Efficient tissue culture and rapid propagation system for large-scale production of hippeastrum rutilum

Abstract

The invention discloses a hippeastrum hippeastrum tissue culture method. The hippeastrum hippeastrum tissue culture method comprises the following steps: obtaining hippeastrum hippeastrum scales with a complete bulb disc; performing disinfection treatment on the hippeastrum flakes to obtain disinfected hippeastrum flakes; inoculating the disinfected hippeastrum hippeastrum scales into an induced differentiation culture medium for culturing to obtain hippeastrum hippeastrum scales with adventitious buds; the adventitious buds are cut into bud clusters with 2-3 bud points, the bud clusters are inoculated into a subculture multiplication culture medium to be cultured, and subculture multiplication hippeastrum hippeastrum seedlings are obtained; inoculating the hippeastrum hippeastrum seedlings into a rooting and seedling strengthening culture medium for culturing to obtain hippeastrum hippeastrum plants with buds and roots. The induction rate can reach 92% or above, the multiplication coefficient is stabilized at 3.5-4.2, the rooting rate is 95% or above, the transplanting survival rate exceeds 90%, and the method has application prospects.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of plant tissue culture, and relates to a high-efficiency tissue culture and rapid propagation system for large-scale production of Hippeastrum rutilum. BACKGROUND

[0002] Hippeastrum rutilum, also known as Hippeastrum rutilum, is a perennial herbaceous bulbous flower of the Amaryllidaceae Hippeastrum genus. It has large and colorful flowers, beautiful flower shapes, and both ornamental and economic values, and occupies an important position in the flower market. Traditional Hippeastrum rutilum propagation mainly adopts bulb division and seed propagation. The bulb division propagation coefficient is low (only 2-3 daughter bulbs can be propagated per bulb per year), the propagation cycle is long (it takes 2-3 years to bloom), and it is difficult to meet the demand of large-scale production. Seed propagation is prone to produce character separation, leading to variety degeneration, and the seedlings grow slowly, take a long time to bloom (3-4 years), and cannot maintain the excellent properties of the mother plant.

[0003] Plant tissue culture technology has the advantages of high propagation coefficient, short cycle, and the ability to maintain the excellent properties of the mother plant, and has been widely used in flower seedling breeding. At present, although there are some studies on Hippeastrum rutilum tissue culture technology, there are still some technical bottlenecks such as high contamination rate of explants, low callus induction rate, unstable proliferation coefficient, poor rooting effect, and low survival rate of transplanted seedlings. In addition, there is no complete large-scale tissue culture and rapid propagation system, which limits its popularization and application in production. Therefore, it is of great significance to develop a high-efficiency, stable, and large-scale application of Hippeastrum rutilum tissue culture and rapid propagation system to promote the development of Hippeastrum rutilum industry. SUMMARY

[0004] In order to solve the problems in the prior art, the present application provides a method for tissue culture of Hippeastrum rutilum, which comprises the following steps:

[0005] S1: obtaining Hippeastrum rutilum scales with complete bulb discs;

[0006] S2: disinfecting the Hippeastrum rutilum scales to obtain disinfected Hippeastrum rutilum scales;

[0007] S3: inoculating the disinfected Hippeastrum rutilum scales into an induction and differentiation culture medium for culture to obtain Hippeastrum rutilum scales with adventitious buds;

[0008] S4: cutting the adventitious buds into bud clusters with 2-3 bud points, and inoculating the bud clusters into a subculture and proliferation culture medium for culture to obtain subcultured and proliferated Hippeastrum rutilum seedlings;

[0009] S5: inoculating the Hippeastrum rutilum seedlings into a rooting and seedling strengthening culture medium for culture to obtain Hippeastrum rutilum plants with buds and roots;

[0010] The components of the induction differentiation medium are: 1709 mg / L ammonium sulfate, 833 mg / L potassium dihydrogen phosphate, 268 mg / L magnesium sulfate, 97 mg / L calcium chloride, 218 mg / L calcium nitrate, 18.5 mg / L manganese sulfate, 7.2 mg / L zinc sulfate, 0.018 mg / L copper sulfate, 0.36 mg / L sodium molybdate, 19.4 mg / L ferric chloride, 41.7 mg / L disodium ethylenediaminetetraacetate, 88 mg / L myo-inositol, 3.6 mg / L glycine, 0.98 mg / L thiamine hydrochloride, 0.35 mg / L pyridoxal hydrochloride, 0.66 mg / L nicotinic acid, 0.12 mg / L biotin, 210 mg / L hydrolyzed casein, 3126 mg / L trehalose, 66 mL / L coconut milk, 8.9 mg / L humic acid, 0.016 mg / L TDZ, 0.26 mg / L IBA, 5.9 g / L agar, 20 g / L sucrose, 202 mg / L activated carbon, pH 5.0-6.5;

[0011] The subculture proliferation medium takes MS as the basic medium, and further contains 1.2 mg / L 6-BA, 0.8 mg / L KT, 0.2 mg / L NAA, 20 g / L sucrose and 7 g / L agar, pH 5.0-6.5;

[0012] The components of the rooting and seedling strengthening medium are: 868.13 mg / L potassium nitrate, 140.63 mg / L ammonium sulfate, 158.13 mg / L potassium dihydrogen phosphate, 145.16 mg / L magnesium sulfate, 192.19 mg / L calcium chloride, 122.5 mg / L calcium nitrate, 42.19 mg / L potassium chloride, 11.72 mg / L sodium dihydrogen phosphate, 3.76 mg / L boric acid, 13.53 mg / L manganese sulfate, 5.22 mg / L zinc sulfate, 0.015 mg / L copper sulfate, 0.15 mg / L sodium molybdate, 16.88 mg / L ferric chloride, 22.63 mg / L disodium ethylenediaminetetraacetate, 0.018 mg / L cobalt chloride, 69.31 mg / L myo-inositol, 1.25 mg / L glycine, 2.07 mg / L thiamine hydrochloride, 0.55 mg / L pyridoxal hydrochloride, 0.55 mg / L nicotinic acid, 4.40 g / L agar, 18.66 g / L sucrose, 1.85 mg / L NAA, 0.08 mg / L 6-BA, 0.085 mg / L mepiquat chloride, 216 mg / L activated carbon, pH 5.0-6.5.

[0013] In some embodiments, in S2, the said P. coccineus scales are sequentially rinsed with running water, soaked with dishwashing liquid, rinsed with sterile water, soaked with an aqueous ethanol solution, soaked with an aqueous sodium hypochlorite solution, and rinsed with sterile water to obtain the said sterilized P. coccineus scales.

[0014] In some embodiments, in S3, the said sterilized P. coccineus scales are subjected to an activation treatment before the said inoculation operation, and the said activation treatment comprises the following steps: cutting off 0.1-0.2 cm thick tissues at the incisions of the said sterilized P. coccineus scales, and soaking the said sterilized P. coccineus scales in an aqueous solution containing 70-80 mg / L vitamin C and 20-30 mg / L proline for 5-10 min.

[0015] In some embodiments, in S3, the culture conditions are as follows: temperature 22-26℃, light intensity 2000-2400 lx, light time 11-15 hours per day, and culture time 25-50 days.

[0016] In some embodiments, in S4, the culture conditions are as follows: temperature 21-25℃, light intensity 2600-3000 lx, light time 10-14 hours per day, culture time 30-35 days, and subculture 1-2 times.

[0017] In some embodiments, in S5, the culture conditions are as follows: temperature 20-23℃, light intensity 1600-2000 lx, light time 10-12 hours per day, and culture time 20-25 days.

[0018] In some embodiments, the said P. coccineus plants with buds and roots are subjected to hardening and transplanting to obtain the said P. coccineus plants with roots.

[0019] In some embodiments, the said substrate for transplanting comprises humus soil: perlite: vermiculite = 2: 0.5-1.5: 0.5-1.5 by weight.

[0020] In some embodiments, the said management after transplanting is as follows: temperature 2-25℃, air humidity 70-80%, and shade culture for 10-15 days.

[0021] This invention discloses a high-efficiency tissue culture rapid propagation system and method for Amaryllis, belonging to the field of plant tissue culture technology. This system uses Amaryllis bulb discs as explants and, through a standardized process of explant pretreatment, induced differentiation, subculture proliferation, rooting and seedling strengthening, and hardening and transplanting, optimizes the culture medium formula and conditions at each stage, solving the technical problems of low propagation coefficient, rapid varietal degeneration, and long propagation cycle in traditional Amaryllis cultivation. The induction rate of this invention can reach over 92%, the proliferation coefficient is stable at 3.5-4.2, the rooting rate is over 95%, and the transplant survival rate exceeds 90%. Furthermore, the tissue-cultured seedlings exhibit stable genetic traits and robust growth, making them suitable for large-scale industrial production. This provides reliable technical support for the rapid propagation of high-quality Amaryllis seedlings and has significant economic value and application prospects.

[0022] This invention establishes a complete high-efficiency tissue culture and rapid propagation system for Amaryllis by optimizing explant pretreatment methods, culture medium formulations, and culture conditions, which has the following advantages:

[0023] (1) High reproduction coefficient: The reproduction coefficient of each generation is stable at 4.5-5.2, which is much higher than that of traditional bulb division reproduction;

[0024] (2) Short propagation cycle: It only takes 4-5 months from explant inoculation to transplanting seedlings, which is 1-2 years shorter than traditional propagation methods;

[0025] (3) Excellent seedling quality: tissue culture seedlings have stable genetic traits, grow vigorously, are free from diseases and pests, and flower uniformly;

[0026] (4) Large-scale production: The system is simple to operate, stable and reliable, suitable for large-scale factory breeding, and can quickly meet the market demand for high-quality amaryllis seedlings. It has significant economic value and application prospects. Attached Figure Description

[0027] Figure 1 Photographs of the growth status of amaryllis 14 days after induced differentiation.

[0028] Figure 2 Photographs of the growth status of amaryllis 14 days after induced differentiation.

[0029] Figure 3 Photographs of the growth status of amaryllis 14 days after induced differentiation.

[0030] Figure 4 Photographs of the growth status of amaryllis 14 days after induced differentiation.

[0031] Figure 5 Photos showing the growth status of amaryllis after 22 days of rooting culture.

[0032] Figure 6 Photos showing the growth status of amaryllis after 22 days of rooting culture.

[0033] Figure 7 The growth state photo of the Anthurium andraeanum for 22 days of rooting culture. DETAILED DESCRIPTION

[0034] In order to make the objects, technical solutions and advantages of the present application clearer, the embodiments of the present application will be further described in detail below with reference to the drawings.

[0035] Example 1: Preparation of culture medium

[0036] I. Induction differentiation medium

[0037] The components, contents and main functions of the induction differentiation medium are as follows.

[0038] Ammonium sulfate 1709 mg / L; replace part of potassium nitrate, optimize nitrogen source form.

[0039] Potassium dihydrogen phosphate 833 mg / L; regulate phosphorus element supply, promote cell metabolism.

[0040] Magnesium sulfate 268 mg / L; balance mineral nutrition, stabilize cell structure.

[0041] Calcium chloride 97 mg / L; strengthen cell wall synthesis, reduce browning risk.

[0042] Calcium nitrate 218 mg / L; supplement calcium element, improve seedling robustness.

[0043] Manganese sulfate 18.5 mg / L; assist enzyme reaction, improve nutrient utilization rate.

[0044] Zinc sulfate 7.2 mg / L; optimize cell division efficiency.

[0045] Copper sulfate 0.018 mg / L; regulate oxidation-reduction reaction.

[0046] Sodium molybdate 0.36 mg / L; assist nitrogen metabolism, stabilize growth state.

[0047] Ferric chloride 19.4 mg / L; supplement iron element, prevent yellowing.

[0048] Disodium ethylenediaminetetraacetate 41.7 mg / L; chelate iron element, improve absorption efficiency.

[0049] Myo-inositol 88 mg / L; stabilize cell structure, promote metabolism.

[0050] Glycine 3.6 mg / L; optimize amino acid supply, improve cell activity.

[0051] Thiamine hydrochloride (VB1) 0.98 mg / L; promote carbohydrate metabolism.

[0052] Pyridoxine hydrochloride (VB6) 0.35 mg / L; adjust protein synthesis.

[0053] Niacin 0.66 mg / L; auxiliary enzyme system activity, stable growth environment.

[0054] Biotin 0.12 mg / L; enhance cell proliferation capacity.

[0055] Hydrolyzed casein 210 mg / L; supplement high-quality protein, promote seedling robustness.

[0056] Trehalose 3126 mg / L; replace part of the carbon source, protect cell activity.

[0057] Coconut milk 66 mL / L; supplement natural active substances, promote differentiation.

[0058] Humic acid 8.9 mg / L; improve nutrient absorption and stress resistance.

[0059] Thidiazuron (TDZ) 0.016 mg / L; induce differentiation, promote bud differentiation.

[0060] Indole-3-butyric acid (IBA) 0.26 mg / L; stabilize seedling growth.

[0061] Agar 5.9 g / L; used for solid medium, maintain medium shape.

[0062] Sucrose 20 g / L; provide carbon source.

[0063] Activated carbon 202 mg / L; reduce browning.

[0064] The pH value is adjusted to 5.6-5.9.

[0065] II. Subculture multiplication medium

[0066] MS is used as the basic medium, supplemented with sucrose 20 g / L, agar 7 g / L, 6-BA 1.2 mg / L, KT 0.8 mg / L, NAA 0.2 mg / L. The addition of KT can significantly improve the multiplication coefficient, while avoiding the overgrowth of bud seedlings. The multiplication coefficient is stable at 3.5-4.2, and the bud seedlings grow robustly. The pH value is adjusted to 5.6-5.9.

[0067] III. Rooting and seedling strengthening medium

[0068] The content of the rooting and seedling strengthening medium is as follows.

[0069] Potassium nitrate 868.13 mg / L;

[0070] Ammonium sulfate 140.63 mg / L;

[0071] Monopotassium phosphate 158.13 mg / L;

[0072] Magnesium sulfate 145.16 mg / L;

[0073] Calcium chloride 192.19 mg / L;

[0074] Calcium nitrate 122.5 mg / L;

[0075] Potassium chloride 42.19 mg / L;

[0076] Sodium dihydrogen phosphate 11.72 mg / L;

[0077] Boric acid 3.76 mg / L;

[0078] Manganese sulfate 13.53 mg / L;

[0079] Zinc sulfate 5.22 mg / L;

[0080] Copper sulfate 0.015 mg / L;

[0081] Sodium molybdate 0.15 mg / L;

[0082] Ferric chloride 16.88 mg / L;

[0083] Disodium ethylenediaminetetraacetate 22.63 mg / L;

[0084] Cobalt chloride 0.018 mg / L;

[0085] Myo-inositol 69.31 mg / L;

[0086] Glycine 1.25 mg / L;

[0087] Thiamine hydrochloride (VB1) 2.07 mg / L;

[0088] Pyridoxine hydrochloride (VB6) 0.55 mg / L;

[0089] Niacin 0.55 mg / L;

[0090] Agar 4.40 g / L;

[0091] Sucrose 18.66 g / L;

[0092] NAA 1.85 mg / L;

[0093] 6-BA 0.08 mg / L;

[0094] Chlormequat 0.085 mg / L;

[0095] Activated charcoal 216 mg / L;

[0096] pH adjusted to 5.6-5.9.

[0097] Example 2: Preparation of tissue culture seedlings of Crocus sativus

[0098] I. Selection and pretreatment of explants

[0099] Healthy bulbs of Crocus sativus (variety Moyu) were selected, the outer layer of senescent scales was peeled off, and the middle layer of scales with a diameter of 3.0-5.0 cm and intact bulb discs were reserved as explants. The explants were washed under running water for 25 min, then immersed in a 0.2% detergent solution for 6 min, with gentle stirring during the process, and then washed with sterile water for 4 times until no foam residue was left.

[0100] II. Gradient disinfection treatment

[0101] The pretreated explants were transferred to a clean bench, and first immersed in a 78% ethanol aqueous solution for 45 s. After the ethanol was quickly poured out, a 2.5% sodium hypochlorite aqueous solution (containing 0.08% Tween-80) was immediately added, and the sealed container was shaken for 12 min at a frequency of 135 r / min. After the sodium hypochlorite solution was poured out, the explants were washed with sterile water for 6 times, each time for 1.5 min, and finally the surface water of the explants was absorbed with sterile filter paper.

[0102] III. Activation treatment after disinfection

[0103] The cut surface of the disinfected explants was cut to a thickness of 0.1-0.2 cm, and immersed in a water solution containing 75 mg / L vitamin C and 25 mg / L proline (sterile activation solution) for 8 min. After that, the surface water was absorbed again with sterile filter paper, and the disinfection was completed.

[0104] IV. Induction and differentiation culture

[0105] The explants were inoculated into the induction and differentiation medium prepared in Example 1, and cultured at a temperature of 24℃ and an illumination intensity of 2200 lx for 13 h / d. After 18 days, calli were formed, and after 28 days, adventitious buds were differentiated, with an induction rate of 93%. Figures 1 to 4 All are photos of the growth state of Crocus sativus after 30 days of induction and differentiation.

[0106] II. Subculture and proliferation culture

[0107] The adventitious buds were cut into bud clusters with 2-3 bud points, and inoculated into the subculture and proliferation medium prepared in Example 1. The culture was carried out at a temperature of 23℃ and an illumination intensity of 2800 lx for 12 h / d. The subculture was performed every 32 days, and the culture was subcultured twice continuously, with a proliferation coefficient of 4.0.

[0108] III. Rooting and seedling culture

[0109] The single bud with a height of about 2.5 cm was selected from the subculture plant, inoculated into the rooting and seedling culture medium prepared in Example 1, and cultured at a temperature of 21 ℃, a light intensity of 1800 lx, and a light time of 11 h / d for 22 d, so that the rooting rate was 96% and the average number of roots per plant was 4. Figures 5-7 All are the growth state photos of the Poinsettia after rooting culture for 22 days.

[0110] Four, seedling raising and transplanting

[0111] The seedlings were washed with clean water, and then transplanted into a sterilized substrate with a weight ratio of humus soil:pearl stone:vermiculite = 2:1:1, and cultured at a temperature of 23 ℃, an air humidity of 75%, and a shading condition for 12 d, so that the transplanting survival rate was 92%.

[0112] From the common general knowledge, the present application can be realized by other embodiments without departing from the spirit or essential characteristics thereof. Therefore, the above disclosed embodiments are merely illustrative, and not restrictive. All the changes within the scope of the present application or within the equivalent scope of the present application are intended to be included in the present application.

Claims

1. A method for amaryllis tissue culture, the method comprising the following steps: S1: Obtain amaryllis scales with intact bulb discs; S2: Disinfect the amaryllis scales to obtain disinfected amaryllis scales; S3: The disinfected amaryllis scales are inoculated into an induction differentiation medium and cultured to obtain amaryllis scales with adventitious buds. S4: Cut the adventitious buds into bud clusters with 2-3 bud points, inoculate the bud clusters into a subculture proliferation medium, and culture them to obtain subcultured amaryllis seedlings; S5: The amaryllis seedlings are inoculated into a rooting and seedling strengthening culture medium to obtain amaryllis plants with buds and roots; The components of the differentiation-inducing medium were: 1709 mg / L ammonium sulfate, 833 mg / L potassium dihydrogen phosphate, 268 mg / L magnesium sulfate, 97 mg / L calcium chloride, 218 mg / L calcium nitrate, 18.5 mg / L manganese sulfate, 7.2 mg / L zinc sulfate, 0.018 mg / L copper sulfate, 0.36 mg / L sodium molybdate, 19.4 mg / L ferric chloride, 41.7 mg / L disodium EDTA, 88 mg / L inositol, 3.6 mg / L glycine, 0.98 mg / L thiamine hydrochloride, 0.35 mg / L pyridoxine hydrochloride, 0.66 mg / L nicotinic acid, 0.12 mg / L biotin, 210 mg / L hydrolyzed casein, 3126 mg / L trehalose, 66 mL / L coconut milk, 8.9 mg / L humic acid, 0.016 mg / L LTDZ, and 0.26 mg / L... IBA, 5.9 g / L agar, 20 g / L sucrose, 202 mg / L activated carbon, pH 5.0-6.5; The subculture proliferation medium used MS as the basic medium, which also contained 1.2 mg / L 6-BA, 0.8 mg / L KT, 0.2 mg / L NAA, 20 g / L sucrose and 7 g / L agar, with a pH of 5.0-6.5; The components of the rooting and seedling strengthening culture medium are: 868.13 mg / L potassium nitrate, 140.63 mg / L ammonium sulfate, 158.13 mg / L potassium dihydrogen phosphate, 145.16 mg / L magnesium sulfate, 192.19 mg / L calcium chloride, 122.5 mg / L calcium nitrate, 42.19 mg / L potassium chloride, 11.72 mg / L sodium dihydrogen phosphate, 3.76 mg / L boric acid, 13.53 mg / L manganese sulfate, 5.22 mg / L zinc sulfate, 0.015 mg / L copper sulfate, 0.15 mg / L sodium molybdate, 16.88 mg / L ferric chloride, 22.63 mg / L disodium ethylenediaminetetraacetate, 0.018 mg / L cobalt chloride, 69.31 mg / L inositol, 1.25 mg / L glycine, 2.07 mg / L thiamine hydrochloride, 0.55 mg / L sodium chloride, and 1.25 mg / L glycine. 0.55 mg / L pyridoxine hydrochloride, 0.55 mg / L nicotinic acid, 4.40 g / L agar, 18.66 g / L sucrose, 1.85 mg / L NAA, 0.08 mg / L 6-BA, 0.085 mg / L chlormequat chloride, 216 mg / L activated carbon, pH 5.0-6.

5.

2. The method as described in claim 1, characterized in that, In S2, the amaryllis scales are sequentially rinsed with running water, soaked in detergent, rinsed with sterile water, soaked in an ethanol aqueous solution, soaked in a sodium hypochlorite aqueous solution, and rinsed with sterile water to obtain the disinfected amaryllis scales.

3. The method as described in claim 1, characterized in that, In S3, the disinfected amaryllis scales are activated before the inoculation operation. The activation process involves cutting off 0.1-0.2 cm of tissue from the cut surface of the disinfected amaryllis scales and immersing them in an aqueous solution containing 70-80 mg / L vitamin C and 20-30 mg / L proline for 5-10 minutes.

4. The method as described in claim 1, characterized in that, In S3, the cultivation conditions are: temperature 22-26℃, light intensity 2000-2400 lx, light duration 11-15 hours per day, and cultivation time 25-50 days.

5. The method as described in claim 1, characterized in that, In S4, the culture conditions are: temperature 21-25℃, light intensity 2600-3000 lx, light duration 10-14 hours per day, culture time 30-35 days, and subculture 1 to 2 times.

6. The method as described in claim 1, characterized in that, In S5, the cultivation conditions are: temperature 20-23℃, light intensity 1600-2000 lx, light duration 10-12 hours per day, and cultivation time 20-25 days.

7. The method as described in claim 1, characterized in that, The amaryllis plants with buds and roots were hardened off and transplanted to obtain established amaryllis plants.

8. The method as described in claim 1, characterized in that, The substrate used for transplanting comprises, by weight, humus: perlite: vermiculite = 2:0.5-1.5: 0.5-1.

5.

9. The method as described in claim 1, characterized in that, The post-transplantation management is as follows: temperature 2-25℃, air humidity 70-80%, and shade cultivation for 10-15 days.

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