Chimeric Plants Research Articles

MicroRNA399 is a long-distance signal for the regulation of plant phosphate homeostasis.

The presence of microRNA species in plant phloem sap suggests potential signaling roles by long-distance regulation of gene expression. Proof for such a role for a phloem-mobile microRNA is lacking. Here we show that phosphate (Pi) starvation-induced microRNA399 (miR399) is present in the phloem sap of two diverse plant species, rapeseed and pumpkin, and levels are strongly and specifically increased in phloem sap during Pi deprivation. By performing micro-grafting experiments using Arabidopsis, we further show that chimeric plants constitutively over-expressing miR399 in the shoot accumulate mature miR399 species to very high levels in their wild-type roots, while corresponding primary transcripts are virtually absent in roots, demonstrating shoot-to-root transport. The chimeric plants exhibit (i) down-regulation of the miR399 target transcript (PHO2), which encodes a critical component for maintenance of Pi homeostasis, in the wild-type root, and (ii) Pi accumulation in the shoot, which is the phenotype of pho2 mutants, miR399 over-expressers or chimeric plants with a genetic knock-out of PHO2 in the root. Hence the transported miR399 molecules retain biological activity. This is a demonstration of systemic control of a biological process, i.e. maintenance of plant Pi homeostasis, by a phloem-mobile microRNA.

Leaf anatomy of the mosaic Ficus benjamina cv. Starlight and interaction of source and sink chimera components

Leaf anatomy was studied in the mosaic Ficus benjamina cv. Starlight and non-chimeric Ficus benjamina cv. Daniel. The number of chloroplasts in a white, chlorophyll-deficient tissue declines as compared to the green tissue. However, their functional activity is retained. The leaf of the mosaic F. benjamina contains two or, sometimes, three subepidermal layers. Mesophyll forms one layer in the green and white parts of leaf palisade and one white and one green layer in the transitional zone (edge). In the transitional zone, green spongy mesophyll is located between two white spongy layers and the proportion of photosynthesizing cells varies. In cv. Daniel, there are two subepidermal layers and one layer of columnar mesophyll cells. According to the morphometry data, the proportion of white zone in the leaf correlates with the leaf position in the whole shoot: the higher the branch order, the larger the proportion of white zone. The total leaf area depends also on its position in the shoot. No such correlation was found in non-chimeric F. benjamina cv. Daniel. In the mosaic chimera, the source-sink status appears to depend on the leaf position in the shoot. Experiments with individual shoots of the same order and elimination of all lateral shoots have shown that the proportion of white zone in new leaves on the shoot increases with the total area of green zone. Thus, the area of assimilating shoot surface affects the formation of leaves in the meristem. A hypothesis was put forward that the source-sink state affects the ratio of green and white parts in the leaf primordium. Products of photosynthesis (carbohydrates) are a possible metabolic signal affecting the meristem. It cannot be excluded as well that the hormonal state undergoes changes in the chimeric plant.

MNU-induced mutant pools and high performance TILLING enable finding of any gene mutation in rice

Mutant populations are indispensable genetic resources for functional genomics in all organisms. However, suitable rice mutant populations, induced either by chemicals or irradiation still have been rarely developed to date. To produce mutant pools and to launch a search system for rice gene mutations, we developed mutant populations of Oryza sativa japonica cv. Taichung 65, by treating single zygotic cells with N-methyl-N-nitrosourea (MNU). Mutagenesis in single zygotes can create mutations at a high frequency and rarely forms chimeric plants. A modified TILLING system using non-labeled primers and fast capillary gel electrophoresis was applied for high-throughput detection of single nucleotide substitution mutations. The mutation rate of an M(2) mutant population was calculated as 7.4 x 10(-6) per nucleotide representing one mutation in every 135 kb genome sequence. One can expect 7.4 single nucleotide substitution mutations in every 1 kb of gene region when using 1,000 M(2) mutant lines. The mutations were very evenly distributed over the regions examined. These results indicate that our rice mutant population generated by MNU-mutagenesis could be a promising resource for identifying mutations in any gene of rice. The modified TILLING method also proved very efficient and convenient in screening the mutant population.

Transgenic plants from shoot apical meristems of Vitis vinifera L. “Thompson Seedless” via Agrobacterium-mediated transformation

Shoot apical meristem explants of Vitis vinifera "Thompson Seedless" were used for Agrobacterium-mediated genetic transformation. It was determined that the meristems had to be subjected to a dark growth phase then wounded to obtain transgenic plants. Morphological and histological studies illustrated the role of wounding to expose apical meristem cells for transformation. A bifunctional egfp/nptII fusion gene was used to select kanamycin resistant plants that expressed green fluorescent protein (GFP). Kanamycin at a concentration of 16 mg L(-1) in selection medium resulted in recovery of non-chimeric transgenic plants that uniformly expressed GFP, whereas 8 mg L(-1) kanamycin allowed non-transgenic and/or chimeric plants to develop. Polymerase chain reaction (PCR) and Southern blot analyses confirmed the presence of transgenes and their stable integration into the genome of regenerated plants. Up to 1% of shoot tips produced stable transgenic cultures within 6 weeks of treatment, resulting in a total of 18 independent lines.

The unstable tubular ray flower allele of sunflower: inheritance of the reversion to wild‐type

AbstractThe tubular ray flower (turf) mutant of sunflower (Helianthus annuus L.) is characterized by a change from a zygomorphic corolla to a nearly actinomorphic tubular‐like corolla of ray flowers that also achieves the ability to differentiate fertile stamens and ovules. The recessive turf mutation is found to be unstable, spontaneously reverting to a wild‐type or nearly wild‐type phenotype. Reversion frequencies were 1.70% in the original inbred line R1634 (where turf was first identified), 0.17% and 3.20% in two turf/turf homozygous F3 progenies, derived from crossing a turf mutant with two different inbred lines. As no chimeric plant was found it is likely that the reversion had occurred either in the germ line or within the zygote. Self‐pollinated wild‐type reverted plants segregated into normal and mutant phenotypes fitting a monogenic 3 : 1 ratio, thus indicating that the reversion affected a single allele. The hypothesis of an epigenetic mechanism is discussed.

Cucumber mosaic virus as a presentation system for a double hepatitis C virus-derived epitope

Chimeric plant viruses are emerging as promising vectors for use in innovative vaccination strategies. In this context, cucumber mosaic virus (CMV) has proven to be a suitable carrier of the hepatitis C virus (HCV)-derived R9 mimotope. In the present work, a new chimeric CMV, expressing on its surface the HCV-derived R10 mimotope, was produced but lost the insert after the first passage on tobacco. A comparative analysis between R10- and R9-CMV properties indicated that R9-CMV stability was related to structural features typical of the foreign insert. Thus, in order to combine high virus viability with strong immuno-stimulating activity, we doubled R9 copies on each of the 180 coat protein (CP) subunits of CMV. One of the chimeras produced by this approach (2R9-CMV) was shown to systemically infect the host, stably maintaining both inserts. Notably, it was strongly recognized by sera of HCV-infected patients and, as compared with R9-CMV, displayed an enhanced ability to stimulate lymphocyte IFN-gamma production. The high immunogen levels achievable in plants or fruits infected with 2R9-CMV suggest that this chimeric form of CMV may be useful in the development of oral vaccines against HCV.

Oryzalin Treatment of Potato Diploids Yields Tetraploid and Chimeric Plants from which Euploids could be Derived by Callus Induction

Shoot apices from six diploid potato genotypes for polyploidisation were treated with oryzalin and colchicine solutions at different concentrations and incubation times. In addition to chimeric plants, diploids, one octoploid and completely ploidy doubled plants (4x) were regenerated in these experiments. Tetraploid plants were obtained for three of the six genotypes treated with oryzalin, and no tetraploid plants were obtained after colchicine treatments of any of the genotypes. The best result of 43% tetraploid plants was achieved using 28.8 μM oryzalin and 24 h of incubation with the L37 genotype. Moreover, we determined that doubled plants should not be selected on the basis of a single measurement and require additional ploidy checks in successive clonal generations. Some L37 plants that were initially identified as tetraploids by flow cytometry produced diploid or mixoploid plants in the next clonal generation. Morphological characters of doubled L37 plants were compared with the original genotype. The PVY resistance present in a parental clone was maintained in all regenerants and significant differences among ploidy variants were found for some quantitative characters. Molecular AFLP analyses did not show any differences among genotypes. Since polyploidizing substances may produce a large proportion of chimeric plants, we applied a method to increase the number of tetraploid plants. Callus culture of chimeric leaf explants was performed on regeneration medium and tetraploid shoots were obtained in four of the six genotypes tested. The best result of 40% tetraploid plants was achieved with explants from the L37 genotype. This method was successful in producing additional tetraploid genotypes from mixoploid plants obtained after oryzalin treatments.

In vitro Induction of Colchiploidy in an Intergeneric Hybrid of Sorghum*Saccharum

A rare hybrid of Sorghum×Sugarcane was produced at Sugarcane Breeding Institute during 1996 which had a chromosome number 2n=66. This hybrid lacked vigor and was poor with respect to agronomic traits. Attempts were made to create genetic variation in this hybrid through tissue culture and colchicine treatment. Calli induction and their differentiation to plantlets were obtained on modified MS medium. Different concentrations of colchicine (25 mg/l, 50 mg/l, 75 mg/l and 100 mg/l) were added to the differentiation medium. There was no differentiation in the medium containing 75 mg/l and 100 mg/l colchicine, while calli treated with 25 mg/l and 50 mg/l colchicine produced 230 plants. The regenerated plants obtained were rooted, hardened and established in pots as well as in the field and cytomorphologically characterized. Though the somaclones were morphologically similar to the original hybrid in almost all the characters, the cytological analysis revealed the occurrence of polyploidy in these calli clones. Chimeric plants were also obtained during in vitro polyploidization. The results indicate that colchicine could be efficiently utilized for induction of polyploidy in Saccharum hybrids.

In liquid medium colchicine treatment induces non chimerical doubled-diploids in a wide range of mono- and interspecific diploid banana clones

Introduction . The worldwide production of banana and plantain, which is estimated to 106 Mt·year–1 , relies on a narrow genetic basis. Thus, banana production is fragile regarding emergent diseases, and creation of new varieties of banana stands out as a real necessity for its sustainability. In banana, various improvement strategies aim to create triploid hybrids. The objective of this report is to present a simple in vitro methodology to induce stable tetraploid plants by colchicine treatment, usable as parent for obtaining triploid varieties. Materials and methods . Twenty-one diploid M. acuminata clones and three interspecific M. acuminata / M. balbisiana diploid clones were treated as proliferating culture in liquid medium with 1.25 mM of colchicine for 48 h. Plant screening has been performed by morphological identification in greenhouse, chromosome counts on root tips and flow cytometry on leave blades on vitroplants using propidium iodide staining. Results . Chromosome counts led to distinguish diploid and tetraploid plants but did not afford to detect chimeras. Flow cytometry allowed an early screening of a larger number of plants leading to detect rapidly chimerical plants. It was observed that some stable 2x / 4x cytochimeras are periclinal. Tetraploid clones were obtained with all diploid genotypes. General behaviour of tetraploids in the field was globally weaker than the corresponding diploids. Nevertheless, all the doubled-diploids flowered and crossed with diploid plants to obtain triploid progenies. Conclusion . This study has clearly shown that induction of stable doubled-diploid plants can be obtained from a wide range of genetically different bananas. These results open the way to the systematic use of doubled-diploids by banana breeding programs for the release of enhanced triploid varieties.

CUCUMBER (CUCUMIS SATIVUS L.) DIHAPLOID LINE PRODUCTION USING IN VITRO RESCUE OF IN VIVO INDUCED PARTHENOGENIC EMBRYOS

Homozygous dihaploid lines (DHLs) from new cucumber (Cucumis sativus L.) accessions, source of resistance to diseases, would be useful to accelerate breeding for new resistant varieties. DHLs have been generated by in vitro rescue of in vivo induced parthenogenic embryos. The protocol developed involves the following: 1) induction of parthenogenic embryos by pollination with pollen irradiated at 0.5 KGy with a Co 60 γ-ray source; 2) in vitro rescue of putative parthenogenic embryos identified by their morphology and localized under a dissecting scope or X-ray radiography; 3) discrimination of undesirable zygotic individuals from the homozygous plants using cucumber and melon SSR markers; 4) determination of ploidy level from homozygous plants by flow cytometry; 5) in vitro chromosome doubling of haploids; and 6) acclimation and selfing of lines. Codominant markers and flow cytometry confirmed the gametophytic origin of plants regenerated by parthenogenesis, since all homozygous lines were haploids. No spontaneous doubled haploid plants were rescued. Chromosome doubling of haploid plants was accomplished by an in vitro treatment with 500 μM colchicine. Rescue of diploid or chimerical plants was confirmed by flow cytometry, previous to their acclimation and plant growth in the greenhouse. Selfing of colchicine-treated haploid plants allowed the perpetuation by seed of homozygous lines. Seeds were harvested from almost all lines after selfing in the greenhouse. The high rate of seed production facilitated the recovery of inbred lines. Despite some limiting factors, parthenogenesis is routinely used in a cucumber-breeding program to achieve complete homozygosity in one generation, and accelerate breeding for new commercial varieties. DHLs have been used as parental lines for the production of commercial hybrids.

Adventitious regeneration in vitro occurs across a wide spectrum of squash (Cucurbita pepo) genotypes

Cucurbita pepo L. (squash, pumpkin) is a highly polymorphic vegetable species of major importance. Our study characterized a spectrum of C. pepo germplasm for the ability to regenerate in vitro by direct organogenesis from cotyledon explants. Cultivars tested included both cultivated subspecies, texana and pepo, and nearly all of their respective cultivar-groups. Direct shoot regeneration occurred in all accessions, and was generally high (56–94%), with a single exception of 22% (‘Bolognese’). There was no significant difference between the percentage regeneration of the two subspecies. Shoot regeneration per responding explant was uniform (1.2–1.6 shoots per explant). Only ‘True French’ produced statistically more shoots (3.9 per explant) than other accessions. The morphology of regeneration varied. Most cultivars produced long shoots, often fasciated, amid a few small buds. Some subspecies pepo cultivars (Beirut, Yugoslavia 7, Ma’yan and True French) produced short, massive, hollow shoots, sometimes accompanied by shoots that were more normal. Two subspecies texana cultivars (Creamy Straightneck and Small Bicolor) produced single (sometimes double) shoots without other buds. The production of chimeric (mixoploid) regenerants varied and there was a tendency to regenerate chimeric plants from the widest-fruited accessions (i.e. lowest length-to-width ratio) in each subspecies. Subspecies pepo Pumpkin Group ‘Tondo di Nizza’ showed significantly greater production of chimeric regenerants. In comparison with the great range of variation observed in fruit shape, the variation of in vitro responses (mostly less than 2-fold in regeneration and shoot production) was less than expected.

Artificial synthesis of interspecific chimeras between tuber mustard (Brassica juncea) and cabbage (Brassica oleracea) and cytological analysis

Interspecific chimeras between tuber mustard and red cabbage were obtained by in vitro graft-culture method. Before grafting, 6-day-old seedlings of tuber mustard and red cabbage were vertically half-cut and treated with different concentrations of 6-BA and NAA for 1 min, then, they were symmetrically fit together. As a result, sectorial chimeras were initially produced from the united shoot tips. The maximum frequency of chimeral bud formation reached 6.33% when the vertical sections of tuber mustard and cabbage were treated with 2 mg/l 6-BA and 1 mg/l NAA. When sectorial chimeras were propagated on MS medium containing 1 mg/l 6-BA, periclinal and mericlinal chimeras gradually developed. Chimeral shoots were rooted on half-strength MS medium containing 0.1 mg/l NAA. The rooted chimeras were acclimatized and transferred to the field for cytological and morphological analysis. The results showed that stomata density in the chimeras was significantly higher than that of their parents, while chloroplast size, starch grain size and number were intermediate between the two parents. The chimeras were further analyzed by flow cytometry, and the results indicated that they contained both sets of parental chromosomes. Moreover, chimeral plants possessed valuable characters from the two parents.

Optimization of Cucumber Doubled Haploid Line Production Using In Vitro Rescue of In Vivo Induced Parthenogenic Embryos

Homozygous doubled haploid lines (DHLs) from new cucumber (Cucumis sativus L.) accessions could be useful to accelerate breeding for resistant varieties. DHLs have been generated by in vitro rescue of in vivo induced parthenogenic embryos. The protocol developed involves the following: 1) induction of parthenogenic embryos by pollinating with pollen irradiated with a Co60 γ-ray source at 500 Gy; 2) in vitro rescue of putative parthenogenic embryos identified by their morphology and localized using a dissecting scope or X-ray radiography; 3) discrimination of undesirable zygotic individuals from the homozygous plants using cucumber and melon SSR markers; 4) determination of ploidy level from homozygous plants by flow cytometry; 5) in vitro chromosome doubling of haploids; and 6) acclimation and selfing of selected lines. Codominant markers and flow cytometry confirmed the gametophytic origin of plants regenerated by parthenogenesis, since all homozygous lines were haploids. No spontaneous doubled haploid plants were rescued. Chromosome doubling of haploid plants was accomplished by an in vitro treatment with 500 μm colchicine. Rescue of diploid or chimeric plants was shown by flow cytometry, prior to their acclimation and planting in the greenhouse. Selfing of colchicine-treated haploid plants allowed for the perpetuation by seed of homozygous lines. The high rate of seed set, 90% of the lines produced seed, facilitated the recovery of inbred lines. Despite some limiting factors, parthenogenesis is routinely used in a cucumber-breeding program to achieve complete homozygosity in one generation. Breeding for new commercial hybrid cultivars will be accelerated. DHLs are ideal resources for genomic analyses.

Transformation of Recalcitrant Melon (Cucumis melo L.) Cultivars is Facilitated by Wounding with Carborundum

AbstractTransformation of the recalcitrant melon (Cucumis melo L.) cultivars Kιrkağaç 637 and Noi Yarok was accomplished by wounding cotyledon explants by vortexing with carborundum prior to inoculation with Agrobacterium tumefaciens. The addition of silver nitrate to the regeneration‐selection medium reduced the transformation efficiency, as the percentage of the explants forming putative transgenic calli and bud‐like protuberances was decreased and no transgenic shoots were produced. Chimeric transgenic plants were obtained after the regeneration of putatively transformed callus, bud‐like protuberances, buds and shoots on selective medium with kanamycin. The treatments producing the most buds or shoots from explants after 30–40 days of cultivation were the most successful for the production of transgenic plants. Only treatments where explants were vortexed with carborundum produced transgenic melon shoots of either cultivar. Subculture every 18–20 days on fresh regeneration‐selection medium containing 50 mg/L kanamycin after either a relatively high (100 mg/L) or low level (50 mg/L) of kanamycin in the first regeneration‐selection medium was necessary for the successful transformation of cultivar Kιrkağaç 637. These techniques are now being used in breeding programs for the production of melon lines bearing resistances to zucchini yellow mosaic virus and cucumber mosaic virus, important viruses limiting agricultural production.

Characterisation of regenerants obtained under selective conditions after Agrobacterium-mediated transformation of citrus explants reveals production of silenced and chimeric plants at unexpected high frequencies

Genetic transformation has been achieved for several citrus genotypes. However, regeneration of escapes at high frequency is a major problem, making the available procedures rather inefficient. Attempts to improve selection by increasing the concentration of kanamycin, used as the selective agent, or substituting it by geneticin have been unsuccessful. Here, we have critically assessed the actual frequency and origin of escapes in citrus by using visual screening with β-glucuronidase (gusA) and green fluorescent protein (gfp) markers, by studying the persistence of engineered Agrobacterium in the explants, and by characterising through Southern blot analysis all the regenerants obtained under kanamycin selection. Our results show that inefficient selection could be attributed to the protection of the non-transformed cells from the selective agent by the surrounding transformed cells, and to the persistence of kanamycin-resistance Agrobacterium in explant tissues over long periods of time after co-cultivation. This also explained the high frequency (12%) of chimeric shoots that were commonly recovered. High frequency regeneration of chimeras that resulted from the fusion of different transformation events is reported for the first time. On the other hand, molecular analysis of all the regenerants reveals that transformation frequency is underestimated when based on the expression of a screenable marker gene, and that low expressors and silenced lines could account for at least 25% of those plants considered escapes based on selectable and screenable marker analysis. Consequences of these results at the practical level are also discussed.

412 A chimeric plant virus as a potential candidate for hepatitis C vaccine

412 A chimeric plant virus as a potential candidate for hepatitis C vaccine

Potato lectin: an updated model of a unique chimeric plant protein.

A complete cDNA encoding a potato tuber lectin has been identified and sequenced. Based on the deduced amino acid sequence, the still enigmatic molecular structure of the classical chimeric potato lectin could eventually be determined. Basically, the potato lectin consists of two nearly identical chitin-binding modules, built up of two in-tandem arrayed hevein domains that are interconnected by an extensin-like domain of approximately 60 amino acid residues. Although this structure confirms the 'canonical' chimeric nature of the Solanaceae lectins, it differs fundamentally from all previously proposed models. The new insights in the structure are also discussed in view of the physiological role of the Solanaceae lectins.

A chemical-regulated inducible RNAi system in plants.

Constitutive expression of an intron-containing self-complementary 'hairpin' RNA (ihpRNA) has recently been shown to efficiently silence target genes in transgenic plants. However, this technique cannot be applied to genes whose silencing may block plant regeneration or result in embryo lethality. To obviate these potential problems, we have used a chemical-inducible Cre/loxP (CLX) recombination system to trigger the expression of an intron-containing inverted-repeat RNA (RNAi) in plants. A detailed characterization of the inducible RNAi system in transgenic Arabidopsis thaliana and Nicotiana benthamiana plants demonstrated that this system is stringently controlled. Moreover, it can be used to induce silencing of both transgenes and endogenous genes at different developmental stages and at high efficiency and without any detectable secondary affects. In addition to inducing complete silencing, the RNAi can be produced at various times after germination to initiate and obtain different degrees of gene silencing. Upon induction, transgenic plants with genetic chimera were obtained as demonstrated by PCR analysis. Such chimeric plants may provide a useful system to study signaling mechanisms of gene silencing in Arabidopsis as well as other cases of long-distance signaling without grafting. The merits of using the inducible CLX system for RNAi expression are discussed.

Instability of the ploidy level in autotetraploid sorghum plants from a line with variable male fertility

It was found that, in some of the plants in generations C1-C5 of induced tetraploids in the semisterile sorghum line AS-1-30, more than 30% of pollen grains (PGs) have sizes typical of haploid PGs. Pollen of these plants was used to pollinate different lines of sorghum with cytoplasmic male sterility (CMS). Diploid hybrids were obtained, which confirms the presence of haploid PGs in the tetraploids studied. When tetraploid plants with an increased frequency of haploid PGs were pollinated with pollen of plants from fertile diploid lines, diploid hybrids were also obtained. This demonstrates that the tetraploids studied had haploid ovicells. In generation C4, a chimeric plant was found; one of its shoots was diploid (judging from morphological characters) and produced as many as 99% of haploid PGs. It is assumed that haploid gametes in autotetraploids are formed through somatic reduction of chromosomes at different ontogenetic stages.

Efficient production of doubled haploid plants by immediate colchicine treatment of isolated microspores in winter Brassica napus

The effect of colchicine on embryogenesis induction and chromosomedoubling during microspore culture was evaluated in two F1 hybridsofwinter oilseed rape (Brassica napus L.). Colchicinetreatment (50 and 500 mg/L) of isolated microspores during thefirst 15 h in culture stimulated embryogenesis and produced large amounts ofhealthy-looking embryos. These normal embryos germinated well at 24°C after being transferred to solid regeneration medium and aninitial period of low temperature (2 °C) for 10 days, andcoulddirectly and rapidly regenerate vigorous plants. A high doubling efficiency of84–88% was obtained from 500 mg/L colchicine treatment for15h with low frequency of polyploid and chimeric plants. Acolchicinetreatment duration of 6 h was less effective on embryogenesis anddoubling efficiency. The present experiment also showed that changing of induction medium 15h after microspore isolation produced higher spontaneous doublingefficiency, as compared with medium change 6 h after isolation.