Improvement of staining method for observation of mitotic chromosomes in octoploid strawberry plants

An important strategy to conduct intentional breeding of octoploid strawberry plants is to recognize the functions of every chromosome. To do so, a methodology must be developed to distinguish chromosomes one by one. We reported the possibility of distinguishing chromosomes using light microscopy when somatic cells of octoploid strawberry plants were stained using ordinary methods with lacto-propionic orcein (LPO). However, karyotype analysis of octoploid strawberry plants required clearer chromosome images. This study obtained clearer chromosome images of octoploid Fragaria × ananassa and F. chiloensis plants. Three staining methods were examined: 60% acetic acid (AA) alone, 1.5% LPO alone, and two-step treatments with 60% AA and 1.5% LPO. Collected root tips of the plants were placed in 2 mM 8-hydroxyquinoline solution for 1 h and were subsequently stored at 4 °C for 15 h. The samples were then fixed in a 3:1 absolute alcohol: glacial acetic acid solution for 40 min, followed by mixture with 1N HCl solutions at room temperature for 2 h and then at 60 °C for 10 min. For separate staining using 60% AA and 1.5% LPO, the root tip was expelled on the glass slide with a drop of each solution for a few minutes to stain the chromosomes. For the two-step staining method, the samples stained with 60% AA were frozen at −80 °C for at least 5 min. The cover slip was removed using a razor blade. Subsequently, the specimens were air-dried and stained with the 1.5% LPO for 3 min. Digital images of chromosomes were obtained using light microscopy. Samples of the two-step staining method produced the clearest chromosome images in both F. × ananassa and F. chiloensis. Furthermore, the greatest color difference between the chromosomes and the cytoplasm was obtained from images of the two-step staining method among the three staining methods. These results demonstrate that the two-step staining method is useful for chromosome counting and karyotype analysis in strawberry plants.     

Aneuploid strawberry (2n = 8x + 2 = 58) was developed from homozygous unreduced gamete (8x) produced by second division restitution in pollen

Unreduced gamete formation is significant in the evolutionary development of complex polyploidy series found in wild strawberry, genus Fragaria (Rosaceae). Also, it is important for genetics and breeding in strawberry plants to elucidate the mechanism of unreduced gamete formation. The objective of this study was to search for ploidy anomalies resulting from artificial diploid × octoploid crosses, and examine the mechanism through which these unreduced gametes were produced. Five everbearing cultivars of Fragaria vesca L. diploid (2n = 2x = 14) were crossed with pollen from six June-bearing cultivars of Fragaria × ananassa Duch., octoploid (2n = 8x = 56). A total of 3000 mature seeds, 100 from each of the 30 parental combinations were sown at 23 °C/20 °C (day/night) under artificial lighting with a 16 h day. The seedlings were transplanted to pots and grown in a greenhouse. Reproductive and morphological observations, flow cytometry analyses, chromosome counts and DNA analyses using CAPS markers were performed to identify the genetic background of the offspring. Most of the seed (79%) did not germinate or died soon after germination. Of the seedlings produced, 7% seemed to be pure F. vesca based on morphological characteristics, flow cytometry analyses and chromosome counts; 14% were pentaploids (2n = 5x = 35), 0.1% were hexaploids (2n = 6x = 42), and 0.03% (one individual) was aneuploid (2n = 8x + 2 = 58). Electrophoresis banding patterns obtained by CAPS marker analysis were heterozygotic in the 8x pollen parent but homozygotic in the aneuploid progeny. Judging from the chromosome counts and the CAPS marker analysis, the aneuploid was the result of a homozygous unreduced pollen grain (8x) crossed with an incomplete chromosome compliment from the egg. Because of the homozygosity, the unreduced male gamete must have been derived from second division restitution (SDR) in the octoploid pollen parent.     

Dissemination of Phytophthora cactorum, cause of crown rot in strawberry,in open and closed soilless growing systems and the potential for control using slow sand filtration

Closed (recirculating) growing systems provide a greater potential for the dispersal of water-borne plant pathogens and disease expression compared to open (run-to-waste) systems. Here we studied the effects of three soilless growing systems (open, closed, and closed with slow sand filtration) on the dispersion of Phytophthora cactorum propagules and the severity of the crown rot disease in strawberry (Fragaria × ananassa Duch.). The plant-growth medium used was coir fiber. The three growing systems showed the same density of P. cactorum propagules in the water drained from the growing media. However, propagules of this pathogen were not detected by the baits in the filtered solution recovered from slow sand filtration. In all systems Phytophthora propagules dispersed from the inoculated plant to adjacent uninoculated plants. At the end of the first crop no differences in the severity of crown rot were found between the different systems of crop culture. However, at the end of the second crop cycle, crown rot in the closed soilless system without slow sand filtration was more severe than in the other two systems. These results demonstrated that the commercial potential of slow sand filtration to prevent propagule dispersal and hence suppress crown rot in strawberry crops grown in a closed culture system.     

Parental chromosome differentiation in citrangequat [Fortunella sp. × (Citrus sinensis × Poncirus trifoliata)] using CMA banding patterns

The efficiency of chromomycin A₃ (CMA) staining was examined for parental chromosome differentiation in citrange [Citrus sinensis (L.) Osbeck × Poncirus trifoliata (L.) Raf.] and citrangequat (Fortunella sp. × citrange). All of the accessions analyzed had the same chromosome number of 2n = 18. CMA staining revealed six characteristic banding patterns on the basis of the number and position of CMA positive bands (CMA⁺) as follows; A: two terminal and one proximal band, B: one terminal and one proximal band, C: two terminal bands, D: one terminal band, E: no band, and F: one proximal band. Chromosome CMA banding patterns of the accessions were 1A + 1B + 2C + 13D + 1F in Fortunella margarita, 2B + 2C + 7D + 7E in ‘Fukuhara’ orange, 2B + 10D + 6E in Poncirus trifoliata, 1B + 1C + 10D + 6E in citrange and 1A + 1C + 11D + 4E + 1F in citrangequat. The results of this study confirmed the intergeneric and tri-generic hybridity of citrange and citrangequat, respectively.     

Effect of plant growth temperature on membrane lipids in strawberry (Fragaria × ananassa Duch.)

Changes in membrane lipid composition are important in the acclimation of plants. The influence of four day/night growing temperature combinations (18/12, 25/12, 25/22, and 30/22 °C) on membrane lipids of ‘Earliglow’ and ‘Kent’ strawberry (Fragaria × ananassa Duch.) were studied. The monogalactosyl diglyceride (MGDG) and digalactosyl diglyceride (DGDG) were the major galactolipids in the strawberry leaves. ‘Earliglow’ contained a higher amount of galactolipids in the leaves than ‘Kent’. The major phospholipids in the strawberry leaves, roots, and fruit were phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylglycerol (PG), and phosphatidylethanolamine (PE). PC and PE were the two predominant phospholipids in the strawberry. The leaves and fruit of ‘Earliglow’ contained higher amounts of phospholipids compared to those of ‘Kent’, whereas ‘Kent’ strawberry roots had higher phospholipids. Palmitic (C16:0), stearic (C18:0), oleic (C18:1), linoleic (C18:2), and α-linolenic (C18:3) acids were major fatty acids in galacto- and phospholipids of the ‘Earliglow’ and ‘Kent’ strawberry. PC is very rich in linolenic acid in leaves compared to the fruit and root tissues. PC had the highest ratio of unsaturated to saturated fatty acids among all phospholipids. There was a significant increase in the content of galactolipids (MGDG, DGDG) and phospholipids (PC, PI, PG and PE) and unsaturation of their fatty acids in the cool day/night growth temperature. Increasing day/night growth temperatures decreased MGDG/DGDG ratios. The shifts in saturation and composition of fatty acids observed with strawberry may be an adaptation response of plants to the temperature changes.     

Effect of hot acetic acid solutions on postharvest decay caused by Penicillium expansum on Red Delicious apples

The objective of this research was improvement of apple hot water treatment efficiency by using acetic acid. The apples (cultivar Red Delicious) were treated using hot acetic acid solutions (1, 2 and 3%) at 50 °C for 1, 2 and 3 min. The results of in vitro study showed that treatment with acetic acid at 50 °C can significantly reduce the growth of Penicillium expansum spores. The treatment of apples with 50 °C acetic acid solutions, in particular 2% acetic acid solution for 3 min or 3% acetic acid solution for 2 min, had significant impacts in reducing the extent of decay of the fruit during the short time storage experiment, while this effect was not significant in the long-time storage.     

An inbred line of the diploid strawberry Fragaria vesca f. semperflorens for genomic and molecular genetic studies in the Rosaceae

Background  

The diploid woodland strawberry (Fragaria vesca) is an attractive system for functional genomics studies. Its small stature, fast regeneration time, efficient transformability and small genome size, together with substantial EST and genomic sequence resources make it an ideal reference plant for Fragaria and other herbaceous perennials. Most importantly, this species shares gene sequence similarity and genomic microcolinearity with other members of the Rosaceae family, including large-statured tree crops (such as apple, peach and cherry), and brambles and roses as well as with the cultivated octoploid strawberry, F. ×ananassa. F. vesca may be used to quickly address questions of gene function relevant to these valuable crop species. Although some F. vesca lines have been shown to be substantially homozygous, in our hands plants in purportedly homozygous populations exhibited a range of morphological and physiological variation, confounding phenotypic analyses. We also found the genotype of a named variety, thought to be well-characterized and even sold commercially, to be in question. An easy to grow, standardized, inbred diploid Fragaria line with documented genotype that is available to all members of the research community will facilitate comparison of results among laboratories and provide the research community with a necessary tool for functionally testing the large amount of sequence data that will soon be available for peach, apple, and strawberry.     

Fruit calcium content and lipoxygenase activity in relation to albinism disorder in strawberry

Nutrient elements and lipoxygenase (LOX) activity were determined in strawberry fruit to establish a relationship, if it exists, between nutrient ratios (N/Ca and K/Ca), and lipoxygenase activity with albinism disorder. About 33% strawberry fruit were affected by albinism. Etna had highest incidence of albinism (48.6%) and Sweet Charlie the lowest (16.2%). Dry matter content (%) was lower in albino fruit (5.23%) than normal fruit (7.36%). The concentration of N, P, and Mg did not differ significantly, but that of K (1.87 mg g⁻¹ fresh weight) was notably higher and of Ca (0.105 mg g⁻¹ fresh weight) was lower in albino fruit than normal fruit. Consequently, the nutrient ratios, N/Ca (9.78) and K/Ca (16.96) were higher in albino fruit than normal fruit. Cultivars differed widely in respect to dry matter (%), mineral content and nutrient ratios. LOX activity determined on dry weight or fresh weight basis was significantly higher in albino fruit than normal fruit, with significant differences among cultivars. Positive correlations existed between nutrient ratios and albinism incidence (r = +0.338), LOX activity and albinism incidence (r = +0.412), and LOX and nutrient ratios (r = +0.448). Thus, it appears from the study that calcium and LOX activity may not the basic cause of albinism in strawberry, but these may be involved in senescence or fruit ripening process, as LOX activity was lower in albino than in normal fruit.     

Direct regeneration of plants derived from in vitro cultured shoot tips and leaves of three Lysimachia species

The regenerability of three ornamental species—Lysimachia christinae, Lysimachia rubinervis and Lysimachia nummularia ‘Aurea’, were investigated using in vitro leaves and shoot tips. 6-Benzylaminopurine (BAP) and α-naphthalene acetic acid (NAA) added to Murashige and Skoog (MS) medium were tested for their effect on organogenesis. On the medium, shoot regeneration occurred directly without callus formation. In these species, L. christinae developed the highest regeneration rate and numbers of shoots/explant from shoot tips (100%, 12.25) and leaf bases (100%, 13.01) on the MS medium containing 3.0 mg l⁻¹ BAP and 0.1 mg l⁻¹ NAA. For L. rubinervis, the highest shoot induction rate and number of shoots/explant were obtained from shoot tip (100%, 16.87–17.20) on the MS medium with 0.1 mg l⁻¹ NAA and 3.0–5.0 mg l⁻¹ BAP. L. nummularia ‘Aurea’, however, showed the highest regeneration rate and number of shoots/explant (100%, 12.73) from leaf bases on MS medium supplemented with 1.0 mg l⁻¹ BAP and 0.1 mg l⁻¹ NAA. All in vitro shoots rooted well on half macronutrient MS medium containing 0.1 mg l⁻¹ NAA. After acclimatization, transplanted plantlets grew normally and flowered in the field.     

Pollination with laser-irradiated pollens breaks cross-incompatibility between zicaitai (Brassica campestris var. purpurea) and ornamental kale (Brassica oleracea var. acephala) to produce hybrids with the aid of ovule culture

The objective of this study was to produce interspecific hybrids between an Ogura-cytoplasmic male sterile (CMS) line of zicaitai (Brassica campestris var. purupurea, 2n = 20) and cultivars of ornamental kale (Brassica oleracea var. acephala, 2n = 18) to develop a CMS system for hybrid seed production. Pollination with pollen grains of ornamental kales irradiated at a power output of 9.0 mW with a He–Ne laser for 3 min could overcome the cross-incompatibility between the species concerned. Intact hybrids could be efficiently produced from ovules cultured on Murashige and Skoog media supplemented by 0.2 mg l⁻¹ 6-benzyladenine. Chromosome number of hybrids was confirmed to be 2n = 19. Hybrids resembled ornamental kales in leaf morphology and in vernalization response. Pollens of hybrids had a sterile appearance. Moreover the hybridity of the putative hybrids was confirmed by RAPD data on a DNA fragment of 820 bp.     

Effects of plant growth promoting bacteria (PGPB) on yield,growth and nutrient contents of organically grown strawberry

The effects of plant growth promoting bacteria (PGPB) on the fruit yield, growth and nutrient element content of strawberry cv. Fern were investigated under organic growing conditions between 2006 and 2008. The experimental plot was a completely randomized design with 3 replicates. Three PGPB strains (Pseudomonas BA-8, Bacillus OSU-142 and Bacillus M-3) were used alone or in combination as bio-fertilizer agent in the experiment. Data through 3 years showed that the use of PGPB significantly increased fruit yield, plant growth and leaf P and Zn contents. Root inoculation of M3 and floral and foliar spraying of OSU-142 and BA-8 bacteria stimulated plant growth resulting in significant yield increases. M3 + BA-8, BA-8 + OSU-142, M3, M3 + OSU-142 and BA-8 applications increased cumulative yield by 33.2%, 18.4%, 18.2%, 15.3% and 10.5%, respectively. Number of fruits per plant significantly increased by the applications of M3 + BA-8 (91.73) and M3 (81.58) compared with the control (68.66). In addition, P and Zn contents of strawberry leaves with bacterial inoculation significantly increased under organic growing conditions. Available P contents in soil were increased from 0.35 kg P₂O₅/da at the beginning of the study to 2.00, 1.97 and 1.82 kg P₂O₅/da by M3 + OSU-142, M3 + BA-8 and M3 + BA-8 + OSU-142 applications, respectively. Overall, the results of this study suggest that root inoculation of Bacillus M3 alone or in combination with spraying Bacillus OSU-142 or Pseudomonas BA-8 have the potential to increase the yield, growth and nutrition content of strawberry plant under organic growing conditions.     

Photoperiodic reaction of sexual and asexual reproduction in Fragaria chiloensis L. CHI-24-1 plants grown at various temperatures

Floral initiation of a wild strawberry strain, Fragaria chiloensis CHI-24-1, is strongly induced by a 24 h day-length (DL) treatment for 40 days consisting of natural daylight and continuous lighting at night by an incandescent lamp. To use the characteristics of floral initiation in CHI-24-1 as a genetic resource for breeding of cultivated strawberries, the photoperiodic reactions of sexual and asexual reproductive growth under various temperature conditions should be clarified. For that purpose, we examined: (1) floral initiation, inflorescence emergence and runner production seasons of CHI-24-1 plants grown under natural climatic conditions in an open field at the Faculty of Agriculture, Kagawa University and (2) the effects of various DLs and temperatures on floral initiation and runner production of CHI-24-1 plants. When the CHI-24-1 plants were grown under natural conditions, the floral initiation, inflorescence emergence and runner production were observed, respectively, in late autumn, spring, and from spring to autumn. Floral initiation of CHI-24-1 plants was induced strongly by 24 h DL at mean temperatures greater than 20 °C. The maximum floral initiation rates were 90% in the parent plant and 94% in the daughter plants, which were linked by runners to the parent plant. The floral initiation of the daughter plants occurred under 20, 22, and 23 h DL at mean temperatures greater than 20 °C, but not for the parent plants. Floral initiation was induced in 100% of the parent plants by the 8 h DL and the lowest mean-temperature conditions. Results of those experiments indicated that CHI-24-1 was an absolute long day plant having critical DL of about 20 h at mean temperatures greater than 20 °C, even though it was a June-bearing strawberry plant. In addition, CHI-24-1 was a facultative short-day plant at mean temperatures of less than 15 °C.     

Development of seedless and Mal Secco tolerant mutant lemons through budwood irradiation

Mal secco (caused by Phoma tracheiphila (Petri) Kantsch. and Gik.) is the most destructive fungal disease of lemon plantations worldwide and seedless lemons would be preferred by most consumers. Five dosage levels, 0, 3, 5, 7, and 9 kiloradian (krad), of cobalt (⁶⁰Co) gamma irradiation were applied to budstick of ‘Kutdiken’ lemon (Citrus limon (L.) Burm. f.) clone KT-2A. Mutations were stabilized in three vegetative generations. Three hundred fifty-eight and 478 M1V3 (mutation one and vegetation three) plants were evaluated for seed number and mal secco tolerance in the field and the greenhouse, respectively. LD₅₀ was around 5 krad gamma irradiation for ‘Kutdiken’ lemon. The seed number varied from 0 to 34 per fruit. The level of mal secco tolerance also varied significantly among the plants from 1.0 (no symptom) to 4.3 (high level of disease occurance). The stable seedless and mal secco tolerant plants were obtained from 5 and 7 krad irradiation: the three mutants from 5 krad irradiation gave more lemon-like fruits, while 7 krad irradiation caused altered tree morphology and early maturation of fruits. This study shows considerable potential for lemon cultivar improvement aiming to obtain seedless and mal secco tolerant lemons.     

Production of inter-section hybrids between Primula filchnerae and P. sinensis through ovule culture

Inter-section hybrids were obtained in the reciprocal crosses between Primula filchnerae (2n = 2x = 24) of Sect. Pinnatae and P. sinensis ‘Fanfare’ (2n = 2x = 24) of Sect. Auganthus by rescuing ovules on half-strength (1/2) Murashige and Skoog's (MS) medium supplemented with 50 g l⁻¹ sucrose, 2.5 g l⁻¹ gellan gum, 0.1 mg l⁻¹ α-naphthaleneacetic acid (NAA), 0.1 mg l⁻¹ 6-benzyladenine (BA) and 50 mg l⁻¹ gibberellic acid (GA₃). In ovule culture, germination occurred with radicle elongation but no plumule was observed. The radicle kept on the initial medium showed root proliferation with callus formation. When the calluses were transferred to (1/2)MS media containing 30 g l⁻¹ sucrose and 3 g l⁻¹ gellan gum, without plant growth regulators (PGRs) or with 1 mg l⁻¹ zeatin and 0.1 mg l⁻¹ NAA, plantlets were regenerated. The plants thus obtained were confirmed to be hybrids through flow cytometry (FCM) and random amplified polymorphic DNA (RAPD) analyses. The hybrid obtained when P. filchnerae was used as the maternal parent was diploid, whereas hexaploid hybrid was obtained when using P. sinensis as the maternal parent. The hexaploid hybrid might be produced through chromosome doubling of a triploid originated from the fertilization of P. sinensis with unreduced pollen of P. filchnerae.     

Relationships between yield components in first cropping year and average yield of short-day strawberries over two main seasons

Fruit productivity of the garden strawberry, Fragaria × ananassa Duch., is a result of interrelationship of yield components contributing to it directly or indirectly. The main aim of the investigation was to find out whether there exists any general regularity, connecting relationships between main yield components in first cropping year to average yield of two main seasons. Two successive studies which primarily included 23 genotypes, differing in yield components and productivity, each, were planted in field conditions in spring 1999 and in spring 2000, respectively, and used for data collection to explore the material obtained in them. Four key yield components and average yields were measured in both cropping years, after which all possible ratios between the components were calculated to find out: first, the relationships to be influential on average yield of the two main seasons; second, type of the probable yield dependence upon the ratios; and, third, their values leading to high yields. Two ratios between the key yield components in first season, i.e.: inflorescence number per plant to branch crown number per plant, named ratio 1, and fruit weight (g) to flower count per inflorescence, named ratio 2, have been found to be related to average yield. An irregular surface, with a single peak falling approximately in its centre, describes average yield dependence upon the ratios. Values of the two ratios falling in the range of 1.0–1.6 each, provided that they are close, have enabled plants to produce high yields cumulatively over two first years. The paper highlights the most important differences in plant behaviour and yield dynamics of strawberry genotypes, depending on combinations of the ratios in first year, discerning those, which have been particularly advantageous to obtain very high yields, and are influenced by negative environmental factors least of all. Based on the findings, a method of predicting of average yield has been developed. Following this, usefulness of the method and possibilities of its using in strawberry breeding and variety trial are discussed.     

Analysis of the taxonomic subdivision within the genus Helleborus by nuclear DNA content and genome-wide DNA markers

Helleborus is a genus of herbaceous perennials belonging to the family Ranunculaceae. Within this genus six sections with a total of 22 species are found. The largest section Helleborastrum contains 16 species for which genetic relationships are still unclear. This study represents the first genetic analysis in the genus Helleborus, including the two newly described species H. liguricus and H. abruzzicus based on multilocus amplified fragment length polymorphism (AFLP) markers with a genome-wide distribution in combination with nuclear DNA content data. Chromosome analyses of roots tips revealed a number of 2n = 32 for the selected species, which was congruent with previous observations. The nuclear DNA content of Helleborus was estimated by flow cytometry applying propidium iodide staining and varied between 18 and 33 pg/2C, depending on the species. For AFLP analyses, 19 out of the 22 Helleborus species were studied using 10 AFLP primer combinations, resulting in a total of 1109 polymorphic bands among all species including the outgroup. The genetic distances between species varied between 0.034 and 0.330. Based on genetic distances a phenogram using the Neighbor-joining cluster method with bootstrap analysis was calculated. The results support the previously suggested division of the genus into six sections and thereby approve AFLP data to be applicable for phenetic analyses. Moreover, this genetic information is significant for the development of future Helleborus breeding strategies.     

Taxonomic revision of Dendrobium moniliforme complex (Orchidaceae)

Taxonomic revision of Dendrobium moniliforme complex is presented. D. moniliforme complex is characterized by the even slim stems, bracts with brownish zone, semi-spherical anther cap and the hairy disc of lip. Dendrobium tosaense, Dendrobium officinale and Dendrobium guangxiense were excluded by having membranous bracts lacking brownish zone, anther cap conical and bifid. Two species are recognized in this complex, i.e., D. moniliforme and Dendrobium wilsonii. D. wilsonii differs from D. moniliforme by having elliptic leaves about 1.3–2 cm wide, dorsal sepal 3.0–4.0 cm long, 0.6–0.9 cm wide, petals elliptic to oblong, 3.0–4.0 cm long, 1.0–1.5 cm wide, lip elliptic to ovate–lanceolate, 2.6–3 cm long, 1.2–1.5 cm wide.     

Fruit yield and quality of strawberry plants transformed with a fruit specific strawberry pectate lyase gene

Two transgenic strawberry lines (Pel 1 and Pel 3) containing the open reading frame of a fruit specific strawberry pectate lyase gene (FaplC) under the control of the CaMV35S promoter have been obtained to evaluate the role of this gene on fruit softening. Ripen fruits from both lines showed a significant down-regulation of FaplC, being the percentage of silencing of 84 and 71% on Pel 1 and Pel 3, respectively. The agronomic behaviour of transgenic plants was evaluated during two consecutive years. Fruit set increased in the two transgenic lines when compared with control plants, although Pel 1 showed a significant reduction on fruit weight. Firmness of full ripen fruits from Pel lines was significantly higher than control fruits, while color and soluble solids were not affected. The increase of firmness in Pel lines was maintained when ripe fruits were stored for 3 days at 25 °C. Histological analysis of ripe fruits showed lower intercellular spaces and a higher degree of cell to cell contact area in transgenic fruits when compared with controls. Altogether, these results suggest a direct relationship between pectate lyase gene expression and strawberry fruit softening.     

Regeneration and characterization of plants derived from leaf in vitro culture of two sweet orange (Citrus sinensis (L.) Osbeck) cultivars

In the current work attempts were made to investigate culture of leaf explants derived from in vitro seedlings of two sweet orange (Citrus sinensis (L.) Osbeck) cultivars, Bingtangcheng and Valencia. Effects of several factors, including culture medium, lighting condition, explant age and genotype on regeneration response were examined based on three parameters, percentage of explants producing shoots, mean number of shoots per explant and shoot forming capacity. Culture of the explants on shoot-inducing media (SIM) composed of MT salts supplemented with different growth regulators gave rise to disparate shoot regeneration, in which SIM₁ (MT + 0.5 mg L⁻¹ BA + 0.5 mg L⁻¹ Kinetin + 0.1 mg L⁻¹ NAA + 3% sucrose + 0.8% agar, pH 5.8) was shown to be the most effective medium for direct induction of shoots from leaf explants. Highly significant difference in the response of shoot bud regeneration was noted between the two cultivars, with Bingtangcheng being more responsive than Valencia. Culture of explants from fully developed leaves led to better shoot regeneration capacity in comparison to undeveloped ones. However, the two lighting conditions used herein did not cause significant difference in shoot regeneration. Phenotypic observation and randomly amplified polymorphic DNA (RAPD) analysis confirmed that all the regenerated plants from both genotypes were genetically identical to their donor plants, suggesting absence of detectable genetic variation in the regenerated plants. The data presented here demonstrated that direct initiation of plants from leaf explants has been successfully accomplished. To our knowledge, this is the first report on direct regeneration of shoots from leaf explants in Citrus, which will provide an alternative source for citrus genetic manipulation in the future.     

Seed inoculation with Azospirillum mitigates NaCl effects on lettuce

Data on the growth-promoting effects of Azospirillum on lettuce exposed to either normal or saline conditions, is scarce. Lactuca sativa L., cv Mantecosa seeds were colonized with A. brasilense Sp245 cells during imbibition. Germination percentages were determined after 7 d treatments with 0, 30, 50 or 80 mol m⁻³ NaCl. In another experiment, seeds germinated in Hoagland were irrigated for 30 d with 0, 30, 50 or 80 mol m⁻³ NaCl supplemented media. Vegetative growth proceeded in a growth chamber with a 13–11 h day–night cycle. Buffer-imbibed seeds were considered non-inoculated controls. Plant samples were taken at 0, 14, 20, and 30 d after the onset of NaCl treatments and dissected in aerial and root portions. The weights of both tissues were measured. Azospirillum-inoculated seeds had significantly higher germination percentages than controls in all treatments. Inoculated dried seeds stored up to 30 d maintained such characteristic in most of the treatments, particularly at 80 mol m⁻³ NaCl. Plants grown from inoculated seeds and irrigated with saline media displayed higher total fresh and dry weights and biomass partition to the aerial portion, than non-inoculated controls. Azospirillum-inoculated lettuce seeds had better germination and vegetative growth than non-inoculated controls after being exposed to NaCl.