Adventitious shoot regeneration and Agrobacterium tumefaciens-mediated transformation of leaf explants of sweet cherry (Prunus avium L.)

Sweet cherry (Prunus avium L.) remains recalcitrant for genetic transformation due to the lack of efficient plant regeneration systems via organogenesis or somatic embryogenesis. In this study, in vitro shoot cultures were derived from a single mature embryo (open pollinated) of ‘Selah’ sweet cherry. Leaf explants were cultured on Woody Plant Medium supplemented with different plant growth regulators to induce shoot regeneration. The optimal regeneration at a frequency of 32.5% and an average of 1.1 shoots per explant occurred on the medium containing 4.54 µM thidiazuron (TDZ) and 2.95 µM indole-3-butyric acid (IBA). Transient transformation showed an efficient delivery of the β-glucuronidase (GUS) reporter gene (gusA) using Agrobacterium tumefaciens strain EHA105. Under the optimal gene delivery conditions, stable transformations were conducted using pGA643 and pBI-VcFT containing a blueberry FLOWERING LOCUS T (VcFT). A total of 500 leaf explants, 250 for each construct, were used for transformation. After 10-week selection, three leaf explants transformed with the pGA643 produced four kanamycin-resistant shoots, in which stable integration and expression of the nptII were confirmed by Southern blot and RT-PCR analysis, respectively. This study demonstrated that it was possible to produce stable transgenic sweet cherry using Agrobacterium tumefaciens-mediated transformation of leaf explants.     

Micropropagation of fastigiate bird cherry (Prunus padus L.) and adventitious shoot formation from leaves

A shoot tip of a mature clone of fastigiate bird cherry (Prunus padus L.) was successfully established in vitro. Culture of shoot tip explants on a Murashige and Skoog (MS) based medium with phloroglucinol (PG) resulted in micropropagation, but the clonal line gradually became hyperhydric on this medium. This problem was overcome using PG- free medium based on either MS or Driver and Kuniyuki Walnut medium (DKW). Heavier cultures with more shoots were obtained on DKW medium with fructose or glucose rather than sucrose or sorbitol. Leaf explants placed on DKW basal medium with benzyladenine (BA) produced adventitious shoots. Addition of 1-naphthaleneacetic acid to media with BA increased regeneration. More leaves produced shoots on medium with sucrose or sorbitol rather than glucose or fructose. Adventitious shoots were excised and micropropagated. Shoots were rooted by insertion into DKW medium with indol-3yl- butyric acid, followed by transfer to hormone-free DKW. PG increased the proportion of shoots that produced adventitious roots.     

甜樱桃品种吉列玛叶片再生不定梢的研究

在基本培养基NN69或WPM上附加BA和低浓度的NAA，IAA，IBA作培养基，诱导甜樱桃品种吉列玛试管苗的叶片产生了不定梢。以WPM附加BA 3mg/L和IAA0．3mg/L的处理，不定梢再生率最高为33．3％。     

In vitro shoot regeneration from stored mature cotyledons of sweet cherry (Prunus avium L.) cultivars

Shoot regeneration was achieved from stored mature cotyledons of sweet cherry (Prunus avium L.) in vitro. The influences of different cytokinins (thidiazuron (TDZ) and benzylaminopurine (BA)) and their levels, the dark incubation for the first 10 days of the culture and TDZ pretreatments on shoot regeneration were determined. All varieties regenerated in the presence of TDZ if cotyledons were maintained in darkness for the first 10 days of the culture; however, only three varieties regenerated with low frequencies in the absence of dark incubation. Dark incubation at the early stage of culture was critical for obtaining higher regeneration efficiencies from stored cherry cotyledons. TDZ was more effective than BA in inducing shoot regeneration. The highest regeneration efficiencies were obtained with intermediate concentrations of TDZ (3.6 and 7.2 μM) in combination with dark incubation and the best regeneration frequencies for ‘Vista’, ‘Sunburst’, ‘Tehranivee’, ‘Vouge’ and ‘Heidelfingen’ cotyledons were 70.0%, 53.3%, 23.3%, 30% and 26.6%, respectively.     

S-genotyping of 25 sweet cherry (Prunus avium L.) cultivars from the Czech Republic

Sweet cherry (Prunus avium L.) is a self-incompatible species. Determination of the S-genotypes of cherry cultivars is crucial for breeding and to select appropriate cultivars for cross-fertilisation and fruit set. In this study, we characterised the S-genotypes of 25 sweet cherry cultivars, some of which had being bred at the Research and Breeding Institute of Pomology (RBIP), Holovousy, Czech Republic, and others were European cultivars in the RBIP collection. S-genotyping was carried out by polymerase chain reaction using consensus primers for the S-RNase and SFB genes, and capillary electrophoresis. Nine different known S-haplotypes (S₁, S₂, S₃, S₄, S₅, S₆, S₉, S₁₃, and S₁₆) were identified and the cultivars were assigned to 12 incompatibility groups. One local cultivar, ‘Pta?ka z Plzně’, originated from a wild forest seedling and used as a pollinator, was assigned to Group 0 of universal donors. The pedigree of some cultivars was confirmed by their S-genotype. This study represents the ?rst comprehensive S-genotype screening of sweet cherry genetic resources in the Czech Republic and will be useful for the design of crossing programmes and orchard management of these sweet cherry cultivars.     

Detection of genetic diversity among populations of sweet cherry (Prunus avium L.) by AFLPs

Summary

The high degree of polymorphism of AFLPs provides an efficient system for identification and genome analysis of sweet cherry (Prunus avium) cultivars and selections. The cultivars of sweet cherry have usually been characterized by assessment of phenotypic and pomological traits. AFLP markers were employed to identify 38 sweet cherry accessions and estimate the genetic diversity among this material. Ten of 18 tested primer combinations were informative with up to 80 bands per primer combination. Seven to 33% of the amplfied bands were polymorphic depending upon primer combination. Allcultivars and selections tested could be clearly identified. The objective of this work was to demonstrate the usefulness of molecular markers in revealing the genetic diversity among different sweet cherry genotypes.     

Genetic relatedness of sweet cherry (Prunus avium L.) cultivars from Ukraine determined by microsatellite markers

Microsatellite (SSR) markers were used to characterise 23 sweet cherry cultivars of Ukrainian, and four cultivars of non-Ukrainian, origin. To assess their genetic diversity and relatedness, 11 pairs of primers were applied to microsatellite loci, resulting in amplification of 66 SSR alleles. The mean value of the number of different alleles, and the polymorphic index content, amount to 7.333 and 0.700, respectively, demonstrating a significant genetic diversity of the investigated sweet cherry cultivars. Four highly polymorphic SSR loci (EMPAS02, EMPAS06, PceGA34, UDP98-412), which belong to the list recommended by the European Cooperative Program for Plant Genetic Resources, can be used as a minimum genetic marker set for identification of the majority of the studied cultivars; however, for successful discrimination of the most similar cultivars, more markers, located on all chromosomes of sweet cherry, appear to be necessary. Application of unweighted variable-group method using averages clustering allowed elucidation of the relatedness among the sweet cherry varieties, and showed that the Ukrainian cultivars combine genetic material of local, western European, and probably Caucasian origin; however, the origin of several cultivars still remains unclear, and should be studied additionally.     

黑色培养基促进中国樱桃试管苗生根的研究

以中国樱桃抗病毒优系39号试管苗为试材，进行了在培养基中滴厍碳素墨水促根的试验，结果表明，不同蔗糖浓度、不同IBA浓度条件下碳素墨水皆有十分明显的促根作用，最佳组合为1/2MS IBA0.2-0.7mg.L^-1 蔗糖1%，每40-50mg培养基中滴加1滴碳素墨水（约0.04mdl)。     

甜樱桃果实果肉Ca~(2+)质量浓度变化规律及其与裂果的关系

通过对拉宾斯(Lapins),滨库(Bing)和红丰(Hongfeng)3个抗裂性不同的甜樱桃(Prunus avium L.)品种进行果实发育期外源补钙处理,研究了甜樱桃果实整个发育过程中果肉Ca2+质量浓度变化规律,及其对甜樱桃裂果的影响。结果表明,1)拉宾斯果实在成熟后期果肉Ca2+质量浓度呈稳定的上升趋势,而滨库和红丰在果实成熟期果肉Ca2+质量浓度则呈下降趋势。2)外源补钙处理可明显提高果实中的钙含量,所有处理的果实在整个发育过程中果肉Ca2+质量浓度都明显高于对照,其中拉宾斯比对照高12%、比滨库高13%、比红丰高40%。3)在裂果试验中,高抗裂品种拉宾斯对于外源补钙不敏感,叶面补钙处理后裂果率由7%降为4%;而抗裂性较差的滨库和红丰则非常敏感,裂果率滨库由49%降为28%、红丰由92%降为60%,证明果肉Ca2+质量浓度的提高可明显降低果实裂果率。     

Multiple shoot regeneration in seed-derived immature leaflet explants of peanut (Arachis hypogaea L.)

A protocol was developed for organogenesis from immature leaflet explants derived from mature seeds of peanut. Immature leaflets pre-incubated on MS medium supplemented with 13.32 μM BAP + 4.95 μM NAA for 7 days, turned green and enlarged. The enlarged green leaflets produced multiple shoot buds after 1–2 cycles of sub-culture on MS medium supplemented with 13.32 μM BAP. Three cycles of shoot buds on the elongation medium (13.32 μM BAP) produced 6.17 ± 0.47 elongated shoots per explant. The shoot bud formation was genotype independent. All elongated shoots rooted on the medium containing 4.95 μM NAA. The complete protocol gave efficient (>81%) direct organogenesis, leading to the development of plantlets within 4 months.     

An efficient adventitious shoot regeneration system on excised leaves of micropropagated lingonberry (Vaccinium vitis-idaea L.)

Summary

An efficient system to regenerate shoots in vitro on excised leaves of lingonberry (Vaccinium vitis-idaea L.) was developed. Leaf explants from shoot-proliferating cultures produced multiple shoots without an intermediary callus phase on zeatin (ZN)-containing shoot induction media within 3–4 weeks of culture initiation. Cultivars Regal and Splendor, and one clone from a natural stand in Estonia (ECL1), were used in the first experiment. Young expanding leaves with the adaxial side touching the culture medium, and maintained for 7 d in darkness, produced the best results. There were significant genotypic differences in adventitious shoot formation. A second experiment studied the effects of ten concentrations of three cytokinins: ZN at 5, 10, 20, 30 and 40 μM; 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea (thidiazuron, TDZ) at 0.1, 1, 5 and 10 μm; and 6-(γ-γ-dimethylallylamino) purine (2ip) at 25 μM were compared with leaf segments of different polarity in ‘ECL1’. Zeatin was found to be more effective than TDZ or 2iP as an inductive signal for regenerating many vigorous shoots. Zeatin induced multiple shoot formation at all concentrations tested, but maximum morphogenic response was observed at 20 to 30 μM. The media containing TDZ generally promoted more callus formation and suppresed shoot elongation. In a third experiment with the lingonberry cultivar Erntedank and the clone ‘ECL1’, a new medium developed for lingonberry shoot culture proved more effective than the modified Murashige and Skoog medium for regenerating shoots on leaf explants. Elongated shoots were excised and rooted directly on a 2 peat:1 perlite (v/v) medium after dipping in 0.8% indole-3-butyric acid. Rooted plantlets were acclimatized under greenhouse conditions to evaluate somaclonal variation.     

Effects of cultivar,orchard elevation,and storage on fruit quality characters of sweet cherry (Prunus avium L.)

Fruit quality characters were analysed in the sweet cherry cultivars, Burlat, Van, Tragana and Mpakirtzeika, harvested from low (39–59 m), medium (216 m) or high (490–546 m) elevation sites. The effects of storage for 2 or 4 days at 2 °C and 1 day at 20 °C on the fruit antioxidant contents were also evaluated. Tragana and Mpakirtzeika had greater fruit fresh weight (FW) and total soluble solid content compared to Van and Burlat, the latter being the most red colored. Tragana and Burlat had greater total phenolic content and total antioxidant capacity, measured by DPPH extinction, compared to Mpakirtzeika and Van (mean values 204.4 mg vs. 103.7 mg gallic acid equivalent 100 g⁻¹ FW, and 176.1 mg vs. 79.3 mg ascorbic acid equivalent 100 g⁻¹ FW, respectively). The geographic elevation had a marked influence on the cherry antioxidant content in all studied cultivars, apart from Van, with high elevation orchards producing cherries with greater contents of antioxidant compounds compared to lower elevation orchards. Changes in the antioxidant contents during storage were depended on the cultivar and some times on the orchard elevation. Total antioxidant capacity was significantly correlated with total phenolic content in Tragana, Burlat and Mpakirtzeika, but not in Van; nevertheless this was not the case during storage.     

In vitro regeneration and genetic transformation of common bean (Phaseolus vulgaris L.)—Problems and progress

Common bean is one of the most cultivated species in the Leguminosae family. Thus far, progress in bean improvement has been achieved mainly by conventional breeding methods. These methods, however, in addition of being time-consuming and labor-intensive, are met with a wide range of problems including traits associated with low genetic variation, low survivability of interspecific hybrids, specific inheritance of some valuable characteristics such as yield, disease, and pest resistance, as well as harvesting characteristics. Plant biotechnology offers different strategies to overcome these difficulties. With some exceptions, species belonging to the Leguminosae are difficult to regenerate in vitro. In this review, we discuss the potential and limitations of in vitro cultivation of Phaseolus vulgaris L. based on the existing literature data. Different tissue culture methods like somatic regeneration are discussed and evaluated as well as gene transfer and other approaches in bean modification in vitro.     

Cold and heat requirements of the apricot tree (Prunus armeniaca L.)

Summary

The cold units (CU) and heat units (HU) required for the flowering of 13 apricot cultivars (Prunus armeniaca L.) cultivated in AbaraÂn (Murcia) have been established. The end of the dormancy period was determined on the basis of the dry-weight increase of the flower buds. The tests in which the dry weight was measured after forcing the flower buds at 208C turned out to be more precise in detecting the beginning of the reactivation phase than those in which they were not forced.     

Adventitious shoot regeneration from hypocotyl slices of mature apricot (Prunus armeniaca L.) seeds: A feasible alternative for apricot genetic engineering

Adventitious shoot regeneration from hypocotyl slices of mature apricot seeds has been achieved with regeneration percentages of 31.7%, 44.4%, and 46.9% for the cultivars ‘Canino’, ‘Dorada’, and ‘Moniqui’, respectively. Regeneration was significantly affected by the parental origin of the explants (P < 0.05) but not by thidiazuron or 3-indolebutyric acid for any of the three cultivars, at the levels tested. None of the other factors studied (basal medium, 2,4 dichlorophenoxy-acetic acid pulses, dark incubation period, or addition of silver thiosulfate) affected significantly shoot regeneration percentage from ‘Canino’ hypocotyl sections. The effect of paromomycin on regeneration was genotype-dependent and different dose–response curves were obtained for each cultivar. While 40 μM paromomycin completely inhibited regeneration from ‘Canino’ sections, some buds were obtained from ‘Dorada’ and ‘Moniqui’ explants. The two aminoglycoside antibiotics tested, kanamycin and paromomycin, showed differing toxicity on ‘Canino’. A lower concentration of kanamycin (20 μM) than of paromomycin inhibited totally adventitious regeneration from ‘Canino’ explants. Agrobacterium-mediated transformation experiments, with the non-oncogenic strain AGL1 harboring the binary plasmid p35SGUSINT, were performed and GUS assays were carried out after four weeks to determinate stable transformation events. The utilization of paromomycin (10 μM) as the selective agent increased significantly both the number of explants that presented at least one transformation event (P < 0.05) and the number of large area or calli expressing the gus gene (P < 0.001), compared with the addition of kanamycin (10 μM). Moreover, when 10 μM paromomycin was added to the medium some massively transformed explants were observed and a chimerical bud was regenerated.     

发根农杆菌介导山定子遗传转化及发根再生植株

为了提高山定子的生根能力,用发根农杆菌转化山定子,诱导产生发根,并由发根诱导出再生植株。通过对不同发根农杆菌株系、培养基、共培养时间以及添加乙酰丁香酮（AS）的研究,优化了发根农杆菌转化体系。当用添加AS（100μmol﹒L-1）的菌液侵染植株切段30 min,共培养3 d,在无植物生长调节剂的固体1/4MS培养基上诱导产生发根时,发根诱导率最高,达到88.89%。将发根根段在MS+BA 2.0 mg﹒L-1 +IBA 0.1 mg﹒L-1+GA3 0.1 mg﹒L-1的再生培养基上,通过愈伤组织的诱导得到再生植株,再生率3%。     

矮化蟠桃的育种实践与连锁遗传分析

 利用乔化异质型蟠桃(DwdwSs) 与矮化型圆桃( dwdwss) 或乔化异质型圆桃(Dwdwss) 进行有性杂交, 培育杂种实生苗, 对子代相关性状的分离情况进行了分析。结果显示, 乔化对矮化为一对等位基因, 且乔化对矮化为完全显性, 蟠桃与圆桃为一对等位基因, 蟠桃对圆桃为完全显性。在乔化异质型蟠桃(DwdwSs) 与矮化型圆桃( dwdwss) 的正反交后代中, 没有出现矮化型蟠桃和乔化型圆桃, 乔化蟠桃与矮化圆桃的比例为1∶1; 在乔化异质型蟠桃(DwdwSs) 与乔化异质型圆桃(Dwdwss) 的杂交后代中, 也同样没有出现矮化型蟠桃, 乔化蟠桃、乔化圆桃与矮化圆桃的分离比例为2∶1∶1, 据此得出以下推论, 果实扁平性状与树体乔化性状完全连锁, 树体矮化性状与果实非扁平性状完全连锁, 即矮化突变与扁平果形突变分别发生在不同的同源染色体上, 两者之间没有发生过交换, 不产生dwS配子。