Plant regeneration from in vitro leaves of mature black cherry (Prunus serotina)

A regeneration system was developed for Prunus serotina from a juvenile (F) and two mature genotypes (#3 and #4). Adventitious shoots regenerated from leaves of in vitro cultures on woody plant medium with thidiazuron (TDZ) and naphthaleneacetic acid (NAA). The best regeneration for genotype F (91.4%) was observed on medium with 9.08 μM TDZ and 1.07 μM NAA. The highest mean number of shoots (8.2) was obtained on medium containing 9.08 μM TDZ and 0.54 μM NAA. Genotype #3 had the highest regeneration (41.7%) with a mean number of shoots (4.8) on 9.08 μM TDZ and 1.07 μM NAA, whereas genotype #4 had a 38.8% regeneration with a mean of 3.3 shoots. Genotype #4 had the highest mean number of shoots (4.8) on 4.54 μM TDZ and 1.07 μM NAA. Silver thiosulphate at 60 or 80 μM increased the percent regeneration of the mature genotypes #3 (75%) and #4 (58%). Adventious shoots were rooted (70–76%) and rooted plantlets survived after acclimatization to the greenhouse. The effect of kanamycin concentration on adventitious shoot regeneration was also evaluated.     

In vitro plant regeneration from leaves and internode sections of sweet cherry cultivars (Prunus avium L.)

Regeneration of adventitious shoots from leaves and, for the first time, from internode sections were compared and optimized for five economically important sweet cherry cultivars, i.e. Schneiders, Sweetheart, Starking Hardy Giant, Kordia and Regina (Prunus avium L.). The influence of basal media, carbon source, combination and dosage of phytohormones, ethylene inhibitor such as silver thiosulfate and a 16 h:8 h light:dark photoperiod versus complete darkness were evaluated. Both, DKW/WPM (1:1) and Quoirin/Lepoivre (QL) basal media stimulated organogenesis more than QL/WPM (1:1), Chee and Pool (CP), Murashige Skoog (MS), Driver and Kuniyuki (DKW) or woody plant (WPM) media did. An induction phase in darkness resulted in lower or zero regeneration rates. The best regeneration efficiencies were generally obtained with thidiazuron in combination with indole-3-butyric-acid. The addition of silver thiosulfate resulted in a similar or reduced regeneration efficiency. Significant genotypic variability in adventitious bud formation was evident for both explant sources, leaf and internode section. Adventitious shoots were obtained from 11% of leaf explants and 50% of internode sections indicating that shoot regeneration from internodes was significantly more efficient than from leaves.     

Six-year field test results of micropropagated black cherry (Prunus serotina)

Summary A field test was established in 1987 to evaluate the growth of micropropagated black cherry plantlets and control seedlings. The study also evaluated effects of two container types on initial survival and growth and of pruning on stem form and growth. At the time of field establishment, plantlets had more extensive root systems than the control seedlings. Survival and height growth were not influenced by container size. Through the first three growing seasons, seedlings were larger than micropropagated plants, but growth differences diminished in the fourth through sixth seasons. Pruning increased the length of clear stem by nearly five-fold but adversely affected diameter growth. Although all clones were from ortet trees more than 50 yr old, none showed plagiotropic growth. Six-year results showed that in a well-prepared and maintained plantation, black cherry trees derived from tissue culture can have at least 80% survival, and growth rates in excess of 1 m per year.     

Shoot regeneration from cultured leaves of Japanese pear (Pyrus pyrifolia)

Several experiments were conducted to investigate in vitro regeneration of adventious shoots from cultured leaves of Japanese pear (Pyrus pyrifolia). A protocol was developed and regeneration achieved from six cultivars. Leaves harvested from shoot cultures which had been preconditioned on B5 medium with 5 μM thidiazuron plus 0.25 μM gibberellic acid were placed on regeneration medium of the same composition. Frequency of regeneration per leaf was as high as 23% but cultivar and environmental factors influenced the result. More mature (basal) leaves regenerated more frequently than younger ones from the shoot tip. Leaf orientation during regeneration and photoperiod was not a strong influence but regeneration from leaf pieces was less than from uncut leaves. An alternative regeneration procedure was developed in which first, shoot cultures were grown on the preconditioning medium. Leaves of the intact shoot cultures were then induced to regenerate directly when adventitious shoots formed on leaves of the intact shoot culture leaves without excision. Adventitious shoots from both procedures developed into typical shoot cultures when transferred to shoot culture maintenance medium. This revised version was published online in June 2006 with corrections to the Cover Date.     

Delayed flowering and reduced branching in micropropagated mature wild cherry (Prunus avium L.) compared with rooted cuttings and seedlings

The development of micropropagated wild cherry (Prunus avium L.) was compared in the nursery and the field with cuttings and seedlings, over a total period of 6 years. Summer semi-hardwood cuttings tended to produce moderate numbers of branches in the season following propagation, whereas micropropagules and seedlings produced significantly fewer or none at all. Removal of branches from cuttings resulted in taller trees. A greater proportion of cuttings than micropropagules flowered in the first year in which trees produced flowers. In the second flowering year, there were no differences in flowering habit between cuttings and micropropagules. Propagation by cuttings or micropropagation did not consistently affect increments in stem diameters or heights. These results are discussed in terms of the suitability of micropropagation and cuttings to produce clonal wild cherry. Received: 23 February 1997 / Revision received: 3 January 1998 / Accepted: 5 February 1998     

Shoot regeneration from tissue-cultured leaves of the American cranberry (Vaccinium macrocarpon)

A method for shoot regeneration from leaf explants in two cultivars of cranberry (Vaccinium macrocarpon Ait.) is described. Modified Anderson's medium supplemented with combinations of thidiazuron (TDZ) with or without 1 M NAA (-naphthaleneacetic acid) was used to optimize shoot regeneration. The effect of light or dark incubation was also determined. Maximum regeneration was obtained in the light in the presence of 10 M TDZ and 1 M NAA. While this medium was suitable for leaf explants obtained from shoot cultures, regeneration did not occur from leaves collected from greenhouse-grown plants. Elongation of the regenerated shoot tips did not occur until explants were transferred to growth regulator-free medium at which time only a minority of shoots elongated. Elongated shoots could be dissected away from leaf tissue, rooted easily, and acclimitized to ambient conditions.Abbreviations NAA -naphthaleneacetic acid - TDZ 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea     

Shoot regeneration from organogenic callus of sweet cherry (<Emphasis Type="Italic">Prunus avium</Emphasis> L.)

A complete method to regenerate adventitious shoots and to produce field-ready trees from three commercial cultivars of sweet cherry (Prunus avium L.) is described. The effects of explant types, pre-treatments, basal media, and phloroglucinol on cultivars Bing, Sweetheart, and Lapins were investigated. Callus developed on four explant types: apical shoot tips isolated from orchard trees; and punctured shoot tips, stem sections, and shoot bases of in vitro shoot cultures. Callus formed on Bing (5%), Sweetheart (8%), and Lapins (20%) shoot tips from orchard trees after 4 months on Murashige and Skoog medium (MS) at half-strength with 3 μM benzylaminopurine (BA). In vitro-derived explants formed callus after 3 months on Woody Plant Medium with 3 μM BA (W3B): punctured shoot tips (Sweetheart and Lapins 67%), stem sections (Sweetheart 31%, Lapins 27%), and shoot bases (Sweetheart 10%, Lapins 17%). Pre-treatment of shoot cultures on MS with 3 μM BA and 1 mM phloroglucinol increased callus formation three-fold on shoot base explants. Callus was separated from parental explants and maintained on MS with 3 μM BA. Shooting was induced by transferring callus to W3B. At 2 weeks, shoot development approached 100%. By 4 weeks, 7–17 shoots had formed on each explant. Callus was maintained for 1.5 years with no decrease in shoot production. Shoots were grafted onto Mazzard (P. avium) rootstocks with 54% (Sweetheart), 57% (Lapins), and 21% (Bing) success after 5 weeks.     

Gibberellin A3 stimulates adventitious rooting of cuttings from cherry (Prunus avium)

We have examined the effect of exogenous gibberellin A₃(GA₃) on adventitious rooting of Prunus avium(cherry) cultivars Stella, F12/1 and Charger. We show that GA₃pre-treatment of P. avium stock plants causes an increasein shoot growth rate and also improves the rooting of cuttings subsequentlytaken from the treated plants. Approximately 37% of cuttings from controlshootsrooted, whereas the percentage rooting could be increased to 80% or more withGA₃ pre-treatment. The number of roots per rooted cutting was alsoincreased by GA₃ pre-treatment. The stimulation of adventitiousrooting could be partially explained by the increase in shoot growth rate.Cultivar Charger responded better than the other cultivars to the lowest levelof GA₃ treatment. In vitro cultures of cultivarCharger were also treated with GA₃. However, the stimulation ofadventitious rooting was less marked than in the GA₃-treated stockplants: percentage rooting increased from 70% to 85%. The results are discussedin the context of 'rejuvenation' effects of GA₃.     

Distribution and fine-scale spatial-genetic structure in British wild cherry (Prunus avium L.)

Insights into the within-population spatial-genetic structure (SGS) of forest tree species, where little is known regarding seed and pollen dispersal patterns, enhance understanding of their ecology and provide information of value in conservation and breeding. This study utilised 13 polymorphic simple sequence repeat loci to investigate the impact of asexual recruitment, management regime and tree size on the development of SGS in wild cherry (Prunus avium L). Only 246 genotypes were identified in the 551 trees sampled, reflecting significant levels of clonal reproduction in both managed and unmanaged populations. Naturally regenerated wild cherry was spatially aggregated under both management regimes. However, in the managed population, sexually derived trees accounted for a greater proportion of the smaller size classes, whereas vegetatively produced trees dominated the smaller size classes in the unmanaged population. High overall SGS values (Sp 0.030-Sp 0.045) were observed when considering only sexually derived genets and kinship coefficients were significant up to the 120 m distance class for both populations. The inclusion of clonal ramets in the analysis significantly increased the overall SGS (Sp 0.089-Sp 0.119) as well as kinship coefficients in the 40-80 m distance classes, illustrating the dramatic impact of vegetative propagation on SGS in this species. Increased spatial aggregation and regeneration appeared to be concomitant with increased SGS in the 40 m distance class in the unmanaged population. Neighbourhood size estimates were relatively small for both populations and kinship coefficients were found to decline with distance under both management regimes, suggesting that common mechanisms may restrict gene dispersal in wild cherry.     

Predicting patterns of invasion by black cherry (Prunus serotina Ehrh.) in Flanders (Belgium) and its impact on the forest understorey community

The design of cost-efficient control strategies for invasive species that are too widespread and abundant for complete eradication, at least in the short term, will benefit from a rigorous analysis of invasion patterns and associated effects on native biodiversity. In this paper, the case of the invasive North American tree Prunus serotina in Flanders (Belgium) is presented. Our main objectives were to determine the susceptibility of forest stands to invasion by P. serotina and the subsequent effects of invasion on the understorey community. We used the large database of the first Flemish Forest Inventory. Multiple logistic regressions indicated that P. serotina occurred more frequently in privately owned, younger forest on coarse-textured, dry soils (podzols), and the combination of these factors allowed us to correctly predict presence/absence of P. serotina in 70% of the validation plots. However, locational variables proved to be important as well, indicating that the invasion process is still ongoing. Prediction of P. serotina densities by means of multiple linear regressions was less successful. Effects on the understorey richness were analysed by comparing the number of species and the mean Ellenberg values between pairs of plots, only differing by the presence of P. serotina in the shrub layer. A reduction of the understorey richness following invasion was only pronounced on the more moist soils, while compositional changes mainly occurred on drier soils. It is concluded that priority for control should be given to landscapes with a low fraction of invaded stands and to forest stands located on more moist soils. However, using its potential to threaten native biodiversity as an argument for control should be done with care as further research is needed whether the observed negative effects are due to a species (i.e. native vs. non-native) or a density effect (high vs. low).     

Characterisation of novel S-alleles from cherry (Prunus avium L.)

In plant populations exhibiting gametophytic self-incompatibility, individuals harbouring rare S alleles are likely to have a reproductive advantage over individuals having more common alleles. Consequently, determination of the self-incompatibility haplotype of individuals is essential for genetic studies and the development of informed management strategies. This study characterises six new S alleles identified in wild cherry (Prunus avium L.). Investigations to determine the S genotype of individuals in recently planted woodland through length polymorphisms of introns associated with the stylar S-RNase gene and the pollen SFB gene revealed six S intron profiles which did not correspond to those of known S alleles. These are now attributed to S ₂₇ to S ₃₂ . Consensus primers, annealing in the S-RNase sequence coding for the signal peptide and C5 regions, were used to isolate the S-RNase alleles associated with the novel S intron profiles. The proteins corresponding to the new alleles were separated by isoelectric focusing from stylar extracts and their pI values determined. Similarities between the deduced amino acid sequence for the new alleles isolated and other cherry S-RNase sequences available on the databases ranged from 40% to 86%. Amplification products for SFB introns ranged from 172 to 208bp. New sequence regions exposed to positive selection were identified and the significance of the PS3 region reinforced. A phylogenetic relationship between P. avium S-RNases for S ₁₀ and S ₁₃ and between corresponding SFB alleles may indicate co-evolution of allele specificities of these two genes. The nucleotide sequences reported in this paper have been submitted to the EMBL/GenBank database under the following accession numbers: S ₂₇ (DQ266439), S ₂₈ (DQ266440), S ₂₉ (DQ266441), S ₃₀ (DQ266442), S ₃₁ (DQ266443), S ₃₂ (DQ266444).     

High-frequency plant regeneration from leaf-disc cultures of Jatropha curcas L.: an important biodiesel plant

A simple, high-frequency and reproducible protocol for induction of adventitious shoot buds and plant regeneration from leaf-disc cultures of Jatropha curcas L. has been developed. Adventitious shoot buds were induced from very young leaf explants of in vitro germinated seedlings as well as mature field-grown plants cultured on Murashige and Skoog’s (MS) medium supplemented with thidiazuron (TDZ) (2.27 μM), 6-benzylaminopurine (BA) (2.22 μM) and indole-3-butyric acid (IBA) (0.49 μM). The presence of TDZ in the induction medium has greater influence on the induction of adventitious shoot buds, whereas BA in the absence of TDZ promoted callus induction rather than shoot buds. Induced shoot buds were multiplied and elongated into shoots following transfer to the MS medium supplemented with BA (4.44 μM), kinetin (Kn) (2.33 μM), indole-3-acetic acid (IAA) (1.43 μM), and gibberellic acid (GA₃) (0.72 μM). Well-developed shoots were rooted on MS medium supplemented with IBA (0.5 μM) after 30 days. Regenerated plants after 2 months of acclimatization were successfully transferred to the field without visible morphological variation. This protocol might find use in mass production of true-to-type plants and in production of transgenic plants through Agrobacterium/biolistic-mediated transformation.     

Promotion by phloroglucinol of adventitious root formation in micropropagated shoots of adult wild cherry (Prunus avium L.)

Micropropagated shoots of wild cherry (Prunus avium L.) produced roots in auxin-free medium. Phloroglucinol (PG) increased the proportion of shoots that rooted, while phloretic acid reduced this response in medium with or without PG, and cancelled the promotive effect of PG. Concentration of PG also significantly affected rooting in media with and without auxin. The proportion of shoots rooting in media containing auxin, or auxin plus PG, increased with the number of successive subculture, but the proportion that rooted with PG alone was unaffected by the number of subcultures. Before the shoots had become responsive to auxin, 1 mM PG was more effective than auxin in inducing root formation.     

Characterization of the S-RNase promoters from sweet cherry (Prunus avium L.)

Genomic DNA fragments containing the S(3)-, S(4)-, and S(6)-RNase genes were isolated from the sweet cherry (Prunus avium L.) and sequenced. Comparison of the 5'-flanking sequences of these three S-RNases indicated that a highly conserved region (designated CR) existed just upstream from the putative TATA boxes. We postulate that CR contains cis-regulatory element(s) involved in pistil expression. To examine the activity of the isolated S-RNase promoters of sweet cherry in the pistil, we transiently introduced approximately 650-bp fragments of the S(4)- and S(6)-RNase promoters fused to beta-glucuronidase (GUS) gene into the pistil of the petunia using a particle bombardment technique. Histochemical analysis showed that the 5'-flanking region of each S-RNase was active in the pistil. This suggests that cis-regulatory element(s) for pistil-specific expression may exist(s) within the 650-bp region upstream from the TATA box in the sweet cherry S-RNase promoter.     

Multiple introductions boosted genetic diversity in the invasive range of black cherry (Prunus serotina; Rosaceae)

Background and Aims

Black cherry (Prunus serotina) is a North American tree that is rapidly invading European forests. This species was introduced first as an ornamental plant then it was massively planted by foresters in many countries but its origins and the process of invasion remain poorly documented. Based on a genetic survey of both native and invasive ranges, the invasion history of black cherry was investigated by identifying putative source populations and then assessing the importance of multiple introductions on the maintenance of gene diversity.

Methods

Genetic variability and structure of 23 populations from the invasive range and 22 populations from the native range were analysed using eight nuclear microsatellite loci and five chloroplast DNA regions.

Key Results

Chloroplast DNA diversity suggests there were multiple introductions from a single geographic region (the north-eastern United States). A low reduction of genetic diversity was observed in the invasive range for both nuclear and plastid genomes. High propagule pressure including both the size and number of introductions shaped the genetic structure in Europe and boosted genetic diversity. Populations from Denmark, The Netherlands, Belgium and Germany showed high genetic diversity and low differentiation among populations, supporting the hypothesis that numerous introduction events, including multiple individuals and exchanges between sites, have taken place during two centuries of plantation.

Conclusions

This study postulates that the invasive black cherry has originated from east of the Appalachian Mountains (mainly the Allegheny plateau) and its invasiveness in north-western Europe is mainly due to multiple introductions containing high numbers of individuals.     

Probing behavior of bird cherry-oat aphid Rhopalosiphum padi (L.) on native bird cherry Prunus padus L. and alien invasive black cherry Prunus serotina Erhr. in Europe and the role of cyanogenic glycosides

The probing behavior of bird cherry-oat aphid Rhopalosiphum padi was studied on its natural winter host in Europe, the bird cherry Prunus padus, and on the invasive black cherry Prunus serotina, on which spring generations of R. padi do not survive. The EPG-recorded behavior of R. padi on bird cherry and black cherry showed differences in crucial aspects of probing and feeding. The period of the pre-phloem penetration was twice as long and rarely interrupted in aphids on bird cherry as opposed to aphids on black cherry. On black cherry, there was a considerable delay between finding and accepting the phloem. Aphids that had sampled phloem sap either refused to ingest it or the ingestion periods were very short. Amygdalin and prunasin (cyanogenic glycosides present in leaves of Prunus) seriously impeded ingestion activities when applied in pure sucrose diet. The role of amygdalin and prunasin in winter host plant selection and host alternation in R. padi is discussed.     

In vitro Shoot Regeneration from Leaves of Sweet Cherry (Prunus avium) 'Lapins' and 'Sweetheart'

Shoot regeneration was achieved from leaves of in vitro cultures of Prunus avium L. cv. 'Lapins' and 'Sweetheart' using woody plant medium (WPM) supplemented with 1-naphthalene-acetic acid (NAA) and thidiazuron (TDZ) or benzyladenine (BA). Percent regeneration was influenced by plant growth regulators and by explant type, orientation and wounding. Optimal regeneration was observed with whole-leaf explants wounded by transverse cuts along the midrib and incubated abaxial surfaces uppermost, on media supplemented with 2.27 or 4.54 µM TDZ plus 0.27 µM NAA. The percent regeneration of the two cultivars was not significantly different. Optimum conditions for regeneration resulted in 71.4% of 'Lapins' and 54% of 'Sweetheart' explants producing one or more shoots per explant.