CN102388805A - Spathiphyllum somatic embryo inducing and plant regenerating method - Google Patents

Spathiphyllum somatic embryo inducing and plant regenerating method Download PDF

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CN102388805A
CN102388805A CN2011102670841A CN201110267084A CN102388805A CN 102388805 A CN102388805 A CN 102388805A CN 2011102670841 A CN2011102670841 A CN 2011102670841A CN 201110267084 A CN201110267084 A CN 201110267084A CN 102388805 A CN102388805 A CN 102388805A
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medium
somatic embryo
milligram
somatic
plant
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CN102388805B (en
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于波
刘金梅
刘晓荣
廖飞雄
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FLORICULTURE RESEARCH INSTITUTE OF GUANGDONG ACADEMY OF AGRICULTURAL SCIENCES
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FLORICULTURE RESEARCH INSTITUTE OF GUANGDONG ACADEMY OF AGRICULTURAL SCIENCES
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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

The method of a kind of Spathiphyllum kochii somatic embryo inducement and regeneration plant
 
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 the white palm again or sailing right before the wind, originating in Central and South America, is important tropical ornamental plants, enjoys great popularity in American-European 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 the seventies in 20th century in succession to some extent; Mainly utilize explant inductions such as terminal bud and stem section to obtain to grow thickly bud; With the mode expanding propagation quantity of adventitious buds proliferation, obtain whole plant with culture of rootage again, its objective is and realize exsomatizing breeding fast; Enlarge the individual amount of rare variety resource, satisfy the needs of breeding and production.
Cao waits (1995) quietly and Zhu Genfa (2004) is an explant with the tender spathe inflorescence of children, 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; Explant collection also needs joint during the blossom season, and it is bigger to receive the restriction of growth and development of plants time limit.
At present, on Spathiphyllum kochii, also do not see from the report of explants such as nutrition organs such as blade or petiole success inductor blast and regeneration plant.
Summary of the invention
The objective of the invention is Spathiphyllum kochii somatic embryo induction plant regeneration technology is innovated from material and method, 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 is adopted is 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; Being transferred to then 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, be inoculated in then 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.With the somatic embryo successive transfer culture that obtains 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 said inducing clumping bud medium of step (1) is to be minimal medium with 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 said test-tube plantlet proliferated culture medium of step (1) is to be minimal medium with 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 said somatic embryo inducement medium of step (1) is to be minimal medium with 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 said somatic embryo proliferated culture medium of step (2) is to be minimal medium with 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 said plant regeneration medium of step (3) is to be minimal medium with 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; Said 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.Though the prior art report is that explant somatic embryo inducement rate can reach 96.5% ~ 100% with the tender floral organ of children, forms tens individual cells embryos at the most on every explant surface.The present invention is an explant with blade or petiole, 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 through a large amount of expansions of tissue culture, it is very convenient to draw materials.
Embodiment
Embodiment 1: present embodiment carries out through 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; Being transferred to then 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: with the somatic embryo successive transfer culture that obtains 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; In 24 ~ 30 ℃ of temperature, intensity of illumination is 1000 ~ 1400 lux; 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 to be minimal medium with 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 said test-tube plantlet proliferated culture medium of step (1) is to be minimal medium with 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 to be minimal medium with the MS medium; Also contain 1.0 ~ 3.0 milligrams 6-benzylamino adenine, 0.5 ~ 2.0 milligram 2,4 dichlorophenoxyacetic acid, the sucrose of 20 ~ 40 grams and the agar of 6 ~ 9 grams simultaneously in per 1 liter of medium, the pH value is 5.6 ~ 6.0; Somatic embryo proliferated culture medium in the step (2) is to be minimal medium with 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 to be minimal medium with 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: what present embodiment and embodiment 1 were different is in the step (1) the test-tube plantlet petiole to be cut into 1 ~ 10 millimeter intercept, is inoculated in then 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 with embodiment 1 or 2 is that the somatic embryo inducement medium is a minimal medium with the MS medium in the step (1); 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 a minimal medium with the MS 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: what present embodiment and embodiment 1,2 or 3 were different is that the plant regeneration medium is a minimal medium with the MS medium in the step (3); 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 are identical with embodiment 1,2 or 3.

Claims (5)

1. the method for Spathiphyllum kochii somatic embryo inducement and regeneration plant utilizes explant material to induce, and it is characterized in that the explant material that is adopted is Spathiphyllum kochii blade or petiole or its mixing.
2. the method for claim 1 is characterized in that comprising 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, be inoculated in then 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: with the somatic embryo successive transfer culture that obtains 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.
3. method as claimed in claim 2; It is characterized in that; The said somatic embryo inducement medium of step (1) is for being minimal medium with the MS medium; Also contain 1.0 ~ 3.0 milligrams 6-benzylamino adenine or 0.05 ~ 0.25 milligram Thidiazuron, 0.5 ~ 2.0 milligram 2,4 dichlorophenoxyacetic acid, 20 ~ 40 gram sucrose and 6 ~ 9 gram agar simultaneously in per 1 liter of medium, the pH value is 5.6 ~ 6.0.
4. method as claimed in claim 2; It is characterized in that; The said somatic embryo proliferated culture medium of step (2) is to be minimal medium with the MS medium; Also contain 1.0 ~ 3.0 milligrams 6-benzylamino adenine or 0.05 ~ 0.25 milligram Thidiazuron, 0.5 ~ 1.0 milligram 2,4 dichlorophenoxyacetic acid, the sucrose of 20 ~ 40 grams and the agar of 6 ~ 9 grams simultaneously in per 1 liter of medium, the pH value is 5.6 ~ 6.0.
5. method as claimed in claim 2; It is characterized in that; The said plant regeneration medium of step (3) is for being minimal medium with 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.
CN 201110267084 2011-09-09 2011-09-09 Spathiphyllum somatic embryo inducing and plant regenerating method Expired - Fee Related CN102388805B (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
CN109362567A (en) * 2018-12-03 2019-02-22 广东省农业科学院环境园艺研究所 A kind of method of Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait blade somatic embryos and regeneration plant
CN109892225A (en) * 2019-03-07 2019-06-18 三明市农业科学研究院 A kind of method of white palm germ plasm resource Plantlet in vitro

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
CN109362567A (en) * 2018-12-03 2019-02-22 广东省农业科学院环境园艺研究所 A kind of method of Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait blade somatic embryos and regeneration plant
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
CN109892225A (en) * 2019-03-07 2019-06-18 三明市农业科学研究院 A kind of method of white palm germ plasm resource Plantlet in vitro
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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