CN101946704A - Method for regenerating Chinese rose plant by using immature seed as explant - Google Patents
Method for regenerating Chinese rose plant by using immature seed as explant Download PDFInfo
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Abstract
The invention belongs to the technical field of tissue and cell culture of plants and in particular relates to a method for regenerating a plant in Chinese rose somatic embryogenesis. The method comprises the follow three cultivating stages of: (1) cultivating a Chinese rose explant on an induction culture medium and directly inducing somatic embryo from the explant, wherein the explant is an immature zygotic embryo of a Chinese rose; (2) inoculating the somatic embryo to a proliferating culture medium for proliferation and promoting the further differentiation of the somatic embryo; and (3) inoculating the somatic embryo subjected to the proliferating culture to a somatic embryo seedling culture medium so as to regenerate an integrated plant. In the Chinese rose somatic embryo subjected to isolated culture, roots and shoots can grow at the same time during the regeneration of the plant, so that the integrated plant can be formed without rooting culture. The method has the advantages of wide sources of the explant, readily available raw materials and the like. The obtained somatic embryo is a good agrobactrium rhizogenes-mediated and genetically transformed receptor. The method can also be applied to the research of the genetic transformation of Chinese roses.
Description
Technical field
The invention belongs to plant tissue and cell culture technology field, being specifically related to the Chinese rose immature zygotic embryos is explant, carries out the method for Chinese rose plant regeneration by the somatic cell embryogenesis path.
Background technology
Chinese rose (Rosa hybrida) belongs to the rose family (Rosaceae) Rosa (Rosa) perennial evergreen woody plant.The Chinese rose cultivation history is long, is extensively planted by countries in the world, is very important ornamental flower and has important commercial and be worth, and plays an important role in afforestation.Chinese rose is gorgeous because of its attitude grace, pattern enrich, strong, florescence of fragrance is long, adaptability is strong, breeding easily and convenient management be described as and spend middle queen.The purposes of Chinese rose is very extensive, for example can arrange gallery and arched door with the liana Chinese rose, decorate major trunk roads with dendriform China rose, the shrub Chinese rose is made hedgerow, intersperse flower bed with poly-flower Chinese rose, the fragrant Chinese rose of tea can be used for exhibition, and miniature Chinese rose is used as furnishings, and Chinese rose of cut flower can be applied to various places.
It is shorter that but the bottle of China rose is inserted the life-span, easily suffers from powdery mildew, black spot, and these have all influenced its ornamental value and economic worth.On these character improvements, traditional breeding way exists significant limitation.Plant gene engineering technology can utilize foreign gene that its proterties of controlling is carried out orderly improvement on the metastable basis of other proterties that keeps kind, thereby provides new approach for cultivating the Chinese rose new varieties.And the foundation of Chinese rose regenerating system and genetic conversion system is the important foundation of utilizing the technique for gene engineering breeding.
The somatic embryo of plant is that the somatic cell of plant is under isolated condition, by obtaining new individual process with the similar development pathway of zygotic embryo.Somatic cell embryogenesis path regenerating system to establish the test-tube plantlet that helps Chinese rose numerous soon, the production of artificial seed and stored refrigerated, cells,primordial is the best acceptor that carries out the Chinese rose genetic transformation simultaneously, can be the research that acceptor carries out the Chinese rose genetic transformation with the somatic embryo.
Different modern rose cultivarses and explant and experimental technique are applied to the research that the Chinese rose somatic embryo takes place, the result shows because genotypic restriction is difficult to find a kind of method in common to carry out (the Marchant et al. that induces of Chinese rose somatic embryo, Somatic embryogenesis and plant regeneration in floribunda rose (Rosa hybrida L.cvs.Trumpeter and Glad Tidings) .Plant Science, 1996,120:95-105).Excised leaf or stem section with Chinese rose Rosa hybrida cv.Carl Red and R.canina are that explant can obtain embryo callus (Visessuwan et al., Plant regeneration systems from leaf segment culture through embryogenic callus formation of Rosa hybrida and R.canina.Breed Sci, 1997,47:217-222).DeWit et al. (Somatic embryogenesis and regeneration of flowering plants in rose.Plant Cell Rep, 1990,9:456-458) blade with R.hybrida cv.Domingo and R.hybrida cv.Vicky Brown is that explant has also obtained embryo callus.Rout et al. (Somatic embryogenesis in callus culture of Rosa hybrida L.cv.Landora.Plant Cell Tiss Org Cult, 1997,27:65-69) in the research of Chinese rose R.hybrida cv.Landora somatic embryo inducement, induce immature blade or stem section can obtain somatic embryo.Marchant et al. (Somatic embryogenesis and plant regeneration in floribunda rose (Rosa hybrida L.cvs.Trumpeter and Glad Tidings) .Plant Science, 1996,120:95-105) petiole and the root with Chinese rose R.hybrida cv.Trumpeter and R.hybrida cv.Glad Tidings is that explant induction has obtained somatic embryo.Utilize immature seed to induce and obtain somatic embryo (Kim et al., Control of direct and indirect somatic embryogenesis by exogenous growth regulators in immature zygotic embryo cultures of rose.Plant Cell Tiss Org Cult, 2003,74:61-66; Kamo et al., Dispersal and size fractionation of embryogenic of callus increases the frequency of embryo maturation and conversion in hybrid tea roses.Plant Cell Rep, 2004,22:787-792).In addition, somatic embryo can obtain (Arene et al. by the petal of inducing Chinese rose, A comparison of the somaclonal variation level of Rosa hybrida L.cv.Meirutral plants regenerated from callus of direct induction from different vegetative and embryonic tissues.Euphytica, 1993,71:83-90).At present, both at home and abroad also less than report about Chinese rose ' purple mist ' somatic embryo inducement.
Summary of the invention
The objective of the invention is to overcome the defective of prior art, providing a kind of is material with Chinese rose ' purple mist ' immature zygotic embryos, by the somatic cell embryogenesis path, sets up the method for Chinese rose regenerating system.The somatic embryo that simultaneously the inventive method obtained can long-term subculture and is kept its differentiation capability, can be used as the material of Chinese rose genetic transformation.
Technical scheme of the present invention and step are as follows:
A kind of Chinese rose is regenerated as the method for whole plant, it comprises the following steps:
1. be explant with the Chinese rose immature zygotic embryos, described explant is directly induced somatic embryo in inducing culture;
2. the somatic embryo of step in 1. is inoculated in the somatic embryo proliferated culture medium propagation and makes its further differentiation, obtain mature somatic embryo;
3. the mature somatic embryo that 2. step is obtained is inoculated into somatic embryo and becomes in the seedling medium to be regenerated as whole plant;
Wherein:
Described inducing culture composed as follows: 1/2MS minimal medium, 2,4 dichlorophenoxyacetic acid 0-3.0mg/L, 6-benzyladenine 0-1.0mg/L, glucose 30g/L, plant gel 2.5g/L;
Described somatic embryo proliferated culture medium composed as follows: MS minimal medium, 2,4 dichlorophenoxyacetic acid 1.0mg/L, 6-benzyladenine 0.05mg/L, glucose 60g/L, plant gel 2.5g/L;
Described somatic embryo becomes the composed as follows of seedling medium: the MS minimal medium, 6-benzyladenine 0-1.0mg/L, glucose 30g/L, plant gel 2.5g/L or MS minimal medium, azoles diazobenzene stem urea (TDZ) 1.0mg/L, 6-benzyladenine 0.5mg/L, glucose 30g/L, plant gel 2.5g/L.
As preferred version, the 2,4 dichlorophenoxyacetic acid in the described inducing culture is 2.0mg/L, and 6-benzyladenine is 0.5mg/L.
As preferred version, the thin blast of described body becomes the composed as follows of seedling medium: MS minimal medium, 6-benzyladenine are 0.5mg/L.
The present invention is an explant with modern rose cultivars " purple mist " immature zygotic embryos, and directly the inductor blast takes place, and can keep somatic embryo constantly propagation and the characteristic that becomes seedling.These somatic embryos provide good acceptor for the genetic transformation of agriculture bacillus mediated Chinese rose; Simultaneously, this regenerating system also is the desirable acceptor of Chinese rose genetic transformation and is that the artificial seed for preparing transgenosis Chinese rose new varieties has been established certain technical foundation.
Good effect of the present invention is:
1, explant of the present invention is originated and is not subject to seasonal restrictions, and can carry out the tissue and the cell culture of Chinese rose ' purple mist ' anniversary.
2, the Chinese rose somatic embryo under the cultured in vitro can constantly be bred by secondary embryo, has good cultivation effect.
3, the Chinese rose somatic embryo under the cultured in vitro can the long-term subculture preservation also can keep it further to be divided into the ability of plantlet.
Description of drawings
Fig. 1: the propagation of Chinese rose among the present invention ' purple mist ' somatic embryo.
Fig. 2: the sprouting of Chinese rose among the present invention ' purple mist ' somatic embryo.
Fig. 3: Chinese rose among the present invention ' purple mist ' somatic embryo is sprouted the whole plant that the back forms tool root and bud.
Fig. 4: the regeneration plant of the Chinese rose ' purple mist ' that field condition is grown down among the present invention.
Embodiment
Embodiment 1
Test material is selected, medium designs and inoculated and cultured
1, test material source and processing thereof:
Test material among the present invention is selected from the robust growth of taking to be grown in the Hua Zhong Agriculture University flower and plant base, the immature fruit (8 weeks after the open pollination) of the good modern rose cultivars of no damage by disease and insect " purple mist " (the primary source kind is the commercial variety available from Wuchang, Wuhan City, Hubei Province Hubei Prov. Acdemy of Agricutural Sciences flower plants and nursery stock market).Fruit processing method: the immature fruit of the Chinese rose ' purple mist ' of gathering is cut, take out seed in it, select the full seed that in clear water, can sink.Put into the mixed liquor (concentrated sulfuric acid by volume: the 30min that soaks seed water=3: 2) behind the running water flushing 30min.On superclean bench with 70% alcohol disinfecting 40s, and then with 0.1% mercuric chloride sterilization 10min, aseptic water washing 3-4 time.Strip immature zygotic embryos as explant.
2, medium design:
Table 1 is the composition and the consumption thereof of the various medium among the present invention.
The cultured in vitro base design of table 1 Chinese rose
Annotate: the preparation of MS minimal medium is referring to Murashige T.and F.Skoog.Physiol.Plant, 1962,15:473-497; The 1/2MS minimal medium is that the macroelement in the MS minimal medium reduces by half, trace element reduces by half, Fe salt reduces by half, organic element is constant.
Somatic embryo subculture medium in the table 1 as general subculture medium, also had been the somatic embryo long-term subculture medium of indication of the present invention both.
The code name of various compositions is as follows in the medium: and 2,4 dichlorophenoxyacetic acid (2,4-D), 6-benzyladenine (6-BA), plant gel (GEL) all can be from commercial purchase.
3, condition of culture
24 ± 2 ℃ of culturing room's cultivation temperature, intensity of illumination 1000-1500lx, periodicity of illumination are 14h; 24 ± 2 ℃ of dark culturing temperature.
4, inoculation and cultivation:
On the superclean bench immature zygotic embryos of Chinese rose ' purple mist ' is being inoculated on the middle inducing culture (medium is as shown in table 1) 10-12 explant of inoculation in each culture dish, dark culturing.Have somatic embryo to produce after two months on the immature zygotic embryos, then browning death of the explant that has fails to induce somatic embryo.
Somatic embryo is inoculated on the somatic embryo proliferated culture medium (being shown in Table 1), under illumination condition, cultivates.These somatic embryos can be on the somatic embryo proliferated culture medium fast breeding (see figure 1), somatic embryo is inoculated on the somatic embryo subculture medium (being shown in Table 1), but long preservation.Mature somatic embryo is inoculated into somatic embryo becomes on the seedling medium (being shown in Table 1), the somatic embryo maturation is also sprouted (see figure 2).The somatic embryo of sprouting is inoculated into fresh somatic embryo becomes on the seedling medium, the somatic embryo of Chinese rose ' purple mist ' can become direct one-tenth seedling (see figure 3) on the seedling medium at somatic embryo owing to have the radical bud bipolar structure.
After 1 month, the Chinese rose regeneration plant that root system development is good moves on under the natural conditions of scattered light, one week of hardening, the taking-up plantlet of taking root, clean the root medium, be transplanted to then peat soil is housed: in the plastic cup of perlite (volume ratio 1: 1), the low light level was cultivated 3-4 days down, make it grow new root, under high light, cultivate again.When in plastic cup, seeing obviously new root, it is transplanted in the earthen basin that peat soil is housed, make its healthy growth (see figure 4).
Embodiment 2
2, the influence that 4-D and the combination of 6-BA variable concentrations take place Chinese rose ' purple mist ' somatic embryo
Immature zygotic embryos with Chinese rose ' purple mist ' is the generation of explant induction somatic embryo.When 2, when 4-D concentration was 2mg/L, the highest inductivity of ' purple mist ' somatic embryo was 11.11%.It is dead not generate brownization of generation that the immature zygotic embryos of somatic embryo has, the generation non-embryonic callus tissue that has.Its experimental result is as shown in table 2:
Table 22, the influence that 4-D and the combination of 6-BA variable concentrations take place Chinese rose ' purple mist ' somatic embryo
Embodiment 3
Different medium are to the influence of sprouting of Chinese rose ' purple mist ' somatic embryo and plant regeneration
Different medium is as shown in table 3 to Chinese rose ' purple mist ' sprouting of somatic embryo and the influence of plant regeneration: in the somatic embryo sprouting test, there is significant difference in the influence that 9 kinds of medium are sprouted the Chinese rose somatic embryo.On MS+0.5mg/L 6-BA+3%Glucose+0.25%GEL medium, ' purple mist ' somatic embryo germination rate is the highest, reaches 86.67%, and unit bodies blast planting percent is up to 98.88%.6-BA does not have obvious facilitation to the sprouting and the plant regeneration of Chinese rose ' purple mist ' somatic embryo, and the effect of 6-BA is better than TDZ.The plant regeneration process of Chinese rose ' purple mist ' somatic embryo, root and bud take place simultaneously, can form the whole plant of tool root and bud, do not need to carry out culture of rootage.
The different one-tenth of table 3 seedling medium is to the influence of sprouting of Chinese rose ' purple mist ' somatic embryo and plant regeneration
Annotate: data show is mean value ± standard error, and different letter representations are significant difference on P＜0.05 level.Somatic embryo sum * 100% of the somatic embryo number/inoculation of somatic embryo germination rate (%)=sprouting; Somatic embryo sum * 100% of unit bodies blast planting percent (%)=one-tenth seedling sum/inoculation.
Claims (4)
1. one kind is regenerated as the method for whole plant with Chinese rose, and it comprises the following steps:
1. be explant with the Chinese rose immature zygotic embryos, described explant is directly induced somatic embryo in inducing culture;
2. the somatic embryo of step in 1. is inoculated in the somatic embryo proliferated culture medium propagation and makes its further differentiation, obtain mature somatic embryo;
3. the mature somatic embryo that 2. step is obtained is inoculated into somatic embryo and becomes in the seedling medium to be regenerated as whole plant;
Wherein
Described inducing culture composed as follows: 1/2MS minimal medium, 2,4 dichlorophenoxyacetic acid 0-3.0mg/L, 6-benzyladenine 0-1.0mg/L, glucose 30g/L, plant gel 2.5g/L;
Described somatic embryo proliferated culture medium composed as follows: MS minimal medium, 2,4 dichlorophenoxyacetic acid 1.0mg/L, 6-benzyladenine 0.05mg/L, glucose 60g/L, plant gel 2.5g/L;
Described somatic embryo becomes the composed as follows of seedling medium: the MS minimal medium, 6-benzyladenine 0-1.0mg/L, glucose 30g/L, plant gel 2.5g/L or MS minimal medium, azoles diazobenzene stem urea (TDZ) 1.0mg/L, 6-benzyladenine 0.5mg/L, glucose 30g/L, plant gel 2.5g/L.
2. method according to claim 1, the 2,4 dichlorophenoxyacetic acid in the wherein said inducing culture are 2.0mg/L, and 6-benzyladenine is 0.5mg/L.
3. method according to claim 1, it is 0.5mg/L that the thin blast embryo of wherein said body becomes the 6-benzyladenine in the seedling medium.
4. method according to claim 1, wherein the 2. described method of step also comprises described somatic embryo is inoculated on the somatic embryo subculture medium, makes its long-term subculture and keeps differentiation capability.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102405830A (en) * | 2011-06-07 | 2012-04-11 | 河南科技学院 | Induction method of rosa chinensis receptacle callus tissues |
| CN102870681A (en) * | 2012-10-22 | 2013-01-16 | 延安大学 | Method for flowering test-tube plantlets of Chinese roses in test tube and carrying out pistil monosexual flowering and culture medium thereof |
| CN102884984A (en) * | 2012-11-05 | 2013-01-23 | 湖南农业大学 | Somatic embryogenesis and plant regeneration method of Rosa chinesis |
| CN103355174A (en) * | 2013-08-05 | 2013-10-23 | 黑龙江省农垦科学院 | Low-cost high-efficiency industrialized seedling production method for cold-resistant Chinese rose |
| CN113455395A (en) * | 2021-07-30 | 2021-10-01 | 辽宁省经济作物研究所 | Device for stripping immature embryo of Chinese rose and culture method thereof |
-
2010
- 2010-08-17 CN CN2010102569019A patent/CN101946704B/en not_active Expired - Fee Related
Non-Patent Citations (2)
| Title |
|---|
| 《Plant Cell Rep》 20041231 Kamo et al Dispersal and size fractionation of embryogenic of callus increases the frequency of embryo maturation and conversion in hybrid tea roses 787-792 1-4 , 第22期 2 * |
| 《Plant Cell Tiss Org Cul》 20031231 Kim et al Control of direct and indirect somatic embryogenesis by exogenous growth regulators in immature zygotic embryo cultures of rose 61-66 1-4 , 第74期 2 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102405830A (en) * | 2011-06-07 | 2012-04-11 | 河南科技学院 | Induction method of rosa chinensis receptacle callus tissues |
| CN102870681A (en) * | 2012-10-22 | 2013-01-16 | 延安大学 | Method for flowering test-tube plantlets of Chinese roses in test tube and carrying out pistil monosexual flowering and culture medium thereof |
| CN102870681B (en) * | 2012-10-22 | 2015-08-19 | 延安大学 | The method that Tissue Culture Shoot of Rosa Chinensis Jacq tubers in vitro and gynoecium unisexuality are bloomed and medium thereof |
| CN102884984A (en) * | 2012-11-05 | 2013-01-23 | 湖南农业大学 | Somatic embryogenesis and plant regeneration method of Rosa chinesis |
| CN103355174A (en) * | 2013-08-05 | 2013-10-23 | 黑龙江省农垦科学院 | Low-cost high-efficiency industrialized seedling production method for cold-resistant Chinese rose |
| CN113455395A (en) * | 2021-07-30 | 2021-10-01 | 辽宁省经济作物研究所 | Device for stripping immature embryo of Chinese rose and culture method thereof |
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