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1.
Somatic hybrids were obtained from electrofused protoplasts derived from embryogenic suspension cultures of tetraploid cotton (G. hirsutum L. cv. Coker 201) and embryogenic callus of diploid wild cotton G. davidsonii. The regenerants were initially identified as hybrids by RAPD (random amplified polymorphic DNA) analysis. Subsequently, observation on chromosome counting, morphology and SSR (simple sequence repeat) confirmed the hybrid status. Cytological investigation of the metaphase root-tip cells of the regenerated plants revealed there were 74 to 84 chromosomes in the plants, close to the expected 78 chromosomes. SSR analysis revealed the regenerated plants contained specific genomic fragments from both fusion partners, further confirmed their hybridity. The morphology of the plants was intermediate between the two fusion partners. The regenerants were difficult to develop into mature plants because their roots browned and they wilted from the stem apex before forming 3 to 5 true leaves. The hybrid plants were transferred to soil by grafting in vitro onto rootstocks.  相似文献   

2.
Random amplified polymorphic DNA (RAPD) markers were used to evaluate genetic stability of regenerants of soybean plants obtained through somatic embryogenesis using 180 μM 2,4‐dichloro‐phenoxy acetic acid. Twenty primers were used to screen 44 regenerants from the cultivar ‘Spring’ and 28 from the cultivar ‘CAC‐1’. Three primers were polymorphic for two of the ‘Spring’‐derived regenerants, with a somaclonal frequency of 4.5%. Four primers were polymorphic for the ‘CAC’‐l‐derived regenerant, with a somaclonal frequency of 3.57%. The results indicate the usefulness of RAPD markers to detect genetic instability in soybean primary regenerant plants derived from somatic embryogenesis, and as a certification tool for monitoring genetic stability during the regeneration process.  相似文献   

3.
The effect of regeneration of Lolium perenne and Festuca rubra from embryogenic suspension cells and protoplasts on fertility and growth was evaluated. Embryogenic suspension cultures were either routinely subcultured or cryopreserved and re-established. Phenology, morphology and fertility of regenerated plants were studied for two growing seasons in a replicated field experiment. Most regenerated L. perenne and F. rubra plants showed a delay in inflorescence emergence, a reduced seed yield and differences in morphological traits when compared with seed-grown plants. For L. perenne, performance of plants regenerated from cryopreserved suspension cultures and protoplasts was similar to that of respective plants regenerated from routinely maintained suspension cultures. However, differences in performance were observed for respective regenerants in F. rubra. The phenotypic deviation observed was partly reflected in the randomly amplified polymorphic DNA (RAPD) analysis performed. However, regenerants of both species showing similar, or even superior performance to the seed-grown plants were also found. Embryogenic suspension cells and corresponding protoplasts of L. perenne and F. rubra have the potential for producing fertile, well-performing plants which can be integrated in breeding programs.  相似文献   

4.
Five populations of lemon plants [Citrus limon (L.) Burm] obtained from undeveloped ovules through different tissue culture procedures were examined for the presence of somaclonal and irradiation-induced genetic variation. Tested groups were: (1) nucellar seedlings; (2) organogenic, regenerated via adventitious buds from nucellar seedling internodes; (3) embryogenic population, regenerated from non-irradiated nucellar callus via somatic embryogenesis; (4) embryogenic population, regenerated from irradiated nucellar callus via somatic embryogenesis; and (5) protoplast-derived, regenerated via somatic embryogenesis. Genomic DNA samples from 360 plants (72 from each group) were screened for polymorphism among RAPD fingerprints amplified by 10 decamer primers. Among all tested plants, genetic variation was detected only within the group of plants recovered from irradiated embryogenic calli. Out of 72 plants from that group, three had RAPD fingerprints different from the rest of the population, and fourth plant was found to be cytochimeric, consisting of diploid and tetraploid cells as revealed by flow cytometry. In all other populations of regenerated plants, we did not come across any plants with changed ploidy level.  相似文献   

5.
Genetic modification from selfed progenies of 18 rice (Oryza sativa L.) plants regenerated from callus tissues which survived desiccation, were investigated at the DNA level using the random amplified polymorphic DNA (RAPD) method. Twelve 10-mer random primers were used to amplify DNA of progenies from the regenerated plants, and a total of 228 PCR products and 1780 DNA fragments were obtained by primers, generating between four to thirteen major bands. The size of the amplified fragments ranged from 0.2 to 2.55 kb. The results showed that 10 out of 12 primers produced polymorphic bands, two primers (RA31 and RA185) showed no polymorphism among plants tested. A dendrogram of the genetic distance was constructed based on their polymorphism, demonstrating that somaclonal variation exists in rice plants regenerated from callus which survived the desiccation treatment. Part of this variation can be useful in rice breeding. This revised version was published online in August 2006 with corrections to the Cover Date.  相似文献   

6.
In order to assess the genetic variability among regenerants. field trials were conducted with 64 barley lines derived from tissue culture (TCD lines) of four Bulgarian barley varieties. The results indicate that the agronomic performance was altered by tissue culture regeneration and the frequency of variation was cultivar dependent. Seven TCD lines showing desirable agronomic characters were selected in the SC16 progeny. Molecular markers (protein, restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) have used applied to determine the variability induced by tissue culture. Polymorphism was detected in sequences coding for C-hordeins in line 70 derived from cv.‘Jubiley’Two lines from cv.‘Ruen’exhibited polymorphic bands after hybridization with a mitochondrial DNA probe. RAPD assays have been carried out using 20 different l0-mer primers. Heritable polymorphisms in several TCD lines have been observed.  相似文献   

7.
Summary Somatic embryogenesis was initiated from immature embryos on Murashige-Skoog (MS) medium plus 2 mg.l-1 2,4-dichlorophenoxyacetic acid, 2% sucrose and 0.6% agarose. Somatic embryos were isolated and regenerated into whole green plants on MS medium devoid of 2,4-D. These regenerants were previously demonstrated to differ in their mitochondrial DNA organization. In order to estimate their characteristics three progenies of short-term culture regenerants and three progenies of long-term culture regenerants were analyzed and compared to the parental line. These somaclones obtained from the wheat variety Chinese Spring were evaluated for variation of 13 agronomic and morphological quantitative characters in comparison to the parental line. Significant variation was observed for plant height, spike length, main tiller diameter, between the somaclones regenerated from long-term culture and their parent. Differences were observed to increase with the duration of culture, leading to a significant modification of the structure of the plants. Several changes occurred during the somatic tissue cultures, but to a lesser extent than has previously been described in the literature.  相似文献   

8.
Plants were regenerated from intergeneric somatic hybridization between embryogenic protoplasts of Microcitrus papuana Swingle and leaf-derived protoplasts of sour orange (Citrus aurantium L.) via electrofusion. The regenerated plants were morphologically similar to the leaf parent in growth vigor, leaf and branch structure. FCM analysis showed that they were diploids. Simple-sequence-repeat (SSR) and cleaved-amplified-polymorphic-sequence(CAPS) were employed for hybridity characterization. SSR banding patterns of the regenerated plants were identical to the leaf parent, sour orange, indicating that they possessed nuclear component derived from sour orange. DNA amplification with chloroplast and mitochondrial universal primers, followed by restriction endonuclease digestion, revealed polymorphism between the fusion parents. Therefore, this method was used to determine the cytoplasmic compositions of the regenerated plants. Banding patterns for all the polymorphic primer/enzyme combinations of the regenerated plants were similar to those of the embryogenic parent, M. papuana, suggesting that only the cytoplasmic components derived from the embryogenic parent were present in the regenerated plants. FCM, SSR and CAPS demonstrated that intergeneric diploid cybrids have been successfully obtained by symmetric fusion. Related results concerning nuclear and cytoplasmic composition of previous diploid somatic hybrids and potential mechanism for regeneration of such kind of plants are discussed herein. This revised version was published online in July 2006 with corrections to the Cover Date.  相似文献   

9.
A plant regeneration system from rice protoplasts using calli derived from mature embryos was established for the two Brazilian modern rice cultivars IAC-201 and IAC-165. After 30 to 40 days of in vitro culture it was possible to obtain on average 6 million protoplasts per gram of callus. Microscopic selection of embryogenic calli was a key step for protoplast isolation. The production of embryogenic calli increased when L-proline and casein hydrolysate were used in the callus induction medium. The Oc or IR52 nurse cell lines were essential for protoplast division. Different regeneration media were studied and 139 plants were regenerated which set seed. Some of the regenerated plants showed morphological variation such as the presence of awns in spite of the short time of the in vitro culture. This revised version was published online in July 2006 with corrections to the Cover Date.  相似文献   

10.
Summary Intergeneric symmetric and asymmetric somatic hybrids have been obtained by fusion of metabolically inactivated protoplasts from embryogenic suspension cultures of tall fescue (Festuca arundinacea Schreb.) and unirradiated or 10–500 Gy-irradiated protoplasts from non-morphogenic cell suspensions of Italian ryegrass (Lolium multiflorum Lam.). Genotypically and phenotypically different somatic hybrid Festulolium mature flowering plants were regenerated.Species-specific sequences from F. arundinacea and L. multiflorum being dispersed and evenly-represented in the corresponding genomes were isolated and used for the molecular characterization of the nuclear make-up of the intergeneric, somatic Festulolium plants recovered. The irradiation of Italian ryegrass protoplasts with 250 Gy X-rays prior to fusogenic treatment favoured the unidirectional elimination of most or part of the donor chromosomes. Irradiation of L. multiflorum protoplasts with 500 Gy produced highly asymmetric (over 80% donor genome elimination) nuclear hybrids and clones showing a complete loss of donor chromosomes.The RFLP analysis of the organellar composition in symmetric and asymmetric tall fescue (+) Italian ryegrass regenerants confirmed their somatic hybrid character and revealed a bias towards recipient-type organelles when extensive donor nuclear genome elimination had occurred.Approaches aimed at improving persistence of ryegrasses based on asymmetric somatic hybridization with largely sexually-incompatible grass species (F. rubra and Alopecurus pratensis), and at transferring the cytoplasmic male sterility trait by intra- and inter-specific hybridization in L. multiflorum and L. perenne, have been undertaken.Abbreviations cpDNA chloroplast DNA - CMS cytoplasmic male sterility - 2,4-D 2,4-dichlorophenoxy-acetic acid - IOA iodoacetamide - mtDNA mitochondrial DNA - RFLP restriction fragment length polymorphism  相似文献   

11.
C. Halldén    B. Gertsson    T. Säll  C. Lind-Halldén   《Plant Breeding》1993,111(3):185-191
In order to improve regenerative potential in Beta vulgaris L. cultural conditions were established for efficient plant regeneration from cotyledon-derived calli. Organogenic calli were initiated at frequencies of 8–59 % with six sugarbeet lines. N6-benzyl-ammopurine (1 mg/1) associated with 2,3,5 triiodobenzoic acid (0.5 mg/1) was found to be the best combination to alter the endogenous phytohormonal balance in 21 ± 1 day-old seedlings. Flow cytometry was used to determine nuclear DNA Content (ploidy) in cotyledons, calli and regenerated plants, and suggested that 88 % of the regenerants were diploids. The relatively high cytological stability of the regenerants was attributed to the rapid cell differentiation within initiated calli. The problem of vitrification was overcome by using phloridzin and high agar concentration.  相似文献   

12.
Pollen embryos were obtained from eight genotypes and viable green plants were regenerated from four genotypes in an anther-culture experiment with 165 genotypes of Phleum pratense L. Formation of proembryos inside the cultured anthers during the first 10—12 days was significantly influenced by genotypes and by the type of nutrient media. Primary embryos developed into multiple secondary embryos before regeneration of plants. Among a total of 62 plants regenerated, only 13 were albinos. Of the green regenerants, 11 were triploid while 35 were hexaploid when DNA-content was measured by flow cytometry. Eight plants with a triploid DNA-content did possess the triploid chromosome number of 21. Triploid and hexaploid regenerants from two different parents showed simplified isozyme (GPI and PGD) banding patterns relative to that of their parents.  相似文献   

13.
In the present study, embryogenic calli of sugarcane variety BL4 were induced using 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin in different concentrations and combinations. In contrast to earlier studies, embryogenic callus sectors were identified and isolated microscopically within 1–2 weeks. Subsequently, 51 media formulations were used for regeneration of proliferated embryogenic callus, using MS medium supplemented with three different cytokinins [kinetin, 6-Benzylamino purine (BAP), and thidiazuron (TDZ)] and auxins (IAA/NAA and IBA) in different combination and concentrations. After acclimatization, the genomic DNA of regenerated plants was studied to explore the insertion polymorphism of transposable elements in order to ascertain the variation among somaclones. Though low concentration of kinetin with 2,4-D was found supportive to embryogenic callus development, the highest number of regenerated plantlets was observed using BAP (1 μM), however the plantlets had very low fresh weight (2.2 g). Conversely, TDZ alone supported a significant increase in the number of plantlets regenerated (38–40) with higher fresh weight. The somaclones generated during this study showed considerable positional polymorphism of activator-like transposable elements possibly due to the stress associated with in vitro culture. This study provides a procedure to produce regenerated sugarcane plants from embryogenic callus in a relatively short time.  相似文献   

14.
Summary Plant regeneration from cultured immature inflorescence segments of Eleusine coracana was obtained by direct shoot development and somatic embryogenesis. Direct development of shoots from cultured inflorescence segments occurred on MS medium supplemented with 2,4-D in combination with zeatin. Inflorescences with well developed spikelets differentiated at a low frequency (<5%) from callus cultures initiated on media supplemented with 2,4-D in combination with zeatin or coconut water or picloram + kinetin. Somatic embryogenesis was also induced in callus cultures growing on MS + picloram + kinetin at the end of four passages. Supplementation of the media with different concentrations of sucrose showed 3% sucrose as the best concentration for plant differentiation from somatic embryos. The majority of the regenerated plants showed the diploid chromosome constitution in their root tips. The regenerants were in general shorter with an increased number of tillers compared to the control.Abbreviations CW Coconut water - 2,4-D 2,4-dichloro phenoxyacetic acid - Kn Kinetin - Z Zeatin  相似文献   

15.
B. Javornik    B. Bohanec  B. Campion 《Plant Breeding》1998,117(3):275-278
Haploid induction in onion via gynogenesis provides inbred lines that can be used in production of hybrid cultivars. Despite several successful reports, the gynogenic induction rate is still relatively low, ranging mainly from 0.5 to six embryos per 100 cultured flowers. In the present study, gynogenesis was induced in selfed progeny of three genetically homozygous and stable fertile doubled haploid lines previously obtained through in vitro gynogenesis. The aims were to acquire information on whether the gynogenic responsiveness of a particular heterozygous cultivar is correlated with higher or lower gynogenic capacity in the second cycle of gynogenesis, to evaluate the non-genetic component of the variability, and to monitor by random amplified polymorphic DNA (RAPD) analysis the onset of eventual gametoclonal variation induced during the gynogenic processes. The results from the gynogenic induction of 9708 flowers showed significant differences among three lines, one of them being extremely responsive, showing a mean yield of 118.3 haploid embryos per 100 cultured flowers (118.3%). The high embryo yield of this line was determined by the line's particular genotype. However, the induction rate of gynogenic embryos obtained within 24 genetically uniform donor plants of this responsive line varied from 67 to 196% and this variation can be attributed to uncontrolled environmental conditions. A total of 157 plants sampled from two lines and obtained through a first (RI) and a second (SCG) cycle of gynogenesis, was analysed by RAPDs. The single band profiles were compared either between or within the RI and SCG plants of each line. A total number of 4600 RAPD fragments were scored and no variation was found in the highly responsive line, while in the less responsive one, minor changes were detected for the presence/absence of two RAPD bands. These results indicate high genetic stability of onion homozygous lines passed through two cycles of gynogenesis.  相似文献   

16.
高效的水稻基因枪转化和可育转基因植株再生   总被引：7，自引：0，他引：7  
许新萍  胡明 《作物学报》1999,25(6):691-699
从水稻品种台北309成熟胚诱导生长3~4周的愈伤组织,经过1~3次继代培养后,可以形成大量颗粒状胚性愈伤组织,适用于基因枪转化实验。将分别含有雪花莲凝集素(GNA)基因、h pt基因的质粒pIP860和p35H混合包裹在金粉上轰击转化上述胚性愈伤组织,在含30~50mg/L潮霉素的培养基上进行筛选、预再生、再生及长根培养。使用干燥处理  相似文献   

17.
Summary We have used random amplified polymorphic DNA (RAPD) analysis to characterize eleven cultivars of the five economically most important yam species grown in Jamaica (Dioscorea alata, D. cayenensis, D. rotundata, D. trifida and D. esculenta). Amplification of genomic DNA samples with nine different arbitrary 10mer primers revealed a total of 338 different band positions, ranging in size from 0.3 to 2.5 kb. RAPD patterns proved to be highly reproducible and somatically stable. While no variation was observed among plants belonging to the same cultivar, a large number of intervarietal and interspecific polymorphisms enabled us to reliably discriminate between all Jamaican cultivars investigated.  相似文献   

18.
The morphological and genetic variations in somaclones of chili pepper (Capsicumannuum L.) derived from tissue culture were evaluated. Cotyledonary node explants of cultivars, Shishitou and Takanotsume, were cultured on Murashige and Skoog (MS) medium supplemented with benzyladenine (BA)5 mg/l for shoots regeneration and regenerated shoots were rooted on MS medium supplemented with naphthalene acetic acid(NAA) 0.1 mg/l and indol-3-butyric acid(IBA) 0.05 mg/l. The regenerated plants(R0) were selfed to obtain seeds for next generation (R1 lines). Qualitative characters were studied in R0generation and both qualitative and quantitative characters were studied in R1 generation. In R0 generation, variations were noticed in plant growth habit, stem color, flower color and color of unripe fruits, and expression of anthocyanin in unripe fruits. Comparison among the R1 lines and their parents were made for morphological and agronomic characters. Significant variation among R1 lines and differences between R1 lines and their parents were observed. Genetic variations among three somaclones were revealed by random amplified polymorphic DNA (RAPD) analysis. Variation, such as early flowering and increase of yield components, is an indication of the response to selection for any specific character. Occurrence of productive variants among somaclones of established cultivars, like Shishitou and Takanotsume, indicates the possibility of their improvement through somaclonal variation. This revised version was published online in July 2006 with corrections to the Cover Date.  相似文献   

19.
F. J. Novak    S. Daskalov    H. Brunner    M. Nesticky    R. Afza    M. Dolezelova    S. Lucretti    A. Herichova  T. Hermelin 《Plant Breeding》1988,101(1):66-79
Sixteen inbred lines and one hybrid of manse were tested for their capability of somatic embryogenesis, and fully developed plants could be regenerated, from ten inbred, lines. The highest frequency of plant regeneration was expressed in the inbred line CHI 31, and when this line was crossed with a recalcitrant, non-regenerating line, the F1 and BC hybrids were regenerable. The results of reciprocal crosses demonstrated that dominant nuclear genes and cytoplasmic factors are primarily responsible for the heritable determination of embryogenic callus proliferation and in vitro regeneration of maize plants. Somaclonal and radiation-induced variability was studied in maize to assess their nature and potential contribution to plant breeding., The inbred line CHI 31 possessing a high in vitro capacity of somatic embryo formation was used as experiments.] material. CHI 31 plants were selfed and twelve-day old zygotic embryos irradiated with 60Co gamma radiation in situ. Mature caryopses were harvested and assigned as M1 material. In another series, immature zygotic embryos (size 1.2—1.5 mm) were cultured in vitro on N-6 medium supplemented with 2,4-D (2.5 μM), and somatic embryos regenerated into plants; these were transplanted into soil and self-pollinated. Regenerants from non-irradiated cultures were grown as R1 generation, while regenerants from irradiated explants were considered as M1R1 generation. The genetic variability was evaluated in the M2, R2 and M2R2 generations, respectively, and compared with a non-treated seed control. Irradiation induced a variety of chlorophyll and morphological variants segregating in the M; generation; however, the frequency of deviant types obtained in the R: generation (somaclonal variation) was significantly exceeding the one derived from the M2 populations. The combination of expert irradiation and in vitro regeneration was most effective for the manifestation of chlorophyll and morphological o if types in the M2R2 generation, and increased drastically the frequency of early flowering variants. Differences in the segregation patterns of mutant phenotypes amonsister somaclones in the R3 and M3R3 generations indicate a different genetic basis, of plants originating from the same explant. This phenomenon suggests a mutational sectoring of the callus during culture. Radiation induced and somaclonal variation exerted a similar spectrum of chlorophyll and morphological deviants.  相似文献   

20.
甘薯胚性细胞悬浮培养系的建立   总被引：5，自引：0，他引：5  
地甘薯胚性细胞悬浮增减系的进行了研究。将１２个基因的长约０．５ｍｍ的茎尖培养在含有０．２ｍｇ／Ｌ或２．０ｍｇ／Ｌ２，４－Ｄ的ＭＳ培养基上，形成了胚性愈伤组织。胚性愈伤组织的形成率因基因型和２，４－Ｄ深度不同而很大差异，为０－７５．７％。一方面，将胚性愈伤组织继续增减在含有２，４－Ｄ的ＭＳ培养基上，它们形成了处于各发育时期的体细胞胚。将具有体细胞胚的胚性愈伤组织转移到ＭＳ基本培养基上，体细胞胚发育成  相似文献   

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