Young Leaf Tissue Research Articles

Improvement in efficiency of microspore culture to produce doubled haploid canola (Brassica napus L.) by flow cytometry

The efficiency of production of doubled haploid plants in canola (Brassica napus L.) breeding programmes is reduced when large numbers of haploid and infertile plants survive until flowering. We used flow cytometry to determine ploidy status and predict subsequent fertility of microspore-derived plantlets from three canola genotypes, with or without colchicine treatment of microspore suspensions. Young leaf tissue was sampled from microspore-derived plantlets within 1 week of transfer to soil, and processed immediately by flow cytometry. The process was repeated on the same plants 3–5 weeks later. Of the 519 plants transferred to soil, 57.2% were consistently haploid at both sample times, 33.5% were consistently diploid at both sample times, and the remainder (9.2%) were uncertain or inconsistent in ploidy status across sampling times. Of the 518 plants that survived to flowering, 32.4% were diploid at both times of sampling and fertile (set seed) and 46.3% were haploid at both sampling times and infertile. Another 10.8% were haploid at both sampling times and fertile, but had low pollen viability and seed set, and some were triploid or of uncertain ploidy level. Colchicine treatment of microspore suspensions significantly increased the proportion of diploid plants from 9.7 to 69.7%, with significant variation among genotypes. Evidence from simple sequence repeat marker loci indicated that diploid and fertile plants from the control treatment (no colchicine) were derived from spontaneously doubled haploid gametes, rather than unreduced gametes or somatic tissue. Flow cytometry at the first sample time was very efficient in detecting diploid plants of which 94.2% were subsequently fertile.

A simplified, cost- and time-effective procedure for genotyping pearl millet in resource-limited laboratories

Procedures for DNA extraction and genotyping of large plant populations are cumbersome and expensive for resource-limited laboratories. Through eliminating or changing several steps used in DNA extraction, PCR amplification and PAGE electrophoresis in pearl millet [Pennisetum glaucum (L.) R. Br.], we developed a modified procedure that reduced the cost of consumables and required less time without compromising data quality. In the revised procedure, DNA was extracted by incubating 0.5 - 0.7 g ground young leaf tissue in 2% CTAB/
-mercaptoethanol followed by refrigerated differential centrifugations with phenol:chloroform:isoamylalcohol. Steps such as additional phenol/chloroform treatments, DNA pellet drying followed by RNase treatments and incubation were eliminated, reducing use of costly and corrosive chemicals and saving time. DNA produced from 174 genotypes exhibited an average concentration of 640 ng/iL and average optical density ratio of 1.9. PCR amplification of SSR markers with this DNA produced clear and scorable bands following ethidium bromide stained agarose and silver stained polyacrylamide gel eletcrophoresis. Post PCR duplexing of two or more microsatellites based on different lengths of base pairs reduced the time and cost per unit data generation by up to half as compared to single marker per PAGE. Cluster analysis performed on the marker data generated 11 SSR primers following these procedures formed two main groups from genotypes of the U.S. origin. In summary, the procedures reported are simplified, shortened and economical and well suited for resource limited laboratories engaged in molecular breeding requiring large volume of genotyping.

High Frequency Plant Regeneration from Leaf Derived Callus of HighΔ9-Tetrahydrocannabinol YieldingCannabis sativaL.

An efficient in vitro propagation protocol for rapidly producing Cannabis sativa plantlets from young leaf tissue was developed. Using gas chromatography-flame ionization detection (GC-FID), high THC yielding elite female clone of a drug-type CANNABIS variety (MX) was screened and its vegetatively propagated clones were used for micropropagation. Calli were induced from leaf explant on Murashige and Skoog medium supplemented with different concentrations (0.5, 1.0, 1.5, and 2.0 µM) of indole- 3-acetic acid (IAA), indole- 3- butyric acid (IBA), naphthalene acetic acid (NAA), and 2,4-dichlorophenoxy-acetic acid (2,4-D) in combination with 1.0 µM of thidiazuron (TDZ) for the production of callus. The optimum callus growth and maintenance was in 0.5 µM NAA plus 1.0 µM TDZ. The two-month-old calli were subcultured to MS media containing different concentrations of cytokinins (BAP, KN, TDZ). The rate of shoot induction and proliferation was highest in 0.5 µM TDZ. Of the various auxins (IAA, IBA, and NAA) tested, regenerated shoots rooted best on half strength MS medium (1/2 - MS) supplemented with 2.5 µM IBA. The rooted plantlets were successfully established in soil and grown to maturity with no gross variations in morphology and cannabinoids content at a survival rate of 95 % in the indoor growroom.

Cloning and characterization of a wheat β-1,3-glucanase gene induced by the stripe rust pathogen Puccinia striiformis f. sp. tritici

b-1,3-Glucanases are a group of pathogenesis related proteins that have been reported to be involved in plant defense against pathogens in many other plant pathogen systems. However, it was not clear if these genes play similar role in wheat (Triticum aestivum L.) against Puccinia striiformis f. sp. tritici (Pst), the stripe rust pathogen. To investigate the role of b-1,3-glucanase (EC3.2.1.39) in the resistance response of wheat (cv. Suwon11) to stripe rust, a wheat b-1,3-glucanase gene induced by Pst, designated as TaGlu, was cloned and characterized.TaGlu was predicted to encode a basic protein of 334 amino acids. Quantitative real-time PCR analyses revealed that the transcription of TaGlu was induced during both compatible and incompatible interactions with Pst, but the transcription level was much higher in the incompatible interaction than that in the compatible interaction. TaGlu also showed noticeable induction of gene expression in young green leaf tissues treated with salicylic acid, methyl jasmonate or ethylene. Immunogold labeling assays showed that the enzyme were localized mainly in the host cell wall and over the extra haustorial matrix, and the labeling densities were found significantly higher in the incompatible interaction than those in the compatible interaction.

Characterization of the 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase multigene family of Malus domestica Borkh

Two 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACO) genes have been cloned from RNA isolated from leaf tissue of apple ( Malus domestica cv. Royal Gala). The genes, designated MD-ACO2 (with an ORF of 990 bp) and MD-ACO3 (966 bp) have been compared with a previously cloned gene of apple, MD- ACO1 (with an ORF of 942 bp) . MD-ACO1 and MD-ACO2 share a close nucleotide sequence identity of 93.9% in the ORF but diverge in the 3′ untranslated regions (3′-UTR) (69.5%). In contrast, MD-ACO3 shares a lower sequence identity with both MD-ACO1 (78.5%) and MD-ACO2 (77.8%) in the ORF, and 68.4% ( MD-ACO1) and 71% ( MD-ACO2) in the 3′-UTR. Southern analysis confirmed that MD-ACO3 is encoded by a distinct gene, but the distinction between MD-ACO1 and MD-ACO2 is not as definitive. Gene expression analysis has shown that MD-ACO1 is restricted to fruit tissues, with optimal expression in ripening fruit, MD-ACO2 expression occurs more predominantly in younger fruit tissue, with some expression in young leaf tissue, while MD-ACO3 is expressed predominantly in young and mature leaf tissue, with less expression in young fruit tissue and least expression in ripening fruit. Protein accumulation studies using western analysis with specific antibodies raised to recombinant MD-ACO1 and MD-ACO3 produced in E. coli confirmed the accumulation of MD-ACO1 in mature fruit, and an absence of accumulation in leaf tissue. In contrast, MD-ACO3 accumulation occurred in younger leaf tissue, and in younger fruit tissue. Further, the expression of MD-ACO3 and accumulation of MD-ACO3 in leaf tissue is linked to fruit longevity. Analysis of the kinetic properties of the three apple ACOs using recombinant enzymes produced in E. coli revealed apparent Michaelis constants ( K m) of 89.39 μM ( MD-ACO1), 401.03 μM ( MD-ACO2) and 244.5 μM ( MD-ACO3) for the substrate ACC, catalytic constants ( K cat) of 6.6 × 10 −2 ( MD-ACO1), 3.44 × 10 −2 ( Md-ACO2) and 9.14 × 10 −2 ( MD-ACO3) and K cat/ K m (μM s −1) values of 7.38 × 10 −4 μM s −1 ( MD-ACO1), 0.86 × 10 −4 M s −1 ( MD-ACO2) and 3.8 × 10 −4 μM s −1 ( MD-ACO3). These results show that MD-ACO1, MD-ACO2 and MD-ACO3 are differentially expressed in apple fruit and leaf tissue, an expression pattern that is supported by some variation in kinetic properties.

Herbivory on Zostera marina by the gastropod Smaragdia viridis

The feeding activity of the gastropod Smaragdia viridis on Zostera marina (eelgrass) was studied under laboratory conditions and from shoots collected in a deep eelgrass bed (12–14 m depth) in southern Spain (Alboran Sea). This gastropod preferentially ingested young leaf tissues, such as those located in the central leaf and first pair of adjacent leaves and at close distances from the junction of the leaves with the sheath. The ingestion rate of this gastropod was size dependent, ingesting up to 40.6 mm 2 of epidermal tissues in 24 h (for large individuals), however this value generally represented a very low percentage of the area of a single shoot (0.3–2.1%). The absorption of eelgrass tissues, in relation to digested/non-digested eelgrass cells in faecal pellets, was not size dependent and reached high values (75–90% cells digested). The grazing impact in an eelgrass bed, based on the affected area (length of radular marks by leaf width), also represented a very low value (0.3–1.1%) in relation to the total LAI (Leaf Area Index) available. A seasonal trend of herbivory was registered with maximum values in summer together with maximum densities of S. viridis.

Quadratic form: A robust metric for quantitative comparison of flow cytometric histograms

Comparison of fluorescence distributions is a fundamental part of the analysis of flow cytometric data. This approach is applied to detect differences between control and test sample and thus analyze a biological response. Comparison of standard test samples over time provides an estimate of instrument stability for quality control. However, application of statistical methods of distribution comparison in flow cytometry is difficult owing to instrument noise and the complex shape of intensity distributions. We applied quadratic form (QF) as a mathematical metric for comparison of flow cytometry histograms. QF operates on histograms as vectors and calculates the total distance in an interbin manner using a matrix of distances between single histogram bins. Euclidean interbin distance and histograms normalized to unity were used. Critical values corresponding to 95% significance level were calculated using Monte-Carlo simulation and single-maximum Gaussian distributions populated with several numbers of events. The QF statistic was then validated for non-Gaussian single-maximum distributions and multiple-maxima distributions. We determined that the critical values for Gaussian distributions depended on standard deviations and number of events in the compared histograms. A simple empirical function was constructed to characterize this dependence. Furthermore, it was verified that critical values (corresponding to 95% significance) for non-Gaussian histograms were similar to values for the Gaussian histograms characterized by the same standard deviation. We applied the QF statistic to estimate the differences between histograms of DNA content (ploidy) in cells of old and young leaf tissue of Brassica campestris. Furthermore, we quantified differences in fluorescence intensity in immunostaining of human lymphocytes. Quadratic form (QF) provides a true (mathematical) metric for estimation of distance between flow cytometry histograms of arbitrary shape. QF can be applied as a statistical test for estimation of significance of the distance measure. The respective critical values depend only on the number of events and standard deviations of compared histograms and are not affected by distribution shape. Therefore, applications of QF do not require assumptions concerning distribution shape and can be easily implemented in practice. This notion was confirmed using empirical distributions of DNA content in plant tissue and distributions of immunofluorescence in human cells.

Biochemical and Genomic Characterization of Terpene Synthases inMagnolia grandiflora

Magnolia grandiflora (Southern Magnolia) is a primitive evergreen tree that has attracted attention because of its horticultural distinctiveness, the wealth of natural products associated with it, and its evolutionary position as a basal angiosperm. Three cDNAs corresponding to terpene synthase (TPS) genes expressed in young leaves were isolated, and the corresponding enzymes were functionally characterized in vitro. Recombinant Mg25 converted farnesyl diphosphate (C(15)) predominantly to beta-cubebene, while Mg17 converted geranyl diphosphate (C(5)) to alpha-terpineol. Efforts to functionally characterize Mg11 were unsuccessful. Transcript levels for all three genes were prominent in young leaf tissue and significantly elevated for Mg25 and Mg11 messenger RNAs in stamens. A putative amino-terminal signal peptide of Mg17 targeted the reporter green fluorescent protein to both chloroplasts and mitochondria when transiently expressed in epidermal cells of Nicotiana tabacum leaves. Phylogenetic analyses indicated that Mg25 and Mg11 belonged to the angiosperm sesquiterpene synthase subclass TPS-a, while Mg17 aligned more closely to the angiosperm monoterpene synthase subclass TPS-b. Unexpectedly, the intron-exon organizations for the three Magnolia TPS genes were different from one another and from other well-characterized TPS gene sets. The Mg17 gene consists of six introns arranged in a manner similar to many other angiosperm sesquiterpene synthases, but Mg11 contains only four introns, and Mg25 has only a single intron located near the 5' terminus of the gene. Our results suggest that the structural diversity observed in the Magnolia TPS genes could have occurred either by a rapid loss of introns from a common ancestor TPS gene or by a gain of introns into an intron-deficient progenote TPS gene.

Expression of Caffeine Biosynthesis Genes in Tea (Camellia sinensis)

Using semi-quantitative reverse transcription-PCR, we studied the expression of genes encoding caffeine synthase (TCS1), inosine-5'-monophosphate dehydrogenase (TIDH), S-adenosyl-L-methionine synthase (sAMS), phenylalanine ammonia-lyase (PAL) and alpha-tubulin (Tua1) in young and mature leaves, stems and roots of 4-month-old tea seedlings and young and old tea tissue cultures. The amounts of transcripts of TCS1 were much higher in young leaves than in other parts of the plant. Expression of TIDH was greater in leaves than in other parts. Little difference in the amounts of transcripts of PAL, sAMS and Tua1 was found between various organs of tea seedlings. Larger amounts of transcripts of TCS1 and PAL were found in young callus tissues than in old tissues. These results support our conclusion deriving from previous enzymatic and metabolic studies that caffeine is synthesized mainly in young leaf tissues. We propose that marked caffeine biosynthesis in young leaves is dependent on a greater expression of the TCS1 gene in the organ.

Tobacco Mosaic VirusReplicase-Auxin/Indole Acetic Acid Protein Interactions: Reprogramming the Auxin Response Pathway To Enhance Virus Infection

The replicase protein of Tobacco mosaic virus (TMV) disrupts the localization and stability of interacting auxin/indole acetic acid (Aux/IAA) proteins in Arabidopsis, altering auxin-mediated gene regulation and promoting disease development (M. S. Padmanabhan, S. P. Goregaoker, S. Golem, H. Shiferaw, and J. N. Culver, J. Virol. 79:2549-2558, 2005). In this study, a similar replicase-Aux/IAA interaction affecting disease development was identified in tomato. The ability of the TMV replicase to interact with Aux/IAA proteins from diverse hosts suggests that these interactions contribute to the infection process. To examine the role of this interaction in virus pathogenicity, the replication and spread of a TMV mutant with a reduced ability to interact with specific Aux/IAA proteins were examined. Within young (4- to 6-week-old) leaf tissue, there were no significant differences in the abilities of Aux/IAA-interacting or -noninteracting viruses to replicate and spread. In contrast, in mature (10- to 12-week-old) leaf tissue, the inability to interact with specific Aux/IAA proteins correlated with a significant reduction in virus accumulation. Correspondingly, interacting Aux/IAA levels are significantly higher in older tissue and the overaccumulation of a degradation-resistant Aux/IAA protein reduced virus accumulation in young leaf tissue. Combined, these findings suggest that TMV replicase-Aux/IAA interactions selectively enhance virus pathogenicity in tissues where Aux/IAA proteins accumulate. We speculate that the virus disrupts Aux/IAA functions as a means to reprogram the cellular environment of older cells to one that is more compatible for virus replication and spread.

A Non-destructive Seed Sampling Method for PCR-based Analyses in Marker Assisted Selection and Transgene Screening

Extraction of quality DNA from peanut (Arachis hypogaea L.) generally requires extensive manipulation in order to remove numerous phenolic compounds and polysaccharides. To reduce the amount of problematic compounds present, routine purification of peanut DNA is normally performed on young leaf tissue which requires time and space for seed germination and growth of the peanut plant for at least two to four weeks. Here, we describe a simple, non-destructive method for extracting genomic DNA from a mature dry peanut seed that is of suitable quality for PCR-based molecular analysis. The method we developed requires only 0.02 g of peanut cotyledon tissue taken directly from the distal end of the mature dry seed and provides 30–46 µg of DNA suitable for use in restriction digests, PCR, SCAR and SSR analyses. This method is the first of its kind developed for DNA extraction from peanut seed. Since this method is non-destructive, seed can be subsequently germinated to produce healthy mature plants, making this technique a useful tool for the application of marker assisted selection in screening segregating populations of putative transgenic seed and in the advancement of breeding populations.

Geminivirus Infection Up-Regulates the Expression of Two Arabidopsis Protein Kinases Related to Yeast SNF1- and Mammalian AMPK-Activating Kinases

Geminivirus Rep-interacting kinase 1 (GRIK1) and GRIK2 constitute a small protein kinase family in Arabidopsis (Arabidopsis thaliana). An earlier study showed that a truncated version of GRIK1 binds to the geminivirus replication protein AL1. We show here both full-length GRIK1 and GRIK2 interact with AL1 in yeast two-hybrid studies. Using specific antibodies, we showed that both Arabidopsis kinases are elevated in infected leaves. Immunoblot analysis of healthy plants revealed that GRIK1 and GRIK2 are highest in young leaf and floral tissues and low or undetectable in mature tissues. Immunohistochemical staining showed that the kinases accumulate in the shoot apical meristem, leaf primordium, and emerging petiole. Unlike the protein patterns, GRIK1 and GRIK2 transcript levels only show a small increase during infection and do not change significantly during development. Treating healthy seedlings and infected leaves with the proteasome inhibitor MG132 resulted in higher GRIK1 and GRIK2 protein levels, whereas treatment with the translation inhibitor cycloheximide reduced both kinases, demonstrating that their accumulation is modulated by posttranscriptional processes. Phylogenetic comparisons indicated that GRIK1, GRIK2, and related kinases from Medicago truncatula and rice (Oryza sativa) are most similar to the yeast kinases PAK1, TOS3, and ELM1 and the mammalian kinase CaMKK, which activate the yeast kinase SNF1 and its mammalian homolog AMPK, respectively. Complementation studies using a PAK1/TOS3/ELM1 triple mutant showed that GRIK1 and GRIK2 can functionally replace the yeast kinases, suggesting that the Arabidopsis kinases mediate one or more processes during early plant development and geminivirus infection by activating SNF1-related kinases.

GhSEM-1 Marker Potentially Associated with Regeneration Ability in Cotton

A marker protein for embryogénie potential could be useful in determining if target tissue for Agrobacterium tumefaciens or microprojectile bombardment has the ability to regenerate plants. Certain varieties of cotton, especially Coker 312, are known to form somatic embryos readily, while others are more recalcitrant. Callus induced from Coker 312–17wa s found to be more embryogénie than that from TM-1. To investigate the molecular basis for this difference in somatic embryo potential, total RNA was isolated from globular stage callus embryos, calli with heart-stage embryos, and non-embryogenic calli. Northern analyses were performed using a developmentally regulated cotton fiber gene as a probe. This probe hybridized to a single band, GhSEM-1 (Gossypium hirsutum somatic embryogénie marker 1), only in globularstage embryos. Genomic DNA was also isolated from young leaf tissue of both embryogénie (Coker 312–17) and non-embryogenic (TM-1) lines and a Southern analysis using the same probe was performed. No differences were found between the genomic DNA of both embryogenie and non-embryogenic lines, indicating that this gene was structurally present in both lines but only expressed in Coker 312–17 at the globular stage. Antibodies against GhSEM-1 were developed and used in immunoblots after SDS-PAGE separation of proteins from embryogenie and non-embryogenic calli. The antibody recognized a -15 kDa protein in the globular and heart-stage somatic embryos from Coker 312–17 and was designated as GEP15 (15 kDa Gossypium embryogénie protein). This protein could be used as a marker for determining if tissue has embryogénie potential. Our results represent the first reported potential association of a marker gene (GhSEM-1) and protein (GEP15) with somatic embryogenesis in cotton.

(63) Effect of Fertilization on Growth of Ornamental Lotus in Containers

Little research is reported on container production of ornamental lotus (Nelumbo nucifera Gaertn.). In this study, fertilization has a critical impact on growth index of lotus `No.7', a numbered clone, in 29 liter (7.5 gallon) containers. Compared to the control treatment (zero fertilization), 1–3 tsp. (4g/tsp.) of 20-10-20 (Pro·Sol) applied every 20 days significantly increased plant height (1.3–1.6 times), fresh biomass (2.4–3.3 times), emerging leaf number (1.9–2.7 times), flower number (2.4–2.7 times), and propagule number (1.3–1.5 times). There was a quadratic response as growth parameters increased with increasing fertilizer rates. Growth indices increased linearly from 0–2 tsp. and then leveled as fertilizer rates reached 3 tsp. No difference was recorded in flower number and plant height for 1–3 tsp. fertilizer treatments. Absorption of nutrition increased with fertilization concentration, an absorption peak value appeared between 13 July and 2 Aug. For 1-3 tsp. treatments, nitrogen is nearly 100% absorbed by lotus every 20 days. However, there is some residue for P and K, especially in 3-tsp. treatment in the earlier and later growth season. Analysis of young leaf tissue indicated that macronutrients N, P, K, and dry mass increased, but Ca decreased with increasing fertilizer rates. In tuber tissue, K, Na, and dry mass increased, while Ca and Fe content decreased. The most efficient rate of fertilizer for 7.5 gallon container production of `No.7' lotus was 2 tsp. per 20 days. Although soluble fertilizer also stimulated proliferation of algae growth in the early growth stage of lotus, this problem dissipated as emerging leaves shaded the water surface.

DECIPHERING CHANGES IN PLANT PHYSIOLOGICAL RESPONSE TO WHITEFLY FEEDING USING MICROARRAY TECHNOLOGY

DECIPHERING CHANGES IN PLANT PHYSIOLOGICAL RESPONSE TO WHITEFLY FEEDING USING MICROARRAY TECHNOLOGY

The 14-3-3 Gene Expression Specificity in Response to Stress is Promoter-Dependent

Genomic clone coding for the 16R isoform of 14-3-3 proteins from potato plants has recently been described. This paper reports on 20R-gene isolation and analysis, and compares two isoforms. The northern blot analysis of mRNA of the 20R 14-3-3 isoform suggests its similarity to 16R. Vascular tissue-specific expression and age-dependent synthesis in potato leaves has been detected in both promoters. Screening of the potato genomic library using 20R cDNA isoform resulted in identification and isolation of the corresponding gene. This gene contains four exons and three introns. Inspecting the promoter sequence of the 20R isoform revealed several boxes important for the regulation of gene expression. The strongest GUS expression in transgenic potato plants transformed with the uidA reporter gene under the 20R promoter has been found in young leaf and stem vascular tissue, root tips, pollen and ovules. Mature fragments exhibit a significant decrease in GUS staining, which suggests age-dependent promoter activity. The analysis of transgenic plants transformed with 20R-GUS in contrast to 16R-GUS has revealed strong activation of the 20R promoter by metal ions and NaCl. Instead the 16R promoter is strongly affected by virus and salicylic acid treatments. The only factor, which strongly induced both promoters, was abscisic acid. It is thus suggested that promoter domain composition is the main factor differentiating the appearance of 14-3-3 isoforms.

Effect of simulated and actual herbivory on alligator weed, Alternanthera philoxeroides, growth and reproduction

Simulated herbivory has often been used as a substitute for actual herbivory when assessing the effects of herbivores on plants. However, mechanical damage does not always produce the same response as herbivore feeding. Similarity of plant response should be determined before mechanical damage is used as a surrogate for actual herbivory. This study compared two types of mechanical leaf tissue removal with actual herbivory on alligator weed [Alternanthera philoxeroides (Martius) Grisebach (Amaranthaceae)], a significant wetland weed in Australia. We contrasted the effects of simulated herbivory with those of actual herbivory by the alligator weed flea beetle, Agasicles hygrophila Selman and Vogt (Chrysomelidae: Alticinae), on plant growth and reproduction. There was no significant difference in the response of plant biomass to the three herbivory treatments. However, stem length and the number of stem nodes were reduced more by beetle damage than by the two simulated treatments. This was likely caused by preferential feeding on young leaf tissue and/or feeding damage to stem tissue as leaf tissue quality decreased at greater defoliation levels. To more closely mimic alligator weed response to A. hygrophila adult feeding for variables other than biomass, simulated damage methods should consider damage to new growth and stem tissue.

Rapid and Simple Procedure for Homogenizing Leaf Tissues Suitable for Mini‐Midi‐Scale DNA Extraction in Rice

We describe a rapid and simple procedure for homogenizing leaf samples suitable for mini/midi‐scale DNA preparation in rice. The methods used tungsten carbide beads and general vortexer for homogenizing leaf samples. In general, two samples can be ground completely within 11.3±1.5 sec at one time. Up to 20 samples can be ground at a time using a vortexer attachment. The yields of the DNA ranged from 2.2 to 7.6 µg from 25–150 mg of young fresh leaf tissue. The quality and quantity of DNA was compatible for most of PCR work and RFLP analysis.

(247) Genetic and Environmental Effects on Glucosinolate Content in Chinese Cabbage

Gluconasturtiin is a glucosinolate (GS) present in Chinese cabbage and its breakdown product, phenelethyl isothiocyanate (PEITC), inhibits phase I enzyme activation of endogenous carcinogenic compounds and enhances phase II enzyme detoxication, reducing cancer risk and promoting health in humans. This study was conducted to evaluate the interaction between the genotype and the environment to influence GSs in Chinese cabbage. Twenty-five accessions were grown in three environments and tissue quantified for GS levels by HPLC. While gluconasturtiin was observed to be the most abundant GS form, 3-indolylmethyl GS (glucobrassicin) and 1-methoxy-3-indolylmethyl-GS (neoglucobrassicin) were also found. Significant differences were observed among tissues, genotypes and environments in GS concentration and composition. Gluconasturtiin ranged from 0.56 μmol·g-1 DW in leaf tissue of Hau No. 2 to 11.89 μmol·g-1 DW in Chilsung. There were dramatic differences among different tissues of the same genotype with young leaf and root tissues having significantly higher concentrations of gluconasturtiin than other tissues. Gluconasturtiin in Sandong No. 5 ranged from 1.69 μmol·g-1 DW in mature leaves to 18.69 μmol·g-1 DW in root tissue. GS content of the same genotypes in three different environments indicated that plants grown in the greenhouse had higher GS content compared to field grown plants. Results of this study indicate that genotypic variation and the growing environment have substantial effects on GS content in Chinese cabbage. This investigation provides important information for future genetic and molecular studies and has identified Chinese cabbage genotypes that offer superior health benefits to consumers.

Involvement of α-Amylase I-1 in Starch Degradation in Rice Chloroplasts

To determine the role of alpha-amylase isoform I-1 in the degradation of starch in rice leaf chloroplasts, we generated a series of transgenic rice plants with suppressed expression or overexpression of alpha-amylase I-1. In the lines with suppressed expression of alpha-amylase I-1 at both the mRNA and protein levels, seed germination and seedling growth were markedly delayed in comparison with those in the wild-type plants. However, the growth retardation was overcome by supplementation of sugars. Interestingly, a significant increase of starch accumulation in the young leaf tissues was observed under a sugar-supplemented condition. In contrast, the starch content of leaves was reduced in the plants overexpressing alpha-amylase I-1. In immunocytochemical analysis with specific anti-alpha-amylase I-1 antiserum, immuno-gold particles deposited in the chloroplasts and extracellular space in young leaf cells. We further examined the expression and targeting of alpha-amylase I-1 fused with the green fluorescent protein in re-differentiated green cells, and showed that the fluorescence of the expressed fusion protein co-localized with the chlorophyll autofluorescence in the transgenic cells. In addition, mature protein species of alpha-amylase I-1 bearing an oligosaccharide side chain were detected in the isolated chloroplasts. Based on these results, we concluded that alpha-amylase I-1 targets the chloroplasts through the endoplasmic reticulum-Golgi system and plays a significant role in the starch degradation in rice leaves.