基于ITS序列的中国外来苋属植物系统关系分析

通过ITS序列对21种中国外来苋属植物进行系统进化关系研究。通过ITS序列种间、种内遗传距离分析,发现苋属种间变异为0~0.055 1,种内变异为0~0.009 2。使用TAXON DNA软件分析ITS序列种间、种内变异的分布图看出规律,结果表明苋属ITS序列的种间变异适中,种间变异明显大于种内变异。采用最大似然法(ML)构建的系统树将中国苋属分为5或6个进化支(根据自展支持率取值不同)。异株苋亚属长芒苋和苋亚属刺苋聚类在一起,西部苋和糙果苋单独成为一个进化支。苋亚属中苋组苋亚组反枝苋和绿穗苋亚组鲍氏苋有着更近的亲缘关系,苋组苋亚组尾穗苋和绿穗苋亚组绿穗苋、繁穗苋等亲缘关系更近。白苋亚属分为2或3个类群,根据自展支持率取值不同,合被苋可以和白苋、北美苋并为一支,也可以单独成为一支。综上所述,该文认为苋属经典分类体系中3亚属或2~3组的分类地位不成立,建议中国苋属采取5组2亚组或6组2亚组的分类体系。5组2亚组分别由长芒苋组、糙果苋组、苋组(苋亚组和绿穗苋亚组)、白苋组和凹头苋组组成。其中,合被苋也可从白苋组分出,单独构成1组,形成6组2亚组的分类体系。表明该序列对苋属大部分种类分类效果较好,对西部苋和糙果苋复合群,绿穗苋复合群以及白苋亚属的分类价值不高。     

Transfer of auxinic herbicide resistance from Brassica kaber to Brassica juncea and Brassica rapa through embryo rescue

Auxinic herbicides are widely used in agriculture to selectively control broadleaf weeds. Prolonged use of auxinic herbicides has resulted in the evolution of resistance to these herbicides in some biotypes of Brassica kaber (wild mustard), a common weed in agricultural crops. In this study, auxinic herbicide resistance from B. kaber was transferred to Brassica juncea and Brassica rapa, two commercially important Brassica crops, by traditional breeding coupled with in vitro embryo rescue. A high frequency of embryo regeneration and hybrid plant establishment was achieved. Transfer of auxinic herbicide resistance from B. kaber to the hybrids was assessed by whole-plant screening of hybrids with dicamba, a widely used auxinic herbicide. Furthermore, the hybrids were tested for fertility (both pollen and pistil) and their ability to produce backcross progeny. The auxinic herbicide-resistant trait was introgressed into B. juncea by backcross breeding. DNA ploidy of the hybrids as well as of the backcross progeny was estimated by flow cytometry. Creation of auxinic herbicide-resistant Brassica crops by non-transgenic approaches should facilitate effective weed control, encourage less tillage, provide herbicide rotation options, minimize occurrence of herbicide resistance, and increase acceptance of these crops.     

普通小麦与鹅观草属间杂种F1及BC1的分子细胞遗传学、育性和赤霉病抗性研究

通过胚拯救, 成功获得鹅观草Roegneria kamoji (2n=6x=42, SSHHYY)和普通小麦中国春Triticum aesti-vum (2n=6x=42, AABBDD)的正反交属间杂种F1, 并对这些杂种F1及其BC1的形态学、减数分裂配对行为、育性和赤霉病抗性进行研究。结果表明, (鹅观草×中国春)F1和(中国春×鹅观草)F1的形态介于双亲之间。杂种F1花粉母细胞减数分裂中期I染色体构型分别为40.33I + 0.78II + 0.03III和40.40I + 0.79II 。杂种F1高度雄性不育, 用中国春花粉与其回交可获得BC1代种子。(鹅观草×中国春) F1×中国春BC1植株的染色体数目主要分布在55~63之间, 单价体较多, 植株高度不育; (中国春×鹅观草)F1×中国春BC₁植株染色体数目也主要分布在55~63之间, 但其中部分植株拥有整套小麦染色体且能正常配对、分离, 可形成部分可育花粉粒, 能收到少量自交结实种子。在 (鹅观草×中国春)F1中有1株穗型趋向中国春, 其染色体数目为2n=63, 经染色体分子原位杂交(GISH)检测, 含有42条小麦染色体和21条鹅观草染色体。该杂种F1在减数分裂中期I平均每个花粉母细胞有26.40I+18.30II, 但植株高度雄性不育, 用中国春花粉回交能收到BC₁种子。(鹅观草×中国春) F₁ (2n=63)×中国春BC₁的染色体数目主要分布在40~59之间, 其中的外源染色体已经逐渐减少, 虽然该BC₁的穗型已接近中国春, 但仍然高度不育。赤霉病抗性鉴定结果显示, 所有杂种F1及大部分BC₁对赤霉病均表现出较好的抗性。     

Seedling herbivory by slugs in a willow hybrid system: developmental changes in damage, chemical defense, and plant performance

We evaluated feeding preference and damage by the slug, Arion subfuscus, on seedlings of two willow species, Salix sericea and S. eriocephala, and their F₁ interspecific hybrids. Trays of seedlings were placed in the field and excised leaves were presented to slugs in choice tests. Slugs preferred feeding on and caused the most damage to S. eriocephala seedlings. S. sericea seedlings were least preferred and least damaged. F₁ hybrid seedlings were intermediate in preference and damage. Slug preference of and damage to these seedlings decreased over time, suggesting developmental changes in resistance. Seedlings were sampled for phenolic glycoside and tannin chemistry weekly to coincide with the field and laboratory experiments. Concentrations of phenolic glycosides and tannins increased linearly with seedling age, coincident with changes in slug preference and damage, indicating a developmental change in defense. Slug deterrence was not detected at low concentrations of salicortin when painted on leaves or discs, but both salicortin and condensed tannins deterred slug feeding at concentrations between 50 and 100 mg/g, levels found in adult willows. Seedling performance was related to damage inflicted by slugs. Due to lower levels of damage when exposed to slugs in the field, S. sericea plants had significantly greater biomass than S. eriocephala plants. Biomass of F₁ hybrids was equal to S. sericea when damaged. However, undamaged S. eriocephala and F₁ hybrid plants had the greatest biomass. Because F₁ hybrid seedlings performed as well as the most fit parent in all cases, slugs could be an important selective factor favoring introgression of defensive traits between these willow species.     

Transformed T0 orchardgrass (Dactylis glomerata L.) plants produced from highly regenerative tissues derived from mature seeds

A highly efficient and reproducible transformation system for orchardgrass (Dactylis glomerata L. cv. Rapido, 2n=42=28) was established using microprojectile bombardment of highly regenerative, green tissues derived from mature seeds. These tissues, induced from embryogenic callus, were bombarded with a mixture of three plasmids containing the hygromycin phosphotransferase (hpt), phosphinothricin acetyltransferase (bar) and #-glucuronidase (uidA; gus) genes. From 147 individual explants bombarded, 11 independent hygromycin-resistant lines (7.5%) were obtained after an 8- to 16-week selection period using 30-50 mg/l hygromycin B. Of the 11 independent lines, ten (91%) were regenerable. The presence and integration of the transgene(s) were assessed using PCR and DNA blot hybridization. Coexpression frequency of the three transgenes (hpt/bar/uidA) in T₀ plants was 20%, and of two transgenes, either hpt/bar or hpt/uidA, 45-60%. Due to greenhouse conditions optimized for the growth of other species, T₁ seed has not been obtained from these plants. While the inability to analyze progeny plants precludes the conclusive demonstration of stable transformation, the results of all molecular and biochemical analyses of T₀ plants are consistent with the production of stably transformed plants. Frequent change in ploidy level was observed in transformed T₀ orchardgrass plants. Plants from only three of the ten independent lines analyzed had the normal tetraploid number of chromosomes (2n=42=28), while plants from seven lines (70%) were octaploid (2n=82=56). The octaploid plants had abnormal morphological features, such as narrower, thicker and more upright leaves.     

Amaranthus hybridus can be pollinated frequently by A. tuberculatus under field conditions

Recent studies have confirmed that weedy Amaranthus species are capable of interspecific hybridization, and such hybridization may foster the evolution of herbicide resistance. However, the extent to which hybridization among these species occurs in nature is unknown. The purpose of this study was to determine the frequency under field conditions at which A. hybridus, a monoecious and predominantly self-pollinated species, would be pollinated by A. tuberculatus, a dioecious species. To do this, parents carrying different alleles at the ALS locus, which encodes a herbicide target site, were used. Male A. tuberculatus parents were homozygous for a dominant herbicide-insensitive allele, while A. hybridus parents were homozygous for a sensitive form. Hybrid progeny therefore could be detected via herbicide selection. Mean hybridization frequencies between 0.4 and 2.3% were obtained, depending on the proximity between parents (P=0.02). The robustness of the hybrid selection assay was verified using a molecular marker and DNA content analyses. Using these techniques, more than 99% of the progeny that survived the herbicide were confirmed to be hybrids. Frequencies obtained in this study were many times higher than the generally expected rate of mutation. Therefore, even minimal fertility in hybrid progeny would support the view that hybridization could play a role in adaptive evolution of weedy Amaranthus species.     

Herbicide-resistance conferred by expression of a catalytic antibody in Arabidopsis thaliana

Engineering herbicide resistance in crops facilitates control of weed species, particularly those that are closely related to the crop, and may be useful in selecting lines that have undergone multiple transformation events. Here we show that herbicide-resistant plants can be engineered by designing an herbicide and expressing a catalytic antibody that destroys the herbicide in planta. First, we developed a carbamate herbicide that can be catalytically destroyed by the aldolase antibody 38C2. This compound has herbicidal activity on all three plant species tested. Second, the light chain and half of the heavy chain (Fab) of the catalytic antibody were targeted to the endoplasmic reticulum in two classes of Arabidopsis thaliana transformants. Third, the two transgenic plants were crossed to produce an herbicide-resistant F1 hybrid. The in vitro catalytic activity of the protein from F1 hybrids corroborates that catalytic antibodies can be constitutively expressed in transgenic plants, and that they can confer a unique trait.     

High-resolution mapping of the bolting gene <Emphasis Type="Italic">B</Emphasis> of sugar beet

Sugar beet (Beta vulgaris L.) is a biennial species. Shoot elongation (bolting) starts after a period of low temperature. The dominant allele of locus B causes early bolting without cold treatment. This allele is abundant in wild beets whereas cultivated beets carry the recessive allele. Fifteen AFLP markers, tightly linked to the bolting locus, have been identified using bulked segregant analysis. The F₂-population consisted of 2,134 individuals derived after selfing a single F₁-plant (Bb). In a first step, a linkage map was established with 249 markers based on 775 F₂-individuals with a coverage of 822.3 cM. The loci are dispersed over nine linkage groups corresponding to the haploid chromosome number of Beta species. Seventeen marker loci were placed at a distance less than 3.2 cM around the bolting gene. In a second step, four of those markers most closely linked to B were mapped with the entire F₂-population. Two of the markers were mapped flanking the B gene at distances of 0.14 and 0.23 cM. The other two markers were mapped at a distance of 0.5 cM from the gene. The tight linkage could be verified by testing 88 unrelated plants from a breeding program. The closely linked markers will enable breeders to select for the non-bolting character without laborious test crossings. Moreover, these markers are being used for map-based cloning of the bolting gene.     

A bentazon and sulfonylurea sensitive mutant: breeding,genetics and potential application in seed production of hybrid rice

The use of a thermosensitive genic male sterility (TGMS) system in two-line hybrid rice breeding is affected greatly by the sterility instability of TGMS lines caused by temperature fluctuation beyond their critical temperatures for fertility reversion. To prevent seed production from self contamination, we have developed a system to secure seed purity using a herbicide-sensitive TGMS mutant, M8077S, obtained by radiation. Genetic analysis, using the F₁, F₂ and F₃ populations derived from this mutant and other normal varieties, revealed that bentazon lethality/sensitivity was controlled by a single recessive gene, which was named bel. The mutant can be killed at the seedling stage by bentazon at 300 mg/l or higher, a dosage that is safe for its F₁ hybrids and all other normal varieties. This mutant is also sensitive to all the tested sulfonylurea herbicides. Response of segregating plants to these two types of herbicide indicated that sulfonylurea sensitivity was also controlled by bel. By crossing this mutant with Pei-Ai 64S, an F₂ population was developed for genetic mapping. Surveying the two DNA pools from sensitive and non-sensitive F₂ plants identified four markers that were polymorphic between the pools. The putative linked markers were then confirmed with the F₂ population. The bel locus was located on chromosome 3, 7.1 cM from the closest microsatellite marker RM168. Phenotypic analysis indicated that the bel gene had no negative effect on agronomic traits in either a homozygous or heterozygous status. The mutant M8077S is valuable in the development of a TGMS breeding system for preventing impurity resulting from temperature fluctuation of the TGMS. Several two-line hybrid rice crosses using this system are under development.     

Inheritance of Arabidopsis DNA in offspring from Brassica napus and A. thaliana somatic hybrids

 Chromosome counts and RFLP markers mapped to Arabidopsis thaliana were used to determine the proportion of eliminated chromosomes and retained A. thaliana DNA in the back-crossed (BC) progeny derived from symmetric and asymmetric somatic hybrids between Brassica napus and A. thaliana. All plants were analysed for the presence of two RFLP markers per chromosome, preferably with one located on each chromosome arm. A reduction in both A. thaliana RFLP markers and chromosome numbers was found in the BC₁ and BC₂ generations of the symmetric hybrids as well as in the BC₁ generation of the asymmetric hybrids. In the symmetric hybrids, two back-crosses to B. napus were required to reduce the frequency of retained A. thaliana loci to 42.4% and mean chromosome number to 39.4. In comparison, the BC₁ progeny of the asymmetric hybrids had 16% of the analysed A. thaliana loci present and an average of 38.4 chromosomes maintained. When the frequency of A. thaliana chromosomes with both analysed loci maintained was compared with the frequency of chromosomes with one locus lost and one kept, a reduction in the number of complete chromosomes between BC₁ and BC₂ derived from the symmetric hybrids was observed. Among the BC₁ plants in the asymmetric group the situation was different, with higher amounts of incomplete donor chromosomes compared to whole chromosomes. The results indicate that A. thaliana chromosome fragments are more often found in the progeny of irradiated hybrids, while back-crossed symmetric hybrids have more complete chromosomes. Received: 2 April 1998 / Accepted: 14 July 1998     

A Second Edible Okra Species, and its Hybrids with Common Okra

Seventeen accessions of a West African okra species were observedin Puerto Rico, and were hybridized with the common okra species,Abelmoschus esculentus. The accessions are larger plants thancommon okra, but with shorter internodes. They also differ innumerous morphological and physiological characteristics. Mostare more daylength sensitive than common okra. The F₁ hybridsof the two species are quite sterile, although in some casesa few germinable F₂ seeds are produced. Back-crosses, on theother hand, are more fertile than the F₁ hybrids, and fertilityis almost complete in the BC₂. Some evidence was found of cytoplasmicinteraction with chromosomes in production of sterile BC hybrids,a possibly useful tool for future use. Meanwhile, the transferof genes from the new species to common okra appears very feasible. Abelmoschus species, okra, species hybrid, hybrid-sterility     

Production of fertile intergeneric somatic hybrids between Brassica napus and Sinapis arvensis for the enrichment of the rapeseed gene pool

Somatic hybrids between Brassica napus (oilseed rape) and its wild relative Sinapis arvensis (Xinjiang wild mustard) from northwestern China were produced by fusing mesophyll protoplasts. Fifty-four plants were identified as symmetric hybrids and four as asymmetric hybrids by random amplified polymorphic DNA analysis and nuclear DNA content. The morphology of investigated 58 hybrid plants resembled characters from both parental species. Highly fertile hybrids were recovered where the fertility was associated with the choice of B. napus genotype. Enhanced disease resistance to Leptosphaeria maculans was found in S. arvensis and in the hybrid offspring. This plant material has great potentials not only for use as a bridge for the introduction of a number of valuable traits from the wild species to Brassica crops but also for breeding new varieties with improved blackleg resistance.     

Transfer of bacterial blight and blast resistance from the tetraploid wild rice Oryza minuta to cultivated rice,Oryza sativa

Summary Oryza minuta J. S. Presl ex C. B. Presl is a tetraploid wild rice with resistance to several insects and diseases, including blast (caused by Pyricularia grisea) and bacterial blight (caused by Xanthomonas oryzae pv. oryzae). To transfer resistance from the wild species into the genome of cultivated rice (Oryza sativa L.), backcross progeny (BC₁, BC₂, and BC₃) were produced from interspecific hybrids of O. sativa cv IR31917-45-3-2 (2n=24, AA genome) and O. minuta Acc. 101141 (2n=48, BBCC genomes) by backcrossing to the O. sativa parent followed by embryo rescue. The chromosome numbers ranged from 44 to 47 in the BC₁ progeny and from 24 to 37 in the BC₂ progeny. All F₁ hybrids were resistant to both blast and bacterial blight. One BC₁ plant was moderately susceptible to blast while the rest were resistant. Thirteen of the 16 BC₂ progeny tested were resistant to blast; 1 blast-resistant BC₂, plant 75-1, had 24 chromosomes. A 3 resistant: 1 susceptible segregation ratio, consistent with the action of a major, dominant gene, was observed in the BC₂F₂ and BC₂F₃ generations. Five of the BC₁ plants tested were resistant to bacterial blight. Ten of the 21 BC₂ progeny tested were resistant to Philippine races 2, 3, and 6 of the bacterial blight pathogen. One resistant BC₂, plant 78-1, had 24 chromosomes. The segregation of reactions of the BC₂F₂, BC₂F₃, and BC₂F₄ progenies of plant 78-1 suggested that the same or closely linked gene(s) conferred resistance to races 2, 3, 5, and 6 of the bacterial blight pathogen from the Philippines.     

The effects of elevated CO2 on seed production and seedling recruitment in a sheep-grazed pasture

Seed production and seedling recruitment were measured over 2 years under ambient (360 ppm) and elevated (475 ppm) atmospheric CO₂ in a free air carbon dioxide enrichment (FACE) experiment, carried out in a sheep-grazed pasture on dry, sandy soil in New Zealand. In both years elevated CO₂ led to more dispersed seeds of the grasses Anthoxanthum odoratum, Lolium perenne and Poa pratensis, the legumes Trifolium repens and T. subterraneum and the herbs Hypochaeris radicata and Leontodon saxatilis. The increased seed dispersal in A. odoratum, H. radicata, Leontodon saxatilis and T. repens reflected both more inflorescences per unit area and more seeds per inflorescence under elevated CO₂. The increased seed dispersal in Lolium perenne, P. pratensis and T. subterraneum was due solely to more inflorescences per unit area. The number of seedlings that emerged and survived to at least 7 months of age was increased by elevated CO₂ for H. radicata, Leontodon saxatilis, T. repens and T. subterraneum in both years and for A. odoratum and Lolium perenne in the first year. For species where increased seedling recruitment was noted, there was a significant positive correlation between seed production in summer and seedling emergence in the following autumn and winter, and sowing 200 extra seeds per species m^-2 resulted in more seedlings compared to unsown controls. Elevated CO₂ did not affect seedling survival in any species. There was no measurable effect of elevated CO₂ on canopy and soil surface conditions or soil moisture at the time of seedling emergence. The results suggest the dominant effect of elevated CO₂ on seedling recruitment in this pasture was an indirect one, reflecting effects on the number of seeds produced. The biomass of H. radicata, Leontodon saxatilis, T. repens and T. subterraneum in the above-ground vegetation was greater under elevated than ambient CO₂. However, the size of individual seedlings and mature plants of these four species was unaffected by elevated CO₂. The results indicate an important way elevated CO₂ influenced plant species composition in this pasture was through changes in the pattern of seedling recruitment.     

Interspecific hybridisations in natural populations of ants by example of a regional fauna (Hymenoptera, Formicidae)

Summary: The identification of hybrids by high-precision stereomicroscopy and chorological data is described in detail by example of the ant species Formica bruni and pressilabris. In a synopsis of heterogenous data, the overall hybrid frequency in the ant fauna of Central Europe is estimated. 17 of the 164 ant species of this region are demonstrated and further 2 species strongly suspected to hybridise. The low-frequency hybridisers, showing overall hybrid ratios < 3%, were native elements of the Central European fauna before the onset of human cultivation and experienced direct interspecific contact for longer periods of their natural history. They developed more effective mechanisms of reproductive isolation beginning at the prezygotic level. Extremely high (12-31%) local hybridisation ratios occurred in species that invaded the area after anthropogenic changes in landscape structure. The segregated distribution of invaders and autochthonous species in the precultural period apparently did not impose the need to evolve more effective mechanisms for reproductive isolation. Prezygotic mechanisms in particular are deficient. In local situations, 19% of Lasius jensi matings and 44% of Leptothorax albipennis matings leading to successful nest foundations were matings with heterospecific partners. Signs for a dissolution of interspecific phenotypic differences are not detectable in most of the species. The dispersal of hybrid genotypes is apparently inhibited. Factors that probably stabilise the genomic integrity of hybridizing parent species are: (a) inability to produce hybrid queens (in L. jensi 2 umbratus), (b) aneuploidy of F₁ females with inability to perform a balanced meiosis (in hybrids of L. albipennis with 3 other species), and (c) selection against hybrids in the epigenetic environment of alleles (Leptothorax nylanderi 2 slavonicus). Extreme ratios of heterospecific matings would mean a dangerous drain of genetic material. A mechanism to reduce these losses is postulated, consisting of a 'cleptogamy' (theft of heterospecific sperm) by queens that missed a conspecific male, an establishment of a functioning colony of F₁ hybrid workers, a depression of the rearing or fertility of hybrid queens, and a maximum production of own sons by the queen. This mechanism could explain the persistence of rare species (Lasius jensi or Leptothorax albipennis) in an environment of more populous heterospecific hybridisation partners.     

Mapping quantitative trait loci controlling early growth in a (longleaf pine × slash pine) × slash pine BC<Subscript>1</Subscript> family

Random amplified polymorphic DNA (RAPD) markers were employed to map the genome and quantitative trait loci controlling the early growth of a pine hybrid F₁ tree (Pinus palustris Mill. 2 P. elliottii Engl.) and a recurrent slash pine tree (P. elliottii Engl.) in a (longleaf pine 2 slash pine) 2 slash pine BC₁ family consisting of 258 progeny. Of the 150 hybrid F₁ parent-specific RAPD markers, 133 were mapped into 17 linkage groups covering a genetic distance of 1,338.2 cM. Of the 116 slash pine parent-specific RAPD markers, 83 were mapped into 19 linkage groups covering a genetic distance of 994.6 cM. A total of 11 different marker intervals were found to be significantly associated with 13 of the 20 traits on height and diameter growth using MAPMAKER/QTL. Nine of the eleven marker intervals were unique to the hybrid parent 488 genome, and two were unique to the recurrent parent 18-27 genome. The amount of phenotypic variance explained by the putative QTLs ranged from 3.6% to 11.0%. Different QTLs were detected at different ages. Two marker intervals from the hybrid parent 488 were found to have QTL by environment interactions.     

Tripsacum dactyloides (Poaceae): a natural model system to study parthenogenesis

Diplosporous apomeiosis, formation of unreduced embryo sacs primarily of the Antennaria type, followed by parthenogenetic embryo development and pseudogamy (fertilization of the central cell) describe gametophytic apomixis within the Tripsacum agamic complex. Tripsacum dactyloides (Eastern gamagrass) is a close relative of domesticated maize and was chosen as a natural model system to investigate gene expression patterns associated with parthenogenesis. The genome size of diploid sexual and polyploid apomictic T. dactyloides was estimated by flow cytometry to be 7.37 pg (2C), 14.74 pg (4C) and 22.39 pg (6C), respectively. The diploid genome size is thus approximately 1.352 larger than that of maize. The apomeiotic-pseudogamous pathway of seed formation was demonstrated at a rate of 92% by the flow cytometric seed screen (FCSS) with single mature seeds in tetraploid accessions. This number includes twin embryos which were detected in 13% of the seeds analyzed. Fertilization of unreduced egg cells (B_III hybrids) was measured in 10% of apomictic seeds. Autonomous (fertilization-independent) embryo development and fertilization-dependent endosperm formation were confirmed by pollination of tetraploid T. dactyloides with a diploid transgenic maize line carrying an actin::#-glucuronidase (GUS) reporter construct. GUS expression was detected after pollination in the developing endosperm, but not in the embryo. In similar intraspecific crossing experiments with maize, GUS expression was detected in both the embryo and endosperm. A protocol was established for microdissection of embryo sacs and early parthenogenetic embryos of T. dactyloides. Together, these techniques provide new tools for future studies aimed at comparing gene expression patterns between sexual maize and sexual or apomictic T. dactyloides.     

Cyanobacterial GR6 glutamate-1-semialdehyde aminotransferase: a novel enzyme-based selectable marker for plant transformation

The mutant glutamate-1-semialdehyde aminotransferase (GSA-AT) enzyme encoded by the hemL gene of the gabaculine-resistant cyanobacterium Synechococcus PCC6301 strain GR6 was expressed in tobacco following Agrobacterium-mediated transformation of leaf discs. When targeted to plastids, the GR6 hemL gene product conveyed gabaculine resistance to transgenic plants. Selection using 50 and 100 µM gabaculine was shown to produce two distinct explant phenotypes: 'greens' and 'whites'. T₁ progeny displayed Mendelian segregation ratios, and PCR analysis demonstrated the 'green' phenotype corresponded with the presence of the GR6 hemL gene. Furthermore, 'whites' could be rescued after 9 days growth on solid media containing between 5 µM and 25 µM gabaculine, allowing the potential use of this system for the isolation of gabaculine-sensitive transformants in mutagenesis screening. The use of GR6 hemL as a selectable marker gene provides a novel enzyme-based method for the selection of transgenic regenerants without the need for antibiotic-resistance markers.     

An Attempt to Detect siRNA-Mediated Genomic DNA Modification by Artificially Induced Mismatch siRNA in Arabidopsis

Although tremendous progress has been made in recent years in identifying molecular mechanisms of small interfering RNA (siRNA) functions in higher plants, the possibility of direct interaction between genomic DNA and siRNA remains an enigma. Such an interaction was proposed in the ‘RNA cache’ hypothesis, in which a mutant allele is restored based on template-directed gene conversion. To test this hypothesis, we generated transgenic Arabidopsis thaliana plants conditionally expressing a hairpin dsRNA construct of a mutated acetolactate synthase (mALS) gene coding sequence, which confers chlorsulfuron resistance, in the presence of dexamethasone (DEX). In the transgenic plants, suppression of the endogenous ALS mRNA expression as well as 21-nt mALS siRNA expression was detected after DEX treatment. After screening >100,000 progeny of the mALS siRNA-induced plants, no chlorsulfuron-resistant progeny were obtained. Further experiments using transgenic calli also showed that DEX-induced expression of mALS siRNA did not affect the number of chlorsulfuron-resistant calli. No trace of cytosine methylation of the genomic ALS region corresponding to the dsRNA region was observed in the DEX-treated calli. These results do not necessarily disprove the ‘RNA cache’ hypothesis, but indicate that an RNAi machinery for ALS mRNA suppression does not alter the ALS locus, either genetically or epigenetically.