Seasonal variation of organogenetic activity and reserves allocation in the shoot apex of Pinus pinaster Ait

BACKGROUND AND AIMS: To understand better the basic growth characteristics of pines and the fundamental properties of the shoot apical meristem (SAM), variations within the shoot apex of buds were studied. METHODS: A detailed structural comparison of meristem dimensions, organogenetic activity, and the presence of lipids, starch grains and tannins was performed on shoot apices of juvenile, and male and female adult Pinus pinaster at five different times in the annual growth cycle. KEY RESULTS: There were significant correlations among traits and differences in the pattern for juvenile and adult shoots. In juvenile shoots, peaks of organogenesis were present in spring and autumn, but not in summer. In adult shoots, one peak, characterized by an increase in meristem dimensions, was present in summer. The accumulation of starch grains beneath the SAM and of tannin in sub-apical pith parenchyma were at their maximum when organogenetic activity was high in spring and autumn in juvenile plants, and in summer in adult plants. In juvenile and adult plants, lipids were stored within the SAM in autumn, filling a large part of the bud in winter, and were depleted in the cortical parenchyma and then in the pith during shoot elongation. CONCLUSIONS: Depending on the sites of accumulation within the SAM and on the stage of the annual growth cycle, lipids, starch and tannins may be involved in different processes. In spring, energy and structural materials released by lipid hydrolysis may contribute to stem elongation and/or cell-to-cell communication. During organogenesis, energy and structural materials released by starch hydrolysis may influence developmental programmes in the SAM and adjacent cells. Tannins may be involved in cellular detoxification. At the end of the growing season, accumulation of lipid and starch is positively correlated with the onset of dormancy.     

Purification and properties of peroxidase from Pinus pinaster needles

Peroxidase (Ec 1.11.1.7) was purified from needles of Pinus pinaster to apparent homogeneity by DE-52 cellulose chromatography with a final recovery of enzyme activity of about 85%. The purified enzyme (A402/A275 = 1.05) had a specific activity of about 948 U/mg of protein and ran as a single protein band both on SDS-PAGE and native PAGE with Mr of 37,000 and 151,000, respectively. Both native PAGE and isoelectric focusing gels of the purified enzyme were stained for activity which coincided with the protein band. The pI of the purified enzyme was found to be 3.2 by isoelectric focusing on an ultrathin polyacrylamide gel. The enzyme has an optimum pH of activity of 5.0 and temperature optimum of 30 degrees C. Stability studies of the enzyme as a function of pH and temperature suggest that it is most stable at pH 5.0 and 0-40 degrees C, respectively.     

Distribution of genetic diversity in Pinus pinaster Ait. as revealed by chloroplast microsatellites

 Using nine chloroplast simple sequence.repeats (cpSSRs) markers, we evaluated haplotypic variation within and among natural populations of Maritime pine (Pinus pinaster Ait.) in order to shed light on the history of this species. Seven out of the nine cpSSRs analysed were polymorphic, giving a total of 24 different variants. The 24 variants combined in 34 different haplotypes. The populations which generally showed the lowest level of haplotypic diversity are those located in Portugal. The Landes (France) and Pantelleria (Italy) populations represent the two main reservoirs of haplotypic diversity. The proportion of genetic differentiation among populations, estimated using R_st, which is a measure based upon a strict stepwise mutation model, was 0.235. The high level of differentiation was also confirmed by the AMOVA analysis (Φ_ST=0.254, P<0.001). Four main groups of populations were identified on the basis of Principal Component Analysis, with the differences being statistically significant (Φ_CT=0.299, P<0.001). Based on our results the presence of refugia located in the South of Portugal, previously proposed for this species, may be excluded, and a different possible recolonization process of Maritime pine in the post-glacial period has been proposed. Populations from North Africa and France might have represented a starting point of the recolonization process of Portugal and of the Italian part of the natural range, respectively. This hypothesis seems to be confirmed by the analysis of the distribution of the pairwise differences among individuals within populations: Landes and Pantelleria populations showed a bimodal distribution, as would be expected for ancient gene pools. Received: 5 November 1997 / Accepted: 5 January 1998     

In vitro adventitious budding on Pinus pinaster cotyledons and needles

Adventitious budding can be induced on two types of Pinus pinaster Ait. organs. Cotyledons (10-mm-long), derived from 8 to 10-day-old seedlings, show morpho-genetic response when an appropriate mineral solution is used (NH₄⁺/K⁺= 1). Of the various cytokinin concentrations added to this optimal mineral medium, 0.8 μM BAP (6-benzylaminopurine), with 5 n M NAA (1-naphtaleneacetic acid), promoted organogenesis best. Buds were induced from outer mesophyll layers.
Short shoots and the elongating needles (70-mm-long) were collected from cuttings of a mature tree (10-years-old). These cuttings benefited from physiological advantages of a well-developed root system (by heating the substrate). In order to stimulate in vitro organogenesis, they had been sprayed every week from March to May with 10 μ M BAP.
When cultivated in the presence of 10 μ M BAP and 25 n M NAA, 79% of the explants produced buds from dome-shaped meristematic cell clusters that pre-existed at the top of the short shoots. Moreover, among these, 42% gave rise to adventitious buds induced from proliferating mesophyll cells at the needle base. The morphologies of the two kinds of shoots were similar. Adventitious budding on these two different explants should allow vegetative multiplication of selected seedlings and elite trees.     

Genetic control of stem form in Pinus pinaster Ait. seedlings exposed to lateral light

 In order to compare family differences in stem form determination, seedlings of eight half-sib progenies of Pinus pinaster were exposed to three different light treatments: fixed lateral light (T1), alternate lateral light (T2), and direct sunlight (T3). From spatial coordinates of several points along the stem we computed four variables: global leaning (GL), apical leaning (APL), flexuosity index (FL) and length of the stem (LEN). Based on comparisons of the four variables, the phototropic stimulus caused different responses in stem form at the familial level. Treatment had an important effect on stem form, although the familial response to treatments was quite variable. Heritability values (h²) of the four analyzed variables showed a different pattern of variation for the three treatments, being very stable for LEN and FL, whereas h² of APL was much greater based on T1 and T2 compared to T3, and h² of GL based on T2 was much greater compared to T3 and T1. Differences in stem form were due not only to photomorphogenetic response to different light stimuli, but also to the straightening processes such as compression wood formation. The results suggest that lateral light treatments may be useful in revealing differences in efficiency of the mechanisms involved in stem form determination. Received: 1 June 1995 / Accepted: 1 April 1997     

Range-wide phylogeography and gene zones in Pinus pinaster Ait. revealed by chloroplast microsatellite markers

Some 1339 trees from 48 Pinus pinaster stands were characterized by five chloroplast microsatellites, detecting a total of 103 distinct haplotypes. Frequencies for the 16 most abundant haplotypes (p(k) > 0.01) were spatially interpolated over a lattice made by 430 grid points. Fitting of spatially interpolated values on raw haplotype frequencies at the same geographical location was tested by regression analysis. A range-wide 'diversity map' based on interpolated haplotype frequencies allowed the identification of one 'hotspot' of diversity in central and southeastern Spain, and two areas of low haplotypic diversity located in the western Iberian peninsula and Morocco. Principal component analysis (PCA) carried out on haplotypes frequency surfaces allowed the construction of a colour-based 'synthetic' map of the first three PC components, enabling the detection of the main range-scale genetic trends and the identification of three main 'gene pools' for the species: (i) a 'southeastern' gene pool, including southeastern France, Italy, Corsica, Sardinia, Pantelleria and northern Africa; (ii) an 'Atlantic' gene pool, including all the western areas of the Iberian peninsula; and (iii) a 'central' gene pool, located in southeastern Spain. Multivariate and AMOVA analyses carried out on interpolated grid point frequency values revealed the existence of eight major clusters ('gene zones'), whose genetic relationships were related with the history of the species. In addition, demographic models showed more ancient expansions in the eastern and southern ranges of maritime pine probably associated to early postglacial recolonization. The delineation of the gene zones provides a baseline for designing conservation areas in this key Mediterranean pine.     

Variation in chloroplast single-sequence repeats in Portuguese maritime pine (Pinus pinaster Ait.)

Genetic variation in 12 Pinus pinaster (maritime pine) populations spanning most of the distribution range of the species in Portugal was evaluated using six polymorphic chloroplast microsatellite (cpSSR) loci. Thirty-two haplotypes were found. There were indications of very weak differentiation among populations (Weir’s θcoefficient, 0.023), and the R _ST value, derived from the stepwise mutation model (SMM), was not significantly different from zero. The pattern, in which similarities in allele size, in base pairs, do not contribute to the genetic structure, may be due to the recent mixing of genetic material from different stands through plantations. Overall, a high level of haplotypic variation within populations was detected. Using the SMM estimator (mean genetic distance of individuals within populations, D ² _sh –––) we divided the populations into two groups, with above and below average values. The first group contained 5 populations, mainly from the central part of the country, which possess, in general, high levels of haplotypic diversity. Among them, 2 populations were divergent from the others based on the pair-wise Nei’s distance. The results indicate that there is no discernible geographic genetic pattern for the Portuguese populations of P. pinaster investigated. The history of expansion of the species range in Portugal during the twentieth century (mainly due to human activity) and extensive gene flow among populations associated with the expansion could explain this finding. Received: 15 February 2000 / Accepted: 14 April 2000     

Clonal variation for shoot ontogenetic heteroblasty in maritime pine (Pinus pinaster Ait.)

Pine seedling shoots undergo sharp heteroblastic changes during the early ontogenetic stages. The rate of these changes has been seen to vary between species and provenances within species, but there is a marked lack of information about its genetic control at the lower hierarchical levels. We used clonal replicates of maritime pine to determine broad-sense heritability of shoot ontogenetic heteroblasty and its correlation to rooting ability. We applied a simple ontogenetic index based on the proportion of basal nodes with secondary needles in rooted cuttings of 15 clones from 9 environmentally contrasting origins. We found a high clonal heritability for shoot ontogenetic index and a moderately high heritability for rooting ability, but both genetic and phenotypic correlations between these two traits were weak and non-significant. These results indicate that both developmental phenomena are genetically controlled, but not strictly associated in this species.     

An estimation of the genome length of maritime pine (Pinus pinaster Ait.)

The genome length, in units of Morgans or centimorgans, is a fundamental feature of a species. It can be calculated from a complete linkage map. However, the genome size can be estimated with partial linkage data. Using linkage data obtained by the analysis of a two-dimensional electrophoresis of the proteins contained in an haploid organ, the megagametophyte, we suggest an estimation and a confidence interval of the genome length of a gymnosperm, the maritime pine (Pinus pinaster Ait.). The results indicate an important gap between the physical and the genetic maps.     

The essential oils of two chemotypes of Pinus sylvestris

The essential oils of Pinus sylvestris from French Massif Central are described. Forty-six compounds have been identified by spectral methods. Two chemotypes are recognized.     

Simplified and improved somatic embryogenesis for clonal propagation of Pinus pinaster (Ait.)

In this study, several improvements and simplifications of SE protocols in Pinus pinaster (Ait.), a species of economic importance in the regions of Western Europe, are described. These improvements pertained to all stages of SE including high initiation frequencies in eight control pollinated seed families, relatively high somatic embryo maturation yield when cells were coated with particles of activated charcoal and a rapid production of plants directly in a shade house. The SE initiation frequency from isolated zygotic embryos was high (up to 100%) and plants were produced from 11 embryogenic lines representing all crosses. Based on these results, the estimated number of somatic embryos required to produce 1,000 plants varied from slightly more than the required number of plants to more than double this number depending on the line. Such an estimate is critical in developing plant production strategy when a number of embryogenic lines are considered for production of clonal plants.     

Physiological aspects underlying the improved outplanting performance of Pinus pinaster Ait. seedlings associated with ectomycorrhizal inoculation

Mycorrhizal inoculation of conifer roots is a key strategy to optimize establishment and performance of forest tree species under both natural and cultivated conditions and also to mitigate transplantation shock. However, despite being a common practice, inoculation in outdoor nursery conditions has been poorly studied. Here, we have evaluated effectiveness of four fungal species (Lactarius deliciosus, Lactarius quieticolor, Pisolithus arhizus, and Suillus luteus) in the production of mycorrhizal Pinus pinaster seedlings in an outdoor commercial nursery and their ability to improve seedling physiology and field performance. All inoculated seedlings showed a significant increase in growth at the end of the nursery stage and these differences remained after 3 years of growth in the field. Differences observed in the content of malondialdehyde, total chlorophyll, carotenoids, anthocyanins, and phenolic compounds from needles of mycorrhizal and control seedlings may reflect a different sensitivity to photo-oxidative damage. We conclude that ectomycorrhizal inoculation improves adaptability to changeable growing conditions of an outdoor nursery and produces a higher quality nursery stock, thereby enhancing seedling performance after planting.     

Genetic variation and migration pathways of maritime pine (Pinus pinaster Ait) in the Iberian peninsula

The genetic variability and migration pathways of Pinus pinaster after glaciation in the Iberian peninsula was studied by means of 18 loci from 12 natural populations of the species. The analysis showed the existence of three groups of populations with different levels of diversity and patterns of recolonization. The southern Iberian group displays a high level of diversity, with a stepping-stone model of variation. The presence of rare alleles in this group and their position in the phylogenetic tree suggest the existence of refugia during glaciations in this zone. The eastern Iberian group also has high levels of diversity but is clearly separate from the first group based on their genetic distances. The Atlantic group displays a low level of diversity that could be interpreted as a rapid recolonization of the entire area by the Eastern group that has not yet developed to a divergence in this area. The southern Iberian range is indicated to be the dispersal centre of the species after the last glaciation. Received: 15 February 1999 / Accepted: 30 April 1999     

Use of molecular markers for estimating breeding parameters: a case study in a Pinus pinaster Ait. progeny trial

The management of a genetic improvement program is based on the knowledge of the genetic parameters and their relationships to determine the genetic gains. Knowledge of the coefficient of coancestry (θ) is a requirement for efficient progeny testing scheme and for estimating additive variance components for any quantitative trait. When using open-pollinated families, most authors assume that the seedlings are related as half-sibs, but this is not always true. Our aim was to estimate a mean value of the coancestry coefficient of the families present in a maritime pine Pinus pinaster Ait. (maritime or cluster pine) progeny trial originating from seed collected in a clonal seed orchard and to study how deviations from the standard assumption of θ = 0.125 affect heritability estimations. Five highly polymorphic microsatellite markers were scored in 125 offspring from a subsample of five families from the progeny trial. The mean value of the coancestry coefficient of the families present in this progeny trial was 0.130. Differences between the unadjusted and adjusted heritability estimates were more pronounced in wood density (0.609 and 0.586, respectively) than in diameter (0.166 and 0.154, respectively). We conclude that in the trial, the associated error in heritability estimates due to the inclusion of full-sibs, when assuming a standard coefficient of relationship among open-pollinated sibs of 0.250, was low and that this result is robust with respect to the number of families sampled, given unbiased estimates of average relationship among offspring within sib families.