Rapid propagation method of garden variety of tigridia pavonia

Abstract

The application discloses a rapid propagation method of a garden variety of iron ladle, and belongs to the technical field of plant propagation. The method comprises the following steps: (1) obtaining and disinfecting explants; (2) primary culture of sprouts; (3) subculture and proliferation of sprouts; and (4) rooting culture. The best culture medium for the tissue culture and rapid propagation of the garden variety of iron ladle is obtained by using different hormone combinations and adjusting different concentration ratios, so that the differentiation and proliferation of iron ladle sprouts, the growth of seedlings and the rooting rate are significantly improved, and a reference for rapid seedling formation in the later stage is provided.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of plant breeding, and relates to a rapid propagation method of a garden variety of iron club. BACKGROUND

[0002] Iron club seeds are extremely difficult to germinate under natural conditions, some varieties do not bear fruit, and seed propagation is difficult. As a commonly used propagation method at present, the propagation coefficient of the stolon propagation is low, and the speed is slow, which cannot meet the production demand. The rapid propagation of the garden variety of iron club through tissue culture can effectively overcome the defects of seed and stolon propagation techniques, greatly shorten the artificial breeding cycle, and has great practical value. Therefore, the present application selects the garden hybrid variety 'Huashen' (Flore) as the test material, and explores the tissue culture and rapid propagation technology, so as to provide technical support for the seedling production of iron club. Helleborus BACKGROUND SUMMARY

[0003] In order to solve the problems of difficult seed germination, low propagation coefficient, slow speed and the like of the existing iron club seeds, the present application provides a rapid propagation method of the garden variety of iron club 'Huashen', and explores the best culture medium for the primary culture, subculture proliferation and rooting culture of 'Huashen', so as to provide a reference for the rapid seedling growth of 'Huashen' in the later period.

[0004] The present application provides a rapid propagation method of the garden variety of iron club 'Huashen', and the specific steps are as follows:

[0005] (1) Disinfection of explants: after flowering, the underground flower buds of 'Huashen' are collected in the field, the surrounding silt is washed clean, then the flower buds are washed with running water for 10 h, placed on a clean bench, soaked in 5% sodium hypochlorite solution for 15 min, then soaked in 75% alcohol for 30 s, washed with sterile water for 3 times, and the excess water is filtered out, and the treated buds are placed for standby.

[0006] (2) Primary culture: the test is carried out on the MS culture medium, and 2 mg·L -1 2IP, 1 mg·L -1 GA3, 1 mg·L -1 KT, 0~2.5 mg·L -1 6-BA, 30 g·L -1 sucrose, 3 g·L -1 plant gel, pH 5.8~6.0 are added. The treated buds are inoculated on the above-mentioned culture medium, and are placed in a culture room with a temperature of 25±2℃, an air relative humidity of 60~70%, a light time of 16 h·d -1 , and a light intensity of 2000~3000 lx for induction culture for 25 d, until the explants differentiate into adventitious buds.

[0007] (3) Subculture: After the bud induction is successful, when the buds grow to 1-2 cm, they are transferred to the proliferation medium. The test is carried out on the MS medium, with the addition of 2 mg·L -1 2IP, 1-2 mg·L -1 GA3, 0.1-0.6 mg·L -1 KT, 1-2 mg·L -1 6-BA, 30 g·L -1 sucrose, 3 g·L -1 plant gel, pH 5.8-6.0. The culture is carried out under the conditions of a culture temperature of 25±2℃, an air relative humidity of 60-70%, a light intensity of 2000-3000 lx, and a light time of 16 h·d -1 , and subculture is carried out every 15 days, which is repeated multiple times to continuously proliferate until 3-4 cm of the cluster buds are obtained.

[0008] (4) Rooting culture: when the cluster buds grow to 3-4 cm, the proliferated buds are cut and transferred to the rooting medium on the super-clean workbench. The rooting culture is induced on the MS medium, with the addition of 0.2-0.6 mg·L -1 NAA, 0.2-0.6 mg·L -1 IBA, 30 g·L -1 sucrose, 3 g·L -1 plant gel, pH 5.8-6.0. The culture is carried out under the conditions of a culture temperature of 25±2℃, an air relative humidity of 60-70%, a light intensity of 2000-3000 lx, and a light time of 16 h·d -1 , and 1-2 cm of the root hairs grow from the base of the plantlets after 30 days of the rooting culture.

[0009] Preferably, the primary culture medium is: MS+2 mg·L -1 2IP+1 mg·L -1 6-BA+1 mg·L -1 GA3+1 mg·L - 1 KT, 30 g·L -1 sucrose, 3 g·L -1 plant gel, pH 5.8-6.0.

[0010] Preferably, the subculture medium is: MS+2 mg·L -1 2IP+1 mg·L -1 6-BA+2 mg·L -1 GA3+1 mg·L - 1 KT, 30 g·L -1 sucrose, 3 g·L -1 plant gel, pH 5.8-6.0.

[0011] Preferably, the rooting medium is: MS+0.6mg·L -1 NAA+0.6mg·L -1 IBA, 30g·L -1 Sucrose, 3g·L -1 Plant gel, pH 5.8~6.0.

[0012] Compared with the prior art, the technical scheme provided by the application has at least the following beneficial effects or advantages:

[0013] The application provides the influence of different hormone combinations and different concentration ratios on differentiation of iron ladle buds, bud proliferation and growth, and rooting, and obtains the best culture medium for tissue culture and rapid propagation of the iron ladle horticultural variety, thereby providing technical support for seedling production. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 The flower bud explant of the iron ladle horticultural variety 'Flower God' is described in the application.

[0015] Figure 2 The schematic diagram of the whole process of tissue culture and rapid propagation of the iron ladle horticultural variety 'Flower God' is described in the application.

[0016] Figure 3 The state of the iron ladle horticultural variety 'Flower God' explant inoculated into the primary culture medium for 50d is described in the application. Wherein, A: 0mg·L -1 6-BA; B: 1mg·L -1 6-BA; C: 1.5mg·L -1 6-BA; D: 2mg·L -1 6-BA; E: 2.5mg·L -1 6-BA.

[0017] Figure 4 The state of the iron ladle horticultural variety 'Flower God' flower bud explant inoculated into the subculture medium for 90d is described in the application. Wherein, A: 1mg·L -1 6-BA+1mg·L -1 GA3+0.2mg·L -1 KT; B: 1mg·L -1 6-BA+1.5mg·L -1 GA3+0.6mg·L -1 KT; C: 1mg·L -1 6-BA+2mg·L -1 GA3+1mg·L -1 KT; D: 1.5mg·L -1 6-BA+1mg·L -1 GA3+0.6mg·L -1KT; E: 1.5 mg L -1 6-BA + 1.5 mg L -1 GA3 + 1 mg L -1 KT; F: 1.5 mg L -1 6-BA + 2 mg L - 1 GA3 + 0.2 mg L -1 KT; G: 2 mg L -1 6-BA + 1 mg L -1 GA3 + 1 mg L -1 KT; H: 2 mg L -1 6-BA + 1.5 mg L - 1 GA3 + 0.2 mg L -1 KT; I: 2 mg L -1 6-BA + 2 mg L -1 GA3 + 0.6 mg L -1 KT.

[0018] Figure 5 For the subculture of the iron ladle gardening variety 'Flower God' of the present application, the proliferation results. Among them, A: 1 mg L - 1 6-BA + 1 mg L -1 GA3 + 0.2 mg L -1 KT; B: 1 mg L -1 6-BA + 1.5 mg L -1 GA3 + 0.6 mg L -1 KT; C: 1 mg L -1 6-BA + 2 mg L -1 GA3 + 1 mg L -1 KT; D: 1.5 mg L -1 6-BA + 1 mg L -1 GA3 + 0.6 mg L -1 KT; E: 1.5 mg L -1 6-BA + 1.5 mg L -1 GA3 + 1 mg L -1 KT; F: 1.5 mg L -1 6-BA + 2 mg L -1 GA3 + 0.2 mg L -1 KT; G: 2 mg L -1 6-BA + 1 mg L -1 GA3 + 1 mg L -1 KT; H: 2 mg L -1 6-BA + 1.5 mg L-1 GA3+0.2mg·L -1 KT;I:2mg·L -1 6-BA+2mg·L -1 GA3+0.6mg·L -1 KT.

[0019] Figure 6 The rooting culture of the tissue culture seedlings of the garden variety ‘Huashen’ of iron ladle under different treatments for 40 days in the present application. Among them, A: 0.2mg·L -1 NAA+0.2mg·L -1 IBA;B:0.4mg·L -1 NAA+0.4mg·L -1 IBA;C:0.4mg·L -1 NAA+0.6mg·L -1 IBA;D:0.6mg·L -1 NAA+0.4mg·L -1 IBA;E:0.6mg·L -1 NAA+0.6mg·L -1 IBA. DETAILED DESCRIPTION

[0020] In the following, the technical solutions of the present application will be described in conjunction with examples, but the present application is not limited to the following examples. The experimental methods and detection methods described in each example are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0021] Some reagents involved in the following examples are BA (cytokinin, 6-benzyladenine), KT (kinetin, furan aminopurine), NAA (α-naphthaleneacetic acid), IBA (3-indolebutyric acid), 2IP (N6-iso-pentenyladenine) and the like.

[0022] Some instruments used in the following examples are super clean bench (AIRTECH SW-CJ-2FD), high pressure sterilization pot (TOMY SX-500), body microscope (Olympus SZX-16), culture container (diameter 7.5cm, 320mL), constant temperature incubator (Dongtuo ZPW-280B) and the like.

[0023] Example 1

[0024] This example provides a disinfection method of explants of the garden variety ‘Huashen’ of iron ladle and primary culture test.

[0025] After flowering, the underground flower buds of 'Huagong' were collected in the field, the surrounding silt was washed clean, then washed with running water for 10 h, placed on a clean bench, soaked in 5% sodium hypochlorite solution for 15 min, then soaked in 75% alcohol for 30 s, washed with sterile water for 3 times, and the excess water was filtered out. The treated buds were placed for standby use.

[0026] The test was carried out on the basic medium (Table 1), and the concentration of plant hormones added was 2 mg·L -1 2 IP, 1 mg·L -1 GA3, 1 mg·L -1 KT, 1 mg·L -1 6-BA. The flower buds of 'Huagong' collected from the underground part were inoculated in the primary culture medium, and after inoculation, they were placed in the tissue culture room for culture (the temperature in the culture room was 25±2℃, the relative humidity of air was 60~70%, the light time was 16 h·d -1 , and the light intensity was 2000~3000 lx), and the growth and differentiation of the buds were observed and recorded. After about 10 d of primary culture, the color of the explants showed signs of turning from white to green. After 25 d, single buds of the explants began to grow, and the color turned green obviously, accompanied by the growth of leaves. After 35 d, the leaves began to stretch, the plants grew, and a second small bud grew. When 50~60 d, the buds increased, and there was different degree of elongation growth, the leaves were emerald green and completely unfolded. After 80 d of induction by the proliferation medium, proliferation buds began to appear, the plants grew well, and the growth was good. Finally, after induction by the rooting medium, 150 d could be transplanted to the flowerpot for growth Figure 2 ).

[0027] Table 1 Configuration table of culture medium

[0028]

[0029] Example 2

[0030] This example provides a test of the influence of different concentrations of 6-BA on the induction of 'Huagong' buds.

[0031] Table 2 Primary culture medium

[0032]

[0033] A single factor experiment was set up with cytokinin 6-BA as the research object to study its influence on bud induction culture. A total of 5 concentration levels (Table 2) were set, 0, 1.0, 1.5, 2.0, 2.5 mg·L -1 . 3 explants were inoculated in each bottle, 30 bottles were inoculated for each treatment, and the experiment was repeated 3 times. After inoculation, they were placed in the tissue culture room for culture (the temperature in the culture room was 25±2℃, the relative humidity of air was 60~70%, the light time was 16 h·d -1, light intensity 2000~3000lx), after 3 weeks, the number of buds, bud induction rate, bud contamination rate were counted, the growth and differentiation of buds were observed and recorded, and the data were processed as follows:

[0034] (1) The number of buds = the number of single bud formed explants;

[0035] (2) The average number of regenerated buds of explants = the total number of regenerated adventitious buds / the number of regenerated adventitious bud explants;

[0036] (3) Induction rate = (the number of single bud formed explants / the number of inoculated explants) x 100%;

[0037] (4) Contamination rate = (the number of contaminated explants / the total number of inoculated explants) x 100%.

[0038] The growth results of flower buds of 'Huashen' inoculated in different 6-BA culture media are shown in Figure 3 From Table 3 and Table 4, it can be seen that different concentrations of 6-BA have different effects on the induction of single buds of 'Huashen', among which the most significant effect is 1mg·L -1 6-BA, with the highest induction rate of 67.03%, the most average number of regenerated buds of explants of 3.87, the most robust growth of buds, the largest and greenest leaves. With the gradual increase of 6-BA concentration, the induction rate shows a downward trend. The effect of 6-BA concentration on the average number of regenerated buds of explants is not significant. Different 6-BA concentrations have no significant effect on the contamination rate and average number of buds of 'Huashen' bud induction. In summary, the most suitable 6-BA concentration for 'Huashen' bud induction is 1mg·L -1 , and the most suitable medium for the primary culture of 'Huashen' is: MS+2mg·L -1 2IP+1mg·L -1 6-BA+1mg·L -1 GA3+1mg·L -1 KT, 30g·L -1 sucrose, 3g·L -1 plant gel, pH 5.8~6.0.

[0039] Table 3 Effects of different concentrations of 6-BA on bud induction

[0040]

[0041] Table 4 Bud morphology of primary induction

[0042]

[0043] Example 3

[0044] This example provides a test of the effect of different hormone types and concentrations on the proliferation of 'Huashen' tissue culture seedlings.

[0045] After the bud induction is successful, the buds are transferred to the proliferation medium when they grow to 1-2 cm. They are subcultured once every 15 days, and the process is repeated to continuously proliferate them.

[0046] The test is carried out on the basic medium (Table 1) with the addition of 2 mg·L -1 2 IP, 6-BA, GA3 and KT at different concentration levels. The subculture is set up as a 3-factor test for the concentration of cytokinin and the concentration of auxin. 30 bottles are inoculated for each treatment, 1 explant is inoculated in each bottle, 3 repetitions are carried out, the bud proliferation multiple is counted every 15 days, and the bud tissue growth and differentiation are observed and recorded. The specific test treatment is shown in Table 5.

[0047] Table 5 Overview of bud proliferation induced by different hormone levels

[0048]

[0049] In this embodiment, the proliferation multiple is calculated as follows: Proliferation multiple = number of buds obtained after inoculation / total number of inoculation.

[0050] The buds of ‘Huashen’ are inoculated in 9 kinds of medium with combinations of 6-BA, GA3 and KT. The effects of different combinations and concentration ratios of hormones on the proliferation of tissue culture seedlings of ‘Huashen’ are shown in Table 6. Figure 4 , Figure 5 Table 6. The effects of different combinations and concentration ratios of hormones on the proliferation of tissue culture seedlings of ‘Huashen’ Figure 4 It can be seen from Table 6 that the tissue culture seedlings of treatment groups B and C have more branches. It can be seen from Table 6 that after 30 days, 60 days and 90 days of culture, the bud proliferation multiple, plant height and leaf number of ‘Huashen’ under different hormone treatments. After 30 days of inoculation, the proliferation phenomenon appears obviously in some treatment groups of ‘Huashen’, and with the extension of time, the proliferation multiple also increases obviously, and the proliferation multiple reaches the maximum value of 3.85 when the concentration of 6-BA+2 mg·L -1 GA3+1 mg·L -1 KT is 1 mg·L -1 KT is 0.6 mg·L -1 KT is 1.5 mg·L -1 KT is 0.6 mg·L -1 KT is 1.5 mg·L -1 The proliferation multiple of the C treatment reaches the maximum value of 8.67, the plant height reaches the maximum value of 39.31 mm, and the number of leaves also reaches the maximum value of 15.66, and the growth and proliferation of the tissue culture seedlings are also the best. The effects of the three different hormone combinations on the proliferation multiple, plant height and leaf number of ‘Huashen’ reach a significant level. In summary, the most suitable medium for the subculture proliferation of ‘Huashen’ is MS+2 mg·L -12 IP + 1 mg·L -1 6-BA + 2 mg·L -1 GA3 + 1 mg·L -1 KT, 30 g·L -1 Sucrose, 3 g·L -1 Plant gel, pH 5.8~6.0.

[0051] Table 6 Effects of different hormone combinations on subculture multiplication of‘Flower God’

[0052]

[0053] Note: A: 1 mg·L -1 6-BA + 1 mg·L -1 GA3 + 0.2 mg·L -1 KT; B: 1 mg·L -1 6-BA + 1.5 mg·L -1 GA3 + 0.6 mg·L -1 KT; C: 1 mg·L -1 6-BA + 2 mg·L -1 GA3 + 1 mg·L -1 KT; D: 1.5 mg·L -1 6-BA + 1 mg·L -1 GA3 + 0.6 mg·L -1 KT; E: 1.5 mg·L -1 6-BA + 1.5 mg·L -1 GA3 + 1 mg·L -1 KT; F: 1.5 mg·L -1 6-BA + 2 mg·L - 1 GA3 + 0.2 mg·L -1 KT; G: 2 mg·L -1 6-BA + 1 mg·L -1 GA3 + 1 mg·L -1 KT; H: 2 mg·L -1 6-BA + 1.5 mg·L - 1 GA3 + 0.2 mg·L -1 KT; I: 2 mg·L -1 6-BA + 2 mg·L -1 GA3 + 0.6 mg·L -1 KT

[0054] Example 4

[0055] This example provides a test of the effects of different hormone types and concentrations on rooting of‘Flower God’ tissue culture seedlings.

[0056] The test was based on the basic medium (Table 1). When the buds were 3-4 cm high, the proliferated buds were cut and transferred to rooting medium with the hormone concentration ratio in Table 7 on a clean bench. Each bottle was inoculated with 1 bud, 20 buds were inoculated for each treatment, repeated 3 times, and after 40 days, the effects of different hormones on the rooting of 'Huashen' sterile seedlings were compared.

[0057] Table 7 Overview of rooting induction with different hormone levels

[0058]

[0059] The rooting rate calculation method in this example is: rooting rate = (number of rooted seedlings / total number of inoculated seedlings) x 100%.

[0060] The effects of different hormone combinations on the rooting culture of 'Huashen' are shown in Figure 6 Table 8. Under the combination treatment of IBA and NAA, 'Huashen' had obvious rooting phenomenon. After about 40 days of rooting induction culture, the bud root primordium protruded in the medium, and the roots began to grow. The rooting rate, root length and root number of 'Huashen' increased significantly with the increase of NAA and IBA concentration. When the NAA concentration was 0.4 mg·L -1 and 0.6 mg·L -1 , the rooting rate of 'Huashen' showed an increasing trend with the increase of IBA concentration. When the NAA concentration was 0.6 mg·L -1 and the IBA concentration was 0.6 mg·L -1 , the rooting rate of 'Huashen' reached the maximum value of 71.14%; the root length reached the maximum value of 13.51 mm; and the average root number reached the maximum value of 16.67. The effects of two different hormone combinations on the rooting rate, root length and rooting rate of 'Huashen' were significant. In summary, the optimal rooting medium for 'Huashen' was MS+0.6 mg·L -1 NAA+0.6 mg·L - 1 IBA, 30 g·L -1 sucrose, 3 g·L -1 plant gel, pH 5.8-6.0.

[0061] Table 8 Effects of different hormone combinations on the rooting culture of 'Huashen'

[0062]

[0063] The above-described embodiments are merely some of the embodiments of the present application, but not all the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the claimed application, but merely represents selected embodiments of the application. All other embodiments obtained by persons of ordinary skill in the art based on the concept of the present application, without making creative labor, are within the scope of the present application.

Claims

1. A rapid propagation method for a horticultural variety of Helleborus, characterized in that, Includes the following steps: (1) Acquisition and disinfection of explants: After flowering, collect the underground flower buds, wash the mud around the flower buds clean, rinse with running water for 10 hours, place them on a clean bench, soak them in 5% sodium hypochlorite solution for 15 minutes, soak them in 75% alcohol for 30 seconds, rinse them with sterile water 3 times, filter out excess water, and set the treated buds aside for later use. (2) Primary culture: The buds treated in step (1) were inoculated onto the primary culture medium and placed in a culture environment with a temperature of 25±2℃, a relative humidity of 60~70%, and a light duration of 16h·d. -1 Induction culture was carried out for 25 days under light intensity of 2000~3000 lx until adventitious shoots differentiated from the explants; (3) Subculture: After successful bud induction in step (2), when the buds grow to 1-2 cm, they are transferred to a subculture medium and placed at a culture temperature of 25±2℃, relative humidity of 60-70%, light intensity of 2000-3000 lx, and photoperiod of 16 h·d. -1 Under the specified conditions, the plants were subcultured every 15 days and repeated multiple times to allow them to proliferate continuously, resulting in 3-4 cm clusters of shoots. (4) Rooting culture: When the shoot clusters in step (3) grow to 3-4 cm in height, the proliferated shoots are cut and transferred to the rooting culture medium. The culture room is placed at a temperature of 25±2℃, a relative humidity of 60-70%, a light intensity of 2000-3000 lx, and a photoperiod of 16 h·d. -1 Under the induced rooting conditions, after 30 days of culture, sterile seedlings with 1-2 cm fibrous roots growing from the base of the plant were obtained; The primary culture medium was: MS + 2 mg·L⁻¹ -1 2IP + 0~2.5mg·L -1 6-BA + 1 mg·L -1 GA3 +1mg·L -1 KT, 30g·L -1 Sucrose, 3g·L -1 Plant-based gel, pH 5.8~6.0; The subculture medium was: MS + 2 mg·L⁻¹ -1 2IP + 1~2mg·L -1 6-BA + 1~2 mg·L -1 GA3 + 0.1~0.6 mg·L -1 KT, 30g·L -1 Sucrose, 3g·L -1 Plant-based gel, pH 5.8~6.0; The rooting medium is: MS + 0.2~0.6 mg·L⁻¹ -1 NAA + 0.2~0.6 mg·L -1 IBA, 30g·L -1 Sucrose, 3g·L -1 Plant-based gel, pH 5.8~6.

0.

2. The rapid propagation method for a horticultural variety of Helleborus according to claim 1, characterized in that, The primary culture medium was: MS + 2 mg·L⁻¹ -1 2IP + 1mg·L -1 6-BA + 1 mg·L -1 GA3 + 1 mg·L -1 KT, 30g·L -1 Sucrose, 3g·L -1 Plant-based gel, pH 5.8~6.

0.

3. The rapid propagation method for a horticultural variety of Helleborus according to claim 1, characterized in that, The subculture medium was: MS + 2 mg·L⁻¹ -1 2IP + 1mg·L -1 6-BA + 2 mg·L -1 GA3 + 1 mg·L -1 KT, 30g·L -1 Sucrose, 3g·L -1 Plant-based gel, pH 5.8~6.

0.

4. The rapid propagation method for a horticultural variety of Helleborus according to claim 1, characterized in that, The rooting medium was: MS + 0.6 mg·L⁻¹ -1 NAA + 0.6 mg·L -1 IBA, 30g·L -1 Sucrose, 3g·L -1 Plant-based gel, pH 5.8~6.0.

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