Abnormal Cotyledons Research Articles

Vacuolar Sugar Transporter TMT2 Plays Crucial Roles in Germination and Seedling Development in Arabidopsis

Vacuolar sugar transporters transport sugar across the tonoplast, are major players in maintaining sugar homeostasis, and therefore play vital roles in plant growth, development, and biomass yield. In this study, we analyzed the physiological roles of the tonoplast monosaccharide transporter 2 (TMT2) in Arabidopsis. In contrast to the wild type (WT) that produced uniform seedlings, the tmt2 mutant produced three types of offspring: un-germinated seeds (UnG), seedlings that cannot form true leaves (tmt2-S), and seedlings that develop normally (tmt2-L). Sucrose, glucose, and fructose can substantially, but not completely, rescue the abnormal phenotypes of the tmt2 mutant. Abnormal cotyledon development, arrested true leaf development, and abnormal development of shoot apical meristem (SAM) were observed in tmt2-S seedlings. Cotyledons from the WT and tmt2-L seedlings restored the growth of tmt2-S seedlings through micrografting. Moreover, exogenous sugar sustained normal growth of tmt2-S seedlings with cotyledon removed. Finally, we found that the TMT2 deficiency resulted in growth defects, most likely via changing auxin signaling, target of rapamycin (TOR) pathways, and cellular nutrients. This study unveiled the essential functions of TMT2 for seed germination and initial seedling development, ensuring cotyledon function and mobilizing sugars from cotyledons to seedlings. It also expanded the current knowledge on sugar metabolism and signaling. These findings have fundamental implications for enhancing plant biomass production or seed yield in future agriculture.

Smooth Elongation ofPavement Cells Induced by RIC1 Overexpression Leads to Marginal Protrusions ofthe Cotyledon inArabidopsis thaliana.

The interdigitated pavement cell shape is suggested to be mechanically rational at both the cellular and tissue levels, but the biological significance of the cell shape is not fully understood. In this study, we explored the potential importance of the jigsaw puzzle-like cell shape for cotyledon morphogenesis in Arabidopsis. We used a transgenic line overexpressing a Rho-like GTPase-interacting protein, ROP-INTERACTIVE CRIB MOTIF-CONTAINING PROTEIN 1 (RIC1), which causes simple elongation of pavement cells. Computer-assisted microscopic analyses, including virtual reality observation, revealed that RIC1 overexpression resulted in abnormal cotyledon shapes with marginal protrusions, suggesting that the abnormal organ shape might be explained by changes in the pavement cell shape. Microscopic, biochemical and mechanical observations indicated that the pavement cell deformation might be due to reduction in the cell wall cellulose content with alteration of cortical microtubule organization. To examine our hypothesis that simple elongation of pavement cells leads to an abnormal shape with marginal protrusion of the cotyledon, we developed a mathematical model that examines the impact of planar cell growth geometry on the morphogenesis of the organ that is an assemblage of the cells. Computer simulations supported experimental observations that elongated pavement cells resulted in an irregular cotyledon shape, suggesting that marginal protrusions were due to local growth variation possibly caused by stochastic bias in the direction of cell elongation cannot be explained only by polarity-based cell elongation, but that an organ-level regulatory mechanism is required.

Genome-Wide DNA Methylation Analysis of Soybean Curled-Cotyledons Mutant and Functional Evaluation of a Homeodomain-Leucine Zipper (HD-Zip) I Gene GmHDZ20.

DNA methylation is a major, conserved epigenetic modification that influences many biological processes. Cotyledons are specialized tissues that provide nutrition for seedlings at the early developmental stage. To investigate the patterns of genomic DNA methylation of germinated cotyledons in soybean (Glycine max) and its effect on cotyledon development, we performed a genome-wide comparative analysis of DNA methylation between the soybean curled-cotyledons (cco) mutant, which has abnormal cotyledons, and its corresponding wild type (WT) by whole-genome bisulfite sequencing. The cco mutant was methylated at more sites but at a slightly lower level overall than the WT on the whole-genome level. A total of 46 CG-, 92 CHG-, and 9723 CHH- (H = A, C, or T) differentially methylated genes (DMGs) were identified in cotyledons. Notably, hypomethylated CHH-DMGs were enriched in the gene ontology term “sequence-specific DNA binding transcription factor activity.” We selected a DMG encoding a homeodomain-leucine zipper (HD-Zip) I subgroup transcription factor (GmHDZ20) for further functional characterization. GmHDZ20 localized to the nucleus and was highly expressed in leaf and cotyledon tissues. Constitutive expression of GmHDZ20 in Arabidopsis thaliana led to serrated rosette leaves, shorter siliques, and reduced seed number per silique. A yeast two-hybrid assay revealed that GmHDZ20 physically interacted with three proteins associated with multiple aspects of plant growth. Collectively, our results provide a comprehensive study of soybean DNA methylation in normal and aberrant cotyledons, which will be useful for the identification of specific DMGs that participate in cotyledon development, and also provide a foundation for future in-depth functional study of GmHDZ20 in soybean.

Vasa prævia : quand et comment rechercher les vasa prævia en échographie et quelle prise en charge proposer aux patientes en cas de découverte d’un vasa prævia ? À propos de quatre cas et revue de la littérature

Vasa praevia is a rare complication that can arise during pregnancy (1 in 2,000-6,000 cases). Rupture of the vessels in the cervical dilation canal can lead to Benckiser’s haemorrhage, a serious fetal haemorrhage associated with a mortality rate of 75–100 %. Screening is carried out where risk factors are present. These are irregularities of the placenta (placenta praevia in the second trimester, placenta bipartita, or one or more abnormal cotyledons), velamentous cord insertion, multiple pregnancies, and in vitro fertilisation. We set out the screening procedures and outline the treatment given to pregnant women when this complication is detected.

Abnormal Somatic Embryo Reduction and Recycling in Grapevine Regeneration

The purpose of the study was to increase plant conversion efficiency and reduce waste of the abnormal somatic embryos; factors influencing both the embryo morphology in germination and the embryogenic callus reinduction rate in secondary embryogenesis were studied in Vitis vinifera L. ‘Thompson Seedless.’ Embryos having mono-, di-, and multiple-normal cotyledons, trumpet-shaped, fascicular, and fused abnormal cotyledons were observed during germination. Normal embryos of one, two, and multiple cotyledons have a relatively higher conversion rate (48.89–64.44%) than abnormal ones (2.22–22.22%). Histomorphology analysis of these embryos showed that normal somatic embryos had a better developed vasculature and shoot meristem than abnormal individuals. A lower percentage of abnormal embryos formed when they germinated on Murashige and Skoog’s medium (MS) and Woody Plant Medium (WPM) both of which contained 15 g/l sucrose and no plant growth regulators. Of all the abnormal embryos, only fused cotyledon embryos were suitable for recycling, and they were reinduced into the embryogenic callus. Cotyledons showed a higher reinduction rate (72.22%) than the other two parts: hypocotyls (4.44%) and roots (0%). In addition, recycled abnormal embryos produced many morphogenic types and this secondary embryogenesis could be used for embryo proliferation.

Inactivation of bacteria on plant seed surface by low-pressure RF plasma using a vibrating stirring device

Studies on applications of plasmas in agriculture have been conducted actively in many countries. The inactivation effect of low-pressure RF oxygen and air plasmas on seed-borne bacteria on plant seed surfaces was investigated. Active species generated in low-pressure plasmas could reach all seed surfaces using a vibrating stirring device, and the inactivation effect would be improved. When cabbage seeds contaminated artificially by Xanthomonas campestris pv. campestris (Xcc), one of seed-borne bacteria that have high pathogenicity, were irradiated by low-pressure oxygen and air plasmas, the inactivation effect with the vibrating stirring device was more than approximately 10 times higher than that without the device. Although the surface of cabbage seeds suffered the crack-like damage, germination rates of cabbage seeds at 3 and 7 days after seeding were not affected by low-pressure oxygen and air plasmas. Also, abnormal cotyledon and root on germinating cabbage were almost not observed after the seeds irradiated by low-pressure plasmas were cultivated for 7 days. Furthermore, occurrence of mold and rot on germinating cabbage was not observed when cabbage seeds were irradiated by low-pressure plasmas and cultivated for 7 days.

English

The objective of study was to verify if desiccation and storage of the seeds affect the morphoanatomy and histochemistry of the seedlings of Campomanesia adamantium. The fruits were processed and seeds were subjected to desiccation to 30, 20, 15, 10 and 5% water contents in laboratory conditions and subsequently subjected to the following storage conditions: In the laboratory (LAB) (25 ± 2°C, 35% RH), cold and dry chamber (CC) (16 ± 2°C, 40% RH), refrigerator (REF) (8 ± 2°C, 35% RH), and freezer (FZ) (-18 ± 1°C, 42% RH) for 0 (newly processed), 30, 60, 90, 120, 150, and 180 days. The evaluation of the seedling survival rate was performed for 42 days, calculated as the survival percentages for seedling shoot and primary root. Anatomical observations and histochemical tests were performed using fixed and non-fixed samples of the median region of xylopodium in normal and abnormal seedlings. In the morphoanatomy of normal seedlings, we observed cotyledons, hypocotyl, xylopodium, and well defined primary root and in abnormal seedlings, cotyledons, hypocotyl and xylopodium (with 20 and 15% water content), and hypocotyl and primary root (with 10% water content). The desiccation and storage of seeds affected the formation of seedlings by preventing the normal development of roots and shoots. The xylopodium of normal and abnormal seedlings showed positive reaction to starch and lipophilic substances. The presence of phenolic compounds and fructans were observed in parenchyma cells of the xylopodium in abnormal seedlings and absent in normal ones. The deleterious effects of desiccation in association with storage induce the production of phenolic compounds and fructans in abnormal C. adamantium seedlings. Key words: Brazilian Savanna, xylopodium, water content, morphoanatomy, histochemistry.

Ectopic expression of the PttKN1 gene in Cardamine hirsuta mediated via the floral dip method

PttKN1 gene (Populus tremula × P. tremuloides KNOTTED1) was isolated from the vascular cambium of a hybrid aspen. Previous studies on transformed plants with the PttKN1 gene suggested that it plays roles in plant development (typically in meristem initiation), maintenance and organogenesis in simple-leaved species. To investigate the gene functions further, sequence analysis of the deduced amino acid was conducted. The results suggested that the gene belongs to the class I KNOX gene (KNOTTED1-like homeobox genes) family and might play important roles in plant development by coding a transcription factor. The gene was introduced into Cardamine hirsuta using the floral dip method mediated via Agrobacterium tumefaciens. The primary transformed plants were obtained via kanamycin selection. Compared to the wild type, the kanamycin resistant plants demonstrated several morphological alterations, such as abnormal cotyledons, abnormal shoot meristem, flattened stem, and lobed and cup-shaped leaves. RT-PCR results showed that the above five types of kanamycin resistant plants expressed the same specific PttKN1 gene band. This suggested that the morphological alterations were caused by the insertion and expression of the gene. However, these phenotypes were similar to other PttKN1 transformed plants, despite the fact that C. hirsuta is a species with compound leaves and the other species have simple leaves. Therefore, the functions of the PttKN1 gene on compound-leaf species have yet to be investigated via the comparison between related species such as Arabidopsis thaliana and C. hirsuta.

English

The present study assessed whether seed germination of Peganum harmala L. is related to population size and whether factors that control three and four cotyledons and difference in cotyledon shape are different and whether abnormality of cotyledons is related to genetic drift. The percentage of final seed germination was highest in Dzungarian Gobian population and followed by Trans-Altai Gobian population and lowest was in East Gobian population (Tukey HSD test, p<0.01). Most seedlings of P. harmala have two cotyledons in our study areas but there were abnormal cotyledons which are three, four and different shape dicotyledons. Three and four cotyledons showed low frequency in biggest (limited by river basins) and bigger (limited by oasis) populations and high frequency in small population (limited by well) whereas pattern of different shape dicotyledon frequency does not follow difference of population size. The different shape dicotyledons showed higher frequency in region with lower elevation, as compared to regions with higher elevation. The present results suggest that percentage of seed germination of P. harmala and frequency of more than two cotyledons might depend on population size, while frequency of different shape dicotyledons might be related to habitat difference which is high soil salinity. Also, our results suppose more than two cotyledons might be related to genetic drift.   Key words: Abnormality of cotyledons, genetic drift, population size, seed germination.

A PP6-Type Phosphatase Holoenzyme Directly Regulates PIN Phosphorylation and Auxin Efflux in Arabidopsis

The directional transport of the phytohormone auxin depends on the phosphorylation status and polar localization of PIN-FORMED (PIN) auxin efflux proteins. While PINIOD (PID) kinase is directly involved in the phosphorylation of PIN proteins, the phosphatase holoenzyme complexes that dephosphorylate PIN proteins remain elusive. Here, we demonstrate that mutations simultaneously disrupting the function of Arabidopsis thaliana FyPP1 (for Phytochrome-associated serine/threonine protein phosphatase1) and FyPP3, two homologous genes encoding the catalytic subunits of protein phosphatase6 (PP6), cause elevated accumulation of phosphorylated PIN proteins, correlating with a basal-to-apical shift in subcellular PIN localization. The changes in PIN polarity result in increased root basipetal auxin transport and severe defects, including shorter roots, fewer lateral roots, defective columella cells, root meristem collapse, abnormal cotyledons (small, cup-shaped, or fused cotyledons), and altered leaf venation. Our molecular, biochemical, and genetic data support the notion that FyPP1/3, SAL (for SAPS DOMAIN-LIKE), and PP2AA proteins (RCN1 [for ROOTS CURL IN NAPHTHYLPHTHALAMIC ACID1] or PP2AA1, PP2AA2, and PP2AA3) physically interact to form a novel PP6-type heterotrimeric holoenzyme complex. We also show that FyPP1/3, SAL, and PP2AA interact with a subset of PIN proteins and that for SAL the strength of the interaction depends on the PIN phosphorylation status. Thus, an Arabidopsis PP6-type phosphatase holoenzyme acts antagonistically with PID to direct auxin transport polarity and plant development by directly regulating PIN phosphorylation.

동백나무 미숙배 배양으로부터 비정상 체세포배 형성과 식물체 재생

동백의 배발생 캘러스는 1 mg/L 2,4-D가 첨가된 MS배지에서 미숙배 배양을 통해 유도하였으며, 동일배지에 계대 배양하여 4주 이상 증식시켰다. 배발생 캘러스로부터 자엽기 체세포배를 생산하기 위하여 2,4-D가 첨가되지 않은 MS기본배지 배양하였다. 동백 미숙배로부터 체세포배 발생율은 25.1%였으며, 형성된 대부분의 체세포배중에서 48.3%가 cup모양, 12.6%가 1개 자엽, 9.4%가 3개자엽, 1.9%가 4개 자엽을 갖는 비정상적인 형태의 체세포배였으며, 반면 2개의 자엽을 갖는 정상적인 체세포배는 27.5%였다. 정상 및 비정상형태의 체세포배를 MS기본배지, <TEX>$\frac{1}{2}$</TEX>MS배지, 0.1 mg/L <TEX>$GA_3$</TEX>가 첨가된 <TEX>$\frac{1}{2}$</TEX>MS배지 및 0.1 mg/L IBA와 <TEX>$GA_3$</TEX>가 조합 첨가된 <TEX>$\frac{1}{2}$</TEX>MS배지에 치상하였을때 2개의 자엽을 갖는 정상체세포배의 식물체 재생율이 11.1%로 0.0 ~ 8.3%를 나타내는 1개 자엽 (0.0 ~ 8.3%), 3개 자엽 (0.0 ~ 5.8%), 4개 자엽 (0.0%) 및 cup 모양 (0.3 ~ 4.2%) 보다 높았다. 이러한 결과로 볼 때 비정상 자엽을 갖는 체세포배 빈도가 높을수록 식물체 재생빈도가 감소될 수 있음을 알 수 있었다. Embryogenic callus was induced from the cultures of immature embryos of Camellia japonica L. on Murashige & Skoog's (MS) solid medium supplemented with 1 mg/L 2,4-dichlorophenoxy acetic acid (2,4-D), and then the embryogenic callus was proliferated on same medium for 4 weeks over. The embryogenic callus was sub-cultured on MS basal medium without 2,4-D to produce coyledonary stage of somatic embryo. The frequency (%) of somatic embryogenesis was 25.1%, and the majority of somatic embryos formed had a abnormal morphology with cupshaped cotyledon (48.3%), one cotyledon (12.6%), three cotyledons (9.4%), four cotyledons (1.9%), whereas was only normal morphology with two cotyledon (27.5%). When the somatic embryos with normal or abnormal cotyledons transfer to MS basal medium or <TEX>$\frac{1}{2}$</TEX> MS medium with/or without plant growth regulators (<TEX>$GA_3$</TEX>, IBA) for regeneration, the frequency (%) of two-cotyledon embryos regenerated into plantlets was higher 11.1% than one cotyledon (0.0~8.3 %), three cotyledons (0.0~5.8%), four cotyledons (0.0%), cup-shaped (0.3~4.2%). These results demonstrated that the anomalous cotyledons of somatic embryos could caused to decrease the rate of plant regeneration.

Microspore culture protocol for Indonesian Brassica oleracea

A microspore culture protocol for Brassica oleracea of Indonesian origin (cv. ‘Kemeh’) has been successfully established. A high number of embryos formed with high microspore density i.e. 15 × 104 cells/ml. Embryo formation was improved by using flower buds (4.5–4.6 mm in length) as explants, a temperature treatment at 30.5°C for 48 h and then transfer to 25°C continuously until embryos formed. A total of 295 embryos were obtained from 189 buds, 30% of which were abnormal (i.e. with an abnormal cotyledon or lacking hypocotyls). All normal embryos that grew and survived, 165 in total, were successfully transferred to soil and grew well in plastic bags (15 cm in diameter) containing a mixture of burned-rice husk and organic manure (1:1, v/v).

Roles of arabinogalactan proteins in cotyledon formation and cell wall deposition during embryo development of Arabidopsis

Arabinogalactan proteins (AGPs) are a class of highly glycosylated, widely distributed proteins in higher plants. In the previous study, we found that the green fluorescence from JIM13-labeled AGPs was mainly distributed in embryo proper and the basal part of suspensor but gradually disappeared after the torpedo-stage embryos in Arabidopsis. And (β-D-Glc)(3) Yariv phenylglycoside (βGlcY), a synthetic reagent that specifically binds to AGPs, could inhibit embryo development. In this study, as a continuous work, we investigated the AGP functions in embryo germination, cotyledon formation, and cell wall deposition in Arabidopsis embryos by using immunofluorescent, immunoenzyme, transmission electron microscopy (TEM), and Fourier transform infrared spectroscopy (FTIR) techniques. The results showed that 50 μM βGlcY caused inhibition of embryo germination, formation of abnormal cotyledon embryos, and disorder of cotyledon vasculature. Compared with the normal embryos in vitro and in vivo, the AGPs and pectin signals were quite weaker in the whole abnormal embryos, whereas the cellulose signal was stronger in the shoot apical meristem (SAM) of abnormal embryo by calcofluor white staining. The FTIR assay demonstrated that the cell wall of abnormal embryos was relatively poorer in pectins and richer in cellulose than those of normal embryos. By TEM observation, the SAM cells of the abnormal embryos had less cytoplasm, more plastid and starch grains, and larger vacuole than that of normal embryos. These results indicated that AGPs may play roles in embryo germination, cotyledon formation, cell wall cellulose and pectin deposition, and cell division potentiality during embryo development of Arabidopsis.

Morphology of somatic embryogenesis and plantlet formation in tissue cultures of lantern tree (Koelreuteria bipinnata var. integrifoliola)

Somatic embryogenesis of Koelreuteria bipinnata var. integrifoliola was observed, plantlet formation in different types of somatic embryos was studied and the effect of abnormal embryos on plantlet formation was identified. Results show that somatic embryos of K. bipinnata var. integrifoliola include normal embryos, embryos with abnormal cotyledons, vitrified embryos, albino embryos, secondary embryos, linked embryos, embryos with abnormal growing points and embryos with expanding hypocotyl. After 40 d of callus culture, the response of normal somatic embryos from K. bipinnata var. integrifoliola was 26.7%, embryos with abnormal cotyledon 30.3% while other types of somatic embryos were below the 20% level. Most of normal embryos developed into plantlets and plantlet formation reached 94.9%. But the percentage of plantlet formation decreased apparently in abnormal embryos: the number of embryos with abnormal cotyledon declined to 76.1%, that of linked embryos to 47.4% and other types of abnormal embryos to below the 20% level. Albino embryos and embryos with abnormal growing point did not develop at all into plantlets. Embryos with abnormal cotyledons, linked embryos and embryos with abnormal growing points were observed during early stages of somatic embryogenesis, but vitrified, secondary and albino embryos and calli of embryos were observed at later stages. Increasing sucrose concentrations can decrease the occurrence of vitrified embryos, but the number of albino embryos decreased with an increasing in sucrose concentration.

Plant SMU-1 and SMU-2 Homologues Regulate Pre-mRNA Splicing and Multiple Aspects of Development

In eukaryotes, alternative splicing of pre-mRNAs contributes significantly to the proper expression of the genome. However, the functions of many auxiliary spliceosomal proteins are still unknown. Here, we functionally characterized plant homologues of nematode suppressors of mec-8 and unc-52 (smu). We compared transcript profiles of maize (Zea mays) smu2 endosperm with those of wild-type plants and identified pre-mRNA splicing events that depend on the maize SMU2 protein. Consistent with a conserved role of plant SMU-2 homologues, Arabidopsis (Arabidopsis thaliana) smu2 mutants also show altered splicing of similar target pre-mRNAs. The Atsmu2 mutants occasionally show developmental phenotypes, including abnormal cotyledon numbers and higher seed weights. We identified AtSMU1 as one of the SMU2-interacting proteins, and Atsmu1 mutations cause similar developmental phenotypes with higher penetrance than Atsmu2. The AtSMU2 and AtSMU1 proteins are localized to the nucleus and highly prevalent in actively dividing tissues. Taken together, our data indicated that the plant SMU-1 and SMU-2 homologues appear to be involved in splicing of specific pre-mRNAs that affect multiple aspects of development.

Roles of extensins in cotyledon primordium formation and shoot apical meristem activity in Nicotiana tabacum.

Extensins are cell wall basic glycoproteins with a polypeptide backbone that is extremely rich in hydroxyproline. In this paper, the function of extensins in embryo development was studied in Nicotiana tabacum. By using Western blot and immunohistochemistry, the extensin JIM20 epitopes were found to express in different developmental stages of embryos, and specifically in the top of the embryo proper (EP) and the suspensor of the late globular embryos. In order to clarify the functions of extensins, a potent hydroxyproline synthesis inhibitor, 3,4-dehydro-L-proline (3,4-DHP), was used in ovule and embryo culture. The results showed that the addition of 3,4-DHP caused abnormal embryos with single, asymmetry and supernumerary cotyledon primordia, and continuous culture led to cotyledon defects in the germinated seedlings. Histological sections showed that the shoot apical meristem (SAM) of the abnormal seedlings was dissimilar from the controls, especially in the seedlings with cup-shaped cotyledons. Furthermore, the vasculature of the abnormal cotyledons was in an out-of-order format and contained at least two main veins. Finally, both the hydroxyproline assay and fluorescent immunolocalization confirmed that 3,4-DHP treatment reduced the level of extensins in the cultured ovules and embryos. These results indicate that extensins may play important roles in the cotyledon primordium formation, SAM activity, and vasculature differentiation during embryo development.

Inhibited polar auxin transport results in aberrant embryo development in Norway spruce

Current hypotheses concerning the role of polar auxin transport in embryo development are entirely based on studies of angiosperms, while little is known about how auxin regulates pattern formation in gymnosperms. In this study, different developmental stages of somatic embryos of Norway spruce (Picea abies) were treated with the polar auxin transport inhibitor 1-N-naphtylphthalamic acid (NPA). Effects of the treatments on auxin content, embryo differentiation and programmed cell death (PCD) were analysed. During early embryo development, NPA-treatment led to increased indole-3-acetic acid (IAA) content, abnormal cell divisions and decreased PCD, resulting in aberrant development of embryonal tube cells and suspensors. Mature embryos that had been treated with NPA showed both apical and basal abnormalities. Typically the embryos had abnormal cotyledon formation and irregular cell divisions in the area of the root meristem. Our results show that polar auxin transport is essential for the correct patterning of both apical and basal parts of conifer embryos throughout the whole developmental process. Furthermore, the aberrant morhologies of NPA-treated spruce embryos are comparable with several auxin response and transport mutants in Arabidopsis. This suggests that the role of polar auxin transport is conserved between angiosperms and gymnosperms.

In Vitro Induction of Aneuploid Forms of Tetraploid Grapes by para-Fluorophynylalanine

Mutation or somaclonal variation is one of the most useful tools in breeding of vegetatively propagated plants such as fruit trees. Mutation mainly occurs at the gene or chromosome level in a given plant. Para–fluorophenylalanine (PFP), which is an amino acid analogue, alters amino acid metabolism (Widholm, 1977), inhibits mitotic spindle formation (Thao et al., 2003) and, subsequently, reduces chromosome number to haploid number in the daughter cells (Lhoas, 1961). The effect of PFP on the reduction of chromosome number has been firstly reported in diploid Aspergillus niger from which haploid cells were induced (Lhoas, 1961). After this report, similar effects of PFP have been described in many plants such as Ribes (Knight et al., 1963), Allium (Sinha and Bhojwani, 1976), Fragaria (Niizeki and Fukui, 1983) and Alocasia (Thao et al., 2003). In Vitis, it has been reported that PFP effected on the change of chromosome number in the root cells of seedlings (Omura and Akihama, 1980; Omura et al., 1985) and induction of a triploid from PFP–treated shoots of tetraploid ‘Kyoho’ (Omura and Akihama, 1981). However, there are no reports as to somaclonal induction of hyper– and hypo–tetraploid grapes by PFP treatment. Hyper– and hypo–tetraploid grapes (2n=4x±1 or 4x±2) resulting from failure or irregularity of meiosis were found as a spontaneous (probably S1) seedling of ‘Kyoho’ (Yamane et al., 1978), appeared in selfed seedlings with abnormal cotyledons in ‘Kyoho’ (Park et al., 1999a) and rarely found in hybrid seedlings from interploid crosses between triploid and tetraploid grapes (Park et al., 1999b, 2002). ‘Takao’, a spontaneous hypotetraploid seedling of ‘Kyoho’, produces almost seedless berries of small size, but it produces large seedless berries with the aid of gibberellin treated at full bloom (Ashikawa, 1972). Thus, the crossing between tetraploid cultivars, followed by selection of the resultant seedlings with abnormal cotyledons, is an excellent method to obtain hyper– and hypo–tetraploid hybrid grapes for breeding of seedless cultivars. On the other hand, direct induction of hyper– and hypo–tetraploid forms from excellent but seeded tetraploid grape cultivars without crossings may be useful for establishing seedlessness in the seeded cultivars without greatly loosing their high tree vigor and other characters such as productivity, large berry size (Reisch and Pratt, 1996; Notsuka et al., 2000) and berry quality, although high parthenocarpy is required for the original cultivars to set and produce large–sized seedless berries without gibberellin treatment. To establish the efficient method to produce hyper– and hypo–tetraploid forms, we studied the effect of in vitro PFP treatment on the small change in tetraploid chromosome number in cultured shoots of ‘Kyoho’, colchiploid of ‘Rozaki’ and ‘Kyoho’ hybrids, all of which are tetraploid plants with relatively high parthenocarpic ability.

Mechanisms of constraints: the contributions of selection and genetic variance to the maintenance of cotyledon number in wild radish

The evolutionary mechanisms underlying the maintenance of invariant traits are poorly understood, partly because the lack of variance makes these mechanisms difficult to study. Although the number of cotyledons that plant species produce is highly canalized, populations of plants frequently contain individuals with abnormal cotyledon numbers. In a garden study with 1857 wild radish plants from 75 paternal half-sibling families, 89 (almost 5%) had cotyledon numbers less or greater than two. We found evidence for direct selection on cotyledon number, but no evidence for additive genetic variation for cotyledon number. In spite of the very large sample size, our power to detect variation and selection was hampered by the small number of individuals (10) producing more than two cotyledons. Thus, our results provide support for both a lack of genetic variation and selection as reasons for the current lack of variation in wild radish cotyledon number.

Altered expression of the Arabidopsis ortholog of DCL affects normal plant development

The DCL ( defective chloroplasts and leaves) gene of tomato ( Lycopersicon esculentum Mill.) is required for chloroplast development, palisade cell morphogenesis, and embryogenesis. Previous work suggested that DCL protein is involved in 4.5S rRNA processing. The Arabidopsis thaliana (L.) Heynh. genome contains five sequences encoding for DCL-related proteins. In this paper, we investigate the function of AtDCL protein, which shows the highest amino acid sequence similarity with tomato DCL. AtDCL mRNA was expressed in all tissues examined and a fusion between AtDCL and green fluorescent protein (GFP) was sufficient to target GFP to plastids in vivo, consistent with the localization of AtDCL to chloroplasts. In an effort to clarify the function of AtDCL, transgenic plants with altered expression of this gene were constructed. Deregulation of AtDCL gene expression caused multiple phenotypes such as chlorosis, sterile flowers and abnormal cotyledon development, suggesting that this gene is required in different organs. The processing of the 4.5S rRNA was significantly altered in these transgenic plants, indicating that AtDCL is involved in plastid rRNA maturation. These results suggest that AtDCL is the Arabidopsis ortholog of tomato DCL, and indicate that plastid function is required for normal plant development.