CN102388805B - Spathiphyllum somatic embryo inducing and plant regenerating method - Google Patents
Spathiphyllum somatic embryo inducing and plant regenerating method Download PDFInfo
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- CN102388805B CN102388805B CN 201110267084 CN201110267084A CN102388805B CN 102388805 B CN102388805 B CN 102388805B CN 201110267084 CN201110267084 CN 201110267084 CN 201110267084 A CN201110267084 A CN 201110267084A CN 102388805 B CN102388805 B CN 102388805B
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Abstract
The invention provides a spathiphyllum somatic embryo inducing and plant regenerating method, which relates to a somatic embryo inducing and plant regenerating method and comprises the following steps of: 1, taking young and tender spathiphyllum test tube seedlings, cutting blades or petioles into small sections to be induced, and obtaining somatic embryos; 2, subculturing the somatic embryos into a somatic embryo multiplication culture medium for mass multiplication; and 3, transferring the somatic embryos onto the plant regeneration culture medium for culture to obtain complete small plants. The method has the advantages that the somatic embryo inducing rate reaches 97 percent to 100 percent, 50 to 200 somatic embryos can be formed on the surface of an explant in the primary culture, the inducing efficiency is obviously improved, and the plant regeneration rate of the obtained somatic embryos reaches 85 to 95 percent. The method solves the problems that the existing spathiphyllum somatic embryo has low inducing efficiency, and the explant material collection is inconvenient.
Description
Technical field
The present invention relates to plant and induce the regeneration techniques field, specifically, relate to the method for a kind of Spathiphyllum kochii somatic embryo inducement and regeneration plant.
Background technology
Spathiphyllum kochii (
SpathiphyllumSchott) for claiming again the white palm or sailing right before the wind, originating in Central and South America, is important tropical ornamental plants, enjoys great popularity at European ﹠ American Market.Introduce China mainland the eighties in last century, develops the formation industry rapidly, becomes one of most important tropical ornamental plants of China.
The Spathiphyllum kochii Plant Tissue Breeding has been reported since 20 century 70s in succession to some extent, mainly utilize the explant inductions such as terminal bud and stem section to obtain Multiple Buds, mode expanding propagation quantity with adventitious buds proliferation, obtain whole plant with culture of rootage again, its objective is the realization Vitro Quick Reproduction, enlarge the individual amount of rare variety resource, satisfy the needs of breed and production.
Cao waits (1995) and Zhu Genfa (2004) quietly take the tender spathe inflorescence of children as explant, obtains somatic embryo and regeneration plant through cultivating.When Werbrouck etc. (2000), Eeckhaut T etc. (2001) and Qin Jingyuan (2005) etc. have reported and cultivated with young tender spathe inflorescence, induce from stamen filigree position to have produced somatic embryo.The culture materials of these inductor blasts is floral organ, belongs to the reproductive organs tissue.The efficient of seeing the inductor blast from report is lower, generally can only produce single on each explant or tens individual cells embryos; The collection of explant also needs during the blossom season joint, and it is larger to be subject to the restriction of growth and development of plants time limit.
At present, yet there are no from the report of the explants such as nutrition organs such as blade or petiole success inductor blast and regeneration plant at Spathiphyllum kochii.
Summary of the invention
The objective of the invention is Spathiphyllum kochii somatic embryo induction plant regeneration technology is innovated from materials and methods, provide a kind of with nutrition organs-young tender test-tube plantlet blade or petiole as explant, the method for high efficiency inductor blast and regeneration plant.
To achieve these goals, the present invention adopts following technical scheme:
The method of a kind of Spathiphyllum kochii somatic embryo inducement and regeneration plant utilizes explant material to induce, and the explant material that adopts is the Spathiphyllum kochii tender test-tube plantlet blade of children or petiole.Specifically comprise the steps:
（1）
The rear pillar somatic embryo inducement.Get the Spathiphyllum kochii platymiscium
S. cannifoliumStem section or terminal bud, sterile-processed, be seeded in the inducing clumping bud medium, obtain aseptic test-tube plantlet, then being transferred in the test-tube plantlet proliferated culture medium, is 1000 ~ 1400 lux in 24 ~ 30 ℃ of temperature, intensity of illumination, in the environment of illumination in 10 ~ 14 hours every day, every 3-4 week subculture once, test-tube plantlet is constantly bred.The tender test-tube plantlet blade of children is cut into the stick of 1 ~ 10 mm wide, or petiole is cut into 1 ~ 10 millimeter intercept, then be inoculated in the somatic embryo inducement medium, in the dark surrounds of 24 ~ 30 ℃ of temperature, cultivated for 3 ~ 6 weeks, obtain somatic embryo.
(2) propagation of somatic embryo.The somatic embryo subculture that obtains is incubated in the somatic embryo proliferated culture medium, in the dark surrounds of 24 ~ 30 ℃ of temperature, cultivated for 2 ~ 4 weeks after, somatic embryo is bred in a large number.
(3) plant regeneration.The somatic embryo that obtains is seeded in the plant regeneration medium, is 1000 ~ 1400 lux in 24 ~ 30 ℃ of temperature, intensity of illumination, and in 2 ~ 4 weeks of illumination cultivation, 10 ~ 14 hours every days, somatic embryo is sprouted, and obtains complete regenerated plant.
In said method, the described inducing clumping bud medium of step (1) is as minimal medium take the MS medium, added 3.0 milligrams 6-benzylamino adenine in per 1 liter of medium, the sucrose of α-naa of 0.2 milligram, 20 ~ 40 grams and the agar of 6 ~ 9 grams, the pH value is 5.6 ~ 6.0; The described test-tube plantlet proliferated culture medium of step (1) is as minimal medium take the MS medium, added 2.0 milligrams 6-benzylamino adenine in per 1 liter of medium, 0.2 the agar of the sucrose of the α-naa of milligram, 20 ~ 40 grams and 6 ~ 9 grams, the pH value is 5.6 ~ 6.0; The described somatic embryo inducement medium of step (1) is as minimal medium take the MS medium, also contain in per 1 liter of medium simultaneously 1.0 ~ 3.0 milligrams 6-benzylamino adenine or 0.05 ~ 0.25 milligram Thidiazuron, 0.5 ~ 2.0 milligram 2, the agar of the sucrose of 4-dichlorphenoxyacetic acid, 20 ~ 40 grams and 6 ~ 9 grams, the pH value is 5.6 ~ 6.0; The described somatic embryo proliferated culture medium of step (2) is as minimal medium take the MS medium, also contain in per 1 liter of medium simultaneously 1.0 ~ 3.0 milligrams 6-benzylamino adenine or 0.05 ~ 0.25 milligram Thidiazuron, 0.5 ~ 1.0 milligram 2, the agar of the sucrose of 4-dichlorphenoxyacetic acid, 20 ~ 40 grams and 6 ~ 9 grams, the pH value is 5.6 ~ 6.0; The described plant regeneration medium of step (3) is as minimal medium take the MS medium, also contain 0.5 ~ 1.0 milligram 6-benzylamino adenine, α-naa of 0.1 ~ 0.2 milligram or 0.1 ~ 0.2 milligram indolebutyric acid in per 1 liter of medium simultaneously, the agar of the sucrose of 20 ~ 40 grams and 6 ~ 9 grams, the pH value is 5.6 ~ 6.0; Described MS medium is 1900 mg L by concentration
-1KNO
3, concentration is 1650 mg L
-1NH
4NO
3, concentration is 170 mg L
-1KH
2PO
4, concentration is 370 mg L
-1MgSO
47H
2O, concentration is 440 mg L
-1CaCl
22H
2O, concentration is 0.83 mg L
-1KI, concentration is 6.2 mg L
-1H
3BO
3, concentration is 22.3 mg L
-1MnSO
44H
2O, concentration is 8.6 mg L
-1ZnSO7H
2O, concentration is 0.25 mg L
-1Na
2MoO
42H
2O, concentration is 0.025 mg L
-1CuSO
45H
2O, concentration is 0.025 mg L
-1CoCl
26H
2O, concentration is 37.25 mg L
-1Na
2EDTA, concentration is 27.85 mg L
-1FeSO
47H
2O, concentration is 100 mg L
-1Inositol, concentration is 2.0 mg L
-1Glycine, concentration is 0.4 mg L
-1Cobastab
1, concentration is 0.5 mg L
-1Cobastab
6, concentration is 0.5 mg L
-1Nicotinic acid form.
Compared with prior art, the present invention has obvious progress:
(1) somatic embryo inducement efficient obviously improves.Because different on organ-tissue structure and Endogenous Hormones of the floral organ of Spathiphyllum kochii and blade or petiole have caused somatic embryo inducement efficient notable difference.Although prior art report take the tender floral organ of children as explant somatic embryo inducement rate can reach 96.5% ~ 100%, form at the most tens individual cells embryos on every explant surface.The present invention is take blade or petiole as explant, and not only the somatic embryo inducement rate can reach 97% ~ 100%, and every explant surface can form nearly 50 ~ 200 individual cells embryos, induces efficient obviously to improve.
(2) explant material obtains easily.After the used test-tube plantlet of the present invention is induced acquisition by stem section or terminal bud, can be numerous by a large amount of expansions of tissue cultivation, it is very convenient to draw materials.
Embodiment
Embodiment 1: present embodiment is undertaken by following steps:
(1) somatic embryo inducement.Get the Spathiphyllum kochii platymiscium
S. cannifoliumStem section or terminal bud, sterile-processed, be seeded in the inducing clumping bud medium, obtain aseptic test-tube plantlet, then being transferred in the test-tube plantlet proliferated culture medium, is 1000 ~ 1400 lux in 24 ~ 30 ℃ of temperature, intensity of illumination, in the environment of illumination in 10 ~ 14 hours every day, every 3-4 week subculture once, test-tube plantlet is constantly bred.The tender test-tube plantlet blade of children is cut into the stick of 1 ~ 10 mm wide, is inoculated in the somatic embryo inducement medium, in the dark surrounds of 24 ~ 30 ℃ of temperature, cultivated for 2 ~ 4 weeks, obtain somatic embryo;
(2) propagation of somatic embryo: the somatic embryo subculture that obtains is incubated in the somatic embryo inducement medium, in the dark surrounds of 24 ~ 30 ℃ of temperature, cultivated for 2 ~ 4 weeks after, somatic embryo is bred in a large number;
(3) plant regeneration: the somatic embryo that obtains is seeded in the plant regeneration medium, be 1000 ~ 1400 lux in 24 ~ 30 ℃ of temperature, intensity of illumination, cultivated for 2 ~ 4 weeks in the environment of illumination in 10 ~ 14 hours every day, somatic embryo is sprouted, and obtains complete regenerated plant.
Inducing clumping bud medium in the above-mentioned steps (1) is as minimal medium take the MS medium, added 3.0 milligrams 6-benzylamino adenine in per 1 liter of medium, 0.2 the agar of the sucrose of the α-naa of milligram, 20 ~ 40 grams and 6 ~ 9 grams, the pH value is 5.6 ~ 6.0; The described test-tube plantlet proliferated culture medium of step (1) is as minimal medium take the MS medium, added 2.0 milligrams 6-benzylamino adenine in per 1 liter of medium, 0.2 the agar of the sucrose of the α-naa of milligram, 20 ~ 40 grams and 6 ~ 9 grams, the pH value is that the somatic embryo inducement medium in 5.6 ~ 6.0 steps (1) is as minimal medium take the MS medium, also contain in per 1 liter of medium simultaneously 1.0 ~ 3.0 milligrams 6-benzylamino adenine, 0.5 ~ 2.0 milligram 2, the agar of the sucrose of 4-dichlorphenoxyacetic acid, 20 ~ 40 grams and 6 ~ 9 grams, the pH value is 5.6 ~ 6.0; Somatic embryo proliferated culture medium in the step (2) is as minimal medium take the MS medium, also contain in per 1 liter of medium simultaneously 1.0 ~ 3.0 milligrams 6-benzylamino adenine, 0.5 ~ 1.0 milligram 2, the agar of the sucrose of 4-dichlorphenoxyacetic acid, 20 ~ 40 grams and 6 ~ 9 grams, the pH value is 5.6 ~ 6.0; Plant regeneration medium in the step (3) is as minimal medium take the MS medium, also contain 0.5 ~ 1.0 milligram 6-benzylamino adenine, α-naa of 0.1 ~ 0.2 milligram in per 1 liter of medium simultaneously, the agar of the sucrose of 20 ~ 40 grams and 6 ~ 9 grams, the pH value is 5.6 ~ 6.0.
Cultivate by above-mentioned condition, have 97% ~ 100% explant can induce the organizator blast, can form 50 ~ 200 individual cells embryos on the every explant surface.In the somatic embryo that obtains, have 85% ~ 95% can sprout the complete plantlet of formation.
Embodiment 2: present embodiment is cut into 1 ~ 10 millimeter intercept with the test-tube plantlet petiole in the step (1) as different from Example 1, then is inoculated in to cultivate in the somatic embryo inducement medium to obtain somatic embryo; All the other are identical with embodiment 1.
Embodiment 3: what present embodiment was different from embodiment 1 or 2 is that the middle somatic embryo inducement medium of step (1) is take the MS medium as minimal medium, and contain in per 1 liter of medium 0.05 ~ 0.25 milligram Thidiazuron, 0.5 ~ 2.0 milligram 2, the agar of the sucrose of 4-dichlorphenoxyacetic acid, 20 ~ 40 grams and 6 ~ 9 grams, the pH value is 5.6 ~ 6.0; The somatic embryo proliferated culture medium is take the MS medium as minimal medium in the step (2), and contain in per 1 liter of medium 0.05 ~ 0.25 milligram Thidiazuron, 0.5 ~ 1.0 milligram 2, the agar of the sucrose of 4-dichlorphenoxyacetic acid, 20 ~ 40 grams and 6 ~ 9 grams, the pH value is 5.6 ~ 6.0; All the other are identical with embodiment 1 or 2.
Embodiment 4: present embodiment and embodiment 1,2 or 3 different be in the step (3) the plant regeneration medium take the MS medium as minimal medium, and contain 0.5 ~ 1.0 milligram 6-benzylamino adenine, α-naa of 0.1 ~ 0.2 milligram, the sucrose of 20 ~ 40 grams and the agar of 6 ~ 9 grams in per 1 liter of medium, the pH value is 5.6 ~ 6.0; All the other and embodiment 1,2 or 3 identical.
Claims (1)
1. the method for a Spathiphyllum kochii somatic embryo inducement and regeneration plant utilizes explant material to induce, and it is characterized in that the explant material that adopts is Spathiphyllum kochii blade or petiole or its mixing;
Described method comprises the steps:
(1) somatic embryo inducement: get the tender test-tube plantlet of Spathiphyllum kochii children, blade is cut into 1 ~ 10 millimeter stick, or petiole is cut into 1 ~ 10 millimeter intercept, then be inoculated in the somatic embryo inducement medium, in the dark surrounds of 24 ~ 30 ℃ of temperature, cultivated for 3 ~ 6 weeks, obtain somatic embryo;
(2) propagation of somatic embryo: the somatic embryo subculture that obtains is incubated in the somatic embryo proliferated culture medium, in the dark surrounds of 24 ~ 30 ℃ of temperature, cultivated for 2 ~ 4 weeks after, somatic embryo is bred in a large number;
(3) plant regeneration: the somatic embryo that obtains is seeded in the plant regeneration medium, is 1000 ~ 1400 lux in 24 ~ 30 ℃ of temperature, intensity of illumination, and cultivate 2 ~ 4 weeks in the photoenvironment at 10 ~ 14 hours every day, and somatic embryo is sprouted, and obtains complete regenerated plant;
The described somatic embryo inducement medium of step (1) is take the MS medium as minimal medium, also contain in per 1 liter of medium simultaneously 1.0 ~ 3.0 milligrams 6-benzylamino adenine, 0.5 ~ 2.0 milligram 2, the agar of 4-dichlorphenoxyacetic acid, 20 ~ 40 gram sucrose and 6 ~ 9 grams, perhaps also contain in per 1 liter of medium 0.05 ~ 0.25 milligram Thidiazuron, 0.5 ~ 2.0 milligram 2, the agar of 4-dichlorphenoxyacetic acid, 20 ~ 40 gram sucrose and 6 ~ 9 grams, the pH value of medium is 5.6 ~ 6.0;
The described somatic embryo proliferated culture medium of step (2) is as minimal medium take the MS medium, also contain in per 1 liter of medium simultaneously 1.0 ~ 3.0 milligrams 6-benzylamino adenine, 0.5 ~ 1.0 milligram 2, the agar of the sucrose of 4-dichlorphenoxyacetic acid, 20 ~ 40 grams and 6 ~ 9 grams, perhaps also contain in per 1 liter of medium 0.05 ~ 0.25 milligram Thidiazuron, 0.5 ~ 1.0 milligram 2, the agar of the sucrose of 4-dichlorphenoxyacetic acid, 20 ~ 40 grams and 6 ~ 9 grams, the pH value of medium is 5.6 ~ 6.0;
The described plant regeneration medium of step (3) is take the MS medium as minimal medium, also contain simultaneously 0.5 ~ 1.0 milligram 6-benzylamino adenine, α-naa of 0.1 ~ 0.2 milligram, the sucrose of 20 ~ 40 grams and the agar of 6 ~ 9 grams in per 1 liter of medium, the pH value is 5.6 ~ 6.0.
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| CN105557525A (en) * | 2016-01-13 | 2016-05-11 | 广东省农业科学院环境园艺研究所 | Breeding method for somaclonal variation of caladium bicolor |
| CN106106181A (en) * | 2016-08-08 | 2016-11-16 | 吴子平 | A kind of tissue culture and rapid propagation method of Spathiphyllum kochii |
| CN109362567B (en) * | 2018-12-03 | 2020-06-05 | 广东省农业科学院环境园艺研究所 | Method for inducing somatic embryo and regenerating plant by using hippeastrum leaves |
| CN109892225B (en) * | 2019-03-07 | 2022-03-04 | 三明市农业科学研究院 | In-vitro preservation method for anthurium andraeanum germplasm resources |
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| CN101238795A (en) * | 2008-03-07 | 2008-08-13 | 阳光国际集团科技发展有限公司 | Artificial breeding method for spathiphyllum floribundum |
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| CN101238795A (en) * | 2008-03-07 | 2008-08-13 | 阳光国际集团科技发展有限公司 | Artificial breeding method for spathiphyllum floribundum |
Non-Patent Citations (4)
| Title |
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| 曹静.白鹤芋花序体细胞胚胎发生及植株再生的研究.《农业生物技术学报》.1995,第3卷(第3期),81-85. |
| 白鹤芋花丝体细胞胚的诱导及快繁技术;秦静远;《中国农学通报》;20050930;第21卷(第9期);67－69 * |
| 白鹤芋花序体细胞胚胎发生及植株再生的研究;曹静;《农业生物技术学报》;19950930;第3卷(第3期);81-85 * |
| 秦静远.白鹤芋花丝体细胞胚的诱导及快繁技术.《中国农学通报》.2005,第21卷(第9期),67－69. |
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