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Rengong Meng, Chad E. Finn, and Robert P. Doss

Knowledge of the chromosome number in Rubus would be valuable when planning crosses and identifying plants, etc., however, preparation of tissue for microscopic evaluation and chromosome counting is difficult and time-consuming. Flow cytometry offers a more-efficient approach to this task. DNA flow cytometry was used to determine the nuclear DNA content in 22 Rubus genotypes. The genotypes represented a range of reported chromosome numbers from 2x to 12x. Six of the genotypes were representatives of Rubus ursinus, which is reported to have both 8x and 12x forms. Samples of nuclei were prepared from leaf discs of newly emerged and mature leaves following published protocols with some modifications. The DNA content was estimated by comparison of the fluorescence of Rubus nuclei with an internal DNA standard. There was an increase in nuclear DNA content concurrent with the increase in chromosome number. In these studies DNA flow cytometry could differentiate genotypes that differed by 2x, such as 6x and 8x, but could not reliably distinguish genotypes that differed by 1x, such as 7x vs. 8x or 6x. Aneuploids cannot be differentiated at this time.

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Chockpisit Channuntapipat, Margaret Sedgley, and Graham Collins

Leaf explants were taken from mature leaves of two almond [Prunus dulcis (Miller) D.A. Webb] cultivars, Ne Plus Ultra and Nonpareil selection 15-1, and maintained in vitro to grow shoot tips. Shoot tips were grown also from a pre-existing in vitro culture of an almond-peach rootstock, P. dulcis `Titan' × P. persica `Nemaguard'. The shoot tips were harvested, cryopreserved, and tested for survival after 3 days and then at intervals of 3 months up to two years. The mean survival was 80% for `Ne Plus Ultra', 54% for `Nonpareil', and 78% for the hybrid rootstock, and there were no significant differences in survival between 3 days and 24 months. The effects of in vitro culture and cryopreservation on DNA integrity were examined by both RAPD-PCR, and restriction enzyme digestion followed by RAPD-PCR, using DNA from the original trees from which the explants were derived, from leaves regrown from cultures that had undergone several passages of in vitro culture, and from leaves regrown from cryopreserved shoot tips. No detectable differences were found between the DNA fingerprints of each DNA sample using RAPD-PCR with seven different 10-mer primers. However, differences were detected when the DNA was first digested with the isoschizomeric pairs, Hpa II/Msp I and Bsp 143 I/Mbo I and then subjected to RAPD-PCR with six different 10-mer primers. Changes in the structure and methylation of DNA were found that were probably related to the process of in vitro culture, and in addition, methylation changes were detected that were probably associated with the cryopreservation process. These changes did not appear to be caused by the vitrification solution used before immersion of shoot tips in liquid nitrogen. While cryopreservation appears to be an ideal method for the long-term storage of almond germplasm, the significance of the alterations to both methylation and structure of DNA needs to monitored in regenerated plants, especially as they relate to agronomic performance when the regenerants become reproductively mature.

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John O. Williams III, Nick Gawel, and Jiang-Tian Ling

The lengthy generation times associated with woody ornamentals has led to the exploration of alternatives to traditional breeding methods for the development of new cultivars. This report summarizes the results of experiments designed to examine the feasibility of altering plant morphology by DNA assimilation following electroporation of intact seedlings. Brassica rapa (a nonwoody plant) was chosen as a model plant for initial experiments due to its rapid development and short generation time. Seedlings were subjected to either one or five 300-V pulses (99 ms) in the presence or absence of foreign DNA. Foreign DNA used was Ulmus parvarvifolia at a concentration of 200 μg·mL–1. Results indicate a lower survival rate of seedlings electoporated in the presence of DNA. Data were recorded after 21 days for plant height and leaf number. No significant differences were noted for plant height. However, significantly more leaves were produced on plants electroporated (five pulses) in the presence of foreign DNA. These results suggest the possible utilization of this technique for induction of variation in other plants.

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W.E. Jones, A.R. Kuehnle, and K. Arumuganathan

Flow cytometry (FC) has proven to be an efficient and reliable method to estimate nuclear DNA content (genome size) in quantifiable units useful for genetic and molecular biology studies. This method also makes possible determination of the variation in nuclear DNA content between related taxa, which gives insights into the process of speciation. In this study, DNA content was determined in nuclei isolated from leaves of 21 Dendrobium species representing each of the major taxonomic groups used in the Univ. of Hawaii breeding program. Nuclei were mechanically isolated, stained with the nucleic acid-specific fluorochrom propidium iodide, and DNA content determined using a Coulter Epics 753 laser flow cytometer. Chicken erythrocyte nuclei (2C = 2.33 pg DNA) were used as an internal standard for direct comparative measurement. The mean diploid genome (2C) values for Dendrobium species ranged from 3.36 to 5.06 pg. Genome sizes were evaluated for possible use as discrete characters for taxonomic group assignment and compared to previous data on breeding compatibility and evolutionary relationship between species.

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Dan E. Parfitt and Maria L. Badenes

PCR amplification and restriction analysis of a 3.2 kilobase hypervariable chloroplast DNA, as well as hybridization of the entire restricted chloroplast genome with tobacco chloroplast DNA probes permitted the development of a phylogeny for 10 Pistacia species. The genus divided into two major groups. P. Vera was most ancestral. P. weinmannifolia, an Asian species, is most closely related to P. texana and P. mexicana, new world species. The 3 sp. are more recently diverged, suggesting that a common ancestor of P. texana and P. mexicana originated in Asia. P. integerrima and P. chinensis are distinct species while species within two tertiary groups were monophyletic, P. vera:P. khinjuk and P. mexicana:P. texana. A general evolutionary trend from large to small nuts and leaves with few, large leaflets to many, small leaflets was documented. Pistacia had an unusually low chloroplast DNA mutation rate, more than 20x less than expected.

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Yuri Nakamura and Shozo Kobayashi

Restriction fragment length analyses of mitochondrial DNA (mtDNA) and chloroplast DNA (cpDNA) were carried out on 11 cultivars of Japanese persimmon (Diospyros kaki Thunb.) and five other Diospyros species. Total genomic DNA was digested with seven restriction endonucleases, Southern blotted, and hybridized with five mtDNA probes (PstI or SalI fragments of Brassica campestris L. mtDNA) and one cpDNA probe [pTBal, BamHI fragment of Nicotiana tabacum (L.) cpDNA]. All Japanese persimmon cultivars displayed identical mtDNA and cpDNA fragment patterns, while polymorphisms among species were observed using both mtDNA and cpDNA probes. A low degree of polymorphism was observed between D. kaki, D. oleifera Cheng., D. kuroiwai Nakai, D. virginiana L., and D. lotus L., suggesting that these species are closely related. A high degree of polymorphism was observed between D. rhombifolia Hemsl. and the other five species, indicating that this species is more distantly related to the other five.

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François Luro, Frédéric Laigret, Joseph-Marie Bové, and Patrick Ollitrault

We used three short repetitive nucleotide sequences [(GTG)5, (TCC)5, and (GACA)4] either as radiolabeled probes for hybridization with restricted citrus DNA or as single primers in polymerase chain reaction amplification experiments with total genomic DNA. We tested the ability of the sequences to discriminate between seedlings of zygotic or nucellar origin in the progeny of a Volkamer lemon (Citrus volkameriana Ten. & Pasq.) tree. The genetic variability within two species [Citrus sinensis (L.) Osbeck (sweet oranges) and Citrus reticulata Blanco and relatives (mandarins)] also was evaluated. DNA amplified fingerprinting with single primers was the more successful technique for discriminating between nucellar and zygotic seedlings. Although we were not able to distinguish among 10 cultivars of C. sinensis, all 10 C. reticulata cultivars tested were distinguishable. However, it still is difficult to identify the putative parents of a hybrid plant when the two parental genomes are closely related.

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Jean-Guy Parent and Danièle Pagé

Five polymorphic random amplified polymorphic DNA (RAPD) markers for 13 red raspberry (Rubus idaeus L.) and two purple raspberry (R. idaeus L. × R. occidentalis L.) cultivars were cloned and their termini sequenced. Sequence-specific 24-mer primer pairs were synthesized as extended RAPD primers and used in sequence characterized amplified region (SCAR) DNA analysis. All primer pairs generated polymorphic SCAR markers of the original RAPD marker sizes and length variants. Markers from four of the primer pairs could be easily scored and were adequate to identify the raspberry cultivars of the certification program of the province of Quebec.

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K. Haghighi and J.F. Hancock

Restriction fragment analyses of chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) were carried out on the principal cytoplasms of northern highbush cultivars and one representative of Vaccinium ashei Reade. Twenty-three restriction enzymes were used to identify variation and clarify mode of organelle inheritance. All species and genotypes displayed identical cpDNA fragment patterns, but high degrees of polymorphism were observed in the mitochondrial genomes. `Bluecrop' and `Jersey' did not appear to have `Rubel' cytoplasm as was previously believed. All hybrids contained maternal-type mtDNA.

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Patricia M. Sweeney and T. Karl Danneberger

The usefulness of random amplified polymorphic DNA (RAPD) in characterizing two perennial ryegrass (Lolium perenne L.) synthetic cultivars, `Accolade' and `Caravelle', was tested. Two out of 10 arbitrary primers produced three RAPD markers that distinguished bulk samples of 30 seedlings. Additional fragments were apparent when DNA from individual seedlings was amplified. Amplification products from bulk samples were not simply the sum of amplification products of individual seedlings and did not reflect all the diversity within or between the cultivars. The study illustrates the need to screen individuals to accurately evaluate the genotypic composition of a synthetic cultivar or heterogeneous population.