Field performance of <Emphasis Type="Italic">Theobroma cacao</Emphasis> L. plants propagated via somatic embryogenesis

Somatic embryogenesis is an in vitro clonal propagation method with potential to contribute to the improvement of cacao varieties. Before using this technology for commercial production, it is essential that somatic embryogenesis-derived plants be tested in field conditions. Therefore, we established a field test at Union Vale Estate, Saint Lucia. Thirty- to 50-yr-old trees were selected for clonal propagation as potentially high yielding based on local farmers observations. Clonal plants were propagated in vitro from immature flowers by embryogenesis and micropropagation. Multiple plants from nine genotypes were acclimated to greenhouse conditions then returned to Saint Lucia and planted in a field. Orthotropic rooted cuttings and locally propagated open pollinated seedlings were also planted for a total of 214 trees. Growth data were collected every 4–6 mo. including: stem diameter, stem height, length of the longest jorquette branch, number of jorquette branches, and dates of first flowering and fruiting. At 4.5 yr after planting in the field there were no major differences in all growth parameters among the propagation methods evaluated with exception of the orthotropic rooted cuttings. Trees grown from seeds were slightly taller then trees propagated by the other methods. Trees propagated as orthotropic rooted cuttings exhibited smaller average stem diameters, shorter stem heights to the jorquette, and shorter jorquette branches. We concluded that somatic embryo-derived plants demonstrated normal phenotypes in field conditions and have growth parameters similar to plants propagated by traditional methods.     

Assessment of expression and inheritance patterns of three transgenes with the aid of techniques for promoting rapid flowering of transgenic apple trees

Rapid flowering of transgenic Royal Gala apple (Malus×domestica) trees was achieved by growing trees under controlled greenhouse conditions. The expression and inheritance of three transgenes were confirmed in the seedling progeny. Grown as single stems on their own roots, the transgenic apple trees produced 80–110 nodes and were 2 m high on average at the end of the 1st year's growth. In the 2nd year, approximately 20% of these trees flowered around node 80. However, when scions collected from the top of 1-year-old trees were grafted onto the dwarfing rootstock Malling 9, 85% produced flowers and fruit within the next year. The grafted trees continued to produce fruit in the following years. Expression of the transgene uidA was monitored by assaying β-glucuronidase (GUS) activity in leaves, flowers and fruit. Inheritance of three transgenes, uidA, neomycin phototransferase II and acetolactate synthase, were demonstrated through the recovery of GUS-positive, kanamycin-resistant and chlorsulfuron-resistant progeny. Segregation patterns fitted a 1:1 ratio in most lines. However, a detailed analysis in one progeny line revealed a complex T-DNA integration pattern. Received: 8 June 1998 / Revision received: 19 November 1998 / Accepted: 26 November 1998     

A draft genome of sweet cherry (Prunus avium L.) reveals genome‐wide and local effects of domestication

Sweet cherry (Prunus avium L.) trees are both economically important fruit crops but also important components of natural forest ecosystems in Europe, Asia and Africa. Wild and domesticated trees currently coexist in the same geographic areas with important questions arising on their historical relationships. Little is known about the effects of the domestication process on the evolution of the sweet cherry genome. We assembled and annotated the genome of the cultivated variety “Big Star*” and assessed the genetic diversity among 97 sweet cherry accessions representing three different stages in the domestication and breeding process (wild trees, landraces and modern varieties). The genetic diversity analysis revealed significant genome‐wide losses of variation among the three stages and supports a clear distinction between wild and domesticated trees, with only limited gene flow being detected between wild trees and domesticated landraces. We identified 11 domestication sweeps and five breeding sweeps covering, respectively, 11.0 and 2.4 Mb of the P. avium genome. A considerable fraction of the domestication sweeps overlaps with those detected in the related species, Prunus persica (peach), indicating that artificial selection during domestication may have acted independently on the same regions and genes in the two species. We detected 104 candidate genes in sweep regions involved in different processes, such as the determination of fruit texture, the regulation of flowering and fruit ripening and the resistance to pathogens. The signatures of selection identified will enable future evolutionary studies and provide a valuable resource for genetic improvement and conservation programs in sweet cherry.     

Introduction of asymbiotically propagated seedlings of <Emphasis Type="Italic">Cephalanthera falcata</Emphasis> (Orchidaceae) into natural habitat and investigation of colonized mycorrhizal fungi

Asymbiotic seedling propagation and introduction of seedlings into a natural habitat were achieved for Cephalanthera falcata. For immature seeds collected 65 days after pollination, high germination rate (av. 50%) was achieved on Hyponex agar medium plates. Root development occurred in about 10% of the protocorms 5 months after seed sowing. Rooted protocorms were transferred to a culture bottle containing 100 ml of the Hyponex agar medium and incubated continually. In about 30% of the transferred individuals, shoot height reached 1.5–2 cm 8 months after the transfer. After acclimatization in wet vermiculite at 4°C for 6 months, 135 individuals were planted in a natural stand of C. falcata in mid February 2001. Shoot appearance rate was 44.4% at the first year and flowering was noted in some plants. At the fifth year, shoots with an average height of 21.6 cm still appeared in four plants, and flowering was noted in three of them. Colonization of mycorrhizal fungi was examined in two of them as well as one wild plant, in which the mycorrhizal fungi were identified to be in Thelephoraceae or Russulaceae. These fungi are known to form ectomycorrhiza with trees, and thus a tripartnership symbiosis consisting of C. falcata, mycorrhizal fungi and trees was suggested. The involvement of ectomycorrhizal fungi might be the reason for the low survival rate in the field experiment, because the distribution of ectomycorrhizal fungi relevant to this orchid is assumed to be uneven. The possibility of introducing artificially propagated orchids into natural habitats was discussed.     

Antibodies to phase related proteins in juvenile and mature Prunus avium

In the search for biochemical and molecular markers of juvenility in trees proteins have been identified which are preferentially or differentially expressed in either the juvenile or the mature phases of Prunus avium cv. Stella. These fall into two classes: those which are phase-related and those which may be affected by root-shoot distance. N-terminal amino acid sequence data from some of these proteins were used to produce polyclonal antibodies to corresponding synthetic peptides in order to determine if they could be used as markers of phase state in woody plants. Western blot analysis was performed on proteins extracted from three sources; juvenile trees, mature trees and rooted cuttings from mature trees. The results showed that the antibodies recognised differentially-expressed proteins. In particular, one antibody to a juvenile specific protein cross-reacted with a polypeptide of approx. 28 kDa which was present in greater amounts in shoot tips of juvenile P. avium cv. Stella seedlings compared with rooted cuttings of mature plants placed in the same growth environment.     

Protocol for in vitro propagation of Excoecaria agallocha L., a medicinally important mangrove species

An in vitro propagation protocol has been developed for Excoecaria agallocha L. (Euphorbiaceae), a mangrove species. Nodal segments were used for axillary shoot proliferation. One shoot from each node of binodal explants was observed 3 weeks after inoculation. The best axillary sprouting was seen on a newly formulated medium containing BA, Zeatin and IBA in concentrations of 13.3 μM, 4.65 μM and 1.23 μM, respectively. The new medium, first used in this study, has a specific composition of major nutrients, MS micronutrients and iron compounds. Nodal segments from rooted cuttings and seedlings responded better than those of mature tree explants. Multiple shoot induction was complemented with efficient shoot elongation, and repeated subculture of binodal segments from axillary shoots resulted in 10–12 shoots per explant in 3 months. Rooting was achieved by growing shoots in the new medium with 0.23 μM IBA. Regenerated plants were successfully acclimatized to the natural environment, and about 85% of plantlets survived under ex vitro conditions. This is the first report of micropropagation in the genus Excoecaria and also in mangrove tree species. Received: 11 August 1997 / Revision received: 10 February 1998 / Accepted: 2 March 1998     

Activity of the CaMV 35S promoter in various parts of transgenic early flowering birch clones

Early flowering together with small size would be useful for various biotechnical or genetic studies on trees. We report here the selection and micropropagation of early flowering birch (Betula pendula) clones (BPM1–12) obtained from seeds of birches bred elsewhere for early flowering. Under conditions that accelerate flowering (a high CO₂ level, strong and continuous illumination), the first male inflorescences emerged in 3–5 months, the trees then being 20–80 cm high. Transgenic lines (CaMV 35S-GUS INT) were produced through Agrobacterium-mediated gene transfer from BPM2, BPM5 and JR1/4 (a normally flowering birch). β-Glucuronidase (GUS) activities in the different lines, assayed 1–1.5 years after transformation, varied greatly. During further in vitro culture for 10 months, the activities decreased to 0.3–7% of the original values. GUS activities were detected in all organs studied, including the developing male inflorescences; the highest activity was in the roots. Received: 28 April 1997 / Revision received: 5 September 1997 / Accepted: 30 November 1997     

Field performance of micropropagated,macropropagated, and seed-derived propagules of three Eucalyptus grandis ortets

Tree size, survival, and coppicing of micropropagated plantlets, macropropagated cuttings, and seedlings of Eucalyptus grandis Hill ex Maiden were monitored through 57 months in a study in southern Florida to assess propagation options. Two plantlet lines developed by direct micropropagation and orchard open-pollinated seedlings from three ortets were compared in the main study. Rooted cuttings from up to four ramets of each of the three ortets and another ortet were examined in an adjacent supplemental study. Freezes at six and 16 months killed most initial and first-coppice stems to the ground. Most developmental differences in the main study were consistent from ages 2 to 57 months. Propagation by ortet interactions were observed beginning at 21 months, due to the poor performance of seedlings of one ortet after the second freeze. At 57 months, no differences in tree height, DBH, volume, or survival were detected between plantlet lines and between rooted cuttings and plantlets, but seedlings were inferior to plantlets and cuttings. Vegetative propagules had more uniform tree size at every age, with typically less than one-half the variability observed among seedlings. Even though plantlets and cuttings may be more expensive to produce, they have numerous advantages over seedlings for E. grandis plantation establishment in Florida.     

Effects of earthworms, nematodes, cultivations and host plants on Verticillium wilt of peach and cherry

Verticillium dahliae but not V. albo-atrum Berth was isolated from eight out of twenty-one stone fruit orchards surveyed for Verticillium wilt disease in western New York. Wilt incidence was related to the cultivation of tomato or legumes as previous or inter-crop with stone fruit trees. A limited cross species inoculation using isolates of V. albo-atrum and V. dahliae from woody and herbaceous plants showed that peach and cherry were susceptible to both species. The effect of V. dahliae on growth of cherry seedlings in the presence of Tylenchorynchus claytoni, Pratylenchus penetrans and Meloidogyne hapla was compared. P. penetrans and M. hapla produced more severe growth reduction than T. claytoni. The adverse effect of Verticillium on the growth of cherry seedlings was greater acting together with any one of the three nematodes than acting alone. V. dahliae was shown to be capable of passage through earthworms without loss of infectivity.     

Delayed inducible resistance against a leaf-chewing insect in four deciduous tree species

Summary Both mechanical damage to mountain birch foliage and rearing of moth larvae on the trees reduced the growth of Epirrita autumnata larvae reared on these trees in the following year. The effects of physical damage and some other cues from insects were additive. On bird cherry the performance of Epirrita larvae was equal on untreated trees and on trees artificially defoliated in the previous year, but larval growth was reduced on previously insect-damaged branches. With mountain ash just physical damage per se reduced the performance of Epirrita larvae. On Salix phylicifolia there were no significant differences in the growth or survival of Epirrita on untreated control bushes and on bushes with partial larval damage during the previous year. Among untreated control trees the growth and survivorship of Epirrita were higher on fast-growing willow and bird cherry than on the slow-growing mountain birch. Mountain birch and mountain ash, the two deciduous tree species adapted to nutrient-poor soils, showed delayed inducible resistance triggered by defoliation (artificial or insect-made). This supports the hypothesis that delayed inducible resistance may be a passive response due to nutrient-stress caused by defoliation. On the other hand, the additional increase in the resistance of mountain birch triggered by specific cues from insects suggests that this response may be an evolved defense against leaf-eating insects.     

Effect of Wild-Type and Mutant Plant Growth-Promoting Rhizobacteria on the Rooting of Mung Bean Cuttings

Mung bean cuttings were dipped in solutions of wild type and mutant forms of the plant growth-promoting rhizobacterium Pseudomonas putida GR12-2 and then incubated for several days until roots formed. The bacteria P. putida GR12-2 and P. putida GR12-2/aux1 mutant do not produce detectable levels of the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase, whereas P. putida GR12-2/acd36 is an ACC deaminase minus mutant. All bacteria produce the phytohormone indole-3-acetic acid (IAA), and P. putida GR12-2/aux1 overproduces it. Treatment of cuttings with the above-mentioned bacteria affected the rates of ethylene production in the cuttings in a way that can be explained by the combined effects of the activity of ACC deaminase localized in the bacteria and bacterial produced IAA. P. putida GR12-2 and P. putida GR12-2/acd36-treated cuttings had a significantly higher number of roots compared with cuttings rooted in water. In addition, the wild type influenced the development of longer roots. P. putida GR12-2/aux1 stimulated the highest rates of ethylene production but did not influence the number of roots. These results are consistent with the notion that ethylene is involved in the initiation and elongation of adventitious roots in mung bean cuttings. Received October 21, 1998; accepted January 3, 1999     

Masting in a temperate tree: Evidence for environmental prediction?

Many plant species produce large fruit crops in some years and then produce few or no fruits in others. Synchronous, inter‐annual variation in plant reproduction is known as ‘masting’ and its adaptive significance has yet to be fully resolved. For 8 consecutive years, I quantified every fruit produced by 22 females of a New Zealand tree species (Dysoxylum spectabile), which has an unusual habit of taking a full calendar year to mature fruits after flowering. Fruit production varied strongly among years and was tightly synchronized among trees. Annual variability in fruit production declined with total reproductive output, indicating trees with lower fecundity exhibited a stronger tendency to mast. Although unrelated to temperature, annual fruit production was positively related to precipitation during annual periods of fruit development, and negatively related to fruit production in the previous year. Seedlings had higher rates of survivorship in a wet, high‐seed year than in a dry, low‐seed year, suggesting that seedlings might be drought sensitive. Therefore, D. spectabile produced large fruit crops during periods of high rainfall prior to fruit maturation, which may enhance survivorship of drought‐intolerant seeds. Results were inconsistent with several hypotheses that are widely believed to be the most likely explanations for masting. Instead, results were consistent with the environmental prediction hypothesis, suggesting that this hypothesis may be more important than previously appreciated.     

Successful crossings with early flowering transgenic poplar: interspecific crossings,but not transgenesis,promoted aberrant phenotypes in offspring

In forest tree species, the reproductive phase is reached only after many years or even decades of juvenile growth. Different early flowering systems based on the genetic transfer of heat‐shock promoter driven flowering‐time genes have been proposed for poplar; however, no fertile flowers were reported until now. Here, we studied flower and pollen development in both HSP::AtFT and wild‐type male poplar in detail and developed an optimized heat treatment protocol to obtain fertile HSP::AtFT flowers. Anthers from HSP::AtFT poplar flowers containing fertile pollen grains showed arrested development in stage 12 instead of reaching phase 13 as do wild‐type flowers. Pollen grains could be isolated under the binocular microscope and were used for intra‐ and interspecific crossings with wild‐type poplar. F1‐seedlings segregating the HSP::AtFT gene construct according to Mendelian laws were obtained. A comparison between intra‐ and interspecific crossings revealed that genetic transformation had no detrimental effects on F1‐seedlings. However, interspecific crossings, a broadly accepted breeding method, produced 47% seedlings with an aberrant phenotype. The early flowering system presented in this study opens new possibilities for accelerating breeding of poplar and other forest tree species. Fast breeding and the selection of transgene‐free plants, once the breeding process is concluded, can represent an attractive alternative even under very restrictive regulations.     

Reduced generation time of apple seedlings to within a year by means of a plant virus vector: a new plant‐breeding technique with no transmission of genetic modification to the next generation

Fruit trees have a long juvenile phase. For example, the juvenile phase of apple (Malus × domestica) generally lasts for 5–12 years and is a serious constraint for genetic analysis and for creating new apple cultivars through cross‐breeding. If modification of the genes involved in the transition from the juvenile phase to the adult phase can enable apple to complete its life cycle within 1 year, as seen in herbaceous plants, a significant enhancement in apple breeding will be realized. Here, we report a novel technology that simultaneously promotes expression of Arabidopsis FLOWERING LOCUS T gene (AtFT) and silencing of apple TERMINAL FLOWER 1 gene (MdTFL1‐1) using an Apple latent spherical virus (ALSV) vector (ALSV‐AtFT/MdTFL1) to accelerate flowering time and life cycle in apple seedlings. When apple cotyledons were inoculated with ALSV‐AtFT/MdTFL1 immediately after germination, more than 90% of infected seedlings started flowering within 1.5–3 months, and almost all early‐flowering seedlings continuously produced flower buds on the lateral and axillary shoots. Cross‐pollination between early‐flowering apple plants produced fruits with seeds, indicating that ALSV‐AtFT/MdTFL1 inoculation successfully reduced the time required for completion of the apple life cycle to 1 year or less. Apple latent spherical virus was not transmitted via seeds to successive progenies in most cases, and thus, this method will serve as a new breeding technique that does not pass genetic modification to the next generation.     

Strength and structure of stems from fast grown Pinus radiata

Summary Segments of living stems from 2-year-old, fast growing Pinus radiata, established on a fertile ex-pasture, were examined in terms of their bending strength parallel to the grain. Stem anatomical and structural variables were examined to identify variables that could explain variation in bending strength. Half of the seedlings were physically supported to minimise the confounding effects of compression wood production in response to environmental stresses. Differences between seedlings and cuttings were examined. Variation in microfibril angle and lignin content was sufficient to account for 81% of the variation in bending strength. A positive correlation between both of these variables and elasticity was evident. Few differences were found to be caused by the support treatment or between seedlings and cuttings. The support treatment resulted in significantly fewer branches in the top most whorl, while cuttings had less branches in this whorl than seedlings.     

Pathogenicity of four Phytophthora Species on Wild Cherry and Italian Alder Seedlings

Inoculation tests were carried out in the greenhouse on wild cherry (Prunus avium) and Italian alder (Alnus cordata) seedlings, to determine their susceptibility to certain Phytophthora species (P. citrophthora, P. alni, P. megasperma and P. cinnamomi) that are commonly present in the soil. Host susceptibility was evaluated in accordance with a disease index, with the lesion length after stem inoculation, and with a root system disease index. Wild cherry was found to be highly susceptible to P. citrophthora, and was also found to be susceptible to P. alni, although to a lesser extent. Italian alder was very susceptible to P. alni, but had only low susceptibility to P. citrophthora. The other Phytophthora species caused only modest symptoms. The danger to alder and wild cherry is all the greater because these trees not only share the same pathogens, but also commonly planted together in mixed stands. The results will now have to be confirmed by using a more natural inoculation method.     

Somatic hybridization of sexually incompatible top-fruit tree rootstocks,wild pear (Pyrus communis var. pyraster L.) and Colt cherry (Prunus avium x pseudocerasus)

Summary Mesophyll protoplasts of wild pear (Pyrus communis var. pyraster L., Pomoideae) were chemically fused with cell suspension protoplasts of cherry rootstock Colt (Prunus avium x pseudocerasus, Prunoideae), following an electroporation treatment of the separate parental protoplast systems. Fusion-treated protoplasts were cultured, on modified K8P medium, where it had been previously established that neither parental protoplasts were capable of division. Somatic hybrid calli were recovered and, following caulogenesis on MS medium with zeatin and after rooting of regenerated shoots, complete trees were obtained and grown in vivo. Hybridity of these trees was confirmed based on morphological characters, chromosome complement and isozyme analysis. Two separate cloned lines of this intersubfamilial rootstock somatic hybrid (wild pear (+) Colt cherry) were produced. This is the first report of the production of somatic hybrid plants of two woody species, of agronomic value, within the order Rosales.     

Survival and growth of eastern white pine shoot apical meristemsin vitro

Shoot apical meristems of seedling and mature eastern white pine trees were excised and grownin vitro. Placing the meristems on filters instead of directly on agarose-solidified nutrient medium enhanced survival of both juvenile and mature meristems. Applying forcing treatments to mature branches improved survival and growth of dissected meristems compared with meristems from non-forced branches in experiments conducted over two years. No consistent differences were observed among 2-, 4-, and 6-week forcing treatments. Including 5.37 nM (0.001 mg l^-1) l-naphthaleneacetic acid in the culture medium did not affect meristem survival or growth. Some meristems from seedlings grew rapidly, produced primary leaves, underwent internode elongation, and in three cases, produced adventitious roots. Meristems from mature trees did not grow as rapidly as seedling meristems. The leaves produced by mature meristems appeared to be scale leaves and a few of these had brachyblast primordia in the axils. The shoots derived from mature meristems did not produce adventitious roots.     

Effects of propagule type on genetic parameters of wood density and growth in a loblolly pine progeny test at ages 10 and 11?years

Nine full-sib families of loblolly pine (Pinus taeda L.) were produced by a 3 × 3 factorial mating design. Rooted cuttings and seedlings of full-sib families were tested together in two field locations. Twelve-millimeter wood increment cores were collected from 10- and 11-year-old test trees. On each of the two sites, there were six blocks and a split-plot design, with propagule type as the whole plot and family as the sub-plot. In addition to the collection of wood samples, height and diameter of 1,600 trees were measured. No significant differences were found between cuttings and seedlings for wood density and growth traits. Significant family variation was found for growth and wood density. Genetic parameters estimated for wood density and growth traits using seedlings and rooted cuttings showed that individual-tree and family heritability estimates from rooted cuttings were similar to or higher than those from seedlings for all traits. Half-sib breeding values for parents were highly correlated based on seedling and rooted cutting estimates for height (0.95) and wood density (0.99) but not for diameter (0.56), which suggests that wood density and height breeding value estimates from rooted cuttings in clonal progeny tests can be estimated by traditional seedling tests, but not for tree diameter.     

Alcohol induced silencing of gibberellin 20-oxidases in Kalanchoe blossfeldiana

Reduction in the amounts of active gibberellic acids (GA) in elongating cuttings from the ornamental crop Kalanchoe blossfeldiana were pursued by genetic manipulation as an alternative to synthetic growth regulators. An alcohol inducible promoter system was used to control silencing of GA activating enzymes. Apart from affecting the stem length, abnormal levels of GA can lead to altered flowering time, lacking seed maturation and changes in morphology. The effects of down regulating a group of GA 20-oxidases were investigated in fast growing cuttings of K. blossfeldiana Poelln. cv. Molly. The transgenic plants were phenotypically indistinguishable from wild type plants until silencing was induced by low concentrations of ethanol. Treated plants were reduced in height but otherwise appeared normal; flowering was delayed but with large variations in time between the transgenic lines. These data indicate that optimisation of the ethanol treatments can enable us to produce more compact growing plants still maintaining normal flowering.