Cytoplasmic Male Sterility in Barley. Xi. the msm2 Cytoplasm

A new cytoplasmic male sterility in barley (Hordeum vulgare s.l.) is described and designated as msm2. The cytoplasm was derived from a selection of the wild progenitor of barley (H. vulgare ssp. spontaneum). This selection, 79BS14-3, originates from the Southern Coastal Plain of Israel. The selection 79BS14-3 has a normal spike fertility in Finland. When 79BS14-3 was crossed by cv. Adorra, the F₁ displayed partial male fertility and progeny of recurrent backcrosses with cv. Adorra were completely male sterile. Evidently 79BS14-3 is a carrier of a recessive or semidominant restorer gene of fertility. The dominant restorer gene Rfm1a for another cytoplasmic male sterility, msm1, is also effective in msm2 cytoplasm. The different partial fertility restoration properties of msm2 and msm1 cause these cytoplasms to be regarded as being distinct. Seventy spontaneum accessions from Israel have been studied for their capacity to produce F₁ restoration of male fertility both in msm1 and in msm2 cytoplasms with a cv. Adorra-like seed parent (nuclear gene) background. The msm2 cytoplasm shows partial restoration more commonly than msm1 in these F₁ combinations. The mean restoration percentage per accession for msm2 is 28, and for msm1 4. Most of the F₁ seed set differences of the two cytoplasms are statistically significant. When estimated with partially restored F₁ combinations, msm2 cytoplasm appeared to be about 50 times more sensitive to the male fertility-promoting genes present in the spontaneum accessions. The spontaneum sample from Central and Western Negev, which has been found to be devoid of restoration ability in msm1 cytoplasm, had only low partial restoration ability in msm2 (mean 0.3%). The female fertility of msm2 appears normal. The new msm2 cytoplasm could be useful in producing hybrid barley.     

Cytoplasmic Male Sterility in Barley : VIII. LIPOXYGENASE ACTIVITY AND ANTHER AMINO NITROGEN IN THE msm1-Rfm1a SYSTEM

The lipoxygenase (LOX) activity was determined in almost isogenic types of barley (Hordeum vulgare L.): normal cv. Adorra, cytoplasmic male sterile (msm1), and msm1 barley with restored fertility, heterozygous for the Rfm1a restorer gene. The LOX activity was lowest in male steriles in the leaf tissue studied at the anthesis stage. The LOX activity in developing anthers was higher than in leaf tissue, and decreased during degeneration of the sterile anthers.     

Marker-assisted identification of restorer gene(s) in iso-cytoplasmic restorer lines of WA cytoplasm in rice and assessment of their fertility restoration potential across environments

Iso-cytoplasmic restorers possess the same male sterile cytoplasm as the cytoplasmic male sterile (CMS) lines, thereby minimizing the potential cyto-nuclear conflict in the hybrids. Restoration of fertility of the wild abortive CMS is governed by two major genes namely, Rf3 and Rf4. Therefore, assessing the allelic status of these restorer genes in the iso-cytoplasmic restorers using molecular markers will not only help in estimating the efficiency of these genes either alone or in combination, in fertility restoration in the hybrids in different environments, but will also be useful in determining the efficacy of these markers. In the present study, the efficiency of molecular markers in identifying genotypes carrying restorer allele of the gene(s) Rf3 and Rf4, restoring male fertility of WA cytoplasm in rice was assessed in a set of 100 iso-cytoplasmic rice restorers using gene linked as well as candidate gene based markers. In order to validate the efficacy of markers in identifying the restorers, a sub-set of selected 25 iso-cytoplasmic rice restorers were crossed with four different cytoplasmic male sterile lines namely, IR 79156A, IR 58025A, Pusa 6A and RTN 12A, and the pollen and spikelet fertility of the F₁s were evaluated at three different locations. Marker analysis showed that Rf4 was the predominant fertility restorer gene in the iso-cytoplasmic restorers and Rf3 had a synergistic effect on fertility restoration. The efficiency of gene based markers, DRCG-RF4-14 and DRRM-RF3-10 for Rf4 (87%) and Rf3 (84%) genes was higher than respective gene-linked SSR markers RM6100 (80%) and RM3873 (82%). It is concluded that the gene based markers can be effectively used in identifying fertility restorer lines obviating the need for making crosses and evaluating the F₁s. Though gene based markers are more efficient, there is a need to identify functional polymorphisms which can provide 100% efficiency. Three iso-cytoplasmic restorers namely, PRR 300, PRR 363 and PRR 396 possessing both Rf4 and Rf3 genes and good fertility restoration have been identified which could be used further in hybrid rice breeding.     

Fine mapping of the restorer gene <Emphasis Type="Italic">Rfp3</Emphasis> from an Iranian primitive rye (<Emphasis Type="Italic">Secale cereale</Emphasis> L.)

Key message

A comparative genetics approach allowed to precisely determine the map position of the restorer gene Rfp3 in rye and revealed that Rfp3 and the restorer gene Rfm1 in barley reside at different positions in a syntenic 4RL/6HS segment.

Abstract

Cytoplasmic male sterility (CMS) is a reliable and striking genetic mechanism for hybrid seed production. Breeding of CMS-based hybrids in cereals requires the use of effective restorer genes as an indispensable pre-requisite. We report on the fine mapping of a restorer gene for the Pampa cytoplasm in winter rye that has been tapped from the Iranian primitive rye population Altevogt 14160. For this purpose, we have mapped 41 gene-derived markers to a 38.8 cM segment in the distal part of the long arm of chromosome 4R, which carries the restorer gene. Male fertility restoration was comprehensively analyzed in progenies of crosses between a male-sterile tester genotype and 21 recombinant as well as six non-recombinant BC₄S₂ lines. This approach allowed us to validate the position of this restorer gene, which we have designated Rfp3, on chromosome 4RL. Rfp3 was mapped within a 2.5 cM interval and cosegregated with the EST-derived marker c28385. The gene-derived conserved ortholog set (COS) markers enabled us to investigate the orthology of restorer genes originating from different genetic resources of rye as well as barley. The observed localization of Rfp3 and Rfm1 in a syntenic 4RL/6HS segment asks for further efforts towards cloning of both restorer genes as an option to study the mechanisms of male sterility and fertility restoration in cereals.

     

Patches and the responses of lake benthos to sunfish nest-building

Summary Males of three sunfish species (Centrarchidae) construct nests for spawning and often share them sequentially in the littoral zone of a 4-hectare lake in New York State. To determine spatial and temporal effects of this reproductive behavior on zoobenthos in patches, I sampled bottom assemblages from inside nests and from adjacent (<1 m from nest perimeter), relatively undisturbed sites during the reproductive season of 1986 and immediately prior to nest-building in 1987. The reproductive behaviors of largemouth bass (Micropterus salmoides), redbreast sunfish (Lepomis auritus), and pumpkinseeds (L. gibbosus) altered relative abundances and significantly decreased benthic invertebrate diversity and density. These effects were extremely pronounced during the reproductive season and were partially detectable the following year. Community changes were probably the result of both bioturbation (modification of sediment size and organic content) and predation. The ecosystem-wide effects of nest-building are evaluated in terms of the number, distribution, and longevity of patches.     

Molecular mapping of a fertility restoration locus (Rfm1) for cytoplasmic male sterility in barley (Hordeum vulgare L.)

The Rfm1a gene restores the fertility of msm1 cytoplasmic male-sterile lines in barley. We identified three RAPD markers linked to the Rfm1 locus (CMNB-07/800, OPI-18/900, and OPT-02/700) using isogenic lines and segregating BC₁F₁ and F₂ populations. Using a previously developed linkage map of barley, we located CMNB-07/800 and OPT-02/700 beside MWG2218 on chromosome 6HS. The linkage between MWG2218 and the Rfm1 locus was demonstrated using the segregating BC₁F₁ and F₂ populations. To confirm the chromosomal locations of these markers, we converted them to STSs and tested against two sets of wheat–barley chromosome addition lines. These STS markers, CMNB-07/800, OPT-02/700, and MWG2218, were amplified only in the addition lines possessing the chromosome 6H, thereby providing additional evidence the Rfm1 locus is located on chromosome 6H. Homoeologous relationships among fertility restoration genes in Triticeae are discussed. Received: 27 March 2000 / Accepted: 25 June 2000     

The Self-Association of Antibiotic Actinocyl-bis(3-dimethylaminopropylamine) in Aqueous Solution: A 1H NMR Analysis

The self-association of the synthetic antibiotic actinocyl-bis(3-dimethylaminopropylamine) was studied in aqueous solution by one- and two-dimensional ¹H NMR spectroscopy at 500 MHz. The two-dimensional homonuclear correlation NMR techniques (TOCSY and ROESY) were used to completely assign all the proton signals of the antibiotic and to quantitatively analyze the mutual arrangement of the antibiotic molecules in their aggregates. The concentration and temperature dependences of proton chemical shifts were used to determine the equilibrium constants and the thermodynamic parameters (H and S) of the self-association, as well as the limiting values of proton chemical shifts in associates. The experimental results were analyzed using both the indefinite noncooperative and cooperative models of the molecular self-association. The calculated value of the cooperativity coefficient ( 1.1) for our synthetic antibiotic confirmed a substantially lower anticooperative effect at the aggregation of its molecules in comparison with that of the antitumor antibiotic actinomycin D ( 1.5). We calculated the most favorable structure of the dimeric associate of the synthetic antibiotic in aqueous solution and found that, like in the actinomycin D dimer, the antiparallel orientation of the phenoxazone chromophore planes of interacting molecules is characteristic of its dimer.     

Cytoplasmic male sterility in barley

The maternal male sterile barley msm1 with or without a dominant gene, Rfmla, which restores male fertility, was studied. Determined with SDS-PAGE, the polypeptide pattern in the anthers of unrestored msm1 plants remains juvenile in the middle of anther development, two major zones being absent or weak. At the stage when anther development stops in msm1 plants, the anther proteins appear to be hydrolyzed to short-chain peptides. Restored plants, heterozygous for the restorer gene, Rfmla, behaved like the near-isogenic normal barley, cv. Adorra. The total leaf protein pattern of young leaf tissue and the chloroplastidic membrane protein pattern are normal in msm1 cytoplasm when studied with this technique. Chlorophyll b is unnecessary for restoration by Rfmla, though the restored plants have a lower chlorophyll a/b ratio than an unrestored plant in the mature stem leaf. Mature stem leaf pieces of unrestored msm1 plants were induced to senesce with 20 mM NaCl solution. This senescence was inhibited by exogenous kinetin. Leaf pieces of restored msm1 plants or those of near-isogenic normal barley behaved in the same way in the NaCl solution as in distilled water. Many features of the physiology of restored plants can be explained as the functions of cytokinins. Kernels of male sterile plants have a more rapid root elongation at germination than near-isogenic normal barley.     

A xyloglucan-oligosaccharide-specific α-d-xylosidase or exo-oligoxyloglucan-α-xylohydrolase from germinated nasturtium (Tropaeolum majus L.) seeds

The -xylosidase which is involved in the postgerminative mobilisation of xyloglucan in nasturtium seed cotyledons has now been purified to apparent homogeneity by a facile procedure involving lectin affinity chromatography. The purified enzyme, a glycoprotein, moved as a single band (apparent molecular weight 85000) on sodium dodecyl sulphate-gel electrophoresis, whilst isoelectric focusing gave a number of enzymatically active protein bands spanning the range pI = 5.0 to 7.1 (maximum activity at pI = 6.1). The enzyme did not hydrolyse the simple -xylosides p-nitrophenyl--d-xylopyranoside and woprimeverose (-d-Xyl(16)-d-Glc), or polymeric tamarind-seed xyloglucan. It released xylose from a complex mixture of oligosaccharides produced by exhaustive hydrolysis of tamarind seed xyloglucan using the xyloglucan-specific endo-(14)--d-glucanase from germinated nasturtium seeds (M. Edwards et al. 1986, J. Biol. Chem., 261. 9489–9494). The three xyloglucan oligosaccharides of lowest molecular size were purified from this mixture and were shown by ¹H-nuclear magnetic resonance (¹H-NMR) and enzymatic analysis to have the structures:Abbreviations Con A Concanavalin A - DEAE diethylaminoethyl - Gal galactose - Glc glucose - HPLC high-performance liquid chromatography - M _r apparent molecular mass - NMR nuclear magnetic resonance - pI isoelectric point - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - Xyl xylose Much of the work reported in this paper was carried out with the aid of the European Community's Science Stimulation Action (Contract No. ST2P-0250-UK), and we wish to record our appreciation of this support.     

Radial transport of water across cortical sleeves of excised roots ofZea mays L.

The stationary radial volume flows across maize (Zea mays L.) root segments without steles (sleeves) were measured under isobaric conditions. The driving force of the volume flow is an osmotic difference between the internal and external compartment of the root preparations. It is generated by differences in the concentrations of sucrose, raffinose or polyethylene glycol. The flows are linear functions of the corresponding osmotic differences ( ) up to osmotic values which cause plasmolysis. The straight lines obtained pass through the origin. No asymmetry of the osmotic barrier could be detected within the range of driving forces applied ( =±0.5 MPa), corresponding to volume-flow densities of j_{v, s}=±7·10^–8 m·s^–1. Using the literature values for the reflection coefficients of sucrose and polyethylene glycol in intact roots (E. Steudle et al. (1987) Plant Physiol.84, 1220–1234), values for the sleeve hydraulic conductivity of about 1·10^–7 m·s^–1 MPa^–1 were calculated. They are of the same order of magnitude as those reported in the literature for the hydraulic conductivity of intact root segments when hydrostatic pressure is applied.Abbreviations and symbols a _s outer surface of sleeve segment - c concentration of osmotically active solute - j _{v, s} radial volume flow density across sleeve segment - Lp_s hydraulic conductivity of sleeves - Lp_r hydraulic conductivity of intact roots - _N thickness of Nernst diffusion layer - reflection coefficient of root for solute - osmotic value of bulk phase - osmotic coefficient     

Xylem development and hydraulic conductance in sun and shade shoots of grapevine (Vitis vinifera L.): evidence that low light uncouples water transport capacity from leaf area

Morphology, water relations, and xylem anatomy of high-light (sun)- and low-light (shade)-grown Vitis vinifera L. shoots were studied to determine the effects of shading on the hydraulic conductance of the pathway for water flow from the roots to the leaves. Shade shoots developed leaf area ratios (leaf area: plant dry weight) that were nearly threefold greater than sun shoots. Water-potential gradients (·m^–1) in the shoot xylem accounted for most of the ·m^–1 between soil and shoot apex at low and high transpiration rates in both sun and shade shoots, but the gradients were two- to fourfold greater in shade-grown plants. Low light reduced xylem conduit number in petioles, but had an additional slight effect on conduit diameter in internodes. The hydraulic conductance per unit length (Kh) and the specific hydraulic conductivity (k_s, i.e. Kh per xylem cross-sectional area) of internodes, leaf petioles, and leaf laminae at different developmental stages leaf plastochron index was calculated from measurements of water potential and water flow in intact plants, from flow through excised organs, and from vessel and tracheid lumen diameters according to Hagen-Poiseuille's equation. For all methods and conductance parameters, the propensity to transport water to sink leaves was severalfold greater in internodes than in petioles. The Kh and k_s increased logarithmically until growth ceased, independent of treatment and measurement method, and increased further in pressurized-flow experiments and Hagen-Poiseuille predictions. However, the increase was less in shade internodes than in sun internodes. Mature internodes of shade-grown plants had a two- to fourfold reduced Kh and significantly lower k_s than sun internodes. Except very early in development, leaf lamina conductance and k_s from shade-grown plants was also reduced. The strong reduction in Kh with only a slight reduction in leaf area (17% of sun shoots) in the shade shoots indicated a decoupling of water-transport capacity from the transpirational surface supplied by that capacity. This decoupling resulted in strongly reduced leaf specific conductivities and Huber values for both internodes and petioles, which may increase the likelihood of cavitation under conditions of high evaporative demand or soil drought.Abbreviations A_c total cross-sectional area (internodes, petioles, leaf laminae) - A_x xylem cross-sectional area - HV Huber value - Kh hydraulic conductance per unit length - k_s specific hydraulic conductivity - LPI leaf plastochron index - LSC leaf specific conductivity - water potential - water-potential gradient - q volume flow of water per unit time Hans R. Schultz was supported in part by the Deutsche Forschungsgemeinschaft (grant Ki-114/8-1). We wish to thank Dr. Thomas Geier, Institut für Biologie, Forschungsanstalt D-6222 Geisenheim, Germany for his advice on sample preparation and microscopy, and two anonomous reviewers for their helpful comments.     

Immunohistochemical localization of adrenocorticotropin and melanocyte stimulating hormone in pars intermedia of rat hypophysis

Summary The sites of production of adrenocorticotropin (ACTH) and melanocyte stimulating hormone (MSH) are studied by the immunoglobulin-peroxidase bridge technique, using antisera prepared against synthetic porcine 1–24 and 17–39 ACTH, and bovine MSH on the rat adenohypophysis. Presence of ACTH all over the pars intermedia (PI) is indicated by staining with antisera _p 1–24 and _p 17-3-9 ACTH. There are darkly stained ACTH cells in the PI and pars tuberalis (PT), similar to those in the pars distalis (PD). With higher dilutions of the ACTH antiserum, staining intensity disappears or reduces markedly in majority of the PI cells, whereas, the ACTH cells in the PI, PD and PT do not vary much in their staining intensity. Therefore, it is concluded that majority of the PI glandular cells (light glandular and dark cells) contain less corticotropin than the ACTH cells. From these observations, it seems to me that the major amount of corticotropin is supplied by the ACTH cells of the PD, PI and PT, and less by the light glandular and dark cells of the PI. The antiserum is ineffective after absorption, so the staining reaction appears to be specific for _p 1–24 and _b 17–39 ACTH.Presence of MSH all over the PI is indicated by staining with antisera to bovine MSH. Majority of the PI cells are highly stained even with higher dilution of the antiserum. The unstained cells in the PI seem to be ACTH cells and/or marginal cuboidal cells. The antiserum was ineffective after absorption, so the staining reaction appears to be specific for _b MSH.Control over the PD corticotropin through the median eminence portal circulation and the PI and PT control through nervous system is also discussed.This study was supported by MRC of Canada Grant nos. MA-3759, and MA-5160.The author gratefully wishes to thank Drs. P. Desaulles and W. Rittel (CIBA, Basle, Switzerland) for the synthetic _p 1–24 ACTH and _b MSH, Dr. R. F. Phifer for _p 17–39 ACTH, and Dr. S. S. Spicer for providing samples of rabbit anti-porcine 17–39 ACTH and anti-human ACTH sera, Drs. George Sétáló and Paul Nakane for their valuable advice. He also acknowledges the help of Mr. Shankar Nayak to prepare the antisera and the skilful technical assistance of Miss. Elise Poiré.     

The fuc1 gene product (20 kDa FUC1) of Pisum sativum has no α-L-fucosidase activity

An -L-fucosidase purified from pea (Pisum sativum L. cv Alaska) epicotyl was previously described as a cell wall enzyme of 20 kDa that hydrolyses terminal -L-fucosidic linkages from oligosaccharide fragments of xyloglucan. cDNA and genomic copies were further isolated and sequenced. The predicted product of the cDNA and the genomic clone (fuc1), was a 20 kDa protein containing a signal peptide and five cysteines. This was the first -L-fucosidase gene to be cloned in plants but its fucosidase activity has not been demonstrated. Here, our biochemical and immuno analyses suggest that fuc1 does not encode an -L-fucosidase. Pea fuc1 expressed in Escherichia coli, insect cells and Arabidopsis thaliana produced recombinant proteins without -L-fucosidase activity. Pea plants had endogenous -L-fucosidase activity, but the enzyme was not recognised by an antibody produced against recombinant FUC1 protein expressed in E. coli. In contrast, the antibody immunoprecipitated a 20 kDa protein which was inactive. By chromatographic analysis of pea protein extracts, we separated -L-fucosidase-active fractions from the 20 kDa protein fractions. We conclude that the -L-fucosidase activity is not attributable to the 20 kDa FUC1 protein. A new function for fuc1 gene product, now named PIP20 (for protease inhibitor from pea) is proposed.     

Cytoplasmic male sterility in barley

Summary The effect of the msm1 cytoplasm of barley (Hordeum vulgare L.) on kernel protein and lysine was studied using the near-isogenic, unrestored derivatives of seven barley varieties. With normal lysine varieties, Adorra, Bomi, CI 4362, and Hankkija's Eero, the msm1 cytoplasm produced an average of one percentage point more protein than did the normal cytoplasm of the same varieties. There was no difference between the two cytoplasms with respect to their effect on the lysine content. With high lysine varieties, Bomi Risø mutant 13, Bomi Risø mutant 1508, and CI 3947, msm1 produced almost one percentage point more protein but protein with a somewhat decreased lysine content.Induced partial spike fertility in normal Adorra was found to be associated with lysine in meal (r=–0.999), with protein in meal (r=–0.984), and with lysine in protein (r=0.941). Removal of the spikes on the secondary tillers affected both the protein and its lysine content. It is suggested that good spike fertility is an important pre-requisite when selecting high lysine and/or high protein segregants or mutants.     

Free Ia Eα chain expression in the E α + :E β − : recombinant strain A.TFR5

Molecular and biochemical techniques have been used to explore the reasons behind low E chain expression in the E ⁺ E ^– I-region recombinant strain, A.TFR5. A.TFR5 (A ^f E ^k, ap5), a recombinant between A.CA (A ^f E ^f) and A.TL (A ^k E ^k), carries the E ^k subregion. Previous results have shown that it expresses the E chain, but at reduced levels relative to E ⁺ E ⁺ strains. No E chains were detected, which is consistent with the A.TFR5E gene being derived from the A.CA parent, which carries the null E ^f allele. In this paper, the defect in E-chain expression is explored. Restriction fragment length polymorphism analysis has localized the recombination event in A.TFR5 approximately 30 kb upstream of E, in the region of the large intervening sequence of E. Northern blot analysis of total RNA from A.TFR5 shows normal amounts of the E message, but no E message. Two-dimensional gel analysis of 15 min pulse-labeled A.TFR5, A.CA, and A.TL E immunoprecipitates shows decreased levels of the intracellular E chain in A.TFR5 relative to A.TL. However, analysis of total cell extracts shows normal levels of this protein. A glycoprotein fraction isolated from total cell extracts of 5 h labeled cells contains normal amounts of intracellular E, but decreased amounts of the mature cell-surface protein. These data suggest that in the absence of E, the E chain (1) takes on an altered conformation that is not as efficiently recognized by alloantibodies, and (2) is found in normal levels as the partially glycosylated intracellular precursor, but is not processed and/or transported efficiently to the cell surface.     

Differences in Leaf δ13C Among Four Dominant Species in a Secondary Succession Sere on the Loess Plateau of China

Differences in leaf ¹³C among four dominant species as well as the species-specific response to the fluctuations of either soil moisture or monthly mean temperature were examined along a secondary succession sere with a time scale from 3 to 149 y on the Loess Plateau in north-western China. We used leaf ¹³C as a surrogate for water use efficiency (WUE) of the mentioned dominant species. Bothrichloa ischaemun as a dominant species in the final succession stage belongs to C₄ photosynthesis pathway, while the other three dominant species occurring in the first three succession stages belong to C₃ pathway. The overall trend of leaf ¹³C variation among the three C₃ species was Artemisia gmelinii (in the third stage) and Lespedeza davurica (in the second stage) > Artemisia scoparia (in the first stage). This suggests that species with higher WUE (more positive leaf ¹³C) would have substantial competitive advantages in the context of vegetation succession. Furthermore, species with highest WUE (i.e. C₄ pathway) have great potential to be dominant in the final succession stage in the habitats (such as the study area) undergoing strong water stress in growing season. The evolution of WUE among the dominant species occurring in different succession stages strongly depends on the time scale of given stage since abandonment. The longer the time scale is, the more significant the differences among them in terms of leaf ¹³C, hence WUE. Our results support the notions that leaf ¹³C may be more positive when water supply is less favourable.     

Pollination by a hugging mechanism inVigna vexillata (Leguminosae-Papilionoideae)

The pollination ofVigna vexillata (Leguminosae-Papilionoideae) by a carpenter bee,Xylocopa gualanensis (Hymenoptera-Anthophoridae) was studied in a secondary vegetation in Costa Rica. The bees were observed foraging onV. vexillata only in early mornings. Visits on individual flowers lasted about 7–8 seconds. Flower—pollen vector interactions are described and illustrated. By its pressure on the left-hand wing- and keel-petal in the asymmetrical flower, the weight of the bee causes the upper bearded part of the style along with the upper free parts of the stamens to slip out of the rigid keel-beak, hugging the bee over the dorsal part of its head and thorax. The occurrence of nototribic pollination inVigna and related genera is discussed.     

Cloning of Maytenus emarginata (Willd.) Ding Hou — a tree of the Indian Desert,through tissue culture

Summary An in vitro method for cloning and mass multiplication of Maytenus emarginata, a highly drought resistant tree of the Indian Desert, has been developed. Shoot segments harvested from a plus tree (30-year-old) were cultured to produce multiple shoots (10–15 shoots/explant) on MS medium containing 0.1 mgl^–1 IAA and 2.5mgl^–1 BAP. In vitro produced shoots were cut into segments and cultured on shoot proliferation medium but with only 1.0 mgl^–1 of BAP to further multiply the shoots. Isolated individual shoots were cultured on a filter paper bridge in half strength MS liquid medium containing 25 mgl^–1 of IBA for 72 h in the dark at 28±2⁰ C for induction of root(s). About 70–80 percent of shoots rooted. The treelets developed were hardened and transferred to pots. Around 20,000 plants can be obtained from a single explant within a period of 6 months. The protocol is highly reproducible and efficient.Abbreviations IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA -naphthalene acetic acid - NOA -naphthoxy acetic acid - BAP 6-benzylaminopurine - Kn 6-furfurylaminopurine - B5 Gamborg et al. (1968) medium - MS Murashige and Skoog (1962) medium     

The location of untranscribed DNA sequences within ras genes essential for eliciting plant growth suppression

Three heterologous ras DNA-coding sequences and their deletion derivatives were introduced into plant cells to investigate the role of the ras-coding sequences, especially conserved regions, in eliciting growth inhibition. All three ras-coding sequences caused a similar inhibition of plant cell growth, and it was the conserved coding regions which were responsible for this inhibitory effect. The 493 bp conserved region within the v-Ha-ras-coding sequence was studied further, and was shown to be responsible for the inhibitory effect. This region is conserved (over 44%) among the three ras genes studied and encodes a catalytic region of the Ras protein. Small deletions at either the 5 or 3 end of this 493 bp sequence could abolish or dramatically reduce the inhibitory effect. A 36 bp region at the 5 end of the 493 bp region was found to be highly conserved between v-Ha-ras and eight different plant ras or ras-related genes based upon analysis of published sequences. Small deletions affecting this highly conserved 36 bp region completely abolished the inhibitory effect, while deletion of a similar number of base pairs in adjacent regions did not. These results indicate that plant growth inhibition by ras DNA requires small regions at both ends of the 493 bp conserved region.     

The molecular basis of genetic diversity among cytoplasms of Triticum and Aegilops

Summary Many related species and strains of common wheat were compared by matching differences among their mitochondrial genomes with their parent nuclear genomes. We examined three species of Aegilops, section Sitopsis (Ae. bicornis, Ae. sharonensis, and Ae. speltoides), emmer wheat (Triticum dicoccoides, T. dicoccum, and T. durum), common wheat (T. spelta, T. aestivum, and T. compaction), and timopheevi wheat (T. araraticum, T. timopheevi, and T. zhukovskyi). A single source of the cytoplasm was used in all the species, except Ae. speltoides (two sources), T. araraticum (two), and T. aestivum (three). Following restriction endonuclease analyses, the mitochondrial genomes were found to comprise seven types, and a dendrogram showing their genetic relatedness was constructed, based upon the percentage of common restriction fragments. MtDNAs from T. dicoccum, T. durum, T. aestivum, and T. compactum yielded identical restriction fragment patterns; these differed from T. dicoccoides and T. spelta mtDNAs in only 2.3% of their fragments. The fragment patterns of T. timopheevi and T. zhukovskyi were identical, and these differed from T. araraticum mtDNA by only one fragment. In both the emmer-dinkel and timopheevi groups, mitochondrial genome differentiation is evident, suggesting a diphyletic origin of each group. MtDNAs from four accessions of the Sitopsis species of Aegilops differ greatly from one another, but those of Ae. bicornis, Ae. sharonensis, and Ae. searsii, belonging to the same subsection Emarginata, are relatively similar. MtDNAs of timopheevi species are identical, or nearly so, to those of Ae. speltoides accession (09), suggesting that the latter was the cytoplasm donor to the former, polyploid group. The origin of this polyploid group seems to be rather recent in that the diploid and polyploid species possess nearly identical mitochondrial genomes. We cannot determine, with precision, the cytoplasm donor to the emmer-dinkel group. However, our results do suggest that mitochondrial DNAs show larger evolutionary divergence than do the ctDNAs from these same strains.Contribution no. 507 from the Laboratory of Genetics, Faculty of Agriculture, Kyoto University, Japan