Genetic analysis of resistance to whitebacked planthopper in twenty-one varieties of rice,Oryza sativa L.

Summary The inheritance of resistance to whitebacked planthopper Sogatella furcifera (Horvath) was studied in 21 rice varieties. Reactions of F₁; F₂ and F₃ progenies of the crosses of 21 resistant varieties with the susceptible variety TN 1 revealed that a single dominant gene governs resistance in Mushkan 41, Santhi, Siahnakidar 195, SM2-34, Tirisurkh 251, Zirijowaian 245, 18, 24A, 39, 76 S, 78, 180, 213 B, 267, 293, CI 6037-4, NP97, S39 JKW and Bansphul. In varieties 65 and 274 A, resistance is governed by one dominant and one recessive gene which segregate independently of each other. Tests for allelism with the Wbph 1 gene originally identified in N 22 revealed that the dominant gene present in all the test varieties is the same as Wbph 1. Further studies are required to determine the allelic relationships of the recessive gene found in varieties 65 and 274 A.     

Transfer of Ogu cytoplasmic male sterility to Brassica juncea and improvement of the male sterile line through somatic cell fusion

Male sterility conferred by ogu cytoplasm of Raphanus sativus has been transferred to Brassica juncea cv RLM 198 from male-sterile B. napus through repeated backcrossing and selection. The male-sterile B. juncea is, however, highly chlorotic and late. It has low female (seed) fertility and small contorted pods. To rectify these defects, protoplasts of the male sterile were fused with normal RLM 198 (green, self fertile). Four dark green, completely male-sterile plants were obtained and identified as putative cybrids. All the plants were backcrossed three times with RLM 198. Mitochondrial and chloroplast DNA analysis of backcross progeny confirmed hybridity of the cytoplasm. The restriction pattern of the chloroplast DNA of progeny plants of three cybrids (Og 1, Og 2, Og 3) was similar to that of the green self-fertile RLM 198 and indicated that the correction of chlorosis resulted from chloroplast substitution. The chloroplast DNA of the lone progeny plant of the fourth cybrid (Og 10) could not be analyzed because the plant was stunted and had only a few leaves. When total cellular DNA was probed with mitochondrial probes coxI and atpA it was found that the cybrids had recombinant mitochondria. The chlorosis-corrected plants were early flowering and had vastly improved seed fertility.     

The sequence surrounding the translation initiation codon of the pea plastocyanin gene increases translational efficiency of a reporter gene

The 5-upstream region of the pea plastocyanin gene (petE) directed 5–10-fold higher levels of -glucuronidase (GUS) activity than the cauliflower mosaic virus 35S promoter in transgenic tobacco plants, although the levels of GUS mRNA were similar. The sequence (AAAAAUGG) around the translation initiation codon of petE enhanced translation of the GUS mRNA 10-fold compared to translation from the GUS translation initiation codon in transgenic tobacco plants and transfected protoplasts.     

Field performance of derived generations of transgenic tobacco

Two inbred cultivars of Nicotiana tabacum (tobacco), Samsun and Xanthi, were transformed with the plasmid pBI 121 using Bin 19 in Agrobacterium tumefaciens. The plasmid carries the nptII gene conferring kanamycin resistance and the uidA gene encoding -glucuronidase (GUS). Progeny carrying the genes in the homozygous condition were identified and selfed over several generations. One line homozygous for the introduced genes and one untransformed control from each cultivar were then selected and crossed reciprocally to give four families per cultivar. Seeds from each family were grown in a replicated field trial and all plants scored for a range of morphological and agronomic characters. In addition, leaf samples were taken and GUS activity measured. In the Samsun material, which contained one copy of the introduced gene at a single locus and showed high levels of GUS expression, the transformed homozygote showed twice the level of GUS activity as the hemizygotes, wheareas in the Xanthi line, which had a lower level of GUS, the hemizygotes showed the same level of GUS activity as the transformed homozygote. The agronomic data showed differences between the families, but the source of such differences could not be ascribed unambiguously. The results are discussed in the light of related information on gene expression and field performance from other transgenic material.     

Microspore cultures as donor tissue for the initiation of embryogenic cell suspensions in barley

We have initiated embryogenic cell suspension cultures of barley (Hordeum vulgare L.) Igri from isolated microspore cultures. Data were obtained on the time required for establishment, frequency of establishment, i.e. number of calluses out of the total number of initiations giving rise to suspensions, and embryogenic capacity of the suspension cultures. For comparison, establishment of embryogenic cell suspensions from callus derived from immature zygotic embryos of Igri, Dissa and Golden Promise was also carried out. The results revealed that embryogenic suspension cultures were established in half the time and with a seven-fold higher frequency from microspore cultures than from zygotic embryo-derived calluses. The suspension cultures were still capable of embryo formation after two years. However, only albino plantlets were regenerated. For comparison, long term callus cultures derived from microspores, anthers and zygotic embryos were established. From the anther and zygotic embryo-derived callus cultures green plants were continuously regenerated, whereas the microspore-derived callus cultures lost this ability after the second subculture.     

The αβ complexes of ATP synthase: the α3β3 oligomer and α1β1 protomer

The basic structures of the catalytic portion (F₁, ₃₃) of ATP synthase are the ₃₃ hexamer (oligomer with cooperativity) and ₁₁ heterodimer (protomer). These were reconstituted from the and subunits of thermophilic F₁ (TF₁), and the ₃₃ hexamer was crystallized. On electrophoresis, both the dimer and hexamer showed bands with ATPase activity. Using the dimer and hexamer, we studied the nucleotide-dependent rapid molecular dynamics. The formation of the hexamer required neither nucleotide nor Mg. The hexamer was dissociated into the dimer in the presence of MgADP, while the dimer was associated into the hexamer in the presence of MgATP. The hexamer, like mitochondrial F₁ and TF₁, showed two kinds of ATPase activity: one was cooperative and was inhibited by only one BzADP per hexamer, and the other was inhibited by three BzADP per hexamer.     

Comparative Analysis of the Mechanisms Underlying Nifedipine-Induced Blockade of Three Subtypes of T-Type Ca2+ Channels

We analyzed the effects of nifedipine on a family of recombinant low-threshold Ca²⁺ channels functionally expressed in Xenopus oocytes and formed by three different subunits (1G, 1H, and 1I). The 1G and 1I channels demonstrated a low sensitivity to nifedipine even in high concentrations (IC₅₀ = 98 and 243 M, maximum blocking intensity A_max = 25 and 47%, respectively). At the same time, the above agent effectively blocked channels formed by the 1H-subunit (IC₅₀ = 5 M and A_max = 41%). The nifedipine-caused effects were voltage-dependent, and their changes depended on the initial state of the channel. In the case of 1G-subunits, the blockade was determined mostly by binding of nifedipine with closed channels, whereas in the cases of 1H- and 1I-subunits this resulted from binding of nifedipine with channels in the activated and inactivated states. The obtained data allow us to obtain estimates of the pharmacological properties of the above three subtypes of recombinant channels and, in the future, to compare these characteristics with the properties of low-threshold Ca²⁺ channels in native cells.     

Transient expression in leaf mesophyll protoplasts of Arabidopsis thaliana

Conditions for maximising transient expression of GUS in leaf mesophyll protoplasts of Arabidopsis thaliana ecotype C24 were investigated. It was found that the factors most influencing expression levels, with optimum levels in parenthesis, were plasmid DNA quantity (100 g per 5 × 10⁵ protoplasts), inclusion of carrier DNA (50 g), PEG pH and amount (pH above 6, and total PEG concentration at least 9% w/w) and the topological form of the DNA. Linearised plasmid DNA with long flanking sequences 3 and 5 to the marker gene yielded the highest levels of GUS expression.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - GUS -glucuronidase - MU methylumbelliferone - PEG polyethylene glycol - X-gluc 5-bromo-4-chloro-3-indolyl--glucuronic acid     

Modification of the properties of a translocation in the German cockroach by selection

Summary A stock of Blattella germanica bearing the interchange T(3; 12)/3;12 was subjected to close inbreeding with selection for random disjunction at metaphase I. After 3–4 generations of selection, interchange quadrivalent chiasma frequency decreased, variability in free bivalent chiasma frequency increased sharply, and individuals with either random or directed disjunction were present in the stock. Random disjunction was modified from a ratio of 2112 (adj.-1; alt.-1; adj.-2; alt.-2) to a ratio of 1111. After 7–8 generations of selection, chiasma frequency appeared to stabilize at lower than normal levels and variability decreased for both quadrivalents and free bivalents. Directed disjunction was modified from a ratio of 2114 to 1112, and no individuals with the original high level of directed disjunction were detected. Chains-of-four tended to orient randomly, especially in individuals where the ring quadrivalents showed directed disjunction. Relaxation of inbreeding, but not selection, produced an increase in chiasma frequency and variability in both free bivalents and quadrivalents, but the modified ratios for both random and directed disjunction were retained. These results are discussed with respect to inbreeding effects and genetic control of chiasma frequency and metaphase I disjunction in interchange quadrivalents.     

Highly efficient transformation and regeneration of aspen plants through shoot-bud formation in root culture

The natural capacity of aspen (Populus tremula L.) roots for direct shoot-bud regeneration was harnessed to establish a highly efficient transformation and regeneration procedure that does not require a pre-selection stage on antibiotics. Aspen stem segments were transformed using wildtype Agrobacterium rhizogenes (LBA9402) with the binary p35SGUSINT plasmid carrying the genes coding for -glucuronidase (GUS) and neomycin phosphotransferase II. High levels of transient GUS expression were found in the basal cut surface of 87% of the segments, and 98% of these formed well-developed adventitious roots. Proliferating root cultures were established in liquid culture, and GUS expression was found in 75% of the roots. Shoot-bud regeneration in root cultures was very high: 99% of the roots yielded shoot-buds (4.3 buds per root), of which 91% expressed GUS. Southern blot analysis and polymerase chain reaction confirmed the transgenic nature of the plants expressing GUS. Kanamycin resistance of transformants was tested with respect to callus growth and bud regeneration. Callus from transgenic plants exhibited a high growth rate in the presence of up to 100 g/l kanamycin, and bud regeneration from transformed roots occurred in the presence of up to 30 g/l kanamycin. Callus and buds from control (non-transformed) plants failed to proliferate or regenerate, respectively, in the presence of kanamycin at concentrations above 10 g/l. Ninety-four independent clones from different transformation events were established, of which 52 were phenotypically true-to-type.Abbreviations NAA -naphthaleneacetic acid - BA 6-benzylaminopurine - GUS -glucuronidase - NPTII neomycin phosphotransferase II - PCR polymerase chain reaction - EtOH ethanol - CTAB cetyltrimethylammonium bromide - SDS sodium dodecyl sulfate - NOS nopaline synthase - CaMV cauliflower mosaic virus     

Hydrogen isotope discrimination in higher plants: Correlations with photosynthetic pathway and environment

Summary The ratio of deuterium to hydrogen (expressed as D) in hydrogen released as water during the combustion of dried plant material was examined. The D value (metabolic hydrogen) determined on plant materials grown under controlled conditions is correlated with pathways of photosynthetic carbon metabolism. C₃ plants show mean D values of-132 for shoots and -117 for roots; C₄ plants show mean D values of -91 for shoots and-77 for roots and CAM plants a D value of-75 for roots and shoots. The difference between the D value of shoot material from C₃ and C₄ plants was confirmed in species growing under a range of glasshouse conditions. This difference in D value between C₃ and C₄ species does not appear to be due to differences in the D value (tissue water) in the plants as a result of physical fractionation of hydrogen isotopes during transpiration. In C₃ and C₄ plants the hydrogen isotope discrimination is in the same direction as the carbon isotope discrimination and factors contributing to the difference in D values are discussed. In CAM plants grown in the laboratory or collected from the field D values range from-75 to +50 and are correlated with ¹³C values. When deprived of water, the D value (metabolic hydrogen) in both soluble and insoluble material in leaves of Kalanchoe daigremontiana Hamet et Perr., becomes less negative. These changes may reflect the deuterium enrichment of tissue water during transpiration, or in field conditions, may reflect the different D value of available water in areas of increasing aridity. Whatever the origin of the variable D value in CAM plants, this parameter may be a useful index of the water relations of these plants under natural conditions.     

Involvement of abscisic acid in bulb dormancy of Allium wakegi Araki. II. A comparison between dormant and nondormant cultivars

The content of abscisic acid (ABA) in bulbs of two Allium wakegi Araki cultivars, Kiharabansei No. 1 (dormant type) and Ginoza (nondormant type), was similar and changed similarly during the development and storage of the bulbs. It increased during bulb development, reached a maximum shortly after bulb harvesting, and gradually decreased during bulb storage. The bulbs of Kiharabansei No. 1 showed dormancy correlated with the change in ABA content, but those of Ginoza did not show significant dormancy throughout the experimental period. The ABA content in the buds of dormant bulbs of Kiharabansei No. 1 did not change after planting of bulbs, but that of nondormant bulbs of Ginoza planted on the same day rapidly decreased after planting. Application of ABA to bulbs delayed sprouting of both cultivars, but dormant bulbs of Kiharabansei No. 1 had higher sensitivity to ABA than the bulbs of Ginoza or the bulbs of Kiharabansei No. 1 partly released from dormancy. These results suggest that the decrease in the ABA content after planting (watering) and low sensitivity to ABA are correlated with the nondormancy of Ginoza.     

Immobilized Lotus tetragonolobus agglutinin binds oligosaccharides containing the Lex determinant

A defined set of oligosaccharides and glycopeptides containing -linked fucose were used to examine the specificity of the immobilized fucose-binding lectin Lotus tetragonolobus agglutinin (LTA1), also known as lotus lectin. Glycans containing the Lewis x determinant (Lex) Gal1-4[Fuc1-3]GlcNAc1-3-R were significantly retarded in elution from high density LTA-Emphaze columns. The lectin also bound the fucosylated lacdiNAc trisaccharide GalNAc1-4[Fuc1-3]GlcNAc. The lectin did not bind glycans containing either sialylLex or VIM-2 determinants, nor did it bind the isomeric Lea, Gal1-3[Fuc1-4]GlcNAc-R. Although 2-fucosyllactose Fuc1-2Gal1-4Glc) was retarded in elution from the columns, larger glycans containing the H-antigen Fuc1-2Gal1-3(4)GlcNAc-R interacted poorly with immobilized LTA. Our results demonstrate that immobilized LTA is effective in isolating glycans containing the Lex antigen and is useful in analyzing specific fucosylation of glycoconjugates. Abbreviations: LTA, Lotus tetragonolobus agglutinin; UEA-1, Ulex europaeus agglutinin-I; LNT, AAL, Aleuria aurantia agglutinin; Gal1-3GlcNAc1-3Gal1-3Glc; LNnT, Gal1-4GlcNAc1-3Gal1-3Glc; Lex, Lewis x antigen; Lea, Lewis a antigen; GDPFuc, guanosine 5-diphosphate--L-fucose     

Genotypic control of pollen plant formation in Nicotiana tabacum L.

Summary Tobacco plants (Nicotiana tabacum L.) of four varieties (Badischer Burley, White Burley, Techne, Kupchunos) were raised at different temperatures and daylengths and the effect of genotype on embryogenic pollen grain formation in situ and on pollen plant formation in anther and pollen cultures from these plants was studied. Genotype controlled embryogenic pollen grain and pollen plant formation by defining productivity under standard growth conditions (long days at 24 °C). Kupchunos was the most productive variety, followed by White Burley, Techne, and Badischer Burley. Furthermore, genotype defined which environmental factor was able to affect embryogenic pollen grain and pollen plant formation and also to which degree. In anther cultures, in addition to these effects, genotype controlled the formation of (an) inhibitory substance(s) in the anther wall in interaction with the plant growth conditions. In Badischer Burley and Techne, inhibitor action could be prevented by isolation of the pollen after one week of anther culture. Finally, direct pollen cultures in Badischer Burley and Techne produced embryos were only when the pollen was isolated from nearly mature anthers, while in White Burley and Kupchunos, embryos also produced at earlier stages and at higher yields. This indicated that genotype controls the time when the embryogenic pollen grains become ready to divide. The results are discussed in relation to strategies to overcome recalcitrance of species and genotypes.     

Transformation of peas

Summary The lateral cotyledonary meristems present in germinating seed were inoculated with a non-oncogenic strain of A. tumefaciens carrying a gene conferring kanamycin resistance as a selectable marker and a -glucuronidase sequence as a reporter gene. Kanamycin resistant plants were derived from the meristems and shown to be transformed on the basis of Southern blots, polymerase chain reaction analysis and tests for -glucuronidase activity. The plants were fertile and tests of their progeny confirmed the transmission of integrated sequences through a sexual generation. This transformation method has the merit of an unlimited supply of material for inoculation and a relatively short time scale from inoculation to the production of rooted plants.Abbreviations BAP benzyl amino purine - GA3 gibberellic acid - GUS =glucuronidase - IBA indole butyric acid - MS Murashige and Skoog medium - NAA naphthalene acetic acid - NPTII neomycin phosphotransferase II - PCR polymerase chain reaction - TO first generation transformants - T1 second generation transformants     

Detection of segregation distortions in an indica-japonica rice cross using a high-resolution molecular map

We have constructed a high-resolution rice genetic map containing 1383 DNA markers covering 1575 cM on the 12 linkage groups of rice using 186 F₂ progeny from a cross between a japonica variety, Nipponbare, and an indica variety, Kasalath. Using this high-resolution molecular linkage map, we detected segregation distortion in a single wide cross of rice. The frequencies of genotypes for 1181 markers with more than 176 genotype data were plotted along this map to detect segregation distortion. Several types of distorted segregation were observed on 6 of the chromosomes. We could detect 11 major segregation distortions at ten positions on chromosomes 1, 3, 6, 8, 9, and 10. The strongest segregation distortion was at 107.2 cM on chromosome 3 and may be the gametophyte gene 2 (ga-2). The Kasalath genotype at this position was transmitted to the progeny with about a 95% probability through the pollen gamete. At least 8 out of the 11 segregation distortions detected here are new. The use of the high-resolution molecular linkage map for improving our understanding of the genetic nature and cause of these segregation distortions is discussed.     

Transgenic plantlets of ‘Chancellor’ grapevine (Vitis sp.) from biolistic transformation of embryogenic cell suspensions

Transgenic plantlets of Chancellor grapevine (Vitis L. complex interspecific hybrid) were produced via biolistic transformation. Embryogenic cell suspensions were bombarded with 1 m tungsten particles coated with pBI426 which encodes a fusion peptide between -glucuronidase (GUS) and neomycin phosphotransferase II (NPTII). The fusion peptide is under the control of a double 35S Cauliflower Mosaic Virus promoter and a leader sequence from Alfalfa Mosaic Virus. The cells were placed on kanamycin-containing media (10, 25 or 50 mg/l) 2 d after bombardment. Activated charcoal reduced cell browning. Embryos were first observed on selective media 14–29 weeks after bombardment. More than 1600 clusters of embryos were germinated and/or assayed for GUS. Of 621 embryos assayed for GUS expression, 182 (29.3%) were positive. PCR confirmed the presence of the NPTII gene in all 5 GUS-positive and 2 GUS-negative (bombarded) embryos tested. In germination experiments, 15% of the embryo clusters produced at least one plant with normal shoot growth. Of 164 normal plants assayed for GUS expression, 37 (22.6%) were positive. The NPTII gene was amplified by PCR in 1 (of 1) GUS-positive and 4 (of 5) GUS-negative bombarded plants, but not in non-bombarded control plants. Southern blotting confirmed integration of the NPTII gene in all 3 of the GUS and PCR-NPTII positive plants tested. Biolistics is an efficient method for transformation of Chancellor and should be applicable to other important grape cultivars.Abbreviations AC activated charcoal - GUS -glucuronidase - 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzylaminopurine - NAA -naphthalene acetic acid - TDZ thidiazuron - NPTII neomycin phosphotransferase II - Km kanamycin - MS Murashige and Skoog (1962) medium - WPM Woody Plant Medium of Lloyd and McCown (1980)     

In planta analysis of the Agrobacterium tumefaciens T-cyt gene promoter: identification of an upstream region essential for promoter activity in leaf,stem and root cells of transgenic tobacco

The promoter region of the Agrobacterium tumefaciens T-cyt gene was fused to a -glucuronidase (gusA) reporter gene and introduced into tobacco plants. Detection of gusA expression in transgenic F1 progeny revealed that the T-cyt promoter is active in many, if not all, cell types in leaves, stems and roots of fully developed plants. Developmental stage-dependent promoter activity was observed in seedlings. Analysis of 5-deleted promoter fragments showed that sequences located between positions–185 and –139 with respect to the T-cyt translational start codon are essential for T-cyt promoter activity in transfected tobacco protoplasts as well as in transformed tobacco plants.     

Molecular cloning and expression analysis of the mevalonate kinase gene from Arabidopsis thaliana

Mevalonate kinase (MVK), the enzyme that catalyzes the phosphorylation of mevalonate to produce mevalonate 5-phosphate, is considered as a potential regulatory enzyme of the isoprenoid biosynthetic pathway. The Arabidopsis thaliana MVK gene corresponding to the MVK cDNA previously isolated has been cloned and characterized. RNAse protection analysis indicated that the expression of the MVK gene generates three mRNA populations with 5 ends mapping 203, 254 and 355 nt upstream of the MVK ATG start codon. Northern blot analysis showed that the MVK mRNA accumulates preferentially in roots and inflorescences. Histochemical analysis, with transgenic A. thaliana plants containing a translational fusion of a 1.8 kb fragment of the 5 region of the MVK gene to the -glucuronidase (GUS) reporter gene, indicated that the MVK 5-flanking region directs widespread expression of the GUS gene throughout development, although the highest levels of GUS activity are detected in roots (meristematic region) and flowers (sepals, petals, anthers, style and stigmatic papillae). The expression pattern of the MVK gene suggests that the role of the encoded MVK is the production of a general pool of mevalonate-5-phosphate for the synthesis of different classes of isoprenoids involved in both basic and specialized plant cell functions. Functional promoter deletion analysis in transfected A. thaliana protoplasts indicated that regulatory elements between positions –295 and –194 of the MVK 5-flanking region are crucial for high-level MVK gene expression.     

Puromycin enhances 20-hydroxyecdysone-induced protein synthesis in wing discs of Drosophila melanogaster

The effect of cycloheximide and puromycin on 20-hydroxyecdysone-induced protein synthesis in wing discs of Drosophila melanogaster has been studied by one-dimensional and two-dimensional SDS polyacrylamide electrophoresis. It is found that puromycin, but not cycloheximide, when applied simultaneously with the hormone enhanced the hormone-induced synthesis of the early and late proteins. However, when puromycin was applied after hormone treatment, only the late proteins were induced. The possible implication of these observations is discussed.