WO2022037017A1 - Series of media for regenerating hedychium hookeri c.b.clarke ex baker plant and application thereof - Google Patents
Series of media for regenerating hedychium hookeri c.b.clarke ex baker plant and application thereof Download PDFInfo
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- WO2022037017A1 WO2022037017A1 PCT/CN2021/073805 CN2021073805W WO2022037017A1 WO 2022037017 A1 WO2022037017 A1 WO 2022037017A1 CN 2021073805 W CN2021073805 W CN 2021073805W WO 2022037017 A1 WO2022037017 A1 WO 2022037017A1
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- the invention relates to a series of culture kits for regenerating Hooke ginger flower plants and applications thereof, belonging to the field of plant tissue culture.
- Hooke ginger flower has a short plant, broad leaves, light yellow flower color and black aril, which has important research and development and utilization value.
- the plant's callus regeneration system is the basic technology platform for further genetic manipulation and breeding improvement of the plant.
- the types of established callus regeneration systems include White Ginger and Red Ginger. These two species belong to the type with thick rhizomes and thin lateral roots, and are distributed at medium and low altitudes, while Hooke ginger flower has small rhizomes, thick lateral roots, and is distributed at high altitudes, and the species are quite different.
- hypocotyls of seedlings germinated from plant seeds are active growth points, and many plants have used hypocotyls as explants to induce callus and regenerate plants.
- the types of plant growth regulators and their concentration ratios are very different among different plants.
- the present invention takes the hypocotyl of the germinated sterile seedling of the seed as the explant, and constructs a simple and efficient callus through systematic experimental research on the conditions of callus induction and re-differentiation.
- Tissue regeneration is a culture system for plants.
- the object of the present invention is to overcome the deficiencies of the prior art and provide a series of culture kits for regenerating Huke ginger flower plants prepared via callus approach with low pollution rate, high induction rate, fast induction speed and easy operation and its applications.
- the technical scheme adopted in the present invention is:
- the present invention provides a series of culture kits for regenerating Huke ginger flower plants, including seed germination medium, callus induction medium, callus proliferation medium and callus redifferentiation culture.
- seed germination medium includes MS medium, sucrose and agar
- callus induction medium includes MS medium, kinetin, 2,4-dichlorophenoxyacetic acid, sucrose and agar
- the callus proliferation medium includes MS medium, kinetin, 2,4-dichlorophenoxyacetic acid, sucrose and agar
- the callus redifferentiation medium includes MS medium, 6-benzylaminopurine, indole -3-acetic acid, sucrose and agar.
- the composition of the callus induction medium and the callus proliferation medium are the same, but the concentrations of kinetin and 2,4-dichlorophenoxyacetic acid in the composition are different.
- the culture kit of the invention has a simple preparation method, can induce callus differentiation without adding complex culture, and does not need to switch back and forth between solid and liquid culture.
- the concentration of sucrose is 28-30 g/L, and the concentration of agar is 7.0-8.0 g/L.
- the concentration of kinetin is 0.1-0.5 mg/L
- the concentration of 2,4-dichlorophenoxyacetic acid is 0.5-1.0 mg/L
- the concentration of sucrose is 28-28 mg/L.
- the concentration of agar is 7.0 ⁇ 8.0g/L.
- the concentration of kinetin is 0.1-0.2 mg/L
- the concentration of 2,4-dichlorophenoxyacetic acid is 0.3-0.5 mg/L
- the concentration of sucrose is 28-28 mg/L.
- the concentration of agar is 7.0 ⁇ 8.0g/L.
- the callus proliferation medium can make callus proliferate rapidly, and obtain callus with more than 60 times the initial inoculation amount.
- the concentration of 6-benzylaminopurine is 1.0-2.0 mg/L
- the concentration of indole-3-acetic acid is 0.1-2.0 mg/L
- the concentration of sucrose is 28 ⁇ 30g/L
- the concentration of agar is 7.0 ⁇ 8.0g/L.
- the callus pieces After being cultured in the callus redifferentiation medium, the callus pieces are transformed into sterile seedlings, which can be directly hardened and transplanted.
- the present invention provides the application of the above-mentioned culture medium in the establishment of a regenerated Hooke ginger flower plant.
- the invention provides the preparation method of the above-mentioned regenerated Huke ginger flower plant, comprising the following steps:
- step (3) when the sterile seedling age obtained in step (2) is more than 7 days, the hypocotyl of the sterile seedling is cut into 1 to 2 sections with a length of 0.3 to 0.5 cm, and inoculated into the callus induction medium, Cultured for more than 30 days to obtain callus;
- step (3) (4) dividing the callus obtained in step (3) into blocks and inoculating on the callus proliferation medium, taking a month as a culture period, and substituting 3 times to obtain callus with an initial inoculation amount more than 60 times;
- the callus obtained by taking the hypocotyl as an explant has high regeneration efficiency, and does not require the steps of seedling strengthening and rooting, and the seedling is formed in one step.
- step (5) The sterile seedlings obtained in step (5) are placed in a place with scattered light for 3 to 4 days, and after cleaning, they are transplanted into a mixed matrix of perlite and peat soil in equal proportions, and the survival rate is observed.
- the hypocotyl is the explant with low contamination rate and high sterilization efficiency. Since the seeds are derived from ovules, they have never been in contact with the outside world from the beginning of development, and are naturally sterile. Therefore, the fruits can be directly inoculated after disinfection, with zero contamination rate, and the disinfection operation steps are simpler. At the same time, the callus induction rate is high and the induction speed is high. faster.
- the culture condition of the step (2) is dark culture, and the temperature is 24-26°C.
- the culture conditions of the steps (3) to (6) are a temperature of 24 to 26° C., an illumination of 12 h/d, and an illumination intensity of 2500 lx.
- the explant contamination rate is low and the aseptic efficiency is high. Since the seeds are protected by the husk during disinfection, it takes longer than ordinary disinfection treatment, and the disinfection is thorough. The seeds are derived from ovules and have never been in contact with the outside world from the beginning of their development. They are naturally sterile, so the fruits can be directly inoculated after disinfection, the pollution rate is zero, and the disinfection operation steps are also simplified.
- the callus induction rate is high and the induction speed is faster.
- the induction rate was 100% at 30 days after hypocotyl inoculation, while the induction of leaves, filaments and anther callus required 60-90 days and the induction rate was 70%.
- the callus induced by the present invention is light yellow, loose and vigorously divided high-quality callus; while the callus induced by leaves, filaments and anthers often needs to undergo 2-3 generations of subculture before screening. to uniform, vigorously dividing callus.
- the regeneration efficiency is high, and the seedlings are grown in one step without the steps of strengthening and rooting.
- the callus was cut into 8 mm-sized callus blocks and transferred to the differentiation medium. After culturing for 120 days, the callus blocks were transformed into sterile seedlings, and 10 to 15 plants with 3 to 4 leaves were differentiated. Rooted plants can be directly hardened and transplanted.
- Figure 1 is a physical picture of the callus induced by the present invention after being cultured in a callus proliferation medium for 30 days.
- Figure 2 shows the regeneration of the callus induced by the present invention after culturing for 120 days on the callus redifferentiation medium with 6-benzylaminopurine of 2.0 mg/L and indole-3-acetic acid of 1.0 mg/L picture.
- the callus regeneration system of Ginger is mainly based on leaves, filaments and filaments as explants, and the efficiency of aseptic treatment is low. Regeneration into plants is inefficient.
- the present invention discloses a simple and efficient method for inducing callus formation and regeneration of Huke ginger flower into plants by taking hypocotyl as explant.
- the present invention is an embodiment of a series of culture kits for regenerating Huke ginger flower plants and its application.
- the present embodiment is used for a series of culture kits for regenerating Huke ginger flower plants, including seed germination medium, callus tissue Induction medium, callus proliferation medium and callus redifferentiation medium.
- the concentration of sucrose is 28 g/L, and the concentration of agar is 7 g/L; in the callus induction medium, the concentration of kinetin is 0.1 mg/L, 2,4-dichloro
- the concentration of phenoxyacetic acid is 0.5mg/L, the concentration of sucrose is 28g/L, and the concentration of agar is 7g/L; in the callus proliferation medium, the concentration of kinetin is 0.1mg/L, 2,4 -
- the concentration of dichlorophenoxyacetic acid is 0.3 mg/L, the concentration of sucrose is 28 g/L, and the concentration of agar is 7 g/L; in the callus redifferentiation medium, the concentration of 6-benzylaminopurine is 1.0 mg/L, the concentration of indole-3-acetic acid was 0.1 mg/L, the concentration of sucrose was 28 g/L, and the concentration of agar was 7.0 g/L.
- step (3) when the sterile seedling age obtained in step (2) is 7 days, the hypocotyl of the sterile seedling is cut into 1-2 sections with a length of 0.3-0.5 cm, inoculated into the callus induction medium, and cultured for 30 sky;
- step (3) (4) dividing the callus obtained in step (3) into 5 mm-sized callus pieces and inoculating them on the callus proliferation medium, taking 30 days as the culture period, and subculture 3 times, the callus proliferates rapidly, and obtains Callus with 60 times the initial inoculation;
- step (4) The callus obtained in step (4) was cut into 8 mm-sized callus blocks and inoculated on the callus redifferentiation medium, and cultured for 120 days to obtain complete 3-4 leaves and roots. vaccine;
- step (6) Place the sterile seedlings obtained in step (5) in a place with scattered light for 3 to 4 days, then open the bottle cap of the bottle seedlings, carefully remove the bottle seedlings, wash the medium attached to the roots, remove the Planted in a mixed matrix of perlite and peat soil (1:1) sterilized with 1% potassium permanganate solution, drenched in enough root-fixing water, sprayed with water and mist every day for moisturizing, and sprayed MS diluted 5 times once a week Nutrient solution with a large number of elements, the survival rate was observed after one month.
- the present invention is an embodiment of a series of culture kits for regenerating Huke ginger flower plants and its application.
- the present embodiment is used for a series of culture kits for regenerating Huke ginger flower plants, including seed germination medium, callus tissue Induction medium, callus proliferation medium and callus redifferentiation medium.
- the concentration of sucrose is 29 g/L, and the concentration of agar is 7.5 g/L; in the callus induction medium, the concentration of kinetin is 0.3 mg/L, 2,4-di
- the concentration of chlorophenoxyacetic acid was 0.8 mg/L, the concentration of sucrose was 29 g/L, and the concentration of agar was 7.5 g/L; in the callus proliferation medium, the concentration of kinetin was 0.15 mg/L, 2 ,
- the concentration of 4-dichlorophenoxyacetic acid was 0.4 mg/L, the concentration of sucrose was 29 g/L, and the concentration of agar was 7.5 g/L; The concentration was 1.5 mg/L, the concentration of indole-3-acetic acid was 1.0 mg/L, the concentration of sucrose was 29 g/L, and the concentration of agar was 7.5 g/L.
- the present invention is an embodiment of a series of culture kits for regenerating Huke ginger flower plants and its application.
- the present embodiment is used for a series of culture kits for regenerating Huke ginger flower plants, including seed germination medium, callus tissue Induction medium, callus proliferation medium and callus redifferentiation medium.
- the concentration of sucrose is 30 g/L, and the concentration of agar is 8 g/L; in the callus induction medium, the concentration of kinetin is 0.5 mg/L, 2,4-dichloro
- the concentration of phenoxyacetic acid is 1.0mg/L, the concentration of sucrose is 30g/L, and the concentration of agar is 8g/L; in the callus proliferation medium, the concentration of kinetin is 0.2mg/L, 2,4 - the concentration of dichlorophenoxyacetic acid is 0.5mg/L, the concentration of sucrose is 30g/L, and the concentration of agar is 8g/L; in the callus redifferentiation medium, the concentration of 6-benzylaminopurine is 2.0 mg/L, the concentration of indole-3-acetic acid was 2.0 mg/L, the concentration of sucrose was 30 g/L, and the concentration of agar was 8.0 g/L.
- aseptic treatment of explants of the present invention select plump, non-damaged, uncracked fruits, under aseptic conditions, soak and sterilize with alcohol and 0.1% mercuric solution successively, rinse with sterile water and use aseptic operation Seeds were inoculated into seed germination medium, and 90 seeds were inoculated.
- a comparative example of a series of culturing kits for regenerating Huke ginger flower plants and an application thereof of the present invention the different concentrations of kinetin and 2,4-dichlorophenoxyacetic acid described in this comparative example are effective for Huke ginger flower.
- the effect of hypocotyl callus induction is as follows:
- the hypocotyls could not induce callus in the induction medium without any hormones.
- the results showed that the ranges of kinetin and 2,4-dichlorophenoxyacetic acid were 0.1 ⁇ 0.5mg/L and 0.5mg/L, respectively. ⁇ 1.0mg/L hypocotyl can induce callus, when kinetin is 0.5mg/L, 2,4-dichlorophenoxyacetic acid is 1.0mg/L, callus induction rate can reach 100%, The growth of the callus is the best, the callus grows fast, and the looseness is good.
- the invention is a comparative example of a series of culture kits for regenerating Huke ginger flower plants and an application thereof.
- the 6-benzylaminopurine and indole-3-acetic acid described in this comparative example are effective for the redifferentiation of Huke ginger flower callus.
- the method of influence is:
- the culture time is 120 days
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Abstract
Provided are a series of media for regenerating a Hedychium hookeri C.B.Clarke ex Baker plant, and an application thereof, relating to the field of plant tissue culture. The media of the present application comprise: a seed germination medium, a callus induction medium, a callus proliferation medium, and a callus redifferentiation medium. A Hedychium hookeri C.B.Clarke ex Baker callus regeneration system of the present invention achieves low explant contamination rate and fast callus induction speed, and enables one-step seedling without strengthening and rooting steps, thus achieving high regeneration efficiency.
Description
本发明涉及一种用于再生虎克姜花植株的系列培养试剂盒及其应用，属于植物组织培养领域。The invention relates to a series of culture kits for regenerating Hooke ginger flower plants and applications thereof, belonging to the field of plant tissue culture.
与同属其他植物相比，虎克姜花植株矮小、叶片宽大、花色鹅黄、假种皮黑色，具有重要的研究和开发利用价值。植物的愈伤组织再生体系是对该植物进行进一步遗传操作和育种改良的基础技术平台。目前，在姜花属中，建立了愈伤组织再生体系的种类包括白姜花和红姜花。这两个种属于根茎粗大、侧根细、分布于中低海拔的类型，而虎克姜花为根茎小、侧根肥厚、分布于高海拔，种类差异较大。在外植体的选择上，目前主要选用叶片、花丝以及花药为外植体，污染率高、无菌处理效率低，愈伤组织诱导速度慢(需60～90天)，需添加复杂的培养物(维生素B5、谷氨酰胺、脯氨酸、麦芽提取物等)，步骤繁琐(需采用液体震荡培养等)，再生效率低，愈伤组织再生为植株的培养体系有待优化。Compared with other plants of the same genus, Hooke ginger flower has a short plant, broad leaves, light yellow flower color and black aril, which has important research and development and utilization value. The plant's callus regeneration system is the basic technology platform for further genetic manipulation and breeding improvement of the plant. At present, in the genus Ginger, the types of established callus regeneration systems include White Ginger and Red Ginger. These two species belong to the type with thick rhizomes and thin lateral roots, and are distributed at medium and low altitudes, while Hooke ginger flower has small rhizomes, thick lateral roots, and is distributed at high altitudes, and the species are quite different. In the selection of explants, currently leaves, filaments and anthers are mainly used as explants, which have high contamination rate, low efficiency of aseptic treatment, slow callus induction (60-90 days), and complex cultures need to be added. (vitamin B5, glutamine, proline, malt extract, etc.), the steps are cumbersome (need to use liquid shaking culture, etc.), the regeneration efficiency is low, and the culture system for callus regeneration into plants needs to be optimized.
植物种子萌发出的幼苗，其下胚轴是活跃的生长点，已有较多植物以下胚轴为外植体诱导出愈伤组织并再生出植株。但在这些技术方案中，在不同植物之间所选用的植物生长调节剂种类及其浓度配比具有很大差异。本发明基于虎克姜花的遗传特点，以种子萌发无菌苗的下胚轴为外植体，经过对愈伤组织诱导和再分化条件进行的***试验研究，构建了简单而高效的愈伤组织再生为植株的培养体系。The hypocotyls of seedlings germinated from plant seeds are active growth points, and many plants have used hypocotyls as explants to induce callus and regenerate plants. However, in these technical solutions, the types of plant growth regulators and their concentration ratios are very different among different plants. Based on the genetic characteristics of Huke ginger flower, the present invention takes the hypocotyl of the germinated sterile seedling of the seed as the explant, and constructs a simple and efficient callus through systematic experimental research on the conditions of callus induction and re-differentiation. Tissue regeneration is a culture system for plants.
发明内容SUMMARY OF THE INVENTION
本发明的目的在于克服现有技术的不足之处而提供一种污染率低、诱导率高、诱导速度快、操作简便的经由愈伤组织途径制备的再 生虎克姜花植株的系列培养试剂盒及其应用。The object of the present invention is to overcome the deficiencies of the prior art and provide a series of culture kits for regenerating Huke ginger flower plants prepared via callus approach with low pollution rate, high induction rate, fast induction speed and easy operation and its applications.
为实现上述目的，本发明采取的技术方案为：To achieve the above object, the technical scheme adopted in the present invention is:
第一方面，本发明提供了一种用于再生虎克姜花植株的系列培养试剂盒，包括种子萌发培养基、愈伤组织诱导培养基、愈伤组织增殖培养基和愈伤组织再分化培养基；其中所述种子萌发培养基包括MS培养基、蔗糖和琼脂；所述愈伤组织诱导培养基包括MS培养基、激动素、2,4-二氯苯氧乙酸、蔗糖和琼脂；所述愈伤组织增殖培养基包括MS培养基、激动素、2,4-二氯苯氧乙酸、蔗糖和琼脂；所述愈伤组织再分化培养基包括MS培养基、6-苄氨基嘌呤、吲哚-3-乙酸、蔗糖和琼脂。In the first aspect, the present invention provides a series of culture kits for regenerating Huke ginger flower plants, including seed germination medium, callus induction medium, callus proliferation medium and callus redifferentiation culture. wherein the seed germination medium includes MS medium, sucrose and agar; the callus induction medium includes MS medium, kinetin, 2,4-dichlorophenoxyacetic acid, sucrose and agar; the The callus proliferation medium includes MS medium, kinetin, 2,4-dichlorophenoxyacetic acid, sucrose and agar; the callus redifferentiation medium includes MS medium, 6-benzylaminopurine, indole -3-acetic acid, sucrose and agar.
所述愈伤组织诱导培养基和所述愈伤组织增殖培养基的成分相同，但成分中激动素和2,4-二氯苯氧乙酸的使用浓度不同。本发明所述培养试剂盒，制备方法简单，不需添加复杂的培养物便能诱导愈伤组织分化完成，无需在固体和液体培养之间来回转换。The composition of the callus induction medium and the callus proliferation medium are the same, but the concentrations of kinetin and 2,4-dichlorophenoxyacetic acid in the composition are different. The culture kit of the invention has a simple preparation method, can induce callus differentiation without adding complex culture, and does not need to switch back and forth between solid and liquid culture.
优选地，所述种子萌发培养基中，蔗糖的浓度为28～30g/L，琼脂的浓度为7.0～8.0g/L。Preferably, in the seed germination medium, the concentration of sucrose is 28-30 g/L, and the concentration of agar is 7.0-8.0 g/L.
优选地，所述愈伤组织诱导培养基中，激动素的浓度为0.1～0.5mg/L，2,4-二氯苯氧乙酸的浓度为0.5～1.0mg/L，蔗糖的浓度为28～30g/L，琼脂的浓度为7.0～8.0g/L。Preferably, in the callus induction medium, the concentration of kinetin is 0.1-0.5 mg/L, the concentration of 2,4-dichlorophenoxyacetic acid is 0.5-1.0 mg/L, and the concentration of sucrose is 28-28 mg/L. 30g/L, the concentration of agar is 7.0～8.0g/L.
下胚轴在不含任何激素的诱导培养基里不能诱导出愈伤组织，在愈伤组织诱导培养基的诱导下，可长出生长快，松散性好的愈伤组织。Hypocotyls could not induce callus in the induction medium without any hormones, but under the induction of callus induction medium, callus with fast growth and good looseness could grow.
优选地，所述愈伤组织增殖培养基中，激动素的浓度为0.1～0.2mg/L，2,4-二氯苯氧乙酸的浓度为0.3～0.5mg/L，蔗糖的浓度为28～30g/L，琼脂的浓度为7.0～8.0g/L。Preferably, in the callus proliferation medium, the concentration of kinetin is 0.1-0.2 mg/L, the concentration of 2,4-dichlorophenoxyacetic acid is 0.3-0.5 mg/L, and the concentration of sucrose is 28-28 mg/L. 30g/L, the concentration of agar is 7.0～8.0g/L.
愈伤组织增殖培养基可以使愈伤组织快速增殖，得到初始接种量60倍以上的愈伤组织。The callus proliferation medium can make callus proliferate rapidly, and obtain callus with more than 60 times the initial inoculation amount.
优选地，所述愈伤组织再分化培养基中，6-苄氨基嘌呤的浓度为1.0～2.0mg/L，吲哚-3-乙酸的浓度为0.1～2.0mg/L，蔗糖的浓度为28～30g/L，琼脂的浓度为7.0～8.0g/L。Preferably, in the callus redifferentiation medium, the concentration of 6-benzylaminopurine is 1.0-2.0 mg/L, the concentration of indole-3-acetic acid is 0.1-2.0 mg/L, and the concentration of sucrose is 28 ~30g/L, the concentration of agar is 7.0~8.0g/L.
经愈伤组织再分化培养基培养后，愈伤组织块转化为无菌苗丛，可直接炼苗移栽。After being cultured in the callus redifferentiation medium, the callus pieces are transformed into sterile seedlings, which can be directly hardened and transplanted.
第二方面，本发明提供了上述培养基在建立再生虎克姜花植株的中的应用。In the second aspect, the present invention provides the application of the above-mentioned culture medium in the establishment of a regenerated Hooke ginger flower plant.
本发明提供了上述再生虎克姜花植株的制备方法，包括以下步骤：The invention provides the preparation method of the above-mentioned regenerated Huke ginger flower plant, comprising the following steps:
(1)选取饱满、无损伤、未开裂的果实作为外植体；(1) Select plump, non-damaged, uncracked fruits as explants;
(2)在无菌条件下，对选取的果实进行消毒，无菌水冲洗后用无菌操作接种种子到种子萌发培养基，得到无菌苗；(2) under sterile conditions, the selected fruit is sterilized, and after rinsing with sterile water, the seed is inoculated into the seed germination medium with aseptic operation to obtain sterile seedlings;
(3)当步骤(2)获得的无菌苗龄为7天以上时，将无菌苗的下胚轴切为0.3～0.5cm长的1～2段，接种到愈伤组织诱导培养基，培养30天以上，得到愈伤组织；(3) when the sterile seedling age obtained in step (2) is more than 7 days, the hypocotyl of the sterile seedling is cut into 1 to 2 sections with a length of 0.3 to 0.5 cm, and inoculated into the callus induction medium, Cultured for more than 30 days to obtain callus;
(4)将步骤(3)得到的愈伤组织分成块接种到愈伤组织增殖培养基上，以月为培养周期，继代3次，得到初始接种量60倍以上的愈伤组织；(4) dividing the callus obtained in step (3) into blocks and inoculating on the callus proliferation medium, taking a month as a culture period, and substituting 3 times to obtain callus with an initial inoculation amount more than 60 times;
(5)将步骤(4)得到的愈伤组织切块接种到愈伤组织再分化培养基上，培养得到无菌苗；(5) inoculating the callus cut pieces obtained in step (4) on the callus redifferentiation medium, and culturing to obtain sterile seedlings;
以下胚轴为外植体得到的愈伤组织再生效率高，不需壮苗和生根步骤，一步成苗。The callus obtained by taking the hypocotyl as an explant has high regeneration efficiency, and does not require the steps of seedling strengthening and rooting, and the seedling is formed in one step.
(6)将步骤(5)得到的无菌苗放置在有散射光的地方炼苗3～4天，清洗后移栽到珍珠岩和泥炭土等比混合基质中，观察成活率。(6) The sterile seedlings obtained in step (5) are placed in a place with scattered light for 3 to 4 days, and after cleaning, they are transplanted into a mixed matrix of perlite and peat soil in equal proportions, and the survival rate is observed.
以下胚轴为外植体污染率低，无菌效率高。由于种子来源于胚珠，从发育之初从未与外界接触，天然无菌，因而果实消毒后可直接接种，污染率为零，消毒操作步骤也更简便，同时愈伤组织诱导率高、诱导速度更快。The hypocotyl is the explant with low contamination rate and high sterilization efficiency. Since the seeds are derived from ovules, they have never been in contact with the outside world from the beginning of development, and are naturally sterile. Therefore, the fruits can be directly inoculated after disinfection, with zero contamination rate, and the disinfection operation steps are simpler. At the same time, the callus induction rate is high and the induction speed is high. faster.
优选地，所述步骤(2)的培养条件为黑暗培养，温度为24～26℃。Preferably, the culture condition of the step (2) is dark culture, and the temperature is 24-26°C.
优选地，所述步骤(3)～(6)的培养条件为温度24～26℃、光照12h/d、光照强度2500lx。Preferably, the culture conditions of the steps (3) to (6) are a temperature of 24 to 26° C., an illumination of 12 h/d, and an illumination intensity of 2500 lx.
与现有技术相比，本发明的有益效果为：Compared with the prior art, the beneficial effects of the present invention are:
1.外植体污染率低、无菌效率高。由于消毒时种子有果壳保护， 使用了比普通消毒处理更长的时间，消毒彻底。而种子来源于胚珠，从发育之初从未与外界接触，天然无菌，因而果实消毒后可直接接种，污染率为零，消毒操作步骤也简化了。1. The explant contamination rate is low and the aseptic efficiency is high. Since the seeds are protected by the husk during disinfection, it takes longer than ordinary disinfection treatment, and the disinfection is thorough. The seeds are derived from ovules and have never been in contact with the outside world from the beginning of their development. They are naturally sterile, so the fruits can be directly inoculated after disinfection, the pollution rate is zero, and the disinfection operation steps are also simplified.
2.愈伤组织诱导率高、诱导速度更快。下胚轴接种后30天时诱导率为100％，而叶片、花丝和花药愈伤组织的诱导需要60～90天、诱导率为70％。本发明诱导出的愈伤组织为淡黄色、疏松、***旺盛的高质量愈伤组织；而由叶片、花丝和花药诱导出的愈伤组织常常需要经历2～3代的继代培养，才能筛选到均匀的、***旺盛的愈伤组织。2. The callus induction rate is high and the induction speed is faster. The induction rate was 100% at 30 days after hypocotyl inoculation, while the induction of leaves, filaments and anther callus required 60-90 days and the induction rate was 70%. The callus induced by the present invention is light yellow, loose and vigorously divided high-quality callus; while the callus induced by leaves, filaments and anthers often needs to undergo 2-3 generations of subculture before screening. to uniform, vigorously dividing callus.
3.愈伤组织的诱导及植株再分化不需要额外添加繁复的培养物，操作步骤简单，无需在固体和液体培养之间来回转换。3. Callus induction and plant re-differentiation do not require additional complicated culture, the operation steps are simple, and there is no need to switch back and forth between solid and liquid culture.
4.再生效率高，不需壮苗和生根步骤，一步成苗。将愈伤组织切为8mm大小的愈伤组织块转接至分化培养基，培养120天后，愈伤组织块转化为无菌苗丛，可分化出10～15株带3～4片叶且已生根的植株，可直接炼苗移栽。4. The regeneration efficiency is high, and the seedlings are grown in one step without the steps of strengthening and rooting. The callus was cut into 8 mm-sized callus blocks and transferred to the differentiation medium. After culturing for 120 days, the callus blocks were transformed into sterile seedlings, and 10 to 15 plants with 3 to 4 leaves were differentiated. Rooted plants can be directly hardened and transplanted.
图1为本发明诱导出的愈伤组织经愈伤组织增殖培养基培养30天后的实物图。Figure 1 is a physical picture of the callus induced by the present invention after being cultured in a callus proliferation medium for 30 days.
图2为本发明诱导出的愈伤组织经6-苄氨基嘌呤为2.0mg/L，吲哚-3-乙酸为1.0mg/L的愈伤组织再分化培养基培养120天后植株的再生情况实物图。Figure 2 shows the regeneration of the callus induced by the present invention after culturing for 120 days on the callus redifferentiation medium with 6-benzylaminopurine of 2.0 mg/L and indole-3-acetic acid of 1.0 mg/L picture.
为更好地说明本发明的目的、技术方案和优点，下面将结合具体实施例对本发明作进一步说明。In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below with reference to specific embodiments.
目前，姜花属植物的愈伤组织再生体系主要以叶片、花丝、花丝为外植体、无菌处理效率低，在愈伤组织诱导阶段培养基的添加物繁复、操作步骤冗长，愈伤组织再生为植株的效率低。为了克服此缺陷，本发明公开了一种以下胚轴为外植体、简单而高效的诱导虎克姜花愈伤组织发生及再生为植株的方法。At present, the callus regeneration system of Ginger is mainly based on leaves, filaments and filaments as explants, and the efficiency of aseptic treatment is low. Regeneration into plants is inefficient. In order to overcome this defect, the present invention discloses a simple and efficient method for inducing callus formation and regeneration of Huke ginger flower into plants by taking hypocotyl as explant.
实施例1Example 1
本发明用于再生虎克姜花植株的系列培养试剂盒及其应用的一种实施例，本实施例用于再生虎克姜花植株的系列培养试剂盒，包括种子萌发培养基、愈伤组织诱导培养基、愈伤组织增殖培养基和愈伤组织再分化培养基。The present invention is an embodiment of a series of culture kits for regenerating Huke ginger flower plants and its application. The present embodiment is used for a series of culture kits for regenerating Huke ginger flower plants, including seed germination medium, callus tissue Induction medium, callus proliferation medium and callus redifferentiation medium.
所述种子萌发培养基中，蔗糖的浓度为28g/L，琼脂的浓度为7g/L；所述愈伤组织诱导培养基中，激动素的浓度为0.1mg/L，2,4-二氯苯氧乙酸的浓度为0.5mg/L，蔗糖的浓度为28g/L，琼脂的浓度为7g/L；所述愈伤组织增殖培养基中，激动素的浓度为0.1mg/L，2,4-二氯苯氧乙酸的浓度为0.3mg/L，蔗糖的浓度为28g/L，琼脂的浓度为7g/L；所述愈伤组织再分化培养基中，6-苄氨基嘌呤的浓度为1.0mg/L，吲哚-3-乙酸的浓度为0.1mg/L，蔗糖的浓度为28g/L，琼脂的浓度为7.0g/L。In the seed germination medium, the concentration of sucrose is 28 g/L, and the concentration of agar is 7 g/L; in the callus induction medium, the concentration of kinetin is 0.1 mg/L, 2,4-dichloro The concentration of phenoxyacetic acid is 0.5mg/L, the concentration of sucrose is 28g/L, and the concentration of agar is 7g/L; in the callus proliferation medium, the concentration of kinetin is 0.1mg/L, 2,4 - The concentration of dichlorophenoxyacetic acid is 0.3 mg/L, the concentration of sucrose is 28 g/L, and the concentration of agar is 7 g/L; in the callus redifferentiation medium, the concentration of 6-benzylaminopurine is 1.0 mg/L, the concentration of indole-3-acetic acid was 0.1 mg/L, the concentration of sucrose was 28 g/L, and the concentration of agar was 7.0 g/L.
本实施例所述再生虎克姜花植株的制备方法，包括以下步骤：The preparation method of the regenerated Hooke ginger flower plant described in the present embodiment comprises the following steps:
(1)挑选饱满、无损伤、未开裂的果实做为外植体；(1) Select plump, non-damaged, uncracked fruits as explants;
(2)在无菌条件下，于超净工作台上用75％酒精溶液对未开裂的果实浸泡消毒1～2分钟，再用0.1％升汞溶液浸泡15～20分钟，无菌水冲洗6次。用消毒好的镊子和刀片剖去果实外壳，把种子接种到种子萌发培养基；(2) Under aseptic conditions, soak and disinfect the uncracked fruit with 75% alcohol solution on the ultra-clean workbench for 1 to 2 minutes, then soak in 0.1% mercuric solution for 15 to 20 minutes, and rinse with sterile water for 6 Second-rate. Cut off the fruit shell with sterilized tweezers and blades, and inoculate the seeds into the seed germination medium;
(3)当步骤(2)获得的无菌苗龄为7天时，将无菌苗的下胚轴切为0.3～0.5cm长的1～2段，接种到愈伤组织诱导培养基，培养30天；(3) when the sterile seedling age obtained in step (2) is 7 days, the hypocotyl of the sterile seedling is cut into 1-2 sections with a length of 0.3-0.5 cm, inoculated into the callus induction medium, and cultured for 30 sky;
(4)将步骤(3)得到的愈伤组织分成5mm大小的愈伤组织块接种到愈伤组织增殖培养基上，以30天为培养周期，继代3次，愈伤组织快速增殖，获得初始接种量60倍的愈伤组织；(4) dividing the callus obtained in step (3) into 5 mm-sized callus pieces and inoculating them on the callus proliferation medium, taking 30 days as the culture period, and subculture 3 times, the callus proliferates rapidly, and obtains Callus with 60 times the initial inoculation;
(5)将步骤(4)得到的愈伤组织切为8mm大小的愈伤组织块接种到愈伤组织再分化培养基上，培养120天，获得带3～4片叶、带根的完整无菌苗；(5) The callus obtained in step (4) was cut into 8 mm-sized callus blocks and inoculated on the callus redifferentiation medium, and cultured for 120 days to obtain complete 3-4 leaves and roots. vaccine;
(6)将步骤(5)得到的无菌苗放置在有散射光的地方炼苗3～4 天，然后将瓶苗瓶盖打开，小心取出瓶苗，洗净附着在根上的培养基，移栽到用l％高锰酸钾溶液消毒过的珍珠岩和泥炭土(1：1)的混合基质中，淋足定根水，每天喷清水薄雾保湿，每周喷一次稀释5倍的MS大量元素的营养液，一个月后观察成活率。(6) Place the sterile seedlings obtained in step (5) in a place with scattered light for 3 to 4 days, then open the bottle cap of the bottle seedlings, carefully remove the bottle seedlings, wash the medium attached to the roots, remove the Planted in a mixed matrix of perlite and peat soil (1:1) sterilized with 1% potassium permanganate solution, drenched in enough root-fixing water, sprayed with water and mist every day for moisturizing, and sprayed MS diluted 5 times once a week Nutrient solution with a large number of elements, the survival rate was observed after one month.
实施例2Example 2
本发明用于再生虎克姜花植株的系列培养试剂盒及其应用的一种实施例，本实施例用于再生虎克姜花植株的系列培养试剂盒，包括种子萌发培养基、愈伤组织诱导培养基、愈伤组织增殖培养基和愈伤组织再分化培养基。The present invention is an embodiment of a series of culture kits for regenerating Huke ginger flower plants and its application. The present embodiment is used for a series of culture kits for regenerating Huke ginger flower plants, including seed germination medium, callus tissue Induction medium, callus proliferation medium and callus redifferentiation medium.
所述种子萌发培养基中，蔗糖的浓度为29g/L，琼脂的浓度为7.5g/L；所述愈伤组织诱导培养基中，激动素的浓度为0.3mg/L，2,4-二氯苯氧乙酸的浓度为0.8mg/L，蔗糖的浓度为29g/L，琼脂的浓度为7.5g/L；所述愈伤组织增殖培养基中，激动素的浓度为0.15mg/L，2,4-二氯苯氧乙酸的浓度为0.4mg/L，蔗糖的浓度为29g/L，琼脂的浓度为7.5g/L；所述愈伤组织再分化培养基中，6-苄氨基嘌呤的浓度为1.5mg/L，吲哚-3-乙酸的浓度为1.0mg/L，蔗糖的浓度为29g/L，琼脂的浓度为7.5g/L。In the seed germination medium, the concentration of sucrose is 29 g/L, and the concentration of agar is 7.5 g/L; in the callus induction medium, the concentration of kinetin is 0.3 mg/L, 2,4-di The concentration of chlorophenoxyacetic acid was 0.8 mg/L, the concentration of sucrose was 29 g/L, and the concentration of agar was 7.5 g/L; in the callus proliferation medium, the concentration of kinetin was 0.15 mg/L, 2 , The concentration of 4-dichlorophenoxyacetic acid was 0.4 mg/L, the concentration of sucrose was 29 g/L, and the concentration of agar was 7.5 g/L; The concentration was 1.5 mg/L, the concentration of indole-3-acetic acid was 1.0 mg/L, the concentration of sucrose was 29 g/L, and the concentration of agar was 7.5 g/L.
本实施例所述再生虎克姜花植株的制备方法同实施例1。The preparation method of the regenerated Hooke ginger flower plant described in this example is the same as that in Example 1.
实施例3Example 3
本发明用于再生虎克姜花植株的系列培养试剂盒及其应用的一种实施例，本实施例用于再生虎克姜花植株的系列培养试剂盒，包括种子萌发培养基、愈伤组织诱导培养基、愈伤组织增殖培养基和愈伤组织再分化培养基。The present invention is an embodiment of a series of culture kits for regenerating Huke ginger flower plants and its application. The present embodiment is used for a series of culture kits for regenerating Huke ginger flower plants, including seed germination medium, callus tissue Induction medium, callus proliferation medium and callus redifferentiation medium.
所述种子萌发培养基中，蔗糖的浓度为30g/L，琼脂的浓度为8g/L；所述愈伤组织诱导培养基中，激动素的浓度为0.5mg/L，2,4-二氯苯氧乙酸的浓度为1.0mg/L，蔗糖的浓度为30g/L，琼脂的浓度为8g/L；所述愈伤组织增殖培养基中，激动素的浓度为0.2mg/L，2,4-二氯苯氧乙酸的浓度为0.5mg/L，蔗糖的浓度为30g/L，琼脂的浓度为8g/L；所述愈伤组织再分化培养基中，6-苄氨基嘌呤的浓度为2.0mg/L，吲 哚-3-乙酸的浓度为2.0mg/L，蔗糖的浓度为30g/L，琼脂的浓度为8.0g/L。In the seed germination medium, the concentration of sucrose is 30 g/L, and the concentration of agar is 8 g/L; in the callus induction medium, the concentration of kinetin is 0.5 mg/L, 2,4-dichloro The concentration of phenoxyacetic acid is 1.0mg/L, the concentration of sucrose is 30g/L, and the concentration of agar is 8g/L; in the callus proliferation medium, the concentration of kinetin is 0.2mg/L, 2,4 - the concentration of dichlorophenoxyacetic acid is 0.5mg/L, the concentration of sucrose is 30g/L, and the concentration of agar is 8g/L; in the callus redifferentiation medium, the concentration of 6-benzylaminopurine is 2.0 mg/L, the concentration of indole-3-acetic acid was 2.0 mg/L, the concentration of sucrose was 30 g/L, and the concentration of agar was 8.0 g/L.
本实施例所述再生虎克姜花植株的制备方法同实施例1。The preparation method of the regenerated Hooke ginger flower plant described in this example is the same as that in Example 1.
对比例1Comparative Example 1
本发明用于再生虎克姜花植株的系列培养试剂盒及其应用的一种对比例，本对比例所述不同外植体无菌处理效率的比较：A kind of comparative example of the present invention for the serial culture kit for regenerating Hooke ginger flower plants and its application, the comparison of the aseptic processing efficiency of different explants described in this comparative example:
(1)本发明外植体无菌处理：挑选饱满、无损伤、未开裂的果实，在无菌条件下，依次用酒精、0.1％升汞溶液浸泡消毒，无菌水冲洗后用无菌操作接种种子到种子萌发培养基，接种90粒。(1) aseptic treatment of explants of the present invention: select plump, non-damaged, uncracked fruits, under aseptic conditions, soak and sterilize with alcohol and 0.1% mercuric solution successively, rinse with sterile water and use aseptic operation Seeds were inoculated into seed germination medium, and 90 seeds were inoculated.
(2)对照试验：以叶片、种子、嫩茎为外植体。依次用酒精、0.1％升汞溶液浸泡消毒，无菌水冲洗。每个处理接种90个外植体(表1)。(2) Control experiment: leaves, seeds and tender stems were used as explants. Soak and disinfect with alcohol and 0.1% mercuric solution in turn, and rinse with sterile water. Ninety explants were inoculated per treatment (Table 1).
表1：不同外植体无菌处理效率比较Table 1: Comparison of aseptic processing efficiency of different explants
结果分析：0.1％升汞溶液消毒处理15～20分钟，未开裂的果实无菌率达到100％，随着升汞溶液处理时间增加，叶片、嫩茎和种子的无菌率虽有上升，但仍不能达到满意的无菌率。与此同时，叶片、嫩茎和种子的死亡率在不断增加，即使再延长0.1％升汞溶液处理时间也无法获得较高的无菌率。Result analysis: 0.1% mercuric chloride solution was disinfected for 15 to 20 minutes, and the sterility rate of uncracked fruit reached 100%. Still can not achieve satisfactory sterility. At the same time, the mortality of leaves, tender stems and seeds was increasing, and even if the treatment time of 0.1% mercuric solution was prolonged, a higher sterility rate could not be obtained.
以种子为外植体，在培养至3～4周时会从萌发孔长出霉菌，导致污染，原因是藏于种子内部的微生物难以被杀灭。以叶片为外植体不仅污染重，褐化也严重。以嫩茎为外植体，因其质地含水量高且暴露于土壤和空气中，污染较难去除，故污染率也高。以开裂的果实为外植体进行消毒，由于消毒时未开裂果实时有果壳保护，长时间消毒果实不会把里面种子消毒致死，且因种子发育于胚珠，从发育之初从未与外界接触，天然无菌，污染率为零，无菌处理效率非常高。Using seeds as explants, mold will grow from the germination hole when cultured for 3 to 4 weeks, resulting in contamination, because the microorganisms hidden inside the seeds are difficult to kill. Using leaves as explants is not only serious pollution, but also serious browning. Taking tender stems as explants, because of its high water content and exposure to soil and air, it is difficult to remove pollution, so the pollution rate is also high. The cracked fruit is used as the explant for disinfection. Since the uncracked fruit is protected by the husk during disinfection, long-term disinfection of the fruit will not kill the seeds inside, and because the seeds develop in the ovule, they have never communicated with the outside world from the beginning of development. Contact, natural sterility, zero contamination rate, very high efficiency of aseptic processing.
对比例2Comparative Example 2
本发明用于再生虎克姜花植株的系列培养试剂盒及其应用的一种对比例，本对比例所述不同浓度的激动素、2,4-二氯苯氧乙酸对虎克姜花下胚轴愈伤组织诱导的影响方法为：A comparative example of a series of culturing kits for regenerating Huke ginger flower plants and an application thereof of the present invention, the different concentrations of kinetin and 2,4-dichlorophenoxyacetic acid described in this comparative example are effective for Huke ginger flower. The effect of hypocotyl callus induction is as follows:
以未开裂的果实进行消毒处理，再无菌条件下取果实内的无菌种子接种于种子萌发培养基，待种子萌发后取下胚轴为外植体诱导愈伤组织并进行增殖，步骤如下：Sterilize the uncracked fruit, then take the sterile seeds in the fruit under aseptic conditions and inoculate it in the seed germination medium, and after the seeds germinate, remove the hypocotyl as an explant to induce callus and proliferate, the steps are as follows :
(1)果实的消毒：挑选饱满、无损伤、未开裂的果实，在无菌条件下，依次用酒精、0.1％升汞溶液浸泡消毒，无菌水冲洗后用无菌操作接种种子到种子萌发培养基，接种90粒。(1) Disinfection of fruits: select plump, non-damaged, and uncracked fruits, soak and disinfect with alcohol and 0.1% mercury chloride solution in order under aseptic conditions, rinse with sterile water, inoculate seeds with aseptic operations until the seeds germinate culture medium, inoculated with 90 grains.
(2)诱导愈伤组织处理：当无菌苗龄为7天时，将无菌苗的下 胚轴切为0.3～0.5cm长的1～2段，接种到愈伤组织诱导培养基，接种30瓶，黑暗培养30天，结果如表2所示。(2) Induced callus treatment: when the sterile seedling age is 7 days, the hypocotyl of the sterile seedling is cut into 1-2 segments of 0.3-0.5 cm long, inoculated into the callus induction medium, and inoculated with 30 The flasks were cultured in the dark for 30 days, and the results are shown in Table 2.
表2不同浓度的激动素、2,4-二氯苯氧乙酸对虎克姜花下胚轴愈Table 2 Different concentrations of kinetin and 2,4-dichlorophenoxyacetic acid heal the hypocotyl of Huke ginger flower
伤组织诱导的影响Tissue-induced effects
注：+，愈伤组织含水量大、致密，生长缓慢；++，愈伤组织生长速度中等、松散性较好；+++，愈伤组织生长快、松散性好；-，没有愈伤组织产生。Note: +, callus with high water content, dense and slow growth; ++, callus with medium growth rate and good looseness; +++, callus with fast growth and good looseness; -, no callus Organization produces.
结果分析：当下胚轴在不含任何激素的诱导培养基里不能诱导出愈伤组织，从结果可知激动素、2,4-二氯苯氧乙酸的范围分别在0.1～0.5mg/L和0.5～1.0mg/L时下胚轴都能诱导出愈伤组织，当激动素为0.5mg/L，2,4-二氯苯氧乙酸为1.0mg/L时愈伤组织诱导率可达100％，愈伤组织生长情况是最好的，愈伤组织生长快，松散性好。Analysis of the results: The hypocotyls could not induce callus in the induction medium without any hormones. The results showed that the ranges of kinetin and 2,4-dichlorophenoxyacetic acid were 0.1～0.5mg/L and 0.5mg/L, respectively. ～1.0mg/L hypocotyl can induce callus, when kinetin is 0.5mg/L, 2,4-dichlorophenoxyacetic acid is 1.0mg/L, callus induction rate can reach 100%, The growth of the callus is the best, the callus grows fast, and the looseness is good.
(3)愈伤组织的增殖：把得到的愈伤组织切成5mm大的愈伤组织块接种到愈伤组织增殖培养基上，以30天为培养周期，请参阅图1，继代3次，愈伤组织增殖60倍。(3) Proliferation of callus: cut the obtained callus into 5mm large callus pieces and inoculate them on the callus proliferation medium, take 30 days as the culture period, see Figure 1, and subculture 3 times , the callus proliferated 60 times.
对比例3Comparative Example 3
本发明用于再生虎克姜花植株的系列培养试剂盒及其应用的一 种对比例，本对比例所述6-苄氨基嘌呤和吲哚-3-乙酸对虎克姜花愈伤组织再分化的影响方法为：The invention is a comparative example of a series of culture kits for regenerating Huke ginger flower plants and an application thereof. The 6-benzylaminopurine and indole-3-acetic acid described in this comparative example are effective for the redifferentiation of Huke ginger flower callus. The method of influence is:
(1)愈伤组织的再分化：把愈伤组织切成8mm×8mm大的愈伤组织块接种到愈伤组织再分化培养基上，1瓶接种5个组织块，接种30瓶。结果如表3所示(1) Redifferentiation of callus: The callus was cut into 8mm×8mm large callus pieces and inoculated on the callus redifferentiation medium. One bottle was inoculated with 5 tissue pieces, and 30 bottles were inoculated. The results are shown in Table 3
表3：6-苄氨基嘌呤(BA)和吲哚-3-乙酸(IAA)对虎克姜花愈伤组织再分化的影响Table 3: Effects of 6-benzylaminopurine (BA) and indole-3-acetic acid (IAA) on redifferentiation of Huke ginger flower callus
注：培养时间120天Note: The culture time is 120 days
结果分析：培养于6-苄氨基嘌呤2.0mg/L，吲哚-3-乙酸1.0mg/L时愈伤组织的再分化效果最好，120天后一块愈伤组织分化出15株带根苗，请参阅图2。结随着吲哚-3-乙酸浓度的增加，出苗数上升，但吲哚-3-乙酸超过1.0mg/L时出苗数下降。原因是较高的吲哚-3-乙酸趋向于诱导植株基部膨大，植株高度降低，根的数量减少。Result analysis: The redifferentiation effect of callus was the best when cultured in 6-benzylaminopurine 2.0mg/L and indole-3-acetic acid 1.0mg/L. After 120 days, one callus differentiated into 15 rooted shoots. See Figure 2. As the concentration of indole-3-acetic acid increased, the number of seedlings increased, but the number of seedlings decreased when indole-3-acetic acid exceeded 1.0 mg/L. The reason is that higher indole-3-acetic acid tends to induce swelling of the plant base, lower plant height, and reduced number of roots.
(2)苗的移栽培养：将在愈伤组织再分化培养基培养了120天的瓶苗放置在有散射光的地方炼苗3～4天，然后洗净附着在根上的培养基，把苗丛分株移栽到用1％高锰酸钾溶液消毒过的珍珠岩和泥炭土等比例的混合基质中，淋足定根水，每天喷清水薄雾保湿，每周喷一次稀释5倍的MS大量元素的营养液，一个月后观察成活率，成活率达90％。(2) Transplantation and culture of seedlings: The bottle seedlings that have been cultured in the callus redifferentiation medium for 120 days are placed in a place with scattered light for 3 to 4 days, and then the medium attached to the roots is washed. The ramets of the seedlings were transplanted into the mixed matrix of perlite and peat soil disinfected with 1% potassium permanganate solution, drenched in enough water to fix the roots, sprayed with water and mist every day to keep moisturizing, and sprayed once a week to dilute 5 times. The survival rate of MS macroelement nutrient solution was observed after one month, and the survival rate was 90%.
最后所应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详 细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit the protection scope of the present invention. Although the present invention is described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that, The technical solutions of the present invention may be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention.
Claims (10)
- 一种用于再生虎克姜花植株的系列培养试剂盒，其特征在于，包括种子萌发培养基、愈伤组织诱导培养基、愈伤组织增殖培养基和愈伤组织再分化培养基；A series of culture kits for regenerating Huke ginger flower plants, characterized in that it comprises a seed germination medium, a callus induction medium, a callus proliferation medium and a callus redifferentiation medium;其中所述种子萌发培养基包括MS培养基、蔗糖和琼脂；所述愈伤组织诱导培养基包括MS培养基、激动素、2,4-二氯苯氧乙酸、蔗糖和琼脂；所述愈伤组织增殖培养基包括MS培养基、激动素、2,4-二氯苯氧乙酸、蔗糖和琼脂；所述愈伤组织再分化培养基包括MS培养基、6-苄氨基嘌呤、吲哚-3-乙酸、蔗糖和琼脂。Wherein the seed germination medium comprises MS medium, sucrose and agar; the callus induction medium comprises MS medium, kinetin, 2,4-dichlorophenoxyacetic acid, sucrose and agar; the callus Tissue proliferation medium includes MS medium, kinetin, 2,4-dichlorophenoxyacetic acid, sucrose and agar; the callus redifferentiation medium includes MS medium, 6-benzylaminopurine, indole-3 -acetic acid, sucrose and agar.
- 根据权利要求1所述的用于再生虎克姜花植株的系列培养试剂盒，其特征在于，所述种子萌发培养基中，蔗糖的浓度为28～30g/L，琼脂的浓度为7.0～8.0g/L。The serial culture kit for regenerating Huke ginger flower plants according to claim 1, wherein, in the seed germination medium, the concentration of sucrose is 28～30g/L, and the concentration of agar is 7.0～8.0 g/L.
- 根据权利要求1所述的用于再生虎克姜花植株的系列培养试剂盒，其特征在于，所述愈伤组织诱导培养基中，激动素的浓度为0.1～0.5mg/L，2,4-二氯苯氧乙酸的浓度为0.5～1.0mg/L，蔗糖的浓度为28～30g/L，琼脂的浓度为7.0～8.0g/L。The serial culture kit for regenerating Huke ginger flower plants according to claim 1, wherein, in the callus induction medium, the concentration of kinetin is 0.1-0.5 mg/L, 2,4 - The concentration of dichlorophenoxyacetic acid is 0.5-1.0 mg/L, the concentration of sucrose is 28-30 g/L, and the concentration of agar is 7.0-8.0 g/L.
- 根据权利要求1所述的用于再生虎克姜花植株的系列培养试剂盒，其特征在于，所述愈伤组织增殖培养基中，激动素的浓度为0.1～0.2mg/L，2,4-二氯苯氧乙酸的浓度为0.3～0.5mg/L，蔗糖的浓度为28～30g/L，琼脂的浓度为7.0～8.0g/L。The series culture kit for regenerating Huke ginger flower plants according to claim 1, wherein, in the callus proliferation medium, the concentration of kinetin is 0.1-0.2 mg/L, 2,4 - The concentration of dichlorophenoxyacetic acid is 0.3-0.5 mg/L, the concentration of sucrose is 28-30 g/L, and the concentration of agar is 7.0-8.0 g/L.
- 根据权利要求1所述的用于再生虎克姜花植株的系列培养试剂盒，其特征在于，所述愈伤组织再分化培养基中，6-苄氨基嘌呤的浓度为1.0～2.0mg/L，吲哚-3-乙酸的浓度为0.1～2.0mg/L，蔗糖的浓度为28～30g/L，琼脂的浓度为7.0～8.0g/L。The series culture kit for regenerating Huke ginger flower plants according to claim 1, characterized in that, in the callus redifferentiation medium, the concentration of 6-benzylaminopurine is 1.0-2.0 mg/L , the concentration of indole-3-acetic acid is 0.1-2.0 mg/L, the concentration of sucrose is 28-30 g/L, and the concentration of agar is 7.0-8.0 g/L.
- 权利要求1～5任一项所述培养基在建立再生虎克姜花植株中的应用。The application of the medium described in any one of claims 1 to 5 in establishing a regenerated Huke ginger flower plant.
- 采用权利要求1～5任一项所述培养基建立再生虎克姜花植株的方法，其特征在于，包括以下步骤：Adopt the described medium of any one of claim 1～5 to establish the method for regenerating Huke ginger flower plant, is characterized in that, comprises the following steps:(1)选取饱满、无损伤、未开裂的果实作为外植体；(1) Select plump, non-damaged, uncracked fruits as explants;(2)在无菌条件下，对选取的果实进行消毒，无菌水冲洗后用无菌操作接种种子到种子萌发培养基，得到无菌苗；(2) under aseptic conditions, the selected fruit is sterilized, and after rinsing with sterile water, the seed is inoculated into the seed germination medium with aseptic operation to obtain aseptic seedlings;(3)当步骤(2)获得的无菌苗龄为7天以上时，将无菌苗的下胚轴切为0.3～0.5cm长的1～2段，接种到愈伤组织诱导培养基，培养30天以上，得到愈伤组织；(3) when the sterile seedling age obtained in step (2) is more than 7 days, the hypocotyl of the sterile seedling is cut into 1-2 sections of 0.3-0.5 cm long, and inoculated into the callus induction medium, Cultured for more than 30 days to obtain callus;(4)将步骤(3)得到的愈伤组织分成块接种到愈伤组织增殖培养基上，以月为培养周期，继代3次，得到初始接种量60倍以上的愈伤组织；(4) dividing the callus obtained in step (3) into blocks and inoculating on the callus proliferation medium, taking a month as a culture period, and substituting 3 times to obtain callus with an initial inoculation amount more than 60 times;(5)将步骤(4)得到的愈伤组织切块接种到愈伤组织再分化培养基上，培养得到无菌苗；(5) inoculating the callus cut pieces obtained in step (4) on the callus redifferentiation medium, and culturing to obtain sterile seedlings;(6)将步骤(5)得到的无菌苗放置在有散射光的地方炼苗3～4天，清洗后移栽到珍珠岩和泥炭土等比混合基质中，观察成活率。(6) The sterile seedlings obtained in step (5) are placed in a place with scattered light for 3 to 4 days, and after cleaning, they are transplanted into a mixed matrix of equal proportions of perlite and peat soil, and the survival rate is observed.
- 根据权利要求7所述的建立再生虎克姜花植株的方法，其特征在于，所述步骤(2)的培养条件为黑暗培养，温度为24～26℃。The method for establishing a regenerated Hooke ginger flower plant according to claim 7, wherein the culture condition of the step (2) is dark culture, and the temperature is 24-26°C.
- 根据权利要求7所述的建立再生虎克姜花植株的方法，其特征在于，所述步骤(3)～(6)的培养条件为温度24～26℃、光照12h/d、光照强度2500lx。The method for establishing a regenerated Hooke ginger flower plant according to claim 7, wherein the culture conditions of the steps (3) to (6) are a temperature of 24 to 26° C., an illumination of 12 h/d, and an illumination intensity of 2500 lx.
- 采用权利要求7～9任一项所述方法制备而得的再生虎克姜花植株。The regenerated Hooke ginger flower plant prepared by the method of any one of claims 7 to 9.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115176706A (en) * | 2022-08-04 | 2022-10-14 | 云南中医药大学 | One-step seedling formation efficient breeding method for pinellia ternata leaves |
| CN115708481A (en) * | 2022-10-25 | 2023-02-24 | 广东省农业科学院环境园艺研究所 | Tissue culture method of dwarf ginger flower |
| CN115885855A (en) * | 2023-01-03 | 2023-04-04 | 贵州大学 | Method for establishing regeneration system by taking hypocotyl of Zikui tea tree as explant |
| CN116508650A (en) * | 2023-04-18 | 2023-08-01 | 河北科技师范学院 | Method for rapidly propagating Jidong' small yellow ginger |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111937746B (en) * | 2020-08-18 | 2022-07-01 | 仲恺农业工程学院 | Series culture kit for regenerating tiger ginger flower plants and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102845309A (en) * | 2012-09-29 | 2013-01-02 | 广东第二师范学院 | Method for efficiently regenerating plant through Hedychium coccineum Buch.-Ham somatic embryogenesis |
| CN111084104A (en) * | 2019-12-30 | 2020-05-01 | 广州普邦园林股份有限公司 | Method for rapidly propagating orange-heart ginger flowers |
| CN111937746A (en) * | 2020-08-18 | 2020-11-17 | 仲恺农业工程学院 | Series culture kit for regenerating tiger ginger flower plants and application thereof |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101983555A (en) * | 2010-08-31 | 2011-03-09 | 北京林业大学 | Method for inducing indirect somatic embryogenesis of chrysanthemum |
| CN102586250A (en) * | 2011-12-30 | 2012-07-18 | 华南农业大学 | Promoter of terpene floral scent gene Hctps1 in hedychium gardneranum and application of promoter |
| CN104170729B (en) * | 2014-09-11 | 2016-05-04 | 广东第二师范学院 | A kind of method of utilizing thin slice culture technique to obtain Bai Jianghua tetraploid plant |
| CN107896922A (en) * | 2017-11-23 | 2018-04-13 | 广西万联重工科技有限公司 | A kind of soilless culture substrate of crimsoned ginger sweetened flower cultivation and preparation method thereof |
| US20190289804A1 (en) * | 2018-03-21 | 2019-09-26 | Canopy Growth Corporation | Methods for inducing polyploidy in cannabis |
| CN109566416B (en) * | 2019-01-10 | 2022-04-19 | 西南科技大学 | Method for inducing adventitious buds to carry out rapid propagation of ginger plant seedlings |
| CN110915446B (en) * | 2019-12-26 | 2021-10-01 | 广州建筑园林股份有限公司 | Ginger flower breeding method based on leaf and stem hidden bud germination promotion |
| CN111264383B (en) * | 2020-03-30 | 2022-09-20 | 广州建筑园林股份有限公司 | Method for synchronously breeding and storing new ginger hybrid line and germplasm |
-
2020
- 2020-08-18 CN CN202010828922.7A patent/CN111937746B/en active Active
-
2021
- 2021-01-26 WO PCT/CN2021/073805 patent/WO2022037017A1/en active Application Filing
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102845309A (en) * | 2012-09-29 | 2013-01-02 | 广东第二师范学院 | Method for efficiently regenerating plant through Hedychium coccineum Buch.-Ham somatic embryogenesis |
| CN111084104A (en) * | 2019-12-30 | 2020-05-01 | 广州普邦园林股份有限公司 | Method for rapidly propagating orange-heart ginger flowers |
| CN111937746A (en) * | 2020-08-18 | 2020-11-17 | 仲恺农业工程学院 | Series culture kit for regenerating tiger ginger flower plants and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| PAN, XUEFENG ET AL.: "Leaf Culture and Regeneration of Alpinia Purpurata", TROPICAL FORESTRY, vol. 30, no. 2, 30 June 2002 (2002-06-30), XP055912522 * |
| XIAO, WANG ET AL.: "Recent Progress in Biotechnological Research on Hedychium", JOURNAL OF GUANGDONG UNIVERSITY OF EDUCATION, vol. 31, no. 5, 31 October 2011 (2011-10-31), pages 74, XP055912521 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115176706A (en) * | 2022-08-04 | 2022-10-14 | 云南中医药大学 | One-step seedling formation efficient breeding method for pinellia ternata leaves |
| CN115708481A (en) * | 2022-10-25 | 2023-02-24 | 广东省农业科学院环境园艺研究所 | Tissue culture method of dwarf ginger flower |
| CN115708481B (en) * | 2022-10-25 | 2023-07-18 | 广东省农业科学院环境园艺研究所 | Tissue culture method of dwarf ginger flowers |
| CN115885855A (en) * | 2023-01-03 | 2023-04-04 | 贵州大学 | Method for establishing regeneration system by taking hypocotyl of Zikui tea tree as explant |
| CN115885855B (en) * | 2023-01-03 | 2023-12-01 | 贵州大学 | Method for establishing regeneration system by taking hypocotyl of tea tree kui as explant |
| CN116508650A (en) * | 2023-04-18 | 2023-08-01 | 河北科技师范学院 | Method for rapidly propagating Jidong' small yellow ginger |
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