Effects of different amino acids on somatic embryogenesis of strawberry (<Emphasis Type="Italic">Fragaria</Emphasis> × <Emphasis Type="Italic">ananassa</Emphasis> Duch.)

This study was conducted to optimize different types and concentrations of amino acids on somatic embryogenesis induction, development and maturation of leaf explants in strawberry cultivars (‘Camarosa’, ‘Paros’ and ‘Kurdistan’). Calli derived from leaf sections were transferred onto MS medium with 1.0 mg/l 2,4-d + 0.5 mg/l BAP supplemented with 0.0, 50, 100, 150 or 200 mg/l concentrations of proline, alanine and glutamine. Stimulation of embryogenesis and embryo development was strictly dependent on the type and concentration of amino acid in the medium. Proline (100 mg/l) was much more effective than glutamine and alanine, on induction and development of somatic embryogenesis in all cultivars. Cultures grown on amino acid-free medium attained lower somatic embryos than cultures grown on amino acid treated medium. Low concentrations (50 mg/l) and high concentrations (200 mg/l) of amino acids tested were inefficient for embryogenesis induction as well as for somatic embryos development.     

An innovative approach to <Emphasis Type="Italic">ex vitro</Emphasis> rooting and acclimatization of <Emphasis Type="Italic">Fragaria</Emphasis> × <Emphasis Type="Italic">ananassa</Emphasis> Duch. microshoots using а biogenic silica- and green-tea-catechin-based mechanocomposite

A new approach for rapid ex vitro rooting and acclimatization of Fragaria × ananassa micropropagated plantlets of two cultivars (“Alpha” and “Festivalnaya”) has been developed using a mechanocomposite based on biogenic silica and green-tea catechins. Two different mechanocomposite treatments were studied: dipping the cut ends of microshoots in the mechanocomposite powder (the dry dip method) and single watering with solutions at concentrations of 0.3, 1.0, and 3.0 g L^?1. These variants were compared with pulse treatment of microplants with 30 mg L^?1 indole-3-acetic acid (IAA) for 4 h and a control group of microshoots that were moistened with hormone-free ¼-strength MS medium. The frequencies of ex vitro rooting at the end of the acclimatization period (30 d) varied from 24.8 to 99.7%. The dry dip treatment was best (rooting frequency about 100%) with up to 7.15?±?0.54-cm root length, and 6.10?±?0.31 roots per plantlet. Moreover, this study showed that the growth-stimulating effect of this mechanocomposite treatment on root formation resulted in increased rosette height, leaf number, leaf area, and dry weight of aerial parts. Histological analysis of the leaf blades revealed decreased mesophyll thickness of microshoots treated with the mechanocomposite (up to 88.77?±?2.95 vs. 111.51?±?3.56 μm for the control). Morphometric analysis of scanning electron microscopy data showed that mechanocomposite treatments led to increased stomata density and stomata length. These structural changes led to normalization of the water regime and indicated successful acclimatization. The combination of ex vitro rooting and acclimatization reduced the procedure time by 4 wk, and may be used for commercial strawberry micropropagation.     

Application of iron nanoparticles and salicylic acid in in vitro culture of strawberries (<Emphasis Type="Italic">Fragaria</Emphasis> × <Emphasis Type="Italic">ananassa</Emphasis> Duch.) to cope with drought stress

The effects of iron nanoparticles and salicylic acid (SA) on strawberry (Fragaria?×?ananassa Duch.) plants in conditions of drought stress were surveyed under in vitro conditions to find the optimum combination for strawberry tissue culture. Cuttings of the Queen Elisa cultivar were surveyed in a three-way factorial experiment with three replications in 2015. The results showed that drought stress significantly affected all measured parameters of strawberry plantlets under in vitro condition in a negative way. SA compensated for the negative effects of drought stress on strawberry plantlets and improved their growth parameters under in vitro culture. Strawberry plantlets treated with iron nanoparticles were able to cope with stressful conditions better than untreated ones. This study found that iron, a micronutrient in plant growth and in vitro development, greatly influenced the plantlets’ growth parameters and other measured traits. These results indicate that the efficiency of tissue culture and in vitro culture of strawberries could be improved by increased application of iron in the form of nanoparticles. The results might also indicate that the application of iron nanoparticles along with SA can be a useful method for providing higher quantity and quantity in the in vitro culture of strawberries, and could be used for adapting strawberry plants to drought before transplanting them in the field.     

A reliable multiplexed microsatellite set for genotyping <Emphasis Type="Italic">Fragaria</Emphasis> and its use in a survey of 60 <Emphasis Type="Italic">F</Emphasis>. × <Emphasis Type="Italic">ananassa</Emphasis> cultivars

We have identified a reliable set of multiplexed microsatellite (SSR) markers for the genotyping of strawberry cultivars and their octoploid progenitors. Over 100 SSRs were screened in two F. × ananassa genotypes and from these, 32 that showed promise for genotyping were selected for further analysis. These SSRs were used to screen a set of 16 strawberry cultivars and a set of fingerprints were produced. Those SSRs that produced reliable, reproducible and easy to interpret fingerprints, that could also distinguish readily between the 16 strawberry cultivars screened, and which could be conveniently included in three multiplex reactions, were selected to form the genotyping set. The genotyping set, consisting of 10 previously-reported SSRs was used to fingerprint a total of 56 cultivated strawberry, and four octoploid Fragaria species accessions. The SSRs used could reliably distinguish between all 60 genotypes surveyed, including sibling cultivars derived from the same parental lines. The primers could be combined for multiplex PCR and represent a useful and convenient genotyping set for Fragaria that will permit fingerprinting data to be shared between laboratories.     

The effect of end of day far-red light on regulating flowering of short-day strawberry (<Emphasis Type="Italic">Fragaria</Emphasis> × <Emphasis Type="Italic">ananassa</Emphasis> Duch. сv. Paros) in a long-day situation

The effects of manipulation of phytochrome state and plant age were tested on flowering strawberry (Fragaria × ananassa Duch. cv. Paros) using end of day far red light (EOD-FR) under undesirable environmental conditions (high temperature and long day). The first and second experiments examined the effects of EOD-FR on flower induction, level of phytochrome and carbohydrates (starch, sucrose, fructose, and glucose), which were variable along with acid invertase enzyme. In the first experiment, there was no flower emergence, but in the second experiment, induction occurred in plants exposed to 6 h of EOD-FR for 32 cycles. The third experiment tested the effect of far red light (6 h + 32 d) on plants of 8, 10, and 12 weeks of age, and induction occurred in 12-week-old plants. The results of experiments II and III indicated that in the induced plants, the amount of phytochrome (Pr) increased. Furthermore, a higher level of sucrose was observed in induced plants, but the level of starch was lower. Nevertheless, amounts of glucose, fructose, and invertase enzyme did not change significantly, although they did show a slight increase in induced plants. These results provide evidence that a light-quality pathway exists that acts on regulation of flowering time in short-day strawberry cultivation.     

Positive responses of strawberry (<Emphasis Type="Italic">Fragaria</Emphasis> × <Emphasis Type="Italic">ananassa</Emphasis> Duch.) explants to salicylic and iron nanoparticle application under salinity conditions

Mucuna bracteata DC. ex Kurz is an important cover crop in plantations across the tropics. However, low germination rate and poor viability of Mucuna seeds pose significant challenges of using the seeds as starting material. To address these limitations, we have optimized seed germination conditions (such as scarification period, surface sterilization protocols and imbibition period) and in vitro propagation protocols for M. bracteata. We found that seeds treated with sulphuric acid for 30 min, imbibed for 6 h and incubated in dark conditions on a wet cotton roll (10 mL of sterile distilled water) supplemented with 0.1% activated charcoal produced the highest percentage of seed germination (44%) and seed vigor index. In vitro-derived cotyledonary nodes showed the highest number of shoots per explant (5.60) and rooting response (92.9%) when cultured on Murashige and Skoog medium containing 4.44 µM 6-benzylaminopurine and 10.7 µM 1-naphthaleneacetic acid, respectively. Of the 100 rooted plantlets acclimatized, 89.0% survived after 4 weeks of transplanting. Single sequence repeat and flow cytometry analysis were performed to confirm the genetic fidelity of the plants. Our protocol offers, for the first time, a simple and effective seed germination and scalable propagation procedures for M. bracteata. Furthermore, we have also estimated the genome size (1448?±?9 Mb) and DNA content (1.48?±?0.01 pg) for M. bracteata that can be used for future cytogenetic studies on genetic diversity and gene exchange.     

Characterization of mixed disomic and polysomic inheritance in the octoploid strawberry (<Emphasis Type="Italic">Fragaria</Emphasis> × <Emphasis Type="Italic">ananassa</Emphasis>) using AFLP mapping

A two-way pseudo-testcross strategy, combined with Single Dose Restriction Fragment (SDRF) marker analysis, was used for genetic mapping in the octoploid cultivated strawberry Fragaria x ananassa (2n = 8 x = 56). Based on a 113 full-sib progeny from a cross between the variety Capitola and the clone CF1116, we generated two parental maps using Amplified Fragment Length Polymorphism (AFLP) markers. Ninety two percent of the markers (727 out of 789) showed ratios corresponding to simplex markers (the majority being SDRF markers), and 8% (62 out of 789) fitted a multiplex ratio. Linkage maps were first established using SDRF markers in coupling phase. The female map comprised 235 markers distributed among 43 co-segregation groups, giving a map size of 1,604 cM. On the male map, 280 markers were assigned to 43 co-segregation groups, yielding a map size of 1,496 cM. Once the co-segregation groups were established, their association was tested using repulsion-phase markers. In total, taking into account associations representing the same linkage groups, 30 linkage groups were detected on the female side and 28 on the male side. On the female map, 68.3% of the pairwise marker linkages were in coupling versus 31.7% in repulsion phase, and the corresponding figures on the male map were 72.2% and 27.8%, respectively. In addition, both groups linked only in the coupling phase and groups linked in the repulsion phase were characterized. The observations suggest that the meiotic behavior of the F. x ananassa genome is neither fully disomic nor fully polysomic, but rather mixed. The genome may not be as completely diploidized as previously assumed.     

<Emphasis Type="Italic">Carex</Emphasis> × <Emphasis Type="Italic">involuta</Emphasis> and <Emphasis Type="Italic">Carex juncella</Emphasis> in the flora of Slovakia

The paper brings information on an isolated occurrence and morphological characters of Carex × involuta and C. juncella populations in the Vel’ká Fatra Mts. Their presence has been known neither from the territory of Slovakia nor from the whole Western Carpathians till now.     

Comparison of suitability of RAPD and ISSR techniques for determination of strawberry (<Emphasis Type="Italic">Fragaria ×</Emphasis><Emphasis Type="Italic">ananassa</Emphasis> Duch.) relationship

Two PCR-based techniques, RAPD and ISSR, were utilized for determination of genetic relationship of 24 strawberry cultivars used in breeding program at the Research Institute of Pomology and Floriculture in Poland. Polymorphism of investigated genotypes was observed in reactions with 23 out of 48 tested RAPD primes and 41 from 90 tested ISSR primers. Relationship, determined on the base of polymorphic products analysis and showed in the form of dendrograms (UPGMA percent method), was generally similar for both techniques, although similarity values based on ISSR data were higher than those based on RAPD. The parallel use of two data sets seems to allow for precise estimation of cultivars relationship and diminishing mistakes connected with methods' technical limitations.     

AFLP and SSR polymorphism in a <Emphasis Type="Italic">Coffea</Emphasis> interspecific backcross progeny [(<Emphasis Type="Italic">C. heterocalyx</Emphasis> × <Emphasis Type="Italic">C. canephora</Emphasis>) × <Emphasis Type="Italic">C. canephora</Emphasis>]

An interspecific cross (BC 1) involving a species with one of the largest genomes in the Coffea genus [Coffea heterocalyx (HET), qDNA = 1.74 pg] and a species with a medium-sized genome [Coffea canephora (CAN), qDNA = 1.43 pg] was studied using two types of molecular markers, AFLP and SSR. One hundred and eighty eight AFLP bands and 34 SSR primer pairs were suitable for mapping. The total map length was 1,360 cM with 190 loci distributed in 15 linkage groups. The results were compared to those obtained previously on an interspecific BC 1 progeny involving a species with a medium-sized genome (Coffea liberica var dewevrei, DEW) and a species with one of the smallest genomes (Coffea pseudozanguebariae, PSE). They are discussed relative to three main points: (1) the relevance of the different marker types, (2) the genomic distribution of AFLP and SSR markers, and (3) the relation between AFLP polymorphism and genome size.Communicated by H.F. Linskens     

Functional expression of the lipase gene Lip2 of <Emphasis Type="Italic">Pleurotus</Emphasis> <Emphasis Type="Italic">sapidus</Emphasis> in <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis>

The lipase Lip2 of the edible basidiomycete, Pleurotus sapidus, is an extracellular enzyme capable of hydrolysing xanthophyll esters with high efficiency. The gene encoding Lip2 was expressed in Escherichia coli TOP10 using the gene III signal sequence to accumulate proteins in the periplasmatic space. The heterologous expression under control of the araBAD promoter led to the high level production of recombinant protein, mainly as inclusion bodies, but partially in a soluble and active form. A fusion with a C-terminal His tag was used for purification and immunochemical detection of the target protein. This is the first example of a heterologous expression and periplasmatic accumulation of a catalytically active lipase from a basidiomycete fungus.     

Cryopreservation of cell suspension cultures of <Emphasis Type="Italic">Taxus</Emphasis> × <Emphasis Type="Italic">media</Emphasis> and <Emphasis Type="Italic">Taxus floridana</Emphasis>

Different lines of cell suspension cultures of Taxus × media Rehd. and Taxus floridana Nutt. were cryopreserved with a two-step freezing method using a simple and inexpensive freezing container instead of a programmable freezer. Four to seven days old suspension cell cultures were precultured in growth medium supplemented with 0.5 M mannitol for 2 d. The medium was then replaced with cryoprotectant solution (1 M sucrose, 0.5 M glycerol and 0.5 M dimethylsulfoxide) and the cells incubated on ice for 1 h. Before being plunged into liquid nitrogen, cells were frozen with a cooling rate of approximately −1 °C per min to −80 °C. The highest post-thaw cell viability was 90 %. The recovery was line dependent. The cryopreservation procedure did not alter the nuclear DNA content of the cell lines. The results indicate that cryopreservation of Taxus cell suspension cultures using inexpensive freezing container is possible.     

Adventitious shoot regeneration from seven commercial strawberry cultivars (<Emphasis Type="Italic">Fragaria </Emphasis>×<Emphasis Type="Italic"> ananassa</Emphasis> Duch.) using a range of explant types

The parameters for optimal regeneration of seven commercial strawberry cultivars were tested using a range of explants and culture conditions. Efficient levels of regeneration--those needed to carry out transformation experiments--with the cultivars Calypso, Pegasus, Bolero, Tango and Emily were achieved with leaf discs, petioles, roots and stipules. Regeneration from cv. Elsanta proved to be difficult from all explant material, although unpollinated ovaries proved to be a promising explant source, with 12% of the explants regenerating shoots. In cv. Eros, regeneration occurred only from root tissue. A comparison of the genetic background suggests that there is a strong genetic component amongst the different cultivars determining their regeneration capacity. The development of these regeneration systems provides a means to use almost the whole stock plant for the efficient genetic transformation of commercial strawberry varieties.     

Somatic hybridization between the diploids of <Emphasis Type="Italic">S.</Emphasis> × <Emphasis Type="Italic">michoacanum</Emphasis> and <Emphasis Type="Italic">S. tuberosum</Emphasis>

Interspecific somatic hybrids between a diploid potato clone DG 81-68 susceptible to Phytophthora infestans (Mont.) de Bary and a resistant diploid tuber-bearing species Solanum × michoacanum were generated and analyzed. About 30 regenerants displaying an intermediate morphology were obtained as a result of three separate PEG-mediated fusion experiments. The RAPD analysis confirmed the hybridity of all the regenerants. About 50% of the hybrid plants exhibited vigorous growth and were stable in culture, while the rest of them rooted poorly and grew slowly in vitro. Most of the hybrid clones were at the tetraploid level (70%), while 30% of the clones examined were at the hexaploid level. The S. × michoacanum (+) DG 81-68 hybrids with growth anomalies were aneuploid. The variation in late blight resistance of the hybrid clones was found in detached leaflet tests, with enhanced resistance characteristic for three tetraploid hybrids.