A method for cultivating seaside hibiscus clump seedlings

Abstract

The present invention provides a method for cultivating clustered seedlings of Hibiscus truncatum, and belongs to the technical field of plant tissue culture. The present invention designs a seed germination medium of Hibiscus truncatum and a clustered bud induction medium of Hibiscus truncatum. By selecting a specific medium formula, the proliferation clustered seedlings can be successfully induced by Hibiscus truncatum seeds. The cultivation method provided by the present invention can make the seed seedlings obtained by cultivating the seed germination medium of Hibiscus truncatum present a brittle and thick state, and the entire material surface has a callus feeling, tending to be a "somatic embryo", and can induce clustered buds after being transferred to a proliferation medium, while seed seedlings in other states cannot be proliferated into clustered seedlings; the clustered bud induction medium of Hibiscus truncatum provided by the present invention can make the "somatic embryo" successfully proliferate into clustered seedlings, and the two culture media are used to form a cultivation method for clustered seedlings of Hibiscus truncatum, which provides a new way for the reproduction of Hibiscus truncatum, and also provides a new tissue culture clustered bud induction way.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, in particular to a method for cultivating seashore hibiscus clump seedlings. Background Art

[0002] Hibiscus truncatum belongs to the genus Hibiscus of the Malvaceae family. It has a dense crown, large and bright golden flowers, a long flowering period, and leaves that change significantly with the seasons. They gradually turn red in autumn. It is an excellent ornamental plant for flowers and leaves. It can be used for greening factories, mines, parks, squares, courtyards, roads, residential areas, etc. It is also an excellent plant material for flower hedges and hedges. Hibiscus truncatum is salt-alkali tolerant, light-loving, has strong wind resistance, has a certain degree of cold and drought resistance, can withstand short-term waterlogging, can withstand high temperatures of 40°C in summer, and can withstand low temperatures of -10°C in winter. It is an excellent windbreak, forest protection and greening tree species.

[0003] At present, scholars at home and abroad have conducted some relevant research on Hibiscus truncatum, mainly focusing on the salt tolerance of Hibiscus truncatum, the effects of salt stress on the physiological characteristics of Hibiscus truncatum, seed germination, reproduction (sowing and cuttings), etc., but there are few tissue culture seedling techniques for Hibiscus truncatum.

[0004] Chinese patent CN107771672A discloses a method for obtaining sterile seedlings of Hibiscus truncatum, which uses mature seeds of Hibiscus truncatum to obtain sterile seedlings through tissue culture. The seeds are first treated with concentrated sulfuric acid, soaked for 15 minutes, and then sterilized. The sterilized seeds are inoculated into solid MS culture medium and cultured in a tissue culture room to obtain healthy and strong sterile seedlings. Summary of the invention

[0005] The object of the present invention is to provide a method for cultivating clump seedlings of Hibiscus syriacus, which can successfully obtain proliferating clump seedlings through induction of Hibiscus syriacus seeds.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides a germination culture medium for seashore hibiscus seeds, comprising a basic culture medium, thidiazuron and NAA.

[0008] Preferably, in the Hibiscus sabdariffa seed germination medium, the added amount of thidiazuron is 0.8-1.2 mg / L.

[0009] Preferably, the amount of NAA added to the Hibiscus sabdariffa seed germination medium is 0.1-0.5 mg / L.

[0010] Preferably, the basal culture medium is WPM culture medium.

[0011] The invention also provides a seaside hibiscus bud cluster induction culture medium, which comprises a basic culture medium, thidiazuron, oligosaccharide chain protein and activated carbon.

[0012] Preferably, in the Hibiscus sylvestris cluster bud induction medium, the added amount of thidiazuron is 0.08-0.12 mg / L.

[0013] Preferably, in the Hibiscus syringae bud induction medium, the added amount of oligosaccharide chain protein is 0.08-0.12 g / L.

[0014] Preferably, in the Hibiscus sylvestris bud induction medium, the amount of activated carbon added is 0.08-0.12 g / L;

[0015] Preferably, the basal culture medium is WPM culture medium.

[0016] The present invention also provides a method for cultivating seashore hibiscus clump seedlings, comprising the following steps:

[0017] S1, inoculating Hibiscus syriacus seeds into the Hibiscus syriacus seed germination medium to obtain Hibiscus syriacus explants;

[0018] S2. Inoculating the explants of Hibiscus syriacus into the above-mentioned Hibiscus syriacus clustered shoot induction medium to obtain Hibiscus syriacus clustered shoots.

[0019] Preferably, the light intensity of the culture is 20-30 μmol / m 2 ·s, photoperiod is 8-12h / d, temperature is 20-24℃.

[0020] Beneficial effects of the present invention:

[0021] The present invention designs a hibiscus seaside bud seed germination medium and a hibiscus seaside bud cluster induction medium. By selecting a specific medium formula, the seed seedlings cultured in the hibiscus seaside bud seed germination medium are in a brittle and thick state, the entire material surface has a callus feeling, tends to be a "somatic embryo", and can induce cluster buds after being transferred to a proliferation medium, while seed seedlings in other states cannot be proliferated into cluster seedlings; the hibiscus seaside bud cluster induction medium provided by the present invention can make the "somatic embryo" successfully proliferate into cluster seedlings, and the two culture media are used to form a hibiscus seaside bud cluster seedling cultivation method, which provides a new way for the reproduction of hibiscus seaside bud, and also provides a new tissue culture cluster bud induction way. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a picture of the "somatic embryo" state of Hibiscus truncatum;

[0023] Figure 2 This is a picture showing the culture results of Hibiscus tiliaceus seed germination medium formula 1;

[0024] Figure 3This is a picture showing the culture results of Hibiscus tiliaceus seed germination medium formula 2;

[0025] Figure 4 This is a picture showing the culture results of Hibiscus tiliaceus seed germination medium formula 3;

[0026] Figure 5 This is a picture showing the culture results of Hibiscus tiliaceus seed germination medium formula 4;

[0027] Figure 6 This is a picture showing the culture results of Hibiscus tiliaceus seed germination medium formula 5;

[0028] Figure 7 This is a picture showing the culture results of Hibiscus tiliaceus seed germination medium formula 6;

[0029] Figure 8 This is a picture showing the culture results of Hibiscus tiliaceus seed germination medium formula 7;

[0030] Fig. 9 This is a picture showing the culture results of Hibiscus tiliaceus seed germination medium formula 8;

[0031] Fig.10 This is a picture showing the culture results of Hibiscus tiliaceus seed germination medium formula 9;

[0032] Fig.11 This is a picture showing the culture results of Hibiscus tiliaceus seed germination medium formula 10;

[0033] Fig.12 This is a diagram showing the culture results of Example 3 in the comparative experiment of germination culture medium for Hibiscus tiliaceus seeds;

[0034] Fig.13 This is a diagram showing the growth of Hibiscus tiliaceus seedlings germinated in Example 3;

[0035] Fig.14 This is a picture showing the culture results of the bud induction medium formula 1 for Hibiscus sylvestris;

[0036] Fig.15 This is a picture showing the culture results of the bud induction medium formula 2 for Hibiscus sylvestris;

[0037] Fig.16 This is a picture showing the culture results of the bud induction medium formula 3 for Hibiscus truncatula;

[0038] Fig.17 This is a picture showing the culture results of Hibiscus truncatus bud induction medium formula 4;

[0039] Fig.18 This is a picture showing the culture results of Hibiscus sylvestris var.

[0040] Fig.19This is a picture showing the culture results of Hibiscus truncatus bud induction medium formula 6;

[0041] Fig. 20 This is a picture showing the culture results of Hibiscus sylvestris bud induction medium formula 7;

[0042] Fig.21 This is a picture showing the culture results of Hibiscus sylvestris var.

[0043] Fig. 22 This is a picture showing the culture results of Hibiscus truncatus bud induction medium formula 9;

[0044] Fig.23 This is a diagram showing the culture results of Example 3 in the comparison experiment of the bud induction medium of Hibiscus truncatum. DETAILED DESCRIPTION

[0045] The invention provides a germination medium for Hibiscus syriacus seeds, comprising a basal medium, thidiazuron and NAA. In the germination medium for Hibiscus syriacus seeds, the addition amount of thidiazuron is preferably 0.8-1.2 mg / L, the addition amount of NAA is preferably 0.1-0.5 mg / L, and the basal medium is preferably a WPM medium.

[0046] The present invention also provides a hibiscus seaside bud induction medium, comprising a basal culture medium, thidiazuron, oligosaccharide chain protein and activated carbon. In the hibiscus seaside bud induction medium, the addition amount of thidiazuron is preferably 0.08-0.12 mg / L, the addition amount of oligosaccharide chain protein is preferably 0.08-0.12 g / L, the addition amount of activated carbon is preferably 0.08-0.12 g / L, and the basal culture medium is preferably WPM culture medium.

[0047] The present invention also provides a method for cultivating seashore hibiscus clump seedlings, comprising the following steps:

[0048] S1. Inoculate the Hibiscus syriacus seeds into the Hibiscus syriacus seed germination medium to obtain Hibiscus syriacus explants, which are subsequently referred to as "somatic embryos". Figure 1 S2, inoculating the seaside hibiscus explant into the seaside hibiscus bud induction medium to obtain seaside hibiscus bud clusters; the light intensity of the culture is preferably 20-30 μmol / m 2 ·s, the photoperiod is preferably 8-12h / d, and the temperature is preferably 20-24℃.

[0049] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0050] Sources of reagents used in the embodiments of the present invention:

[0051] Citric acid: 500g / bottle, brand: Xilong;

[0052] Triiodobenzoic acid (TIBA) 5g / bottle;

[0053] NAA: CP / Shanghai 10g;

[0054] Hydrolyzed casein: 250g / bottle, brand: Mumu Bio;

[0055] Vitamin H: 100g / bottle, brand: Mumu Biotechnology;

[0056] Folic acid: 250g / bottle Brand: Mumu Biotechnology;

[0057] KT: 1g / bottle WPM, brand: Mumu Biotechnology;

[0058] Thidiazuron (TDZ): 1g / bottle, brand: Mumu Biotechnology;

[0059] Preparation of vitamin H: 1L of pure water contains 0.05g of vitamin H;

[0060] Preparation of folic acid: 1L of pure water contains 0.1g of folic acid, first dissolve it with a small amount of sodium hydroxide.

[0061] Example 1

[0062] The germination medium for seaside hibiscus seeds is prepared as follows:

[0063] WPM+TDZ1.0 mg / L+NAA0.3 mg / L.

[0064] Example 2

[0065] The bud induction medium of Hibiscus sylvestris was prepared as follows:

[0066] WPM+TDZ0.1 mg / L+oligosaccharide chain protein 0.1 g / L+activated carbon 1 g / L.

[0067] Example 3

[0068] First, the Hibiscus tiliaceus seeds were induced into "somatic embryos" using the Hibiscus tiliaceus seed germination medium in Example 1, and cultured on a light culture rack with a light intensity of 20 to 30 μmol / m 2 ·s, photoperiod 10h / d, temperature 20-24℃, culture 30d.

[0069] Then, the induced "somatic embryos" were transferred to the Hibiscus truncatus bud induction medium in Example 2 on a clean bench for proliferation induction culture under the same culture conditions for 30 days to obtain Hibiscus truncatus buds.

[0070] Experimental Example 1 Comparison of different germination culture media for Hibiscus sylvestris seeds

[0071] Based on the culture method of Example 3, the formula of the Hibiscus truncatum seed germination culture medium was replaced with the following culture medium, and synchronous comparative culture was performed according to the culture scheme of Example 3:

[0072] Recipe 1: WPM

[0073] Formula 2: WPM + citric acid 10mg / L

[0074] Formula 3: WPM+TIBA0.1mg / L+citric acid 10mg / L

[0075] Formula 4: WPM+6-BA0.5 mg / L+NAA0.1 mg / L

[0076] Formula 5: WPM+6-BA0.5 mg / L+NAA0.1 mg / L+Hydrolyzed casein 0.1 g / L

[0077] Formula 6: WPM + 6-BA 0.5 mg / L + NAA 0.1 mg / L + hydrolyzed casein 0.1 g / L + vitamin H 1 mL / L

[0078] Formula 7: WPM + 6-BA 0.5 mg / L + NAA 0.1 mg / L + hydrolyzed casein 0.1 g / L + folic acid 5 mL / L

[0079] Formula 8: WPM + 6-BA 0.5 mg / L + NAA 0.1 mg / L + hydrolyzed casein 0.1 g / L + folic acid 5 mL / L + vitamin H 1 mL / L + citric acid 10 mg / L + charcoal 0.5 g / L

[0080] Formula 9: WPM + 6-BA 0.5 mg / L + NAA 0.1 mg / L + hydrolyzed casein 0.1 g / L + folic acid 5 mL / L + vitamin H 1 mL / L + citric acid 10 mg / L + charcoal 1 g / L

[0081] Formula 10: WPM+KT0.5 mg / L+NAA0.1 mg / L

[0082] The culture results of formulas 1 to 10 are as follows Figures 2 to 11 As shown, the culture results of Example 3 are as follows Fig.12 As shown in the figure, the growth of the seaside hibiscus seedlings germinated in Example 3 is as follows Fig.13 shown.

[0083] The results show that after the seaside hibiscus seedlings obtained by culturing with different seed germination medium formulas were transferred to the same proliferation formula for culturing, the growth of the seedlings was quite different. The leaves of the seedlings of formulas 5 to 9 showed yellowing and aging, and the seedlings of formula 9 were yellowed to death. The seedlings germinated in the formula with folic acid, vitamins, citric acid and other substances added in the early stage were not conducive to the later proliferation culture. The seed seedlings induced by the seed germination medium provided by the present invention were crisp and thick, and the entire material surface had a callus feeling, tending to be "somatic embryos", and clustered buds could be induced after being transferred to the proliferation medium.

[0084] Experimental Example 2 Comparison of different culture media for inducing clustered buds of Hibiscus truncatus

[0085] Based on the culture method of Example 3, the formula of the Hibiscus syriacus bud induction medium was replaced with the following culture medium, and synchronous comparative culture was performed according to the culture scheme of Example 3:

[0086] Formula 1: WPM + 6-BA 0.1 mg / L + oligosaccharide chain protein 0.1 g / L + activated carbon 1 g / L

[0087] Formula 2: WPM + 6-BA 0.1 mg / L + oligosaccharide chain protein 0.1 g / L

[0088] Formula 3: WPM+6-BA0.1 mg / L+activated carbon 1g / L

[0089] Formula 4: WPM + oligosaccharide chain protein 0.1g / L + activated carbon 1g / L

[0090] Formula 5: WPM + NAA 0.1 mg / L + oligosaccharide chain protein 0.1g / L + activated carbon 1g / L

[0091] Formula 6: WPM+NAA0.1 mg / L+activated carbon 1g / L

[0092] Formula 7: WPM+6-BA0.1 mg / L+NAA0.1g / L+activated carbon 1g / L

[0093] Formula 8: WPM + KT 0.1 mg / L + oligosaccharide chain protein 0.1 g / L + activated carbon 1 g / L

[0094] Recipe 9: WPM

[0095] The culture results of formulas 1 to 9 are as follows Figures 14 to 22 As shown, the culture results of Example 3 of the present invention are as follows Fig.23 shown.

[0096] The growth conditions of the "somatic embryos" obtained by the culture of the present invention under different formulations are shown in Table 1 below:

[0097] Table 1 Growth of somatic embryos under different formulas

[0098] Culture medium type Seedling growth Recipe 1 The seedlings are relatively strong, with only one bud and the plant is dark green. Recipe 2 No significant growth changes Recipe 3 Cotyledons are formed, and the base of the stem is slightly bruised. Recipe 4 Cotyledons and stems are visible, and the cotyledons are thinner Recipe 5 The seedlings are relatively strong, with only one bud and the plant is light green. Recipe 6 No significant growth changes Recipe 7 Cotyledons and stems are seen Recipe 8 Plants have been seen to form with only one bud Recipe 9 No significant growth changes Example 3 See clusters of buds

[0099] The culture results show that the induction of the "somatic embryos" obtained by the present invention under different formulations is different. Only single buds are differentiated in formulations 1, 4 and 7, and no obvious morphological changes occur in the "somatic embryos" under other treatments. The "somatic embryos" under the culture scheme provided in Example 3 proliferate clustered buds.

[0100] From the above, it can be seen that the technology of inducing "somatic embryos" to proliferate cluster seedlings from Hibiscus syriacus seeds is the result of the combined effect of the formulas in the two stages of inducing "somatic embryos" and proliferating "somatic embryos".

[0101] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for cultivating seashore hibiscus clump seedlings, characterized in that: The following steps are involved: S1, inoculating Hibiscus syriacus seeds into Hibiscus syriacus seed germination medium to obtain Hibiscus syriacus explants; S2, inoculating the seaside hibiscus explants into a seaside hibiscus bud induction medium to obtain seaside hibiscus bud clusters; The formula of the germination medium for Hibiscus truncatum seeds is as follows: WPM+TDZ1.0 mg / L+NAA0.3 mg / L; The formula of the bud induction medium of Hibiscus syriacus is as follows: WPM+TDZ0.1 mg / L+oligosaccharide chain protein 0.1 g / L+activated carbon 1 g / L.

2. The method for cultivating seashore hibiscus clump seedlings according to claim 1, characterized in that: The light intensity of the culture is 20-30 μmol / m 2 ·s, photoperiod is 8-12h / d, temperature is 20-24℃.

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