CN102715088A - Coconut mature zygotic isolated culture embryos somatic embryogenisis method - Google Patents
Coconut mature zygotic isolated culture embryos somatic embryogenisis method Download PDFInfo
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Abstract
The invention belongs to the field of agricultural biotechnology, and relates to a coconut mature zygotic isolated culture embryos somatic embryogenisis method. The method comprises the steps of selecting mature coconut jelly, and taking out embryo to be inoculated pre-culture medium for conducting hidden culture so as to lead the embryo to swell; taking the swelled embryo subjected to pre-culture, cutting out the haustorium, longitudinally cutting into two halves, and leading the cut surface to face upward to be inoculated into callus induction culture medium for hidden culture, thus obtaining the embryo callus; transplanting the embryo callus to the somatic embryonal induction culture medium for 80-100 days to form somatic embryo. The method is simple in technique, induces the somatic embryogenisis by utilizing the coconut mature zygotic as explant, solves the problems that the acquisition time for explant of the somatic embryo acquired by utilizing immaturate inflorescence or young embryo and the like of the coconut jelly is limited by seasons, and provides an effective approach for deeply developing research on the genetic engineering, genetic transformation, breed improvement and the like of the coconut.
Description
Technical field
The invention belongs to agricultural biological technical field, be specifically related to a kind of coconut mature zygotic embryos cultured in vitro somatic embryo method for generation.
Background technology
Coconut (Cocos nucifera L.) belongs to Palmae cocoanut monocotyledon, is tropical woody oleiferous plants and the food energy crop that a kind of economically valuable is high, the product utilization rate is high.The most kinds of coconut are cross-pollinatd plant; Growth cycle is long, and reproduction coefficient is low, and hybrid generation's proterties is separated serious; Individual difference is bigger; The same proterties progeny population of very difficult acquisition, and really big, an easy sprouting, not storage tolerance, transportation of coconut palm, these characteristics are brought very big difficulty to germ plasm resource collection, exchange and the preservation of coconut.Utilize fixedly some merit of coconut of tissue culture technique; Then cultivate breeding; On producing, can breed coconut improved seeds such as high yield, cold-resistant, wind resistance in a large number, can make the collection of coconut germ plasm resource simultaneously and preserve easy, improve the safety that coconut germ plasm resource exchanges.
The research work of coconut tissue culture technique starts from twentieth century fifties, and culture in vitro is successively carried out with meristematic tissue, leaf explant, the coconut mature zygotic embryos of flower in countries in the world, is all succeeing in varying degrees.The end of the eighties, Philippine utilized the embryo of soft meat coconut palm fruit to organize training to have obtained success, existing plantlet of transplant land for growing field crops.The beginning of the nineties, Sri Lanka's coconut research institute group training chamber adopted immature coconut embryo to carry out tissue culture also to have obtained new progress.The mid-90, Europe and producing country's cooperation development coconut tissue culture technique tackling key problem, research project has been put into European scientific and technological development plan, and this project is subsidized by the European Community, and purpose is to deepen the technical research to the generation of coconut somatic embryo.Branton and Blake reported first can be carried out somatic embryo through prematurity inflorescence tissue and taken place; Verdeil J L etc. are explant with coconut prematurity inflorescence; Realize plant regeneration through indirect somatic embryo approach; Adkins S W etc. are explant with the coconut immature zygotic embryos; Induce callus and somatic embryo and take place, the embryo that Karunaratne S etc. utilizes 6～7 months coconut nut to cut off is the cultured in vitro material, and cultivation, callus propagation and the somatic embryo of having studied the coconut immature embryo take place.And the relevant coconut mature zygotic embryos that utilizes is the research that the explant induction somatic embryo takes place, and does not all see relevant report at present both at home and abroad.
Summary of the invention
The objective of the invention is to the deficiency of prior art and a kind of coconut mature zygotic embryos cultured in vitro somatic embryo method for generation is provided; The explant acquisition time that has solved acquisition somatic embryos such as utilizing coconut prematurity inflorescence or rataria receives the problem of season limit, for the research of aspects such as the gene engineering of carrying out coconut in a deep going way, genetic transformation, breed improvement provides an effective way.
A kind of coconut mature zygotic embryos cultured in vitro somatic embryo method for generation, the collection that comprises embryo and incubation, embryo callus in advance induce process, somatic embryo generating process, its step is following:
1, the collection of embryo and preparatory incubation
Choose the ripe coconut palm fruit at 10～11 monthly ages; The solid endosperm piece that complete embryo is arranged in the taking-up cleans the back and takes out the embryo sterilization, is inoculated in the pre-culture medium then; Place that relative moisture is 70～80%, secretly cultivated 12～17 days under the condition of 25～29 ℃ of temperature, embryo is expanded.Said pre-culture medium is with Y
3Medium is a minimal medium, and adds active carbon 2.5g/L, sucrose 60g/L.
2, embryo callus induces process
The pre-incubated embryo that expands of learning from else's experience; Its haustorium is excised also rip cutting into two; Cut side up is inoculated in the callus inducing medium, places that relative moisture is 70～80%, secretly cultivates under the condition of 25～29 ℃ of temperature and produced faint yellow granular embryo callus in 170～200 days.Said callus inducing medium is with Y
3Medium is a minimal medium, and adds 2,4-D 25mg/L, active carbon 2.5g/L, PVP 5g/L, agar 4.5g/L, sucrose 30g/L.
3, somatic embryo generating process
Embryo callus is transferred in the body embryonal induction medium, places that relative moisture is 70～80%, cultivate 80～100 days organizator blasts under the condition of 25～29 ℃ of temperature, intensity of illumination 1500～2500lx, illumination 12h/d.Said body embryonal induction medium is with Y
3Medium is a minimal medium, and adds 6-BA 10mg/L, 2,4-D 2mg/L, active carbon 2.5g/L, PVP 5g/L, agar 4.5g/L, sucrose 30g/L.
Technology of the present invention is simple; Utilize the coconut mature zygotic embryos to take place for the explant induction somatic embryo; The explant acquisition time that has solved acquisition somatic embryos such as utilizing coconut prematurity inflorescence or rataria receives the problem of season limit, for the research of aspects such as the gene engineering of carrying out coconut in a deep going way, genetic transformation, breed improvement provides an effective way.
Embodiment
Below in conjunction with embodiment, specific embodiments of the invention describes in further detail.Following examples are used to explain the present invention, but are not used for limiting scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.
Embodiment one
1, the collection of embryo and preparatory incubation
Choose 90 of the ripe coconut palm fruits at 10～11 monthly ages; Remove coconut palm fruit crust and the coconut palm fruit is laterally split with chopper; The use diameter is the solid endosperm piece that complete embryo is arranged in the card punch of 2 cm takes out; With running water flushing 1～2h, on superclean bench, take out embryo then and carry out the embryo surface sterilization, inoculate pre-culture medium (Y
3Medium+active carbon 2.5g/L+ sucrose 60g/L) in, places that relative moisture is 75%, secretly cultivated 15 days under the condition of 26 ℃ of temperature, embryo is expanded.
2, embryo callus induces process
The pre-incubated embryo that expands of learning from else's experience, with its haustorium excision and rip cutting into two, cut side up is inoculated into callus inducing medium (Y
3Medium+2; 4-D 25mg/L+ active carbon 2.5g/L+PVP 5g/L+ agar 4.5g/L+ sucrose 30g/L) in; Place that relative moisture is 70%, secretly cultivated 180 days under the condition of 27 ℃ of temperature, produce faint yellow granular embryo callus, the result sees table 1.
3, somatic embryo generating process
Get 50 of embryo callus and be transferred to body embryonal induction medium (Y
3Medium+6-BA 10mg/L+2; 4-D 2mg/L+ active carbon 2.5g/L+PVP 5g/L+ agar 4.5g/L+ sucrose 30g/L) in; Place that relative moisture is 75%, cultivate 90 days organizator blasts under the condition of 25 ℃ of temperature, intensity of illumination 2300lx, illumination 12h/d, the result sees table 2.
Embodiment two
1, the collection of embryo and preparatory incubation
Choose 90 of the ripe coconut palm fruits at 10～11 monthly ages; Remove coconut palm fruit crust and the coconut palm fruit is laterally split with chopper; The use diameter is the solid endosperm piece of complete embryo to be arranged with running water flushing 1～2h in the card punch of 2 cm takes out; On superclean bench, take out embryo then and carry out the embryo surface sterilization, inoculate pre-culture medium (Y
3Medium+active carbon 2.5g/L+ sucrose 60g/L) in, places that relative moisture is 80%, secretly cultivated 16 days under the condition of 28 ℃ of temperature, embryo is expanded.
2, embryo callus induces process
The pre-incubated embryo that expands of learning from else's experience, with its haustorium excision and rip cutting into two, cut side up is inoculated into callus inducing medium (Y
3Medium+2; 4-D 25mg/L+ active carbon 2.5g/L+PVP 5g/L+ agar 4.5g/L+ sucrose 30g/L) in; Place that relative moisture is 75%, secretly cultivated 190 days under the condition of 25 ℃ of temperature, produce faint yellow granular embryo callus, the result sees table 1.
3, somatic embryo generating process
Get 50 of embryo callus and be transferred to body embryonal induction medium (Y
3Medium+6-BA 10mg/L+2; 4-D 2mg/L+ active carbon 2.5g/L+PVP 5g/L+ agar 4.5g/L+ sucrose 30g/L) in; Place that relative moisture is 70%, cultivate 80 days organizator blasts under the condition of 27 ℃ of temperature, intensity of illumination 2000lx, illumination 12h/d, the result sees table 2.
Embodiment three
1, the collection of embryo and preparatory incubation
Choose 90 of the ripe coconut palm fruits at 10～11 monthly ages; Remove coconut palm fruit crust and the coconut palm fruit is laterally split with chopper; The use diameter is the solid endosperm piece of complete embryo to be arranged with running water flushing 1～2h in the card punch of 2 cm takes out; On superclean bench, take out embryo then and carry out the embryo surface sterilization, inoculate pre-culture medium (Y
3Medium+active carbon 2.5g/L+ sucrose 60g/L) in, places that relative moisture is 80%, secretly cultivated 13 days under the condition of 25 ℃ of temperature, embryo is expanded.
2, embryo callus induces process
The pre-incubated embryo that expands of learning from else's experience, with its haustorium excision and rip cutting into two, cut side up is inoculated into callus inducing medium (Y
3Medium+2; 4-D 25mg/L+ active carbon 2.5g/L+PVP 5g/L+ agar 4.5g/L+ sucrose 30g/L) in; Place that relative moisture is 75%, secretly cultivate under the condition of 26 ℃ of temperature and produced faint yellow granular embryo callus in 200 days, the result sees table 1.
3, somatic embryo generating process
Get 50 of embryo callus and be transferred to body embryonal induction medium (Y
3Medium+6-BA 10mg/L+2; 4-D 2mg/L+ active carbon 2.5g/L+PVP 5g/L+ agar 4.5g/L+ sucrose 30g/L) in; Place that relative moisture is 78%, cultivate 100 days organizator blasts under the condition of 28 ℃ of temperature, intensity of illumination 1800lx, illumination 12h/d, the result sees table 2.
To result when:
Frequency of embryonic callus induction: between culture period, be control group with different switching frequencies, other conditions are with embodiment one, 6 months embryonic callus induction time (180 days).The gained inductivity is seen table 1.
The comparison of the frequency of embryonic callus induction of the different switching of table 1 frequency
The result shows that the process (continuous culture, during do not transfer) of inducing of embryo callus of the present invention can produce more embryo callus, and inductivity is higher.
The inductivity of table 2 somatic embryo
| Project | Callus inoculation number/individual | Somatic embryo number/individual | Inductivity/% |
| Embodiment one | 50 | 36 | 72 |
| Embodiment two | 50 | 39 | 78 |
| Embodiment three | 50 | 37 | 74 |
The result shows that the process of inducing of somatic embryo of the present invention has higher inductivity, can produce more somatic embryo, for the research of aspects such as the gene engineering of carrying out coconut in a deep going way, genetic transformation, breed improvement provides an effective way.
The above only is a preferred implementation of the present invention; Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from know-why of the present invention; Can also make some improvement and retouching, these improvement and retouching also should be regarded as protection scope of the present invention.
Claims (1)
1. a coconut mature zygotic embryos cultured in vitro somatic embryo method for generation is characterized in that, the collection that comprises embryo and incubation, embryo callus in advance induce process, somatic embryo generating process, its step is following:
1), the collection of embryo and preparatory incubation
Choose the ripe coconut palm fruit at 10～11 monthly ages; The solid endosperm piece that complete embryo is arranged in the taking-up cleans the back and takes out the embryo sterilization, is inoculated in the pre-culture medium then; Place that relative moisture is 70～80%, secretly cultivated 12～17 days under the condition of 25～29 ℃ of temperature, embryo is expanded; Said pre-culture medium is with Y
3Medium is a minimal medium, and adds active carbon 2.5g/L, sucrose 60g/L;
2), embryo callus induces process
The pre-incubated embryo that expands of learning from else's experience; Its haustorium is excised also rip cutting into two; Cut side up is inoculated in the callus inducing medium, places that relative moisture is 70～80%, secretly cultivates under the condition of 25～29 ℃ of temperature and produced faint yellow granular embryo callus in 170～200 days; Said callus inducing medium is with Y
3Medium is a minimal medium, and adds 2,4-D 25mg/L, active carbon 2.5g/L, PVP 5g/L, agar 4.5g/L, sucrose 30g/L;
3), somatic embryo generating process
Embryo callus is transferred in the body embryonal induction medium, places that relative moisture is 70～80%, cultivate 80～100 days organizator blasts under the condition of 25～29 ℃ of temperature, intensity of illumination 1500～2500lx, illumination 12h/d; Said body embryonal induction medium is with Y
3Medium is a minimal medium, and adds 6-BA 10mg/L, 2,4-D 2mg/L, active carbon 2.5g/L, PVP 5g/L, agar 4.5g/L, sucrose 30g/L.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109479714A (en) * | 2018-12-06 | 2019-03-19 | 中国热带农业科学院椰子研究所 | A kind of coconut mature zygotic embryos method for tissue culture |
| CN116250482A (en) * | 2023-05-10 | 2023-06-13 | 海南大学三亚南繁研究院 | Method for tissue culture and rapid propagation of coconuts |
| CN116762694A (en) * | 2023-05-17 | 2023-09-19 | 海南大学 | Embryo culture method of maca planoc |
Citations (1)
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| WO1994016551A1 (en) * | 1993-01-29 | 1994-08-04 | Institut Francais De Recherche Scientifique Pour Le Developpement En Cooperation (Orstom) | Process for regenerating the coconut palm from explants, and plants obtained by such process |
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2012
- 2012-07-06 CN CN2012102338935A patent/CN102715088A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994016551A1 (en) * | 1993-01-29 | 1994-08-04 | Institut Francais De Recherche Scientifique Pour Le Developpement En Cooperation (Orstom) | Process for regenerating the coconut palm from explants, and plants obtained by such process |
Non-Patent Citations (5)
| Title |
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| 《Plant Cell Reports》 19841231 P.K.Gupta et al. Somatic embryogenesis and plants from zygotic embryos of coconut (Cocos nucifera L.) in vitro 222-225 1 , 第3期 * |
| KARUNARATNE ET AL.: "Culture of Immature Embryos of Coconut,Cocos nucifera L: Callus Proliferation,and Somatic Embryogenesis", 《COCOS》, vol. 8, 31 December 1990 (1990-12-31), pages 13 - 22 * |
| MONTERO-CORTÉS ET AL.: "Addition Of Benzyladenine To Coconut Explants Cultured In Vitro Improves The Formation Of Somatic Embryos And Their Germination", 《AGROCIENCIA》, vol. 45, no. 6, 31 December 2011 (2011-12-31) * |
| P.K.GUPTA ET AL.: "Somatic embryogenesis and plants from zygotic embryos of coconut (Cocos nucifera L.) in vitro", 《PLANT CELL REPORTS》, no. 3, 31 December 1984 (1984-12-31), pages 222 - 225 * |
| 周焕起等: "椰子愈伤组织诱导与防褐变技术研究", 《江西农业学报》, vol. 22, no. 3, 15 March 2010 (2010-03-15) * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109479714A (en) * | 2018-12-06 | 2019-03-19 | 中国热带农业科学院椰子研究所 | A kind of coconut mature zygotic embryos method for tissue culture |
| CN116250482A (en) * | 2023-05-10 | 2023-06-13 | 海南大学三亚南繁研究院 | Method for tissue culture and rapid propagation of coconuts |
| CN116250482B (en) * | 2023-05-10 | 2023-08-04 | 海南大学三亚南繁研究院 | Method for tissue culture and rapid propagation of coconuts |
| CN116762694A (en) * | 2023-05-17 | 2023-09-19 | 海南大学 | Embryo culture method of maca planoc |
| CN116762694B (en) * | 2023-05-17 | 2024-02-20 | 海南大学 | Embryo culture method of maca planoc |
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Application publication date: 20121010 |