Stable transformation of barley callus using biolistic® particle bombardment and the phosphinothricin acetyltransferase (bar) gene

We have previously studied chromosomal and morphological variation in protoplast cultures of diploid petunia (Petunia hybrida) plants. We found that 85% of the regenerants were tetraploid (2n=4x=28). These plants flowered and set seeds.In the present study, the cytological stability of plants regenerated from leaf mesophyll protoplast cultures derived from the progeny of the self-fertile tetraploid plants was assessed on the basis of mitotic analysis, morphological characters, and protein patterns. When we analyzed the root tip chromosomes of 117 regenerants derived from 39 protoclone calluses, all of the regenerants tested retained the parental chromosome number of 2n=4x=28. One hundred regenerants were further analyzed and displayed normal vegetative morphology and retained the floral characteristics of the seed-derived plants from which they were derived. No significant variations in any character were observed among regenerants. When leaf protein patterns from four regenerated tetraploid protoclones were analyzed by two-dimensional polyacrylamide gel electrophoresis and compared with those of seed-derived plants, the protein patterns exhibited great similarity.The data suggest that tetraploidization of petunia plant increases cytological stability during further in vitro cultures and may play an important role in the genetic stability of regenerant populations.Abbreviations BA benzylaminopurine - IAA indole-3-acetic acid - IEF isoelectric-focusing - PVP polyvinylpyrrolidone - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Variations in the morphology of rice plants regenerated from protoplasts using different culture procedures

Rice protoplasts were cultured using 4 different culture procedures such as agarose embedding (AE) without feeder cells and the use of filter membranes (MEM), one layer of nylon mesh (MS1), or a double layer of nylon mesh (MS2) with the inclusion of Lolium multiflorum as feeder cells. The protoplast plating efficiency was highest on the MEM, followed by MS2, MS1 and AE. However, plant regeneration frequencies were highest for MS1, followed by MS2, MEM and AE. The protoclonal plants differed in the morphology of leaves, flowers, spikelets, and panicles in comparison to seed-derived plants. They varied in almost every phenotypic characters evaluated. In many cases, the variation was significantly different in characteristics such as plant height, flag leaf length and width and ratio, and in panicle characteristics such as panicle length, number of primary branches, and number of spikelets per panicle. The number of seeds per panicle was greatly reduced in protoclonal plants when compared with seed-derived control plants. The seeds showed also significant differences in grain length and width in comparison to the control plants. Among the 4 groups of protoclonal plants derived from the 4 different culturing procedures themselves, there were also variations in almost all the phenotypic characteristics assessed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effects of electric cell fusion treatment among leaf protoplasts of Populus alba and alnus firma on growth, leaf morphology, and rapd pattern of eleven acclimatized plants

Summary This study reports the characterization of 11 plants regenerated from electrically fused protoplasts between Populus alba and Alnus firma. Growth characteristics of five regenerated plants (AP-1-AP-5) in terms of shoot height and leaf color showed small differences compared with those of P. alba grown, in pots, and showed no difference in shoot height and diameter compared with those grown in nursery field. There was also no difference in the RAPD pattern between the plants regenerated from interfamilial protoplast fusion and P. alba. In contrast, the lately regenerated plants (AP-6-AP-11) grown in pots showed a marked difference in leaf morphology and RAPD pattern. There was a variation in the ratio of longitudinal to transverse length of leaves among the 11 plants from interfamilial fusions compared with that of protoclones and intraspecific fused protoplasts of P. alba.     

Uniformity of plants regenerated from orange (Citrus sinensis Osb.) protoplasts

Summary Using 25 plants (protoclones) regenerated from orange (Citrus sinensis Osb.) protoplasts, several characters, including leaf and flower morphology, leaf oil, isozyme patterns and chromosome number, were examined. No significant variations in each character were recorded among the protoclones. Uniformity observed among protoclones was identical to that of nucellar seedlings.     

Somatic hybrids produced by protoplast fusion between S. tuberosum and S. brevidens: phenotypic variation under field conditions

Summary Phenotypic and flowering characteristics of hybrid plants generated by protoplast fusion between a tetraploid S. tuberosum line and diploid S. brevidens were assessed under field conditions. Hybrids were compared to both clonal parental material and protoplast-derived plants of each parent. Almost all of the hybrids were hexaploid. A wide range of variation in morphological characters was observed for hybrids and protoclones. Flowering was markedly reduced in protoclones. The majority of hybrids flowered, had viable pollen and set tubers. Tuber and pollen characteristics of hybrids produced from individual fusion calli also varied. The potential usefulness of fusion hybrids in potato improvement is discussed.     

Protoclonal variation of plant regeneration in rice

Protoplasts were isolated from callus derived from a single homozygous seed of Oryza sativa L. var. Norin 8. Thirty protoclones were randomly selected and these showed variation in regeneration frequency ranging from 0–87% with an average of 52%. The potential for regeneration of each protoclone as reflected in the regeneration frequency was analyzed five times over a period of 250 days and showed that the protoclones can be classified into three types, namely: protoclones with high regeneration frequency; protoclones with low regeneration frequency, both of which maintained their respective levels of regeneration potential; and protoclones with gradually decreasing regeneration frequency. Secondary protoclones established from protoplasts isolated from some of these protoclones and regenerated 2–3 times for a period of 120 days also showed further reduction in regeneration frequency. The polypeptide composition analyzed by two dimensional gel electrophoresis suggests the presence of specific polypeptides related to regeneration potential. Analysis of ploidy level based on plant morphology and pollen size suggests the predominance of tetraploids among the regenerated plants.     

Efficient plant regeneration from cell suspension protoplasts of the woody medicinal plant Solanum dulcamara L. (bittersweet,woody nightshade)

Large populations of viable protoplasts were released from suspension cultured cells of the woody medicinal plant Solanum dulcamara (bittersweet, woody nightshade) when the cells were harvested 3 to 7 months after culture initiation and 4 to 5 days after transfer to fresh medium. A Bio-Gel p6 purified enzyme mixture enhanced the protoplast plating efficiency 6 fold compared to the unpurified mixture, without affecting protoplast yield. Agarose-solidified medium markedly improved protoplast division and colony formation, and enabled protoplasts to be plated at lower densities than in liquid medium. All protoplast-derived tissues produced shoots on MS based medium with 1.0 mgl^-1 zeatin. Shoots rooted readily on medium lacking phytohormones. Cytological examination revealed high chromosome stability of suspension cultured cells, of plants derived from such cells, and of protoplast-derived plants. The implication of these results is discussed in relation to the genetic manipulation of this pharmaceutically important plant.     

Genome modifications in protoplast-derived tobacco plants: Contents of repetitive DNA sequences

Plasticity of the tobacco genome was studied by testing the DNAs of protoplast-derived regenerants with three different repetitive DNA sequences by the method of quantitative DNA/DNA hybridizations. A large population of 91 regenerants belonging to 35 different protoclones was analysed and a high degree of heterogeneity in the contents of the different DNA repeats was detected. The contents of middle repetitive sequences of two types were more stable or changed in the same direction, while the highly repetitive sequence varied independently and displayed a significant reduction in comparison with the two other sequences. Comparing the variation within the subpopulations of plants of the same clonal origin and the variation among the protoclones led to a conclusion that the pre-existing DNA variability in the starting plant material and/or thein vitro stress during the very early stages of protoclone regeneration played a decisive role in the formation of modified genomes in regenerants.     

Transformation of plant mitochondria with mitochondrial DNA plasmids via protoplast fusion

Summary Brassica napus cybrid plants which contain novel nucleus-mitochondria-chloroplast combinations have been constructed, via protoplast fusion. Such fusions resulted in mitochondrial DNA plasmids being lost (at a frequency of 12.5%) or, more surprisingly, being transferred from mitochondria of one protoplast population to mitochondria of the other population (at a frequency of 6.1%). Mitochondria containing their new DNA complement became the dominant organelle population in regenerated plants and were faithfully maternally inherited through successive sexnal generations. No concomitant alterations in mitochondrial chromosome organization or nuclear chromosome number occurred. Protoplast fusion can, therefore, cure plant mitochondria of extrachromosomal DNA and, more importantly, be used to transform plant mitochondria with naturally occurring mitochondrial plasmids. The potential for mitochondrial transformation with recombinant vectors is discussed.     

Geographic and clonal variation in the milkweed-oleander aphid,Aphis nerii (Homoptera: Aphididae), for winged morph production,life history,and morphology in relation to host plant permanence

Summary Populations of the milkweed-oleander aphid,Aphis nerii, were sampled in California, Iowa and Puerto Rico. Among these localities the aphid's host plants differ greatly in permanence. I compared populations for migratory potential, measured as the proportion of winged offspring produced in response to being crowded, and for life history and morphometric traits of the subsequent adult winged aphids. I predicted a negative correlation between degree of host plant permanence and migratory potential. As predicted, aphids from Iowa, where migration on to temporary hosts must occur each year, produce a greater proportion of winged offspring (37.7%) than those from California (25.7%) or Puerto Rico (31.6%) where hosts are more permanent. However, hosts in Puerto Rico appear to be more permanent than those in California, yet the difference between populations for migratory potential was opposite to that predicted. Within California the prediction again held: aphids collected from the most impermanent sites produce the greatest proportion of winged offspring. There were no population differences for any life history or morphometric traits of winged aphids that are important contributors to fitness or migratory ability such as time to reproductive maturity, fecundity or wing length. Nor did any traits covary with migratory potential. Thus, there does not appear to be an association of life history and morphology with migratory potential that could enhance the colonizing ability of migrant aphids. I was unable to detect population differentiation for life history and morphology even though there is ample genetic variation within populations on which selection could act and an absence of constraints arising from genetic correlations that could prevent appropriate evolution of traits within populations. The exploitation of temporary host plants therefore occurs by an increase in the number of colonists produced and not by change in life history or morphology of those colonists.     

Asymmetric protoplast fusion between sweet potato and its relatives,and plant regeneration

Experiments were conducted to asymmetrically fuse protoplasts from sweet potato (Ipomoea batatas L. Lam.) and its wild relativesI. trifida Don. andI. lacunosa L. Protoplasts of sweet potato were treated with iodoacetamide, whereas those ofI. trifida Don. andI. lacunosa L. were irradiated with X-rays. The asymmetric protoplast fusion was carried out by the electrofusion method and by polyethylene glycol treatment. Electrically-fused protoplasts initiated cell division, and then formed calli earlier than the polyethylene glycol-fused protoplasts. Plant regeneration occurred only in electrofused calli, suggesting that polyethylene glycol had some toxic effect on plant regeneration ability. Analysis of peroxidase isozymes confirmed the interspecific hybrid characteristics of both the fusion-derived calli and regenerated plants.     

Influence of plant genotype and environment on oviposition preference and offspring survival in a gallmaking herbivore

Summary Plant resistance to insect herbivores may derive from traits influencing herbivore preference, traits influencing the suitability of the plant as a host, or both. However, the plant traits influencing host-plant selection by ovipositing insect herbivores may not completely overlap those traits that affect larval survival, and distinct traits may exhibit different levels of genetic vs. environmental control. Therefore, resource supply to the host plant could affect oviposition preference and larval performance differently in different plant genotypes. To test this hypothesis, the effects of resistance level, plant genotype, and resource supply to the host plant on oviposition preference and larval performance of a gallmaking herbivore, and on various plant traits that could influence these, were examined. Replicates of four genotypes of Solidago altissima, grown under low, medium, or high levels of nutrient supply in full sun or with medium levels of nutrients in shade, were exposed to mass-released Eurosta solidaginis. The number of plants ovipunctured was significantly affected by plant genotype and the interaction between genotype and nutrient supply to the host plant: one susceptible and one resistant genotype were more preferred, and preference tended to increase with nutrient supply in the more-preferred genotypes. The growth rate of ovipunctured plants during the oviposition period was significantly greater than that of unpunctured plants. Bud diameter (which was strongly correlated with plant growth rate), leaf area, and leaf water content were significant determinants of the percentage of plants ovipunctured, explaining 74% of the variance. The number of surviving larvae was significantly affected by plant genotype, but no effect of nutrient or light supply to the host plant was detected. The ratio of bud diameter to bud length was positively related to the percentage of ovipunctured plants that formed galls, suggesting that the accurate placement of eggs near the apical meristem by ovipositing females may be easier in short, thick buds. No significant correlation was observed between oviposition preference and larval survival at the population level. These results suggest that the plant traits affecting oviposition preference may exhibit different magnitudes of phenotypic plasticity than those affecting larval survival, and that the degree of phenotypic plasticity in plant traits affecting oviposition preference may differ among genotypes within a species.     

Genotyping of somatic hybrids between <Emphasis Type="Italic">Festuca arundinacea</Emphasis> Schreb. and <Emphasis Type="Italic">Triticum aestivum</Emphasis> L.

In order to genotype hybrid genomes of distant asymmetric somatic hybrids, we synthesized hybrid calli and plants via PEG-mediated protoplast fusion between recipient tall fescue (Festuca. arundinacea Schreb.) and donor wheat (Triticum aestivum L.). Seventeen and 25 putative hybrid clones were produced from the fusion combinations I and II, each with the donor wheat protoplast treated by UV light for 30 s and 1 min, respectively. Isozyme and RAPD profiles confirmed that ten hybrid clones were obtained from combination I and 19 from combination II. Out of the 29 hybrids, 12 regenerated hybrid plants with tall fescue phenotype. Composition and methylation-variation of the nuclear and cytoplasmic genomes of some hybrids, either with or without regenerative ability, were compared by genomic in situ hybridization, restriction fragment length polymorphism, and DNA methylation-sensitive amplification polymorphism. Our results indicated that these selected hybrids all contained introgressed nuclear and cytoplasmic DNA as well as obvious methylation variations compared to both parents. However, there were no differences either in nuclear/cytoplasmic DNA or methylation degree between the regenerable and non-regenerable hybrid clones. We conclude that both regeneration complementation and genetic material balance are crucial for hybrid plant regeneration.     

贵州特有药用植物毕节小檗的形态变异

为探讨贵州特有药用植物毕节小檗(Berberis guizhouensis)形态多样性的变异式样及其适应机制,对其野外居群营养器官和花部形态进行研究。对毕节小檗一直空缺的花部性状进行了描述。结果表明,毕节小檗的性状在同一居群或同一性状在不同居群中都存在较丰富的变异,变异系数为0.03~0.57。从总体来看,营养器官性状的变异程度要高于花部性状。单因素方差分析表明居群间部分花部性状存在显著差异(P0.05)。聚类分析表明小白岩居群形成1个独立的分支,这种分化很可能是环境压力选择的结果。主成分分析表明花部性状与营养性状在维持形态结构的稳定性具有协调一致的生态适应。     

Cytogeography and meiotic chromosome configurations of six intraspecific aneuploids ofCarex conica Boott (Cyperaceae) in Japan

Chromosome numbers were determined for 340 plants ofCarex conica from 83 populations in Japan. Six aneuploids, 2n=32, 33, 34, 36, 37 and 38, were found. Plants with even diploid chromosome numbers 2n=32, 34, and 36 were the most common and had different geographical distributions. Individuals with 2n=32 were from islands in the Seto Inland Sea and nearby coastal areas of the Chugoku District of Honshu; those with 2n=34 were from the Kanto, Chubu and Kinki Districts of Honshu; those with 2n=36 were from the mountainous areas of Chugoku, Shikoku and Kyushu Districts. Canonical discriminant analysis of 17 morphological characters demonstrated that the plants with 2n=32 were clearly distinct from those with 2n=34 or 36. All four aneuploids with even chromosome numbers showed normal bivalent pairing at meiotic metaphase I and probably represent cytogenetically stable cytodemes. Plants with 2n=33 had one heteromorphic trivalent and 15 bivalents, indicating a structural mutation. At mitotic metaphase I, one chromosome was markedly larger than the others, suggesting that the 2n=33 plants arose from 2n=34 plants by fusion of two chromsomes. The plant with 2n=37 was intermediate in morphology betweenCarex conica (2n=36) andC. morrowii (2n=38) and probably originated as an interspecific hybrid between these species.     

Population variation in plant traits associated with ant attraction and herbivory in Chamaecrista fasciculata (Fabaceae)

The benefits of ant–plant–herbivore interactions for the plant depend on the abundance of ants and herbivores and the selective pressures these arthropods exert. In plants bearing extrafloral nectaries (EFN), different mean trait values may be selected for by different populations in response to local herbivore pressure, ultimately resulting in the evolution of differences in plant traits that attract ants as defensive agents against herbivory. To determine if variation in traits that mediate ant–plant interactions reflect herbivore selective pressures, we quantified intra- and inter-population variation in plant traits for eight populations of the EFN-bearing annual Chamaecrista fasciculata (Michx.) (Fabaceae). Censuses in rural and urban areas of Missouri and Illinois (USA) showed population differences in ant attendance and herbivore pressure. Seeds were collected from each population, and plants were grown in a common greenhouse environment to measure sugar production, nectar volume and composition, EFN size and time of emergence, leaf pubescence, and leaf quality throughout plant development. Populations varied mainly in terms of nectary size, sugar production, and nectar volume, but to a lesser degree in leaf pubescence. Populations of C. fasciculata within urban areas (low in insect abundance) had small nectaries and the lowest nectar production. There was a positive correlation across populations between herbivore density and leaf damage by those herbivores on the one hand and sugar production and nectar volume on the other. These results, in conjunction with lack of evidence for maternally based environmental effects, suggest that population differences in herbivore damage have promoted differential evolution of EFN-related traits among populations. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The adaptation of Dianthus carthusianorum L. (Caryophyllaceae) to growth on a zinc–lead heap in southern Poland

Plants of Dianthus carthusianorum from a calamine (zinc–lead) waste heap and from unpolluted stands were compared in the field and under controlled laboratory conditions. It was found that the waste-heap plants differed significantly from those in the normal population in respect to the following morphological traits: lower weight of aerial parts, shorter and narrower leaves, smaller number of leaves per plant. In combination with shorter, less numerous shoots, these features reduce the transpiration area of the plants. The general habit (predominance of forms with short and trailing shoots) of the waste-heap population points to adaptation to a xeric environment. Under controlled conditions all of the above traits were maintained through three successive generations. They are thus genetically controlled. Root tolerance tests showed that the waste-heap plants had higher tolerance to zinc and lead. These results indicate that strong selection processes have generated a clear ecotypical differentiation for heavy metal tolerance and drought tolerance in a zinc–lead-rich waste-heap population of D. carthusianorum (variety).     

Geographic differentiation among populations ofArabis serrata Fr. & Sav. (Brassicaceae)

Geographic variation in morphological traits of thirteen populations ofArabis serrata was analyzed to characterize the life history of each particular population in the field. These localities varied in altitude, topography, soil type, humidity, vegetation structure and degree of disturbance. Twelve morphological characters were measured in each plant, and populations were compared using both univariate and multivariate analyses. Populations showed significant differences for most of the traits measured. Principal component analyses revealed a significant differentiation among populations although a continuous variation for some traits was noted. Length of capsules and leaf length of inflorescence's stalks were correlated with the number of frost free days and with temperature, precipitation and number of days of the growing season. The number of rosettes was also correlated with the same variables but negatively. Seed weight was correlated positively with altitude but negatively with temperature and length of the growing season. Populations were also differentiated according to soil conditions and disturbance regimes distinguishing different types of populations: I) populations with many small rosettes, and few heavy seeds per capsule in volcanic soils with low disturbance; ii) plants with many small seeds and few rosettes in very disturbed localities along asphalt roadways and mountain trails; and iii) large plants with intermediate seed size and intermediate number of seeds per plant in limestone and serpentine soils under different conditions of disturbance. Populations ofA. serrata displayed a complex pattern of differentiation in morphological and life history traits in relation to several biotic and abiotic factors. The quantitative nature of the differences among populations ofA. serrata observed in the field deserves further studies (e.g., quantitative genetics and phenotypic plasticity) under controlled conditions in order to assess the extent of differentiation within this species complex.     

Tissue culture in Haworthia

Summary Plants regenerated on two different media (NK and I) from the calluses of simple or cloned subcultures, which were originated from a single stock callus of Haworthia setata derived from its flower bud, were observed for eight characters, i.e., somatic chromosome number in root tips, growth vigor, leaf shape, leaf color, number of stomata per unit leaf area, esterase zymogram, chromosome association at meiotic metaphase I in pollen mother cells, and pollen fertility. From these regenerates plants with different characters from those of the parental plant were obtained. With regards to chromosomal aberrations, tetraploids, aneuploids, plants with a part of the chromosome segment deleted, with reciprocal and non-reciprocal translocations, or with paracentric inversions and those showing sub-chromatid aberrations at meiosis were obtained. The NK medium tended to regenerate more tetraploids and less plants carrying translocation than the I medium.Chromosome variabilities in somatic cells of the regenerates correlated with those of the calluses, from which they regenerated, while they did not correlate with either the meiotic irregularities (chromosome association at MI) or pollen fertility of the regenerates. From these facts, it was concluded that a rather large number of callus cells participate in the regeneration of an individual plant, although, however, only a few limited types of the cells form its germ line.Polyploidy affected growth vigor, leaf shape, stomata number and chromosome association at MI, but its effects were not detected on other characters. Chromosomal aberrations at the diploid level produced no clear changes in the regenerate's phenotype except in meiotic chromosome configuration and pollen fertility.Most chromosomal variants obtained in the present study are already reported in plants collected from wild populations, but plants with the deletion of a whole chromosome (karyotype 7L+6S) or chromosome segment (7L+1M+6S and 14L+2M+12S) have never been reported: this fact suggests that tissue culture is a powerful tool for producing plants with novel karyotypes.Contribution from the Laboratory of Genetics, Faculty of Agriculture, Kyoto University, Japan, No. 436     

In-vitro development of ovules obtained after pollination of cotton (<Emphasis Type="Italic">Gossypium</Emphasis> spp) flowers with pollen from okra (<Emphasis Type="Italic">Abelmoschus esculentus L. Moench)</Emphasis>

The in vitro response of ovules obtained after pollination of cotton flowers with pollen from Abelmoschus esculentus was studied. For this, 492 cotton flowers from five G. hirsutum varieties, four G. barbadense varieties and 10 F1 interspecific hybrids, were pollinated with pollen from A. esculentus and 5,069 ovules were cultured in vitro. From the cultured ovules, 69 embryos were isolated and 16 of them grew into plants. However, only three of them survived after transplantation. Finally, one plant which originated from the interspecific cross (B403 × Acala Sindos) × A. esculentus reached maturity. The mature plant (Pa0) had no morphological traits from A. esculentus. On the contrary, traits from both cotton species were observed. The flowcytometric analysis of the Pa0 plant indicated that it was hypoaneuploid. Root tip chromosome counts of its offsprings revealed a progressive chromosome increase from the Pa1 to Pa4 generation. Plants with 52 chromosomes or hypoaneuploids with a lower level of chromosomes (46–51) could be isolated from the Pa4 generation. These plants exhibited morphological traits from both cotton species and they were fertile. No signs of A. esculentus morphological characteristics were observed in these plants. It was concluded that aneuploid partial interspecific cotton plants could be produced after pollination of cotton interspecific hybrids with pollen from A. esculentus and application of an in-ovule embryo rescue technique.