CN103416302B - Method for culturing regeneration plant of somatic embryo of osmanthus fragrans Lour - Google Patents
Method for culturing regeneration plant of somatic embryo of osmanthus fragrans Lour Download PDFInfo
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- CN103416302B CN103416302B CN201210432277.2A CN201210432277A CN103416302B CN 103416302 B CN103416302 B CN 103416302B CN 201210432277 A CN201210432277 A CN 201210432277A CN 103416302 B CN103416302 B CN 103416302B
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Abstract
The invention belongs to the technical field of plant tissue and cell culture, and particularly relates to a method for a regeneration plant of a somatic embryo of osmanthus fragrans Lour. The method takes zygotic embryo of the osmanthus fragrans Lour as an explant, an osmanthus fragrans Lour regeneration system is built through a body cell embryo generating way so as to solve the problem that a callus can not further differentiate by an osmanthus fragrans Lour tissue culture technology. The method comprises the following steps: taking the zygotic embryo of the osmanthus fragrans Lour as materials; building the osmanthus fragrans Lour somatic embryo regeneration system through five stages: embryonic callus induction, the differentiation of the embryonic callus into somatic embryos, somatic embryos maturity and germination, somatic embryos rootage and seedling of the somatic embryos regeneration plant. According to the invention, through comparing different development states of the zygotic embryo and the different using parts of the zygotic embryo, the development part and state suitable for explant are determined. Due to the adoption of the method, a large number of the regeneration plants of the somatic embryo of osmanthus fragrans Lour, which have the consistent genetic basis, can be obtained, so that a novel method is provided for preservation of sources of the rear osmanthus fragrans Lour varieties, industrialized production of good genetype nursery stocks, and preparation of artificial seeds.
Description
Technical field
The invention belongs to plant tissue and cell culture technical field, be specifically related to the method for sweet osmanthus somatic embryo regeneration plant, taking sweet osmanthus zygotic embryo as explant, set up sweet osmanthus regenerating system by somatic embryo development ways, to solve the problem that in sweet osmanthus tissue culture technique, callus cannot further break up.
Background technology
Sweet osmanthus (Osmanthus fragrans Lour.) belongs to Oleaceae (Oleaceae) Osmanthus (Osmanthus), originates from Asia, is one of tradition ten large famous flowers of China's characteristic.After 18 century 70s, start to import into Europe, the U.S., Japan, Korea S, India, have the cultivation history of more than 2000 year in China.The tree-like grace of sweet osmanthus, the four seasons are evergreen, pleasant aroma, have the good reputation of " monopolize three autumn jobs as harvesting, tilling and planting and press beautiful and fragrant flowers ", have high ornamental value, are usually used as important garden landscape and Afforestation Species for Urban Road.
Along with the exploitation to sweet osmanthus edibility, on market, occur using sweet osmanthus as major ingredient or the food of auxiliary material, as sweet-scented cake, wine fermented with osmanthus flower, sweet osmanthus wine, sweet osmanthus shortcake etc.On edible basis, sweet osmanthus can be acted as expectorant, warm stomach, flat liver, kidney-nourishing, loose cold, and therefore its medicinal health value also starts to attract people's notice gradually.Sweet osmanthus, as a kind of important fragrant plant, does not only have the edible and medical value that person is higher, and it is also a kind of very important fragrant resource simultaneously, is widely used in the high-grade cosmetic industry such as face cream, essential oil.The melanin extracting from the fruit of sweet osmanthus is a kind of natural colouring matter, is that the important natural colouring matter in chemical industry extracts raw material.
Sweet osmanthus is no matter aspect long cultural and historical, and still economic benefit aspect widely, all makes it have large market demand.But because sweet osmanthus selfing is not affine, Natural seed setting rate is low, seed exists again the process of physiology after-ripening, be difficult under field conditions (factors) sprouting; In addition some sweet osmanthus cuttage seeding are difficult to take root, and are subject to the features such as season and environmental influence are large.Therefore, people start to attempt utilizing Techniques of in Vitro Culture, reach the object of breeding seedling in enormous quantities.In addition, tissue culture technique can also be saved sweet osmanthus appreciable variety, preserves existing good sweet osmanthus germ plasm resource, shorten breeding time, not only reproduction coefficient is large, and can keep the merit of kind, is also one of important foundation of higher plant cell engineering, gene engineering and further improvement.But in cultured in vitro process, find, the callus of sweet osmanthus is difficult to differentiation, there is no both at home and abroad so far about the report of setting up sweet osmanthus high-efficiency somatic cell regenerating system method.
Summary of the invention
The object of the invention is cannot further break up for callus in current sweet osmanthus tissue culture procedures, there is the problems such as difficulty in organ, set up efficiently sweet osmanthus somatic embryo renovation process fast of one, for factorial seedling growth and artificial seed preparation from now on provides a new approach and technological means.
The present invention is taking the zygotic embryo of sweet osmanthus select tree seed as starting material, process is to its zygotic embryo developmental condition and the screening comparative studies of taking position, obtain inducing the best explant material of sweet osmanthus somatic embryo regeneration, utilize the modular design containing the plant growth regulator of variable concentrations and combination, induction obtains having the sweet osmanthus embryo callus of further differentiation capability, through differentiation and proliferation, the maturation germination of embryo callus and the cultivation in the stage such as take root, realize the plant regeneration process of sweet osmanthus through somatic embryo generation approach.
The present invention is by the following technical solutions:
By a method for sweet osmanthus somatic embryo regeneration plant, comprise the following step:
(1) pretreatment before the selection of explant and cultivation
Gather the fruit of the adult osmanthus fragrans of high-quality the fine fair-weather morning in annual January to May, remove exosper and pulp, first use 75%(V/V) alcohol disinfecting 15 ~ 60 seconds, then soak 2 ~ 3 times with sterile water, then with 0.1%(W/V) mercuric chloride sterilization 5 ~ 15 minutes, finally use again sterile water wash 3 ~ 5 times, under aseptic condition, peel off the zygotic embryo in sterilization fruit, for subsequent use.
(2) embryonic callus induction
The small fragment that the zygotic embryo of step (1) is cut into 1-3 millimeter is inoculated on callus inducing medium, within every 30 days, change a fresh culture, in culturing room, secretly cultivate, cultivation temperature is 20 ~ 30 DEG C, incubation time is 20 ~ 60 days, until obtain embryo callus.Frequency of embryonic callus induction of the present invention can reach more than 60% ~ 80%.
(3) small fragment that the zygotic embryo of step (1) is cut into 1-3 millimeter by embryo callus clade blast is inoculated on callus inducing medium, within every 30 days, change a fresh culture, in culturing room, secretly cultivate, cultivation temperature is 20 ~ 30 DEG C, incubation time is 20 ~ 60 days, until acquisition embryo callus, its screening technique is: select graininess strong, mellow and full densification and glossiness outward appearance, appearance is the embryo callus of buff, reject and do not there is regular outward appearance, color is white in color, appearance is the non-embryonic callus tissue of light green color or brown.Embryo differentiate rate of the present invention can reach 60% ~ 80%, and each explant average body embryo differentiation number can reach 3 ~ 5.
(4) promote somatic embryo maturation germination
The somatic embryo of the differentiation that step (3) is obtained is chosen, and transfers on germination medium, within every 30 days, changes a fresh culture, in culturing room, secretly cultivates, and cultivation temperature is 20 ~ 30 DEG C, and incubation time is 20 ~ 60 days, makes its further maturation germination;
(5) taking root of inductor blast
The somatic embryo of step (4) maturation germination is transferred to root induction on root media, within every 30 days, change a fresh culture, culturing room's temperature is 20 ~ 30 DEG C, and every day, light application time was 12 ~ 18h, and intensity of illumination is 1500 ~ 3000lux.The rooting rate of the induction of somatic embryo of the present invention is for reaching 70%.
(6) seedling of somatic embryo regeneration plant
Step (5) is transferred to the regeneration plant after taking root in growth medium, within every 30 days, changed a fresh culture, culturing room's temperature is 20 ~ 30 DEG C, incubation time is 20 ~ 60 days, every day, light application time was 12 ~ 18h, and intensity of illumination is 1500 ~ 3000lux, it was further grown healthy and strong; By the Transplantation of Regenerated Plantlets of robust growth in the container that contains sterilized matrix, in greenhouse under natural lighting incubation growth.Test shows that transplanting survival rate of the present invention can reach 70%.
Component and the proportioning of the medium using in above steps are as follows:
Callus inducing medium: MS medium, additional 2,4-dichlorphenoxyacetic acid (2,4-D), 0.1 ~ 5.0mg/L, 6-benzylamino adenine (6-BA) 0.1 ~ 5.0mg/L, sucrose 30g/L and agar 7g/L, pH5.8 ~ 6.5;
Calli Differentiation medium: MS medium, additional methyl α-naphthyl acetate (NAA) 0 ~ 3.0mg/L, 6-benzylamino adenine (6-BA) 0 ~ 3.0mg/L, sucrose 30g/L and agar 7g/L, pH5.8 ~ 6.5;
Germination medium: MS medium, additional methyl α-naphthyl acetate (NAA) 0 ~ 3.0mg/L, 6-benzylamino adenine (6-BA) 0 ~ 3.0mg/L, sucrose 30g/L and agar 7g/L, pH5.8 ~ 6.5;
Root media: 1/2MS medium, additional methyl α-naphthyl acetate (NAA) 0 ~ 3.0mg/L, sucrose 30g/L and agar 7g/L, pH5.8 ~ 6.5;
Growth medium: MS medium, additional 6-benzylamino adenine (6-BA) 0 ~ 3.0mg/L, sucrose 30g/L and agar 7g/L, pH5.8 ~ 6.5;
Transplanting medium: detritus soil: the volume ratio of vermiculite is 2:1.
Medium of the present invention carries out according to Plant Tissue Breeding code, existing technical manual, and the knowledge of document or textbook contributes to enforcement of the present invention, and unless stated otherwise, the present invention carries out with reference to program and the method for existing plant tissue and cell culture.
The preparation of above-mentioned medium is according to the method for routine report, and wherein medium, after each active ingredient polishing, is settled to 1L volume with distilled water routinely, before sterilizing, with 1M NaOH, the pH of medium is adjusted to 5.8 ~ 6.5; By the medium preparing sterilizing 20min under 121 DEG C of high steams, naturally cooling rear for subsequent use.
The effect that the present invention is useful is: the present invention utilizes sweet osmanthus zygotic embryo for explant, set up sweet osmanthus regenerating system by somatic embryo development ways, in solution sweet osmanthus tissue culture technique, callus cannot further break up and obtain by adventitious organogenesis the problem of regeneration plant difficulty.Advantage of the present invention is that the breeding cycle is short, coefficient is high, and this breeding is not subject to the restriction in season, is not only conducive to the preservation of good rare osmanthus cultivars germ plasm resource, has also established and has realized basis for the further genetic improvement of sweet osmanthus and molecular breeding research.In addition, the foundation of the method is also grown seedlings for sweet osmanthus is from now on large-scale industrialized and the preparation of artificial seed provides a new approach and technological means.
Brief description of the drawings
Fig. 1: sweet osmanthus zygotic embryo Divisional sampling pattern figure.In figure: A. radicle B. plumular axis C. plumule D. cotyledon.
Fig. 2: sweet osmanthus zygotic embryo inductor blast regenerative process.In figure: a. solid osmanthus fragrans " Jin Gui " of growing up; B. sweet osmanthus fruit; C. the zygotic embryo explants for inducing; D. the embryo callus obtaining is cultivated in induction; E. the Differentiation and proliferation of somatic embryo; F. the ripe and sprouting of somatic embryo; G. regeneration plant is taken root.
Embodiment
In embodiment below, further illustrate the present invention, this does not limit the scope of the invention.
Each processing in the test of following embodiment at least repeats 4 times, and each processing at least accesses 20 explants.
Embodiment 1: the screening of sweet osmanthus zygotic embryo sampling point
Zygotic embryo for examination material osmanthus fragrans is taken from the fruit (seeing a, b in Fig. 2) in robust growth in Hua Zhong Agriculture University campus, solid many osmanthus fragrans gold osmanthus (this kind is the osmanthus fragrans kind that generally cultivate Wuhan City of Hubei China province).The time of drawing materials is the fine fair-weather morning in March, Fruit returns after indoor and adds a small amount of washing powder running water to rinse 1 ~ 2h, remove exosper and pulp carries out disinfection, be placed in superclean bench 75%(V/V) alcohol disinfecting 30s, aseptic water washing 2 times, use again 0.1%(W/V) mercuric chloride sterilization 10min, aseptic water washing 3 times.
To after sterilization, complete sweet osmanthus zygotic embryo (be shown in and c) be cut into respectively cotyledon (seeing the A in Fig. 1) in Fig. 2, plumular axis (seeing the B in Fig. 1), plumule (seeing the C in Fig. 1) and cotyledon end (D in Fig. 1) four parts, be inoculated into respectively MS, additional 0.5mg/L 2, 4-dichlorphenoxyacetic acid (2, 4-D), 1.0mg/L 6-benzylamino adenine (6-BA), sucrose 30g/L and agar 7g/L, on the calli induction media of pH5.8 ~ 6.5 of medium, in culturing room, at 25 DEG C, secretly cultivating (culture dish of test is placed in carton and is formed in dark subenvironment with conventional black cloth shading) cultivates 30 days, obtain embryo callus (see in Fig. 2 d).
Above-mentioned zygotic embryo each several part material is induced to the embryo callus obtaining, subculture (contains sucrose 30g/L and agar 7g/L to the MS medium that does not add any plant growth regulator respectively, pH5.8 ~ 6.5 of medium) in, at 25 DEG C, secretly cultivate (condition of culture is described above) 30 days, wherein the cotyledon end of zygotic embryo (D in Fig. 1) regeneration rate is significantly higher than other position, and somatic embryo generation rate reaches 80%.The present invention shows, the cotyledon end of sweet osmanthus zygotic embryo is the suitable position of drawing materials of explant in sweet osmanthus Regeneration in Vitro process.
The somatic embryo material that differentiation is obtained (is shown in that the e) subculture in Fig. 2 (contains sucrose 30g/L and agar 7g/L to the MS medium that does not add any plant growth regulator, pH5.8 ~ 6.5 of medium) in, at 25 DEG C, its further maturation germination of (condition of culture is described above) 30 angels is cultivated in dark place.Subsequently by sprout in the somatic embryo of ripe cotyledon shape phase choose (see Fig. 2 f), be inoculated into 1/2MS, additional 2.0mg/L methyl α-naphthyl acetate (NAA), sucrose 30g/L and agar 7g/L, in the root media of pH5.8 ~ 6.5 of medium, under illumination condition, cultivate 45 days (illumination every day is 14h, and intensity of illumination is 3000lux), rooting rate can reach more than 70% (see Fig. 2 g).
The regeneration plant of well developed root system is forwarded in MS medium (containing sucrose 30g/L and agar 7g/L, pH5.8 ~ 6.5 of medium) and under illumination condition, cultivates 30 days (illumination every day is 14h, and intensity of illumination is 3000lux), make plant further grow stalwartness.Subsequently they are transplanted to the humus soil of sterilizing and vermiculite composite soil in (humus soil: vermiculite volume ratio=2:1), under greenhouse natural lighting, cultivate, after 30 days, transplant survival rate and can reach 70%.
Embodiment 2: the screening of the best developmental condition of sweet osmanthus zygotic embryo
Take from robust growth in Hua Zhong Agriculture University campus, solid many osmanthus fragrans " Jin Gui: fruit (this kind is the osmanthus fragrans kind that generally cultivate Wuhan City of Hubei China province) for examination material zygotic embryo.The time of drawing materials is January, and draw materials once the fine fair-weather morning every 30d left and right (in case of rainy weather can suitably regulate), until May, fruit full maturity also came off naturally.
Remove the exosper of fragrans seed, pulp also carries out disinfection, and sterilization method is with embodiment 1.The state zygotic embryo of growing shape taking difference is as explant (table 1), the fragment that is cut into 1-3mm on superclean bench is inoculated into MS in time, additional 0.5mg/L 2,4-dichlorphenoxyacetic acid (2,4-D), 1.0mg/L 6-benzylamino adenine (6-BA), sucrose 30g/L and agar 7g/L, in the callus inducing medium of pH5.8 ~ 6.5 of medium, the same enforcement 1 of condition of culture.
The impact of the different sampling time of explants of table 1 on somatic embryo inducement
The embryo callus that induction is obtained, subculture (contains sucrose 30g/L and agar 7g/L to the MS medium that does not add any plant growth regulator respectively, pH5.8 ~ 6.5 of medium) in, at 25 DEG C, cultivate 30 days dark place, wherein March the immature zygotic embryos in the early stage cotyledon shape phase, its somatic embryo regeneration rate reaches 80%, is significantly higher than other periods.The present invention shows that the immature zygotic embryos of reported first in the early stage cotyledon shape phase is comparatively desirable explant sampling time of explant to sweet osmanthus.
Follow-up maturation germination, take root and transplanting process with embodiment 1 above.
Embodiment 3: sweet osmanthus embryo callus subculture inducing culture screening
Take from the golden osmanthus fruit of robust growth, solid many osmanthus fragrans in Hua Zhong Agriculture University campus for examination material zygotic embryo.The time of drawing materials is the fine fair-weather morning in March, and Fruit is back to after indoor and removes exosper and pulp and carry out disinfection, concrete the same embodiment 1 of sterilization method.The embryo of sterile seed is intactly stripped out, on superclean bench, be cut into the small fragment of 1-3mm, be inoculated in respectively MS, additional 0 ~ 2.5mg/L 2,4-dichlorphenoxyacetic acid (2,4-D), 0 ~ 2.5mg/L 6-benzylamino adenine (6-BA), sucrose 30g/L and agar 7g/L, induce (table 2) in the medium of pH5.8 ~ 6.5 of medium, dark 30 days (condition is with embodiment 1), the acquisition embryo callus cultivated at 25 DEG C.
The impact of table 2 different hormone combinations on embryo callus subculture induction
The embryo callus that induction is obtained, subculture is to not adding in the MS medium (containing sucrose 30g/L and agar 7g/L, pH5.8 ~ 6.5 of medium) of any plant growth regulator respectively, and cultivate 30 days dark place at 25 DEG C, and differentiation obtains somatic embryo.Wherein the frequency of embryonic callus induction of No. 6 medium reaches 88%, and embryo differentiate rate reaches 80%, on average breaks up number and reaches 4.3 individual cells embryos/each explant, is significantly better than other combination formula.Reported first of the present invention, MS adds 0.5mg/L 2,4-dichlorphenoxyacetic acid (2,4-D), 1.0mg/L 6-benzylamino adenine (6-BA), pH5.8 ~ 6.5 of sucrose 30g/L and agar 7g/L(medium) medium be the better culture medium prescription of sweet osmanthus embryonic callus induction.
Follow-up sprouting, take root and transplanting process with embodiment 1 above.
Annex explanation: the preparation of above-mentioned medium is according to the method for routine report, wherein medium, after each active ingredient polishing, routinely, is settled to 1L volume with distilled water, before sterilizing, with 1M NaOH, the pH of medium is adjusted to 5.8 ~ 6.5.By the medium preparing sterilizing 20min under 121 DEG C of high steams, naturally cooling rear for subsequent use.
Claims (2)
1. a method that is obtained regeneration plant by sweet osmanthus somatic embryo, is characterized in that, comprises the following step:
(1) pretreatment before the selection of explant and cultivation
Gather the fruit of the adult osmanthus fragrans of high-quality the fine fair-weather morning in annual January to May, remove exosper and pulp, first use 75% (V/V) alcohol disinfecting 15～60 seconds, then soak 2～3 times with sterile water, then with 0.1% (W/V) mercuric chloride sterilization 5～15 minutes, finally use again sterile water wash 3～5 times, under aseptic condition, peel off the zygotic embryo in sterilization fruit, for subsequent use;
(2) embryonic callus induction
The small fragment that the zygotic embryo of step (1) is cut into 1-3 millimeter is inoculated on callus inducing medium, within every 30 days, change a fresh culture, in culturing room, secretly cultivate, cultivation temperature is 20～30 DEG C, incubation time is 20～60 days, until acquisition embryo callus, its screening technique is: select strong, the mellow and full densification of graininess and glossiness outward appearance, appearance is the embryo callus of buff, reject and do not have regular outward appearance, color is white in color, appearance is light green color or the non-embryonic callus tissue of brown;
(3) embryo callus clade blast
Induce the embryo callus obtaining to transfer on Calli Differentiation medium step (2), within every 30 days, change a fresh culture, in culturing room, secretly cultivate, cultivation temperature is 20～30 DEG C, incubation time is 20～60 days, until obtain the somatic embryo of differentiation;
(4) promote somatic embryo maturation germination
The somatic embryo of step (3) differentiation is chosen, transferred on germination medium, within every 30 days, change a fresh culture, in culturing room, secretly cultivate, cultivation temperature is 20～30 DEG C, and incubation time is 20～60 days, makes its further maturation germination;
(5) taking root of inductor blast
The somatic embryo of step (4) maturation germination is transferred to root induction on root media, within every 30 days, change a fresh culture, culturing room's temperature is 20～30 DEG C, and incubation time is 20～60 days, every day, light application time was 12～18h, and intensity of illumination is 1500～3000lux;
(6) seedling of somatic embryo regeneration plant
Regeneration plant after step (5) is taken root is transferred in growth medium, within every 30 days, change a fresh culture, culturing room's temperature is 20～30 DEG C, incubation time is 20～60 days, every day, light application time was 12～18h, and intensity of illumination is 1500～3000lux, it was further grown healthy and strong, by the Transplantation of Regenerated Plantlets of robust growth in the container that contains sterilized matrix, in greenhouse under natural lighting incubation growth;
Wherein:
The medium using in above steps or the component of matrix and proportioning are as follows:
(1) callus inducing medium: MS, additional 0.5mg/L2,4-D, 1.0mg/L6-BA, sucrose 30g/L and agar 7g/L, the pH of medium is 5.8～6.5;
(2) Calli Differentiation medium: MS, additional saccharose 30g/L and agar 7g/L, the pH of medium is 5.8～6.5;
(3) germination medium: MS, additional saccharose 30g/L and agar 7g/L; The pH of medium is 5.8～6.5;
(4) root media: 1/2MS, additional 2.0mg/L NAA, sucrose 30g/L and agar 7g/L, the pH of medium is 5.8～6.5;
(5) growth medium: MS, additional saccharose 30g/L and agar 7g/L, the pH of medium is 5.8～6.5;
(6) transplanting medium: the volume ratio of detritus soil and vermiculite is 2:1.
2. method claimed in claim 1 application in sweet osmanthus tissue is cultivated.
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| CN103733998B (en) * | 2013-12-25 | 2015-09-16 | 南京林业大学 | A kind of rain city orange osmanthus Calli Differentiation and enrichment procedure |
| CN104351048A (en) * | 2014-10-14 | 2015-02-18 | 深圳文科园林股份有限公司 | Tissue culture rapid propagation method of osmanthus fragrans |
| CN109566126A (en) * | 2018-12-25 | 2019-04-05 | 福建农林大学 | A kind of method of Pearl color osmanthus tissue culture outside sprout-cultivating-bottle radication |
| CN111642403B (en) * | 2020-07-24 | 2021-11-26 | 黑龙江省林业科学研究所 | Phellodendron amurense somatic embryo tissue culture medium and tissue culture method |
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