Method for obtaining regenerated plant through suspension culture of meristematic nodules of peony

Abstract

The invention belongs to the technical field of plant tissue culture, and relates to a method for obtaining regenerated plants through suspension culture of peony meristematic nodules, which comprises the following steps: 1) inducing meristematic nodules; 2) suspension culture multiplication; 3) differentiation culture; and 4) plant regeneration. According to the method, clustered meristematic nodules formed by inducing peony cotyledon calluses are used as explants, the explants are cut and then transferred into a liquid culture medium for suspension culture so as to greatly increase the number of the meristematic nodules, then the proliferated meristematic nodules are transferred into a solid differentiation culture medium for bud and leaf differentiation, and then plant regeneration is achieved; the regeneration plant is obtained by establishing a peony meristematic nodule suspension culture system, and the regeneration efficiency of the peony in-vitro plant is greatly improved.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology and relates to a method for obtaining regenerated plants through suspension culture of peony meristems. Background Technology

[0002] Peony, a traditional Chinese flower, is a deciduous subshrub belonging to the Paeoniaceae family and the Paeonia genus. It possesses medicinal, ornamental, and cultural value. With the discovery of the value of peony seed oil, peony research has surged, leading to significant market demand. Traditional peony propagation methods are inefficient, and cultivating a new peony variety requires more than 10 years. Plant tissue culture for in vitro regeneration is highly efficient, has a short cycle, and maintains genetic stability, making it a necessary means for targeted genetic improvement of peonies through biotechnology and an effective way to commercialize peonies. Since the first report of peony tissue culture in 1984, approximately 40 years of continuous research have revealed that in vitro peony regeneration technology is extremely difficult, and a complete and stable regeneration system has not yet been established for either somatic embryo regeneration or callus organ regeneration.

[0003] Meristematic nodules (MNs) are special structures that resemble somatic embryos in appearance but have significant histological differences. They can proliferate stably over a long period of time and have a high degree of differentiation. Due to their unique structural characteristics and advantages, MNs are defined as the third in vitro regeneration pathway besides organogenesis and somatic embryogenesis. They are an ideal alternative for establishing in vitro regeneration systems, producing metabolites, and conducting research on plant genetic transformation.

[0004] Previous studies have achieved callus differentiation and plant regeneration in peony through the meristematic nodule pathway. This system was conducted on solid culture media. Explants were used to induce callus clusters, which then induced meristematic nodules in a differentiation medium. These nodules further induced adventitious buds, which rooted and were transplanted, resulting in plant regeneration. However, the induced meristematic nodules on solid media could not proliferate in number, only increasing in size, leading to low efficiency. Zhong Yuan found that in the suspension culture of meristematic nodules in *Peony purpurea*, cut nodules could efficiently proliferate in number through suspension culture in liquid media, but could not achieve subsequent nodule differentiation and plant regeneration. Therefore, the existing in vitro regeneration technology for peony callus-induced meristematic nodules is inefficient. The meristematic nodules cannot proliferate in solid media, resulting in a limited number of nodules available for differentiation, leading to low plant regeneration efficiency. Furthermore, the existing peony nodule suspension culture system only achieves nodule proliferation but fails to achieve differentiation.

[0005] Therefore, establishing a peony meristem suspension culture plant regeneration system to significantly increase the number of meristems and thus improve plant regeneration efficiency is a problem that needs to be addressed. Summary of the Invention

[0006] The main objective of this invention is to overcome the deficiencies in the prior art and provide a method for obtaining regenerated plants through suspension culture of peony meristems.

[0007] To achieve the above objectives, the specific technical solution is as follows: This invention provides a method for obtaining regenerated plants through suspension culture of peony meristems, comprising the following steps: 1) Meristematic nodule induction: Callus tissue is induced by cotyledons of peony, which in turn induces the formation of clustered meristematic nodules; 2) Suspension culture proliferation: The above-mentioned clustered meristematic nodules were cut and inoculated into liquid proliferation medium for suspension culture to obtain proliferated meristematic nodules; 3) Differentiation culture: The meristematic nodules proliferated in suspension culture in step 2) are transferred to solid differentiation culture medium to induce bud and leaf tissue differentiation; 4) Plant regeneration: After the nodules that have differentiated bud and leaf tissues are cultured for adventitious bud elongation, they are then cultured for rooting and transplanted for acclimatization to obtain regenerated plants.

[0008] This invention uses clustered meristematic nodules induced from peony cotyledon callus as explants. After cutting, the nodules are transferred to a liquid culture medium for suspension culture to significantly increase the number of meristematic nodules. Then, the proliferated meristematic nodules are transferred to a solid differentiation medium for bud and leaf differentiation, thereby achieving plant regeneration. By establishing a suspension culture system for peony meristematic nodules to obtain regenerated plants, the efficiency of peony in vitro plant regeneration can be greatly improved.

[0009] Further, in step 1), the peony cotyledons are 'Fengdan' peony cotyledons; preferably, the 'Fengdan' peony cotyledons are peony cotyledons grown from 'Fengdan' peony seeds after 15 days of embryo germination culture.

[0010] Furthermore, the specific method for inducing meristematic nodules is as follows: Peony cotyledons are inoculated into callus induction medium and cultured to obtain callus tissue, and then the callus tissue is transferred to meristematic nodule induction medium for subculture to obtain clustered meristematic nodules. The callus induction medium is an MS basal medium supplemented with 0.25-1.0 mg / L chlorpyrifos (CPPU) and 0.5-2.0 mg / L naphthaleneacetic acid (NAA); preferably, an MS basal medium supplemented with 0.5 mg / L chlorpyrifos (CPPU) and 1.0 mg / L naphthaleneacetic acid (NAA). The meristematic nodule induction medium is an MS basal medium supplemented with 0.25-1.0 mg / L thiazuron (TDZ) and 0.25-1.0 mg / L chlorpyrifos (CPPU), preferably an MS basal medium supplemented with 0.5 mg / L thiazuron (TDZ) and 0.5 mg / L chlorpyrifos (CPPU).

[0011] Specifically, the subculture is carried out every 15 days, and the clustered meristematic nodules obtained after 6 subcultures are preferred.

[0012] Further, in step 2), the clustered meristematic nodules are cut into nodule blocks with a diameter of 2.5-6 mm, preferably 2.5-3.5 mm, and more preferably 3 mm; And / or, the liquid proliferation medium is one of the following: MS basal medium supplemented with 0.5-2.0 mg / L TDZ, MS basal medium supplemented with 0.5-2.0 mg / L CPPU, or MS basal medium supplemented with 0.25-1.0 mg / L TDZ and 0.25-1.0 mg / L CPPU; preferably MS basal medium supplemented with 0.5-2.0 mg / L TDZ or MS basal medium supplemented with 0.25-1.0 mg / L TDZ and 0.25-1.0 mg / L CPPU; more preferably MS basal medium supplemented with 0.5-2.0 mg / L TDZ.

[0013] Further, the liquid proliferation medium is one of MS basal medium supplemented with 1.0 mg / L TDZ, MS basal medium supplemented with 1.0 mg / L CPPU, or MS basal medium supplemented with 0.5 mg / L TDZ and 0.5 mg / L CPPU; preferably MS basal medium supplemented with 1.0 mg / L TDZ or MS basal medium supplemented with 0.5 mg / L TDZ and 0.5 mg / L CPPU; more preferably MS basal medium supplemented with 1.0 mg / L TDZ.

[0014] In this invention, the clustered meristematic nodules cut from peony plants mainly proliferate in liquid culture medium. During subculture, the number of nodules with a diameter of 3 mm as explants tends to increase more than that with a diameter of 6 mm. After four subcultures, the number of proliferating nodules with a diameter of 3 mm as explants is higher than that with a diameter of 6 mm.

[0015] The nodules cut in this invention exhibit different states when suspended in liquid culture media with different exogenous hormones. Cytokinin is crucial for the proliferation of nodules in liquid suspension culture, and the liquid proliferation medium with added cytokinin in this invention has a significant effect on the efficiency of suspension culture proliferation.

[0016] Further, in step 2), the rotation speed of the suspension culture is 120-180 rpm; preferably 120-150 rpm; more preferably 150 rpm; And / or, the subculture cycle of the suspension culture is 20 days, and the number of subcultures is not less than 3 times, preferably 4 times, to obtain proliferating meristematic nodules.

[0017] The rotation speed of the liquid culture medium in suspension culture has a significant effect on the proliferation efficiency of suspension culture. Different rotation speeds result in different proliferation effects of meristematic nodules. The rotation speed of the liquid culture medium used in this invention can effectively improve the proliferation efficiency of suspension culture. Among them, considering the nodule biomass, the total number of nodules, and the number of nodules of 3-6 mm, the optimal rotation speed is 150 rpm.

[0018] Further, in step 3), the meristematic nodules are meristematic nodules with a diameter ≥ 3 mm that have been proliferated by suspension culture in step 2); preferably, the meristematic nodules are between 3 and 6 mm. And / or, the solid differentiation medium is WPM basal medium supplemented with 0.5-2.0 mg / L CPPU or WPM basal medium supplemented with 0.25-1.0 mg / L CPPU + 0.25-1.0 mg / L TDZ; preferably WPM basal medium supplemented with 0.5-2.0 mg / L CPPU.

[0019] Further, the solid differentiation medium is WPM basal medium supplemented with 1.0 mg / L CPPU or WPM basal medium supplemented with 0.5 mg / L CPPU + 0.5 mg / L TDZ; preferably WPM basal medium supplemented with 1.0 mg / L CPPU.

[0020] The diameter of the proliferating meristems plays a significant role in nodule differentiation. After 8 subcultures, the bud and leaf differentiation rate of nodules <3mm was low. After suspension culture, nodules with a diameter of 3-6mm showed the highest differentiation rate in solid differentiation medium. Therefore, the optimal treatment was proliferating nodules with a diameter of 3-6mm.

[0021] Exogenous hormones in solid differentiation medium have a significant effect on nodule differentiation. Different exogenous hormones have different effects on nodule differentiation. The solid differentiation medium of the present invention can effectively improve the bud and leaf differentiation rate of proliferating meristematic nodules.

[0022] Furthermore, in step 3), the subculture cycle of the differentiation culture is 20 days, preferably the bud and leaf tissue after 8 subcultures.

[0023] As the number of subcultures increases, the differentiation rate gradually increases, and the bud and leaf differentiation rate of the nodes after 8 subcultures can reach a relatively high level.

[0024] Further, in step 4), the culture medium used for the adventitious shoot elongation culture is WPM basic medium supplemented with 0.2-0.3 mg / L BA and 0.1-0.3 mg / L GA3; preferably WPM basic medium supplemented with 0.25 mg / L BA and 0.2 mg / L GA3. Furthermore, the nodules from which the induced bud and leaf tissue was induced were cultured five times in an adventitious bud elongation medium, with a subculture cycle of 30 days.

[0025] Furthermore, in step 4), the rooting culture includes two stages: rooting induction culture and root formation culture; The rooting induction culture method is as follows: select elongated adventitious buds with stem length ≥1cm, first perform low temperature treatment, and then inoculate them into rooting induction medium and culture them in the dark; the rooting induction medium contains indolebutyric acid (IBA) and putrescine. The method for root formation culture is as follows: adventitious buds after rooting induction are transferred to a root formation medium containing activated carbon (AC) and cultured under light conditions to promote root elongation.

[0026] Furthermore, the rooting induction medium is a 1 / 2 MS basal medium supplemented with 0.5-2.0 mg / L IBA and 0.5-2.0 mg / L putrescine; preferably, a 1 / 2 MS basal medium supplemented with 1.0 mg / L IBA and 1.0 mg / L putrescine. And / or, the root formation medium is a 1 / 2 MS basal medium supplemented with 0.3-0.5 g / L AC; preferably, a 1 / 2 MS basal medium supplemented with 0.4 g / L AC.

[0027] Further, in step 4), the low-temperature treatment involves subjecting the elongated adventitious buds to a cold treatment at 4-8°C for 5-10 days before transferring them to the rooting induction medium; preferably, a cold treatment at 4°C for 8 days.

[0028] Further, in step 4), the substrate for transplanting and acclimatization is a mixture of perlite, vermiculite and peat moss; preferably, it is a mixture of perlite, vermiculite and peat moss in a volume ratio of 1:1:1.

[0029] Compared with the prior art, the present invention has the following significant advantages: (1) In this invention, meristematic nodules induced by callus from the cotyledons of 'Fengdan' peony are used as explants. Large-scale nodule proliferation is achieved through nodule suspension culture. The proliferated nodules are then transferred to a solid culture medium to successfully regenerate the plants and allow them to survive.

[0030] (2) The diameter of the explant, the suspension culture medium and the suspension culture speed used in this invention can enable 50 nodule pieces to proliferate to 183.33 nodules after 4 subcultures (80 days); the proliferated nodules can differentiate in solid culture medium, which greatly improves the efficiency of the peony meristem regeneration system.

[0031] (3) The solid differentiation culture medium and the diameter of the proliferating meristems used in this invention can make the differentiation rate of nodular buds and leaves reach up to 48.89%, the average number of adventitious buds in the adventitious bud elongation culture medium is 12.13, the rooting rate of adventitious buds reaches 43.33%, and the survival rate of rooted seedlings after transplanting reaches 50%. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0033] Figure 1 This invention relates to the explant of clustered meristematic nodules; Figure 2 The nodule fragments cut by this invention reform clustered meristems in liquid culture medium; Figure 3 It is a single free nodule proliferating according to the present invention; Figure 4 These are nodules of different shapes and sizes in the liquid culture medium of this invention; Figure 5 This is the state of suspension culture in the liquid culture medium with different exogenous hormones added according to the present invention; A: Nodule suspension culture under 1.0 mg / L CPPU + 1.0 mg / L NAA hormone conditions; B: Nodule suspension culture under 1.0 mg / L CPPU + 0.25 mg / L NAA hormone conditions; C: Nodule suspension culture under 1.0 mg / L CPPU + 0.125 mg / L NAA hormone conditions; D: Nodule suspension culture under 0.5 mg / L CPPU + 0.5 mg / L TDZ hormone conditions; E: Nodule suspension culture under 1.0 mg / L CPPU hormone conditions; F: Nodule suspension culture under 1.0 mg / L TDZ hormone conditions. Figure 6 This invention relates to the differentiation of nodular bud and leaf tissues; Figure 7 This invention relates to the elongation of adventitious buds; Figure 8This invention relates to the rooting of adventitious buds; Figure 9 This invention enables the transplanting and acclimatization of rooted seedlings to ensure survival. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0035] Unless otherwise specified in the embodiments of the present invention, the techniques or conditions described in the literature in this field or the product instructions shall be followed; if the manufacturers of the reagents or instruments used are not specified, they are all conventional products that can be purchased through legitimate channels.

[0036] The seeds used in this invention were collected from 'Fengdan' peony seeds that had been naturally pollinated for 100 days at the germplasm resource base of Hubei University for Nationalities in August 2022. After sand stratification, they were stored in a refrigerator at 4°C for later use.

[0037] Example 1 This embodiment describes the induction of meristematic nodes in peony cotyledons, including the following steps: (1) After rinsing the peony seeds with running water for 15 minutes, place them on a clean bench and sterilize them with 75% ethanol for 1 minute, then sterilize them twice with 5% sodium hypochlorite solution for 7 minutes each time. Finally, rinse them three times with sterile water. Place the sterilized seeds on sterile filter paper and cut them open along the midline at halfway point with sterile forceps and a scalpel. Remove the seed coat and endosperm and take out the embryo.

[0038] (2) Embryos were inoculated into MS medium supplemented with 0.5 mg / L 6-benzylaminopurine (BA) and 0.5 mg / L gibberellin (GA3) for embryo germination. After 15 days of embryo germination, the cotyledons of peony were cut into 1 cm × 1 cm cubes using a knife and forceps. These cubes were then inoculated into callus induction medium MS + 1.0 mg / L NAA + 0.5 mg / L CPPU with the adaxial surface facing upwards. After 40 days of induction, the callus was transferred to meristematic nodule induction medium MS + 0.5 mg / L TTDZ + 0.5 mg / L CCPPU. Subculture was performed every 15 days, and after 6 subcultures, clustered meristematic nodules were obtained. Figure 1 As shown.

[0039] Example 2 This embodiment describes the suspension proliferation culture of clustered meristematic nodules, including the following steps: (1) Using the clustered basal nodules of peony as explants, they were cut into small nodule pieces with a diameter of about 3 mm; (2) Pour 50 ml of liquid proliferation medium into each 100 ml glass Erlenmeyer flask and inoculate 50 nodule pieces for suspension culture. The rotation speed of the suspension culture is set to 150 rpm. The liquid proliferation medium was cultured in MS liquid medium containing 0.5 mg / L TDZ + 0.5 mg / L CPPU; the MS liquid medium was based on MS basal medium, with 30 g / L sucrose added, and the pH was adjusted to 5.8, and then autoclaved at 120°C for 20 minutes.

[0040] (3) The subculture cycle of suspension culture was selected to be once every 20 days, and the number of meristematic nodules of different diameters (<3mm, 3-6mm, >6mm) was counted after 0-4 subcultures. The biomass (g) of all meristematic nodules was counted after the 4th subculture. Each replicate contained 3 conical flasks and each treatment contained 3 replicates.

[0041] Example 3 This embodiment describes the suspension proliferation culture of clustered meristems. The difference from Embodiment 2 is that in this embodiment, clustered meristems of peony are used as explants, which are cut into small pieces with a diameter of about 6 mm. Other methods and steps are the same as in Embodiment 2, and will not be repeated here.

[0042] Example 4 This embodiment describes the suspension proliferation culture of clustered meristematic nodules. The difference from Example 2 is that the liquid proliferation medium used in this embodiment is MS liquid medium containing 1.0 mg / L CPPU. Other methods and steps are the same as in Example 2, and will not be repeated here.

[0043] Example 5 This embodiment describes the suspension proliferation culture of clustered meristematic nodules. The difference from Example 2 is that the liquid proliferation medium used in this embodiment is MS liquid medium containing 1.0 mg / L TDZ. Other methods and steps are the same as in Example 2, and will not be repeated here.

[0044] Example 6 This embodiment describes the suspension proliferation culture of clustered meristematic nodules. The difference from Example 5 is that the rotation speed of the suspension culture in this embodiment is set to 120 rpm; other methods and steps are the same as in Example 5, and will not be repeated here.

[0045] Example 7 This embodiment describes the suspension proliferation culture of clustered meristematic nodules. The difference from Example 5 is that the rotation speed of the suspension culture in this embodiment is set to 180 rpm; the other methods and steps are the same as in Example 5, and will not be repeated here.

[0046] Example 8 This embodiment describes the differentiation culture of meristematic nodules, including the following steps: (1) The meristematic nodules with a diameter of 3-6 mm obtained after 4 subcultures in Example 5 were placed in a solid differentiation medium WPM containing 0.5 mg / L CPPPU + 0.5 mg / L TTDZ to induce bud and leaf differentiation; the solid medium ratio was 7.5 g / L agar and 30 g / L sucrose, and the pH was adjusted to 5.8.

[0047] (2) The subculture cycle was set at 20 days, and the differentiation rate of shoot and leaf tissue of the nodules was calculated at the 4th, 5th, 6th, 7th and 8th subcultures (number of nodules that differentiated into shoot and leaf tissue / number of inoculated nodules × 100%). Each replicate contained 15 nodules, and each treatment contained 3 replicates.

[0048] Example 9 This embodiment describes the differentiation culture of meristematic nodules. The difference from Example 8 is that the differentiation culture in this embodiment is carried out in WPM solid differentiation medium containing 1.0 mg / L CPPU. Other methods and steps are the same as in Example 8, and will not be repeated here.

[0049] Example 10 This embodiment describes the differentiation culture of meristematic nodules. The difference between this embodiment and Example 9 is that this embodiment uses meristematic nodules with a diameter of >6mm obtained after 4 subcultures in Example 5 for differentiation culture; other methods and steps are the same as in Example 9, and will not be repeated here.

[0050] Example 11 This embodiment describes a method for plant regeneration, including the following steps: (1) Nodules of differentiated bud and leaf tissue of different diameters were inoculated into adventitious bud elongation medium (WPM + 0.25 mg / L BA + 0.2 mg / L GA3) and cultured 5 times, with a subculture cycle of 30 days. The average number of differentiated adventitious buds was counted. Each replicate contained 5 nodules of differentiated bud and leaf tissue, and each treatment contained 3 replicates.

[0051] (2) Adventitious shoots with stem length ≥1cm from different treatments were cut for rooting culture (Wang et al., 2016). Before rooting induction, the shoots were subjected to a 4℃ cold treatment for 8 days. Then, the adventitious shoots with stem length ≥1cm were inoculated into root induction medium (1 / 2MS + 1.0 mg / L IBA + 1.0 mg / L putrescine) and induced to root in the dark for 30 days. They were then transferred to root formation medium (1 / 2MS + 0.4 g / L AC) and cultured under light for 20 days to promote root elongation. The rooting rate was counted. Each replicate contained 10 adventitious shoots, and each treatment contained 3 replicates.

[0052] (3) Wash the obtained rooted test-tube seedlings with warm water to remove the culture medium, and transplant them into square pots filled with substrate. The transplanting substrate (perlite: vermiculite: peat moss = 1:1:1) should be mixed in advance according to the ratio and autoclaved (120℃, 40 minutes). When using, spray with water until it can be formed into a ball by hand without crumbling; the water content should not be too high. The transplanted seedlings are cultured in an artificial climate chamber. The artificial climate chamber conditions are: temperature (22±1℃), humidity 90%, light intensity 25 μmol·m -2 ·s -1 (Incandescent lamp), 16 hours of light per day, and Hogland nutrient solution applied monthly.

[0053] Comparative Example 1 This comparative example is a suspension proliferation culture of clustered meristematic nodules. The difference from Example 2 is that the liquid proliferation medium used in this comparative example is MS liquid medium containing 1.0 mg / L CPPU + 1.0 mg / L NAA. Other methods and steps are the same as in Example 2, and will not be repeated here.

[0054] Comparative Example 2 This comparative example is a suspension proliferation culture of clustered meristematic nodules. The difference from Example 2 is that the liquid proliferation medium used in this comparative example is MS liquid medium containing 1.0 mg / L CPPU + 0.5 mg / L NAA; other methods and steps are the same as in Example 2, and will not be repeated here.

[0055] Comparative Example 3 This comparative example is a suspension proliferation culture of clustered meristematic nodules. The difference from Example 2 is that the liquid proliferation medium used in this comparative example is MS liquid medium containing 1.0 mg / L CPPU + 0.25 mg / L NAA; other methods and steps are the same as in Example 2, and will not be repeated here.

[0056] Comparative Example 4 This comparative example is a suspension proliferation culture of clustered meristematic nodules. The difference from Example 2 is that the liquid proliferation medium used in this comparative example is MS liquid medium containing 1.0 mg / L CPPU + 0.125 mg / L NAA; other methods and steps are the same as in Example 2, and will not be repeated here.

[0057] Comparative Example 5 This comparative example is the differentiation culture of meristematic nodules. The difference between this example and Example 8 is that the differentiation culture of the comparative example is carried out in WPM solid differentiation medium containing 1.0 mg / LTDZ. Other methods and steps are the same as in Example 8, and will not be repeated here.

[0058] Comparative Example 6 This comparative example is the differentiation culture of meristematic nodules. The difference between this comparative example and Example 10 is that this comparative example uses meristematic nodules with a diameter of <3 mm obtained after 4 subcultures in Example 5 for differentiation culture; other methods and steps are the same as in Example 10, and will not be repeated here.

[0059] The specific formulations of the tissue culture basal media MS, 1 / 2 MS, and WPM used in the embodiments and comparative examples of this invention are shown in Tables 1-3. The method for preparing 1L of culture medium is as follows: Prepare a stock solution of macro-elements, micro-elements, vitamins, and growth regulators. Add an appropriate amount of sucrose and 7.5g of agar (not added to liquid culture media) to the distilled water, and bring the volume to 1L. Adjust the pH of all culture media to 5.8-6.0 at room temperature. Autoclave the culture media in containers sealed with plastic film for 20 minutes (121℃, 0.1mPa). In a clean bench, shake the still-unsolidified culture medium (approximately 40℃) for a few seconds, add the heat-sensitive hormone that has been pre-filtered and sterilized, and dispense into sterilized culture containers.

[0060] Table 1 MS formulation of tissue culture basic medium

[0061] Table 2 Formula for Tissue Culture Basic Medium 1 / 2MS

[0062] Table 3 WPM formulation for tissue culture basal medium

[0063] Results analysis: In liquid culture, the clustered nodules of peony primarily proliferate. During the first subculture, the divided nodule fragments show slight volume increase after being transferred to liquid culture, with multiple spherical protrusions (new nodules) appearing on the surface. However, these proliferate without detaching from the original nodule body, and no new, free nodules appear. During the second subculture, the newly proliferated nodules on the original nodule body increase in size and number, completely covering the original nodule and reforming the appearance of a clustered nodule, as shown in the image. Figure 2 As shown. Subsequently, new nodules appear on the surface of the clustered nodules. As the volume further increases, gaps are formed between the clustered nodules and the new nodules. Later, under the action of vibration, these new nodules detach from the clustered nodules and form proliferating individual free nodules, such as... Figure 3 As shown.

[0064] During the third or fourth subculture, multiple new nodules will form on the surface of a single newly proliferating free nodule. As the number of surface nodules increases, new, smaller clusters of nodules will form. These newly formed clusters of nodules will then proliferate into even smaller nodules. The newly formed nodules will not grow indefinitely in liquid culture; after the formation of clusters of nodules, their diameter remains stable as new nodules appear and detach. Because the new clusters of nodules vary in size, nodules of different morphologies and sizes can be observed during subculture, such as... Figure 4 The numbers A, B, C, D, E, and F are shown in the diagram.

[0065] In Example 2, using 3mm diameter nodule segments as explants, the nodule biomass and total number of nodules gradually increased with each subculture during suspension culture. The total number of nodules remained unchanged after the first subculture. During the second subculture, only a few new free nodules (<3mm, 5 nodules) appeared, and the original nodule explants gradually developed into clustered nodules (3-6mm, 50 nodules). During the third subculture, a large number of new free nodules (<3mm, 29.33 nodules) appeared, while the number of newly formed clustered nodules remained stable (3-6mm, 45.33 nodules; >6mm, 4.67 nodules). During the fourth subgeneration, more free small nodules were added (<3mm, 83.33). As the small nodules added during the third subgeneration further developed into new clustered nodules, the number of newly formed clustered nodules increased (3-6mm, 70). As the number of small nodules on the clustered nodules increased and detached, the volume of the clustered nodules remained basically stable (3-6mm), with a few increasing to >6mm (7.33), but generally not exceeding 10mm, as shown in Table 4.

[0066] Table 4. Cut nodules with a diameter of 3 mm under liquid suspension conditions Nodule number and proliferation status at different generations

[0067] In Example 3, the biomass and total number of nodules of 6 mm diameter during suspension culture were basically consistent with those of 3 mm diameter nodules. Although the nodule diameter increased, the total number remained unchanged after the first subculture (>6 mm, 50). During the second subculture, a few new free nodules appeared (<3 mm, 2.67), while the diameter of the newly formed clustered nodules on the original explant was greater than 6 mm. During the third subculture, new free nodules also appeared (<3 mm, 15.33), and some of the free nodules further developed into new clustered nodules (3-6 mm, 4), while the volume of the clustered nodules formed on the original explant remained basically stable as the number and detachment of small nodules increased (>6 mm, 50). During the fourth generation, more free nodules were added (<3mm, 42.67), and the free nodules developed into more new clustered nodules (3-6mm, 34). Some of the newly developed clustered nodules increased in size to >6mm, but generally did not exceed 10mm. The total number of nodules >6mm was 53, as shown in Table 5.

[0068] Table 5. Cut nodules with a diameter of 6 mm under liquid suspension conditions Nodule number and proliferation status at different generations

[0069] Regardless of whether the nodules were 3mm or 6mm in diameter, no new nodules were added during the first subculture. During the second to fourth subcultures, the number of nodules gradually increased, and the increasing trend was progressively stronger. The increase in explants from 3mm diameter nodules was greater than that from 6mm diameter nodules. After four subcultures, the number of explants from 3mm diameter nodules (160.67) was significantly higher than that from 6mm diameter nodules (129.67). Therefore, 3mm diameter nodules are the optimal choice for suspension culture.

[0070] The state of the nodule fragments differed when cultured in suspension in liquid media supplemented with different exogenous hormones. Compared to suspension culture in media supplemented only with cytokinin, in Comparative Example 1, explants in media supplemented with 1.0 mg / L CPPU and 1.0 mg / L NAA directly swelled into callus masses after four subcultures, without nodule proliferation. Figure 5 A, and the biomass of the callus was relatively high (26.08 g), as shown in Table 6. In Comparative Examples 2 and 3, as the auxin ratio decreased (0.5-0.25 mg / L NAA), the number of grooves on the surface of the callus gradually increased, but nodule proliferation still did not occur, such as Figure 5B, and the biomass gradually decreased; in Comparative Example 4, when the auxin ratio dropped to 0.125 mg / L, clustered nodules formed on the surface of the explants after subculture, with a small number of newly added free small nodules (<3 mm, 13.67); however, compared with the nodule state in the medium with only cytokinin added, the clustered nodules in the medium with 0.125 mg / L NAA added were larger in volume and had fewer grooves on the surface of the clustered nodules ( Figure 5 C), the number of newly added nodules is relatively low.

[0071] In Examples 2, 4, and 5, the state of explants cultured in suspension in a culture medium with only cytokinin added ( Figure 5 As described above, nodules D, E, and F all proliferated significantly after four suspension subcultures, with the 1.0 mg / L TDZ in Example 5 (182.00) significantly higher than those in Example 2 (0.5 mg / L CPPPU + 0.5 mg / LTDZ) (159.67) and Example 4 (1.0 mg / L CPPPU) (123.67). In conclusion, cytokinin is crucial for nodule proliferation in liquid suspension culture, and 1.0 mg / LTDZ is more effective than 1.0 mg / L CPPPU and 0.5 mg / L CPPPU + 0.5 mg / LTDZ. The total number of nodules treated with 1.0 mg / LTDZ after four subcultures was 182.00, representing the optimal treatment.

[0072] Table 6. Effects of liquid culture medium with different hormone ratios on meristematic nodule proliferation (after 4 subcultures)

[0073] In Examples 5, 6, and 7, the proliferation effects of meristematic nodules varied under different rotation speeds, as shown in Table 7. The total number of nodules gradually increased with increasing rotation speed, but there was no significant difference in the total number of nodules between the 150 rpm and 180 rpm treatments. Specifically, the number of larger nodules (3-6 mm and >6 mm) decreased with increasing rotation speed, while the number of smaller nodules (<3 mm) increased with increasing rotation speed. There was no significant difference in the number of 3-6 mm nodules between the 120 rpm and 150 rpm treatments. Considering the experimental results on the effect of meristematic nodule diameter on differentiation discussed below, the 150 rpm condition was the optimal choice in this study, resulting in the highest nodule biomass (22.77 g), a relatively high total number of nodules (183.33 g), and a large number of 3-6 mm nodules (74.67 g).

[0074] Table 7. Effects of liquid culture medium at different rotation speeds on meristematic nodule proliferation (after 4 subcultures)

[0075] In Examples 8, 9, and Comparative Example 5, the proliferating meristematic nodules were placed in differentiation media containing three different ratios of cytokinin, resulting in varying differentiation effects, as shown in Table 8. The shoot and leaf differentiation rate of the 1.0 mg / L CTPPU treatment (48.89%) was significantly higher than that of the 1.0 mg / LTDZ treatment (31.11%), while there was no significant difference compared to the 0.5 mg / L CTPPU + 0.5 mg / LTDZ treatment (42.22%). Therefore, the optimal treatment was 1.0 mg / L CTPPU.

[0076] Table 8. Effects of different exogenous hormones on the differentiation of proliferating meristems (after 8 subcultures)

[0077] In Examples 9, 10, and Comparative Example 6, the number of nodules proliferating in liquid culture medium did not increase after being transferred to solid culture medium. The differentiation effects of nodules of different diameters in the differentiation medium varied, as shown in Table 9. Nodules <3 mm in diameter only showed differentiation after 7 subcultures. Nodules with diameters of 3-6 mm and >6 mm were mostly in a clustered nodule state; primordia differentiation was observed starting after 4 subcultures, and obvious bud and leaf tissue differentiation was observed at the end of the 8th subculture. Figure 6 With increasing subculture number, the differentiation rate gradually increased, but there was no significant difference between the two. After 8 subcultures, the bud and leaf differentiation rate of nodules <3mm (15.56%) was significantly lower than that of nodules with diameters of 3-6mm (46.67%) and >6mm (44.45%). This indicates that nodules of 3-6mm showed the highest differentiation rate in solid differentiation medium after suspension culture.

[0078] Table 9. Effect of suspended proliferating nodule diameter on bud and leaf differentiation

[0079] In Example 11, elongated adventitious buds can be obtained by transferring the nodules of differentiated bud and leaf tissue to an adventitious bud elongation medium, such as... Figure 7 As shown in Table 10, nodules with a diameter of 3-6 mm had the highest average number of adventitious buds (12.13), which was higher than that of nodules with a diameter >6 mm (11.73), but the difference was not statistically significant. However, both were significantly higher than those of nodules with a diameter <3 mm (5.73). Transferring the adventitious buds from each treatment to a rooting medium could induce rooting and seedling formation. Figure 8 As shown, the adventitious buds from nodules with a diameter of 3-6 mm had the highest rooting rate (43.33%), but the difference between this and nodules >6 mm (36.67%) and nodules <3 mm (30%) was not significant. The rooted adventitious buds were transplanted, and the survival rate was calculated after transplanting. Figure 9As shown, the transplant survival rate of rooted seedlings with nodules of 3-6 mm in diameter was the highest (50%), but there was no significant difference compared with nodules of >6 mm (46.67%) and nodules of <3 mm in diameter (40%).

[0080] Table 10 Effects of suspension proliferation nodule diameter on adventitious shoot rooting and transplanting

[0081] In summary, this invention successfully established a suspension culture system for peony meristems to obtain regenerated plants. High-efficiency proliferation of peony meristems was achieved through suspension culture, and the proliferated nodules differentiated in a solid culture medium, realizing plant regeneration under in vitro culture conditions. By screening the effects of nodule size and subculture number on nodule suspension culture, it was found that nodule proliferation was basically not achieved after one subculture. From the second subculture onwards, proliferation began to occur, and the proliferation rate gradually increased with the number of subcultures. The number of nodules after four subcultures of nodules with a diameter of 3 mm was significantly higher than that of nodules with a diameter of 6 mm. Nodules with a diameter of 3 mm are the preferred starting nodules for suspension culture. By screening the effects of exogenous hormones and rotation speed on nodule suspension culture, the optimal liquid culture medium for suspension culture was determined to be MS + 1.0 mg / LTDZ of exogenous hormones, and the optimal suspension culture rotation speed was 150 rpm. Under these conditions, the number of nodules after four subcultures (80 days) of nodules with a diameter of 3 mm reached 183.33. The nodules proliferated in suspension culture could differentiate when transferred to a solid differentiation medium. By screening the effects of exogenous hormones on nodule differentiation, WPM + 1.0 mg / L PPU was found to be the most effective. Further screening of the effects of nodule size and subculture number on nodule differentiation revealed that nodules with a diameter of 3-6 mm achieved the highest bud-leaf differentiation rate (48.89%) after 8 subcultures. The highest average number of adventitious buds was 12.13 in the adventitious bud elongation medium, with a rooting rate of 43.33% and a transplant survival rate of 50% for rooted seedlings. This method can significantly increase the number of meristematic nodules through suspension culture, enabling the proliferated nodules to differentiate into adventitious buds, root, and successfully transplant seedlings, thus greatly improving the efficiency of peony in vitro plant regeneration.

[0082] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for obtaining regenerated plants through suspension culture of peony meristems, characterized in that, Includes the following steps: 1) Meristematic nodule induction: Callus tissue is induced by cotyledons of peony, which in turn induces the formation of clustered meristematic nodules; 2) Suspension culture proliferation: The clustered meristematic nodules were cut and inoculated into liquid proliferation medium for suspension culture to obtain proliferated meristematic nodules; 3) Differentiation culture: The meristematic nodules proliferated in suspension culture in step 2) are transferred to solid differentiation culture medium to induce bud and leaf tissue differentiation; 4) Plant regeneration: After the nodules that have differentiated bud and leaf tissues are cultured for adventitious bud elongation, they are then cultured for rooting and transplanted for acclimatization to obtain regenerated plants.

2. The method for obtaining regenerated plants through suspension culture of peony meristems according to claim 1, characterized in that, In step 1), the peony cotyledons are 'Fengdan' peony cotyledons; preferably, the 'Fengdan' peony cotyledons are peony cotyledons grown from 'Fengdan' peony seeds after 15 days of embryo germination culture.

3. The method for obtaining regenerated plants through suspension culture of peony meristems according to claim 1 or 2, characterized in that, The specific method for inducing meristematic nodules is as follows: Peony cotyledons are inoculated into callus induction medium and cultured to obtain callus tissue, and then the callus tissue is transferred to meristematic nodule induction medium for subculture to obtain clustered meristematic nodules. The callus induction medium was MS basal medium supplemented with 0.25-1.0 mg / L chlorpyrifos (CPPU) and 0.5-2.0 mg / L naphthaleneacetic acid (NAA). The meristematic nodule induction medium was MS basal medium supplemented with 0.25-1.0 mg / L thiamethoxam TDZ and 0.25-1.0 mg / L chlorpyrifos CPPU.

4. The method for obtaining regenerated plants through suspension culture of peony meristems according to claim 1 or 2, characterized in that, In step 2), the clustered meristems are cut into nodule blocks with a diameter of 2.5-6 mm, preferably 2.5-3.5 mm, and more preferably 3 mm; And / or, the liquid proliferation medium is one of the following: MS basal medium supplemented with 0.5-2.0 mg / L TDZ, MS basal medium supplemented with 0.5-2.0 mg / L CPPU, or MS basal medium supplemented with 0.25-1.0 mg / L TDZ and 0.25-1.0 mg / L CPPU; preferably MS basal medium supplemented with 0.5-2.0 mg / L TDZ or MS basal medium supplemented with 0.25-1.0 mg / L TDZ and 0.25-1.0 mg / L CPPU; more preferably MS basal medium supplemented with 0.5-2.0 mg / L TDZ.

5. The method for obtaining regenerated plants through suspension culture of peony meristems according to claim 4, characterized in that, In step 2), the rotation speed of the suspension culture is 120-180 rpm; preferably 120-150 rpm; more preferably 150 rpm; And / or, the subculture cycle of the suspension culture is 20 days, and the number of subcultures is not less than 3, preferably 4, to obtain proliferating meristematic nodules.

6. The method for obtaining regenerated plants through suspension culture of peony meristems according to claim 1 or 2, characterized in that, In step 3), the meristematic nodules are meristematic nodules with a diameter ≥ 3 mm that have been proliferated by suspension culture in step 2); preferably, the meristematic nodules are between 3-6 mm. And / or, the solid differentiation medium is WPM basal medium supplemented with 0.5-2.0 mg / L CPPU or WPM basal medium supplemented with 0.25-1.0 mg / L CPPU + 0.25-1.0 mg / L TDZ; preferably WPM basal medium supplemented with 0.5-2.0 mg / L CPPU.

7. The method for obtaining regenerated plants through suspension culture of peony meristems according to claim 1 or 2, characterized in that, In step 4), the culture medium used for the adventitious shoot elongation culture is WPM basic medium supplemented with 0.2-0.3 mg / L BA and 0.1-0.3 mg / L GA3; preferably WPM basic medium supplemented with 0.25 mg / L BA and 0.2 mg / L GA3. And / or, the nodules from which induced bud and leaf tissue were induced were cultured five times in an adventitious bud elongation medium, with a subculture cycle of 30 days.

8. The method for obtaining regenerated plants through suspension culture of peony meristems according to claim 7, characterized in that, In step 4), the rooting culture includes two stages: rooting induction culture and root formation culture; The rooting induction culture method is as follows: select elongated adventitious buds with stem length ≥1cm, first perform low temperature treatment, and then inoculate them in rooting induction medium and culture them in the dark; the rooting induction medium contains indolebutyric acid (IBA) and putrescine; The method for root formation culture is as follows: adventitious buds after rooting induction are transferred to a root formation medium containing activated carbon (AC) and cultured under light conditions to promote root elongation.

9. The method for obtaining regenerated plants through suspension culture of peony meristems according to claim 8, characterized in that, The rooting induction medium is a 1 / 2 MS basal medium supplemented with 0.5-2.0 mg / L IBA and 0.5-2.0 mg / L putrescine; preferably, a 1 / 2 MS basal medium supplemented with 1.0 mg / L IBA and 1.0 mg / L putrescine. And / or, the root formation medium is a 1 / 2 MS basal medium supplemented with 0.3-0.5 g / L AC; preferably, a 1 / 2 MS basal medium supplemented with 0.4 g / L AC.

10. The method for obtaining regenerated plants through suspension culture of peony meristems according to claim 8, characterized in that, In step 4), the low-temperature treatment involves subjecting the elongated adventitious buds to a cold treatment at 4-8°C for 5-10 days before transferring them to the rooting induction medium. And / or, in step 4), the substrate for transplanting and acclimatization is a mixture of perlite, vermiculite and peat moss; preferably, it is a mixture of perlite, vermiculite and peat moss in a volume ratio of 1:1:1.

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