitro embryo culture of CBS-B x BZSY and CBS-H x BZSY hybrid seeds:

[0103] Example 2 In vitro embryo culture of peony hybrid seeds

[0104] 1) The hybrid seeds were obtained by crossing Chuan-shao No. 1 as the female parent and Bozhou peony as the male parent. The pollen of the male parent was applied to the stigma of the female parent, and the hybrid bag was removed 7-10 days later. The hybrid seeds were collected 90 days later when the fruit skin cracked and the seeds were mature and full (Example 2-1). Figure 1 The endosperm was removed (Example 2-2), and the embryo was obtained (Example 2-3). Figure 2 Figure 3

[0105] ​​2) Take the morphologically intact embryos obtained in step 1), inoculate them into the embryo initiation and seedling culture medium, and place them in a culture room with a temperature of 25±2℃, humidity of 40~50%, light intensity of 2500~3000lx, and a photoperiod of 14h (light) / 10h (dark) to cultivate seedlings. When the seedlings grow to 1~2cm, they are subjected to low-temperature treatment and then cultured at a constant temperature to obtain tissue culture seedlings. Figure 8 );

[0106] The embryo initiation and seedling growth medium formula is: WPM medium + 3.0 mg / L 6-benzylpurine (6-BA) + 3.0 mg / L gibberellin (GA3) + 1.0 g / L activated carbon + 30 g / L sucrose + 8.0 g / L agar, pH 5.9;

[0107] The low-temperature treatment method is as follows: seedlings are cultured for 45 days in a constant temperature incubator with a temperature of 4℃, humidity of 30-40%, light intensity of 2500-3000 lx, and a photoperiod of 14h (light) / 10h (dark).

[0108] The conditions for constant temperature incubation are:

[0109] Temperature 25±2℃, humidity 40~50%, light intensity 2500~3000lx, light cycle 14h light / 10h dark;

[0110] 3) Take tissue culture seedlings that are 4-5cm long obtained in step 2) (e.g. Figure 10 (As shown), harden off the seedlings by washing away the root culture medium with a 500 mg / L carbendazim solution, then transplant them into a mixed substrate consisting of nutrient soil, vermiculite, and perlite in a 1:1:1 volume ratio, with a moisture content of 25%–35%. Cultivate in a clean, well-ventilated indoor environment. Figure 12 , Figure 14 );

[0111] The method for hardening off seedlings is as follows:

[0112] Tissue culture seedlings were cultured for 72 hours in a constant temperature incubator with a temperature of 10℃, humidity of 30-40%, light intensity of 2500-3000 lx, and a photoperiod of 14h (light) / 10h (dark).

[0113] Pollen collected from Bozhou peony plants between April 12th and April 15th, 2022, was hybridized with Sichuan peony cultivar 1. The hybrid seeds obtained were cultured in vitro using the method described above. The results are as follows:

[0114] Embryos were cultured from the F1 seed embryos of “Chuanshao No. 1” (CBS-B) × “Bozhou Paeonia (BZSY)”. The seed embryos took 105 days to develop into seedlings. The seedlings were of good quality, with an average seedling length of over 5 cm after hardening off, an average number of true leaves of nearly 2, and a rooting rate of over 93%. The survival rate of F1 seedlings after transplanting was 28.89% 30 days after transplanting.

[0115] Example 3 In-vitro seed embryo culture of Paeonia hybrid seeds

[0116] 1) Cross Paeonia lactiflora Pall. cv. Chuan-shao No. 2 with Paeonia lactiflora Pall. cv. Bo-zhou, pollinate the stigma of the female parent with pollen of the male parent, remove the cross bag 7-10 days later, collect the hybrid seeds (Fig. 3) when the pericarp is cracked and the seeds are mature and full (Fig. 4) 90 days later, wash and sterilize the seeds, remove the endosperm (Fig. 5) and take the embryo to obtain the seed embryo (Fig. 6) of Paeonia hybrid seeds; Figure 1 Figure 2 Figure 3

[0117] 2) Take the morphologically intact seed embryo obtained in step 1) and inoculate it into seed embryo initiation and seedling formation medium, and place it in a culture room with a temperature of 25±2℃, a humidity of 40-50%, a light intensity of 2 500-3 000 lx, and a light cycle of 14 h (light) / 10 h (dark) to culture the seedling, and when the seedling is 1-2 cm long, perform low-temperature treatment and constant-temperature culture to obtain the tissue culture seedling (Fig. 7); Figure 9

[0118] The seed embryo initiation and seedling formation medium has the following formula: 1 / 2MS medium+2.0 mg / L 6-benzylpurine (6-BA)+2.0 mg / L gibberellin (GA3)+1.0 g / L activated carbon+30 g / L sucrose+8.0 g / L agar, and the pH value is 5.9;

[0119] The low-temperature treatment method is as follows: culture in a constant-temperature incubator with a temperature of 4℃, a humidity of 30-40%, a light intensity of 2 500-3 000 lx, and a light cycle of 14 h (light) / 10 h (dark) for 30 days;

[0120] The constant-temperature culture conditions are as follows:

[0121] a temperature of 25±2℃, a humidity of 40-50%, a light intensity of 2 500-3 000 lx, and a light cycle of 14 h (light) / 10 h (dark);

[0122] 3) Take the tissue culture seedling with a length of 4-5 cm obtained in step 2) and acclimate it (Fig. 8), wash the root culture medium with a 500 mg / L carbendazim solution, and transplant it into a mixed substrate composed of nutrient soil, vermiculite and perlite at a volume ratio of 1:1:1 and containing water in an amount of 25%-35% for cultivation in a clean and ventilated room (Fig. 9). Figure 11 Figure 13 Figure 15

[0123] The acclimation method is as follows:

[0124] Acclimate the tissue culture seedling in a constant-temperature incubator with a temperature of 10℃, a humidity of 30-40%, a light intensity of 2 500-3 000 lx, and a light cycle of 14 h (light) / 10 h (dark) for 72 h. ​​​​​​​

[0125] The pollen of Bozhou peony collected from April 12, 2022 to April 15, 2022 was hybridized with No. 2 Sichuan peony, and the hybrid seeds obtained were subjected to in-vitro seed embryo culture according to the above method, and the results were as follows:

[0126] The culture results are as follows: the embryos of the F1 generation of "No. 2 Sichuan peony" (CBS-H) x "Bozhou peony" (BZSY) are subjected to embryo culture, the seed embryo seedling time is 90d, the seedling quality is good, the average seedling length is more than 6cm after hardening, the average true leaf number is close to 3, the rooting rate is more than 86%, and the F1 embryo seedling transplanting survival rate is 35.56% after 30d of transplanting. After 60d of transplanting, it is observed that the leaves are larger and greener, and many new roots grow, and the growth effect is good.

[0127] In summary, the in-vitro seed embryo of the F1 generation of the hybrid of "No. 1 Sichuan peony" (CBS-B) or "No. 2 Sichuan peony" (CBS-H) and "Bozhou peony" (BZSY) is cultured in a specific culture medium, which solves the double dormancy problem of peony hybrid seed, overcomes the low germination rate and high rotting rate of peony hybrid seed and seed embryo abortion, and makes the hybrid peony seedling quantity small and the quality poor. Through the in-vitro seed embryo culture method of the peony hybrid seed of the present application, a hybrid peony seed embryo culture system of Sichuan Zhongjiang white peony and Anhui Bozhou peony can be constructed, new materials for breeding of medicinal peony new varieties are provided, and technical support for factory, scale and industrial production of peony tissue culture seedlings is provided.

Claims

1. A method for in vitro embryo culture of peony hybrid seeds, characterized in that: It includes the following steps: 1) Using Chuan Shao No. 1 or Chuan Shao No. 2 as the female parent and Bozhou peony as the male parent, hybridize the seeds, clean and disinfect them, and extract the embryos to obtain seed embryos; 2) Take the embryos, inoculate them into the embryo starter culture medium, and after the F1 generation embryos emerge, treat them at low temperature and then culture them at a constant temperature to obtain tissue culture seedlings. 3) Harden off the tissue culture seedlings obtained in step 2) and transplant them; When using Chuan Shao No. 1 as the female parent, the composition of the seed embryo initiation and seedling growth medium is: WPM medium + 3.0 mg / L 6-benzylpurine + 3.0 mg / L gibberellin + 1.0 g / L activated carbon + 30 g / L sucrose + 8.0 g / L agar; When using Chuan Shao No. 2 as the female parent, the composition of the seed embryo initiation and seedling growth medium is: 1 / 2 MS medium + 2.0 mg / L 6-benzylpurine + 2.0 mg / L gibberellin + 1.0 g / L activated carbon + 30 g / L sucrose + 8.0 g / L agar.

2. The method for in vitro embryo culture according to claim 1, characterized in that: The pH of the embryo initiation and seedling culture medium is 5.5-6.

0.

3. The method for in vitro embryo culture according to claim 1, characterized in that: Step 2) The conditions for culturing the embryos in the embryo initiation and seedling culture medium are: temperature 25±2℃, humidity 40~50%, light intensity 2500~3000 lx, and photoperiod 14h light / 10h dark.

4. The method for in vitro embryo culture according to claim 1, characterized in that: Step 2) When the seed embryos emerge and the seedlings grow to 1-2 cm in length, they are subjected to low-temperature treatment. The method of low-temperature treatment is as follows: the seedlings are cultivated in the following conditions: temperature 4℃, humidity 30-40%, light intensity 2500-3000 lx, and photoperiod 14 h light / 10 h dark. When using Chuan Shao No. 1 as the female parent, the cultivation time is 45 days, and when using Chuan Shao No. 2 as the female parent, the cultivation time is 30 days.

5. The method for in vitro embryo culture according to claim 1, characterized in that: The conditions for constant temperature culture in step 2) are: temperature 25±2℃, humidity 40~50%, light intensity 2500~3000 lx, and light cycle 14 h light / 10 h dark.

6. The method for in vitro embryo culture according to claim 1, characterized in that: The tissue culture seedlings described in step 3) are 4-5 cm long.

7. The method for in vitro embryo culture according to claim 1, characterized in that: Step 3) describes the seedling hardening method as follows: the tissue culture seedlings are cultured for 72 hours under the conditions of 10℃ temperature, 30-40% humidity, 2500-3000 lx light intensity, and a photoperiod of 14 h (light) / 10 h (dark).

8. The method for in vitro embryo culture according to claim 1, characterized in that: The transplanting process involves washing the root culture medium off the tissue culture seedlings with a carbendazim solution, then transferring them to a substrate composed of nutrient soil, vermiculite, and perlite, and cultivating them in a clean, well-ventilated indoor environment.

9. The method for in vitro embryo culture according to claim 8, characterized in that: The concentration of the carbendazim solution is 500 mg / L.

10. The method for in vitro embryo culture according to claim 8, characterized in that: The substrate has a moisture content of 25% to 35%, and the volume ratio of nutrient soil, vermiculite, and perlite is 1:1:1; the perlite has a particle size of 3-5 mm, and the vermiculite has a particle size of 1-3 mm.

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