CN113115708A

CN113115708A - Method for regenerating peony in-vitro plant and application thereof

Info

Publication number: CN113115708A
Application number: CN202110512437.3A
Authority: CN
Inventors: 成仿云; 徐莉; 钟原
Original assignee: Beijing Forestry University
Current assignee: Beijing Forestry University
Priority date: 2021-05-11
Filing date: 2021-05-11
Publication date: 2021-07-16
Anticipated expiration: 2041-05-11
Also published as: CN113115708B

Abstract

The invention relates to the technical field of peony planting, in particular to a method for regenerating peony in-vitro plants and application thereof. The method comprises the following steps: selecting peony-induced embryonic callus, placing the peony-induced embryonic callus in a differentiation culture medium, and carrying out subculture for 11-13 generations by taking 10-11 days as a subculture period to obtain an adventitious bud primordium; the differentiation medium is as follows: improving WPM + 0.5-1 mg/L CPPU + 0.5-2 mg/L TDZ; and (3) performing adventitious bud elongation and rooting induction on the adventitious bud primordium to obtain the peony seedling. According to the invention, a specific differentiation mode is adopted, the peony embryonic callus is induced in the same differentiation culture medium to form a meristem nodule, and further the adventitious bud is differentiated, so that the problem of low peony meristem nodule differentiation rate in the prior art is solved. The invention constructs a method for regenerating peony in vitro plants, realizes plant regeneration under the in vitro culture condition through a meristem node way, and provides a new technology for peony in vitro rapid propagation and genetic transformation.

Description

Method for regenerating peony in-vitro plant and application thereof

Technical Field

The invention relates to the technical field of peony planting, in particular to a method for regenerating peony in-vitro plants and application thereof.

Background

Peony (Paeonia Sect. Moutan) is a deciduous shrub of Paeonia (Paeonia) of Paeoniaceae, is a famous Chinese traditional flower, and has important cultural, ornamental, medicinal and oil values (Li et al, 2015; Yu et al, 2016). However, the defects of low efficiency and long period of traditional propagation methods such as grafting and plant division limit the industrial development and breeding process. Therefore, the establishment of a highly efficient and stable in vitro regeneration system is urgently needed. Peony has high in-vitro regeneration difficulty, the problems of vitrification, low reproduction rate, poor rooting quality, difficult transplanting and the like still exist in bulbil rapid propagation, embryo culture and callus differentiation (Wen et al, 2019; xuli et al, 2017; Zhu et al, 2015), and somatic embryogenesis also faces the problems of high distortion rate and low germination rate (Du et al, 2020; Zhou XiuMei et al, 2009; Wang Zheng, 2010), and the prior art cannot establish a perfect in-vitro regeneration system all the time.

A meristematic node (mertistem node) is a special structure which is similar to the appearance of a somatic embryo, has obvious histological difference, can be stably proliferated for a long time and has high differentiation capacity, and is an ideal alternative way for establishing in vitro regeneration (McCown et al, 1988; Staphylogen, 2011). Due to its unique structural features and advantages, meristematic nodules are defined as a third in vitro regeneration pathway in addition to organogenesis and somatic embryogenesis (George, 1993). Through screening of culture materials such as different varieties and explants and culture conditions such as exogenous hormones, culture medium types and sucrose, a high-efficiency meristem nodule in-vitro regeneration system of plants such as eucalyptus (Dobrowolska et al, 2017), pineapple (Dal Vesco et al, 2011), hops (Fortes et al, 2010), walnut (Moyo et al, 2009), poplar (Ferreira et al, 2009) and sweetgum (Bayraktar et al, 2015) is established. The meristematic node has stable genetic characteristics and vigorous differentiation capability, the eucalyptus node can still differentiate after subculture for 3 years (Warrag et al, 1991), and the lily meristematic node can still regenerate after suspension culture for more than 4 years and has no change in chromosome content (Godo et al, 1998); transgenic studies on sweetgum and hop were successfully carried out by the gene gun method through the establishment of a meristematic node regeneration system (Kim et al, 1999; Batista et al, 2008), and taxol could be continuously produced by culturing yew nodes with semi-continuous liquid (Ellis et al, 1996).

The research on peony meristematic nodules is started later, and related reports on the peony meristematic nodules are less at present. The origin of the peony meristematic node is indirectly induced by callus (Qinlei et al, 2012). The method is characterized in that a leaf stalk thin layer of Paeonia rockii ' Gao Yuan Shen Huo ' and a subgroup interspecific ' Golden Era ' (P.lemoinei ' Golden Era ') are used as explants to respectively realize the induction rates of 85.7% and 78.0% of meristematic nodes, and the propagation culture is carried out in a liquid culture mode, but the meristematic nodes of ' plateau san Huo ' are not differentiated, the Golden Era ' only obtains the differentiation rate of 16%, and a regenerated plant (Stachyn, 2011; Qin Lei, 2012) is not successfully obtained; in conclusion, the system for inducing and proliferating and culturing the peony meristematic node is relatively perfect, but the problems of low differentiation rate and no differentiation of most varieties exist, and the system becomes a bottleneck for limiting the in-vitro regeneration of the peony.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a method for regenerating a peony in vitro plant and application thereof.

In a first aspect, the present invention provides a method for regenerating a peony in vitro plant, comprising:

placing the embryogenic callus of the peony into a differentiation culture medium, and carrying out subculture for 11-13 generations by taking 10-11 days as a subculture period to obtain an adventitious bud primordium;

the differentiation medium comprises:

improving WPM + 0.5-1 mg/L CPPU + 0.5-2 mg/L TDZ;

and (3) performing adventitious bud elongation and rooting induction on the adventitious bud primordium to obtain the peony seedling.

The method comprises the steps of placing the embryogenic callus of the peony into a differentiation culture medium for culturing, carrying out subculture for 5-6 generations to obtain a meristem nodule, and carrying out subculture for 12-13 generations to differentiate an adventitious bud primordium (in this case, the meristem is in the form of a cluster leaf, and the cluster leaf contains the adventitious bud primordium).

Further, the differentiation medium consists of the following components:

the WPM +0.5mg/L CPPU +0.5mg/L TDZ is improved.

Further, the adventitious bud elongation comprises:

placing the adventitious bud primordium in an adventitious bud elongation culture medium, and subculturing for 1-6 generations by taking 25-35 days as a subculture period;

the adventitious bud elongation culture medium comprises: improved WPM + 0.2-0.3 mg/L BA + 0.1-0.2mg/L GA₃。

In the differentiation medium, the adventitious bud primordium is slow in elongation speed, after the adventitious bud elongation medium is replaced by the adventitious bud elongation medium, the adventitious bud is elongated after 1 subculture, more adventitious buds can be obtained after 5 subcultures, and the adventitious buds are elongated in the process of 4-6 subcultures.

Further, the embryogenic callus of peony is prepared by the following steps:

taking an explant of peony as a raw material, and carrying out induction culture in an embryogenic callus induction culture medium for 25-35 days;

the embryogenic callus induction medium comprises: 0.5-1 mg/L CPPU and 0.5-1 mg/L NAA;

preferably, the explant is one or more of a cotyledon, leaf blade, petiole or stem segment.

Further, the rooting induction comprises:

and placing the elongated adventitious bud into a root induction culture medium, inducing the adventitious bud to root for 25-35 days, transferring the adventitious bud into a root elongation culture medium, and inducing the root to elongate for 15-25 days to obtain the peony seedling.

Further, the root induction medium comprises: 1/2MS +1.0mg/L IBA +1.0mg/L putrescine;

the root elongation medium comprises: 1/2MS +0.4g/L AC.

Further, after obtaining the peony seedlings, the method further comprises the following steps:

transplanting the peony seedlings into a matrix for culture;

the substrate comprises: perlite, vermiculite and turfy soil.

As a preferred embodiment, the present invention provides a method for regenerating a peony isolated plant, comprising:

(1) embryogenic callus induction

Putting the explant of the peony into a callus induction culture medium, and inducing for 25-35 days to obtain embryonic callus;

the embryogenic callus induction medium used included: modified MS + 0.5-1 mg/L CPPU + 0.5-1 mg/L NAA;

the explant is one or more of cotyledon, leaf blade, petiole or stem segment;

(2) induction and differentiation of meristematic nodules

Placing the embryogenic callus obtained in the step (1) into a differentiation culture medium for culturing, and carrying out subculture for 12-13 generations by taking 10-11 days as a subculture period to obtain an adventitious bud primordium; the differentiation medium comprises: improving WPM + 0.5-1 mg/LCPPU + 0.5-2 mg/LTDZ;

(3) elongation of adventitious bud

Placing the adventitious bud primordium obtained in the step (2) in an adventitious bud elongation culture medium, and subculturing for 1-6 generations by taking 25-35 days as a subculture period;

the adventitious bud elongation culture medium consists of the following components:

improved WPM + 0.2-0.3 mg/L BA + 0.1-0.2mg/L GA₃；

(4) Rooting induction of adventitious buds

Placing the adventitious bud treated in the step (3) in a root induction culture medium, inducing rooting for 25-35 days, transferring the adventitious bud into a root elongation culture medium, and inducing root elongation for 15-25 days to obtain a peony seedling;

the root induction medium comprises: 1/2MS +1.0mg/L IBA +1.0mg/L putrescine;

the root elongation medium comprises: 1/2MS +0.4g/L AC;

(5) transplanting domestication

Transplanting the peony seedlings obtained in the step (4) into a matrix for culture;

the substrate comprises: perlite, vermiculite and turfy soil.

Further, the peony is one or more of ' paeonia ostii ', plateau glory ' (p.rockii ' Gao Yuan Shen Huo '), inter-subgroup hybrid ' Golden Era ' (p.lemoinei ' Golden Era '), and ' Noon ' (p.lemoinei ' High Noon ').

The invention further provides application of the method in peony breeding.

The invention has the following beneficial effects:

(1) the peony explant is used as a raw material, the plant regeneration under the in vitro culture condition is successfully realized through a meristematic nodule approach for the first time, and the peony explant is domesticated to survive, so that a new technology is provided for the in vitro rapid propagation and genetic transformation of the peony.

(2) The research of the invention finds that the combination of the auxin and the cytokinin has a promoting effect on the embryogenic callus, and the optimal embryogenic callus induction culture medium of the selected cotyledon explants is improved MS +1.0mg/LCPPU +1.0 mg/LNAA. In the culture medium, the induction rate of cotyledon embryogenic callus is 87.50 percent at most, the biomass is 1.58g at most, and the browning rate is 10.42 percent at least.

(3) The research of the invention finds that the cytokinin combination has a promoting effect on the induction and differentiation of the meristematic nodules, the optimal differentiation medium obtained by screening is improved WPM +0.5mg/LCPPU +0.5mg/LTDZ, the optimal subculture period is 10 days, under the treatment, the induction rate of the meristematic nodules is 100% at most, and the differentiation rate is 45.83% at most.

(4) The research of the invention finds that BA + GA₃Has an effect of promoting the elongation of adventitious buds of nodes differentiating the cluster leaves, and gives a value of 0.25mg/LBA +0.2mg/LGA₃Is the optimum concentration. At this concentration, 13.40 average adventitious bud numbers/explant can be obtained, and 43.33% rooting rate and 45.83% transplanting survival rate are respectively obtained through rooting culture and transplanting domestication.

Drawings

FIG. 1 is a photograph of cotyledonary embryogenic callus provided in example 1 of the present invention;

FIG. 2 is a photograph of a small protrusion (anterior nodule formation) provided in example 1 of the present invention;

FIG. 3 is a photograph showing a large bulge (meristematic nodule) provided in example 1 of the present invention;

FIG. 4 is a photograph of a plexiform nodule provided in accordance with example 1 of the present invention;

FIG. 5 is a photograph of a meristematic nodal differentiation primordium provided in example 1 of the present invention;

FIG. 6 is a photograph of a meristematic nodal differentiated rosette leaf provided in example 1 of the present invention;

FIG. 7 is a photograph showing the elongation of adventitious bud according to example 1 of the present invention;

FIG. 8 is a photograph of a rooted seedling with adventitious bud according to example 1 of the present invention;

fig. 9 is a picture of transplantation acclimatization of rooted seedlings provided in embodiment 1 of the present invention.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1

1.1 Induction of embryogenic callus

1) Test materials

Collecting fruit pods of Paeonia ostii ' peony pollinated for 90 days in Beijing Yanqing national peony garden (40 degrees 45 ' N,115 degrees 97 ' E) in 8 months in 2018, drying in shade at room temperature, and then carrying out low-temperature stratification sand storage on seeds at 4 ℃ for one month for later use.

2) Sterilizing and disinfecting

Cleaning seeds, rinsing with detergent for 5min, rinsing with running water for 15min, and placing on a clean bench. Soaking in 70% ethanol and 2% sodium hypochlorite solution for 30s and 5min respectively, and washing with sterile water for 3 times. Taking out the embryo from the seed with sterilized knife and forceps in a clean bench, inoculating to germination medium (modified MS +0.5mg/L BA +1.0mg/L GA)₃) Culturing in medium.

3) Embryogenic callus induction

The method of Xuli et al (2017) is adopted, the cotyledon of the embryo which is cultured for 15 days is taken as an explant, the explant is cut into squares with the size of 1cm multiplied by 1cm by using a knife forceps, the squares are inoculated in a callus induction culture medium (1.0mg/LCPPU +1.0mg/LNAA) in a paraxial face-up inoculation mode, and the induction culture is carried out for 30 days. Yellow compact callus (embryogenic callus, as shown in FIG. 1) was selected for further differentiation culture.

1.2 Induction and differentiation of meristematic nodules

The cotyledon is taken as an explant, the embryogenic callus induced for 30 days in the obtained optimal callus induction culture medium is inoculated in a differentiation culture medium (improved WPM +0.5mg/L CPPU +0.5mg/L TDZ) for 10 days, cutting is not carried out during subculture, browning caused by wound is reduced, and the whole callus is directly transferred into a fresh culture medium after the culture medium attached to the callus is removed by a knife and forceps. The biomass and volume of the callus are continuously increased in the process of subculture, the subculture is carried out for 3-4 times, small bulge structures (anterior nodule structures, shown in figure 2) are induced on the surface of the callus, the subculture is carried out for 5-6 times, the small bulges develop into large bulge structures (meristematic nodules, shown in figure 3), the subculture is carried out for 7-10 times, the meristematic nodules proliferate in a mode that partial surface regions are retracted to form grooves but the division and detachment do not occur, the nodules are not spherical bulges in appearance, but a plurality of sub-nodules which are not divided and connected together are in a cluster nodule structure (shown in figure 4), and the cluster nodule surface differentiates dome-shaped primordia (shown in figure 5) and leaf primordia. Subculturing 11-12 generations, differentiating macroscopic leaf tissue, and continuing to develop to form cluster leaves.

1.3 adventitious bud elongation

Adventitious buds in the rosette leaves slowly elongate in a differentiation medium, and meristematic nodules which are differentiated into the rosette leaves after 12 subcultures are transferred to the medium (improved WPM +0.25mg/L BA +0.2mg/L GA)₃) Among them, the adventitious bud has a high elongation rate.

The subculture period is 30 days, the extended adventitious bud with stem length of 1-3cm is cut for rooting during subculture, and the rest part is transferred into a fresh adventitious bud extension culture medium for adventitious bud extension continuously to obtain more adventitious buds.

1.4 rooting induction and transplantation domestication of adventitious bud

The extended adventitious buds with stem length of 1-3cm were inoculated into a rooting induction medium (1/2MS +1.0mg/L IBA +1.0mg/L putrescine), cold-treated at 4 ℃ for 8 days, then induced to root in the dark for 30 days (as shown in FIG. 7), and then transferred into a root extension medium (1/2MS +0.4g/L AC) and cultured under the light for 20 days to promote root extension growth (as shown in FIG. 8). The rooting rate (number of adventitious buds rooted/number of adventitious buds inoculated X100%) was counted, respectively. Each flask was inoculated with 10 explants, 30 explants per replicate, and 3 replicates per treatment set.

Taking out the test-tube plantlet from the root from the culture medium (as shown in figure 9), and washing the culture medium adhered to the root to prevent decay after transplanting. The transplanting matrix (perlite: vermiculite: turfy soil: 1: 1: 1) is mixed in advance in proportion, sterilized under high pressure (120 ℃, 40 minutes), and when in use, clear water is sprayed until the transplanting matrix is kneaded into a ball by hand and is not scattered, and the water content is not excessive. The transplanted seedlings are cultured in a phytotron with the following conditions: temperature (24 +/-1 ℃), humidity 90%, and illumination intensity 50 umol.m^-2·s^-1(incandescent lamp), the number of illumination hours is 16h illumination/d, and the Hoagland nutrient solution is poured every month. And (4) counting the survival rate of the transplantation (the total number of the transplanted survived plants/transplanted rooting test-tube seedlings is multiplied by 100%) after 2 months of transplantation culture. 10 rooted tubes were replicated each, and 3 replicates were set up for each treatment.

The formulations of the modified media used in this example and the following experimental examples are as follows:

TABLE 1 improved MS formulation for tissue culture minimal medium

TABLE 2 tissue culture minimal medium 1/2MS formulation

TABLE 3 improved WPM formulation for tissue culture minimal medium

The method for preparing 1L of culture medium comprises the following steps: macroelements, microelements, vitamins and growth regulator are prepared into mother liquor, and a proper amount of sucrose and 7.5g of agar are added into the distilled water, and the volume is fixed to 1L. The pH of all media was adjusted to 5.8-6.0 at room temperature. The medium was filled in a container sealed with a plastic film and sterilized under high pressure for 20 minutes (121 ℃ C., 0.1 mPa). Shaking culture medium (about 40 deg.C) in ultra-clean bench for several seconds, adding sterilized hormone, and packaging into sterilized culture containers.

Experimental example 1

This experimental example was carried out to improve the induction of the embryogenic callus of section (3) in section 1.1 of example 1, and specifically comprises the following steps:

1. the method of Xuli et al (2017) was adopted, and the cotyledons of embryos cultured for 15 days were used as explants, cut into 1cm × 1 cm-sized squares with knife forceps, and inoculated into callus induction medium (modified MS +0.5mg/LBA +1.0mg/LNAA) by paraxial-up inoculation. The embryogenic callus induction rate (number of explants inducing embryogenic callus/number of explants inoculated × 100%), the browning rate (number of explants browned and explants inducing browned callus/number of explants inoculated × 100%) were counted at 20, 30, 40, and 50 days of culture in callus induction medium, respectively. Each flask was inoculated with 8 explants, 16 explants per replicate, and 3 replicates per treatment were set. The research finds that the induced peony callus has two types, and cytology and histology research shows that one type of the induced peony callus is embryonic callus (yellow compact block shape) and the other type of the induced peony callus is non-embryonic callus (brown loose water stain shape, which is generated on the surface of an explant).

With respect to the induction time, the results shown in Table 4 were obtained in this experimental example. The optimal induction time is selected by comprehensively considering the induction rate, browning rate and differentiation effect of the embryogenic callus, 33.33% of embryogenic callus is induced after 20 days, and the browning rate of the callus is lower to 6.25%. The induction rate and browning rate of embryogenic callus increased with the time of induction.

TABLE 4 Effect of induction time on embryogenic callus induction

2. To screen the best embryogenic callus induction medium, the following two-step experiment was set up.

The first step is as follows: taking cotyledons as test materials, setting the tests of 1.0mg/L NAA, 1.0 mg/L2, 4-D of single auxin and different cytokinins (0.5mg/L BA +1.0mg/L NAA, 0.5mg/L CPPU +1.0mg/L NAA, 0.5mg/L TDZ +1.0mg/L NAA, 0.5mg/L BA +1.0 mg/L2, 4-D, 0.5mg/L CPPU +1.0 mg/L2, 4-D, 0.5mg/L TDZ +1.0 mg/L2, 4-D) and selecting proper exogenous hormone combination;

the second step is that: taking cotyledons as test materials, setting a two-factor test of different concentrations of cytokinin and auxin aiming at a proper exogenous hormone combination screened in the first step, wherein CPPU (0.25, 0.5, 1.0mg/L) + NAA (0.25, 0.5, 1.0, 2.0 mg/L); the optimum concentration combination is selected.

The results as shown in tables 5 and 6 were obtained, and table 5 shows that embryogenic callus induction (12.50%, 8.33%) and biomass (1.20g, 1.05g) were significantly lower for NAA and 2,4-D auxin alone than for their combination with CKs, while browning (93.75%, 97.92%) was significantly higher for their combination with CKs. There were no significant differences in the three indices between auxin and different combinations of CKs. The highest induction rate of 0.5mg/L CPPU and 1.0mg/L AA for treating the hypocotyledonous callus is 81.25 percent, the highest biomass is 1.73g, and the lower browning rate is 12.50 percent. Thus, the CPPU + NAA combination is a hormone combination suitable for induction of cotyledonary embryogenic callus.

TABLE 5 Effect of exogenous hormones on embryogenic callus induction

Based on the results, the example further screened the appropriate concentration, and the results are shown in Table 6, the induction rate of 1.0mg/L CPPU +1.0mg/LNAA treatment on the hypocotyledonic callus is 87.50% at the highest, the biomass is 1.58g at the highest, and the browning rate is 10.42% at the lowest. Therefore, the combination of 1.0mg/L CPPU +1.0mg/LNAA is the best treatment mode for inducing cotyledon embryogenic callus.

TABLE 6 Effect of combination of exogenous hormone concentrations on embryogenic callus induction

Experimental example 2

The experimental example is improved aiming at the induction and differentiation of 1.2 node meristematic nodules, and the specific method is as follows:

1. effect of the Secondary Generation period on the Induction and differentiation of meristematic nodules

The cotyledon is taken as an explant, embryogenic callus induced for 30 days in the optimal callus induction culture medium obtained in the step 1.1 is inoculated in a differentiation culture medium (improved WPM +0.5mg/L CPPU), after the subculture is carried out for 12 generations in different subculture periods (10, 15, 20 and 25 days), the induction rate of the meristem nodule (the number of callus inducing the meristem nodule/the number of inoculated callus multiplied by 100%), the differentiation rate (the number of callus of differentiation buds and leaves/the number of inoculated callus multiplied by 100%) and the browning rate (the number of browned callus/the number of inoculated callus multiplied by 100%) are counted, 8 explants are inoculated in each bottle, 16 explants are inoculated in each bottle, and 3 repetitions are arranged for each treatment.

The results shown in Table 7 were obtained, and the results showed that the induction rate and differentiation rate of meristematic nodules were significantly decreased and the browning rate was significantly increased as the subculture cycle was prolonged. The subculture time was 10 days, the induction rate (68.75%) and differentiation rate (54.17%) were the highest, and the browning rate (16.67%) was the lowest.

TABLE 7 Effect of days of subculture on meristematic nodule induction and differentiation

2. Influence of exogenous hormone and induction time on induction and differentiation of meristematic nodules

To study the callus induction time and the effect of exogenous hormones on meristem nodule induction and differentiation, 2 experiments were set up. And (3) counting the induction rate and differentiation rate of the meristematic nodules after 12 generations of subculture, inoculating 8 explants in each bottle, repeating 16 explants in each bottle, and setting 3 repeats in each treatment.

Test 1: the embryonic callus induced by different days (20, 30, 40, 50 days) in the optimal callus induction culture medium obtained by the method is respectively inoculated in improved WPM differentiation culture media (0.5mg/L CPPU, 0.5mg/L CPPU +0.5mg/L TDZ, 0.5mg/L CPPU +0.5mg/L BA, 0.5mg/L CPPU +0.5mg/L NAA, 0.5mg/L CPPU +0.2mg/L GA) containing different exogenous hormones₃) In the method, 20 days are adopted as a subculture period, and callus induction days and exogenous hormone combinations which are suitable for induction and differentiation of meristematic nodules are screened.

Test 2: the cotyledon is taken as an explant, and the embryonic callus which is induced for 30 days in the obtained optimal callus induction culture medium is inoculated in improved WPM differentiation culture media containing different exogenous hormone combinations. A three-factor three-level orthogonal test was set for the appropriate exogenous hormone combination selected in test 1, CPPU (0.25, 0.5, 1.0mg/L), TDZ (0, 0.5, 1.0mg/L), BA (0, 0.5, 1.0mg/L), and the optimum concentration combination was screened using 10 days as the subculture cycle.

The results shown in tables 8 and 9 were obtained in test 1, and the results showed that the callus induction time and exogenous hormone had significant effects on the meristem induction rate and differentiation rate. CPPU and GA₃No meristem node formation and differentiation was observed under the combined treatment. The meristematic nodule induction rate (0-35.42%) and differentiation rate (0-6.25%) of CPPU combined with NAA was significantly lower than CPPU alone and in combination with CKs. The induction rate and differentiation rate of the meristematic node of the combination of CPPU and TDZ and the combination of CPPU and BA are obviously higher than that of the single compositionThe results of the unique CPPU treatment, with no significant difference between the two, suggest that the CPPU + BA + TDZ combination may be beneficial for meristem nodule induction and differentiation. As the induction time is prolonged, the induction rate and differentiation rate of all treated meristematic nodules are obviously reduced, and the influence of the callus induction time on the embryogenic callus induction rate and browning rate is comprehensively considered, so that the optimal meristematic nodule induction time is 30 days.

TABLE 8 callus induction time and Effect of exogenous hormones on the induction rate of growing nodules

TABLE 9 Effect of Induction time and exogenous hormones on meristem nodule differentiation Rate

Based on the results of test 1, orthogonal tests of three CKs were set up, resulting in the results shown in table 10. Table 10 shows that the induction rate of meristematic node (100%), differentiation rate (45.83%) and callus biomass (8.26g) of embryogenic callus induced by cotyledon as explant are highest under the treatment of 0.5mg/L CPPU and 0.5mg/L TDZ. Namely, the culture medium which is most suitable for the differentiation of the growing nodules aiming at the embryogenic callus induced by the cotyledon explants is modified WP M +0.5mg/L CPPU +0.5mg/L TDZ.

TABLE 10 Effect of exogenous CKs combinations on cotyledon meristem nodule differentiation

Experimental example 3

The experimental example improves the elongation of the adventitious bud of the 1.3 section, and specifically comprises the following steps:

inoculating the node of differentiated cluster leaf after subculture for 12 generations (subculture period 10 days) in optimal differentiation medium obtained from section 1.2 in adventitious bud elongation medium (modified WPM), and setting modified WPM +0.25mg/L BA + (0.1, 0.2, 0.3, 0.4mg/L) GA₃The effect of adventitious bud elongation is studied by hormone combination, the subculture cycle is 30 days, the elongated adventitious buds with stem length of 1-3cm are cut for rooting culture during the subculture, the rest part is inoculated in a fresh adventitious bud elongation culture medium for adventitious bud elongation, and the average differentiated adventitious bud number (total number of differentiated adventitious buds/number of differentiated callus) is counted after the subculture for 5 generations. Each replicate 5 explants, with 3 replicates per treatment set.

As shown in Table 11, the cotyledon-induced embryogenic callus induced 12 times after subculture in the differentiation medium resulted in differentiation of clumpy leaves from the induced meristem nodules, whereas adventitious buds in the differentiation medium were elongated slowly and transferred to the medium containing BA and GA₃The adventitious bud in the medium (2) is elongated faster. The adventitious bud is continuously induced and elongated during the subculture of the adventitious bud elongation culture medium for 5 generations, and along with GA₃The increase in concentration means that the number of adventitious buds increases first and then decreases, and at 0.2mg/L, the number of adventitious buds is at most (13.40). However, the rooting rate and the survival rate of transplantation are dependent on GA₃The increase in concentration of (A) was gradually decreased, but there was no significant difference between the concentrations of 0.1-0.2mg/L (Table 11). In conclusion, the improved WPM +0.25mg/L BA +0.2mg/L GA₃The optimal culture medium for adventitious bud elongation.

TABLE 11GA₃Influence on the number of adventitious buds on nodes, rooting and transplanting acclimatization

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. A method for regenerating peony in vitro plants is characterized by comprising the following steps:

the differentiation medium comprises:

improving WPM + 0.5-1 mg/L CPPU + 0.5-2 mg/L TDZ;

2. The method of claim 1, wherein the differentiation medium consists of:

the WPM +0.5mg/L CPPU +0.5mg/L TDZ is improved.

3. The method of claim 1 or 2, wherein the adventitious bud elongation comprises:

4. The method of claim 1, wherein said embryogenic callus of peony is prepared by:

5. The method of claim 1, wherein said rooting induction comprises:

6. The method of claim 5, wherein the root induction medium comprises: 1/2MS +1.0mg/L IBA +1.0mg/L putrescine; and/or the presence of a gas in the gas,

the root elongation medium comprises: 1/2MS +0.4g/L AC.

7. The method of claim 1, further comprising, after obtaining the peony seedlings:

transplanting the peony seedlings into a matrix for culture;

the substrate comprises: perlite, vermiculite and turfy soil.

8. The method according to claim 1, comprising the following steps:

(1) embryogenic callus induction

the explant is one or more of cotyledon, leaf blade, petiole or stem segment;

(2) induction and differentiation of meristematic nodules

(3) elongation of adventitious bud

improved WPM + 0.2-0.3 mg/L BA + 0.1-0.2mg/L GA₃；

(4) Rooting induction of adventitious buds

the root induction medium comprises: 1/2MS +1.0mg/L IBA +1.0mg/L putrescine;

the root elongation medium comprises: 1/2MS +0.4g/L AC;

(5) transplanting domestication

the substrate comprises: perlite, vermiculite and turfy soil.

9. The method of any one of claims 1-8, wherein the peony is one or more of 'Paeonia ostii', 'plateau Saint fire', 'Golden Era', or 'noon'.

10. Use of the method of any one of claims 1-9 in peony breeding.