Selection of haploid cell lines from megagametophyte cultures of maritime pine as a DNA source for massive sequencing of the species

The potential of haploid tissues for genetic studies in conifers is hampered by the lack of abundant and homogeneous plant material suitable for DNA isolation. In this work we have determined factors promoting haploid callus induction and proliferation from megagametophytes of Oria 6, a genotype of Pinus pinaster Aiton (maritime pine) from the natural population Sierra de Oria (Almería, Spain), selected based on its response to extreme drought conditions. The generation of haploid callus was restricted to megagametophytes isolated from light-brown cones with no dehydrated seeds collected in September. Culture medium composition did not significantly affect callus induction, but a modified Murashige and Skoog medium with 2,4-dichlorophenoxyacetic acid and 6-benzyladenine favored further multiplication. The ploidy status of the callus lines was determined by flow cytometry and seven polymorphic microsatellites. A total of sixteen haploid callus lines were established and one of these is being used as a source of DNA for massive sequencing of maritime pine genome.     

Improvement of nutrient medium for growth and embryogenesis of megagametophyte and embryo callus lines of Picea abies

New macro and microelement solutions were formulated for stimulation of growth and embryogenesis in white and chlorophyllous megagametophyte and embryo callus lines of Picea abies (L.) Karst. Macroelement media with different NH₄⁺ and NO₃⁻ ratios (1:2 and 1:4), the increased level of several microelements and the effect of organic nitrogen (100 mg 1⁻¹ casein hydrolysate, 0.25 m M arginine and 0.5 m M glutamine) were tested with 4 combinations of growth regulators (2,4-dichlorophe-noxyacefic acid or indole-3-butyric acid, kinetin). Green chlorophyll-containing, in contrast to white callus lines, grew quite well without exogeneously added organic nitrogen. The ratio between NH₄⁺ and NO₃⁻ was not significant. The increased levels (μ M ) of several microelements: B (200), Zn (50), I (25), Cu (1), Co (0.5) and in addition Ni (0.1) improved callus growth in some lines more than 50% (DW) compared to cultures grown on the micronutrients of Murashige and Skoog. The response of different callus lines varied with the combinations of growth regulators. Embryogenesis did not occur in chlorophyllous callus lines in any of the 24 media combinations tested, but some very good media could be found for white megagametophyte and embryo callus lines. Microelements, favourable combination of growth regulators and organic nitrogen were especially important. A megagametophyte callus subcultured for 4 years was also able to form numerous proembryos.     

An efficient procedure for embryogenic callus induction and double haploid plant regeneration through anther culture of Thai aromatic rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L. subsp. <Emphasis Type="Italic">indica</Emphasis>)

Anther culture is a biotechnology technique that can be used for the production of pure lines. The aims of this investigation were to induce embryogenic callus from major and minor culms of Thai aromatic rice cultivars and to subsequently regenerate double-haploid green plantlets by the application of exogenous polyamines. Embryogenic callus derived from anther culture was successfully induced in varieties KDML105, Homjan (HJ), and Pathumthani 1 (PT1). Production of embryogenic callus from anthers collected from the major culms was greater than those collected from the minor culms, especially in cultivar HJ. Plantlet regeneration in the three rice cultivars was observed from embryogenic callus and was highest, at 12.1%, from variety HJ treated with 0.5 mM spermidine. Plantlet regeneration from anther-derived embryogenic callus was dependent on the plant genotype, the types of exogenous polyamines, and the interactions of these factors. The percentage of haploid plantlets regenerated in PT1, KDML105, and HJ were 68.1%, 70.7%, and 78.5%, respectively. Only haploid plantlets were treated with colchicine for double-haploid production. This investigation has increased the knowledge of both embryogenic callus induction and plantlet regeneration in aromatic rice and has lead to the development of a pure, double-haploid line for the use in rice breeding programs in Thailand.     

Cytokinin autotrophy and differentiation in tissue cultures of haploid Nicotiana tabacum L.

Explants from the apical region (10 cm from the tip) of haploid Nicotiana tabacum cv. Wisconsin-38 were cultured on media with and without kinetin. Cell lines were selected in the dark and in the light. Cytokinins were extracted from the apical region of haploid plants and from callus tissues after 84 days of growth (third transfer culture). Chlorophyll was extracted from callus grown under light after 21 days of growth at each of the four cell line selection steps. Kinetin (+) cell lines and cytokinin autotrophic tissues grown in the light showed a compact growth pattern. Microscopic examination of these callus showed the presence of large numbers of nodules consisting of tracheary elements, parenchymatic cells, sieve elements and meristematic cells. Cytokinin-autotrophic callus grown in the dark showed an irregular growth pattern presenting regions of compact tissue and friable tissue. The compact tissue contained large amounts of nodules similar to those of kinetin (+) tissues and of cytokinin autotrophic tissues grown in the light. Extraction of the compact and the friable callus components showed high cytokinin activity in the compact region and low activity in the friable portion. It is suggested that cytokinin synthesis is related to the differentiation of the nodular structures. The amount of chlorophyll increased during the process of cytokinin autotrophic cell line selection.     

Diploidization in megagametophyte-derived cultures of the gymnosperm Larix decidua

Tested haploid embryogenic lines (n=12) of Larix dedicua Mill, initiated from megagametophyte tissue were maintained on half-strength LM medium without growth regulators. The cultures were analyzed for ploidy level after 1–9 years. All lines tested were found to have doubled (2n=24) their chromosome number at the end of the experiment, though there were a few lines that still gave occasional haploid counts. Flow cytometric data of embryogenic tissue confirmed these results. Protoplasts were stained in ethidium bromide, and cultured human leucocytes and chicken erythrocytes were used as internal standards. Haploid megagametophytes from immature seeds of L. decidua and known diploid culture lines of a related hybrid (L. x eurolepis) were also analyzed by flow cytometry. Haploid reference material had 12.3–13.6 pg DNA per cell, whereas formerly haploid callus lines had an average of 25.0 pg DNA per cell. The one exception was a known, genetically unstable line of L. decidua (34.8 pg DNA per cell). The diploid cell line of L. x eurolepis had 27.6 pg DNA per cell. The results show that spontaneous diploidization of megagametophyte lines is relatively rapid and that both haploid and dihaploid lines are embryogenic in larch.     

An improved system to establish highly embryogenic haploid cell and protoplast cultures from pollen calluses of maize (Zea mays L.)

Regenerable, embryogenic haploid cell suspensions were initiated and established from type II pollen calluses of two selected Chinese maize genotypes (No 592 Y and 592.A2 LY). The induction frequency of friable, embryogenic callus (type II) was highly dependent on three factors: genotype, medium, cold pretreatment, and on their interactions. Repeated callus and cell selection during the culture procedure led to stable haploid suspensions consisting of fine clusters each containing 20–50 cells. The selected cell lines were able to maintain their morphogenic ability during long-term subculture (2 years). Protoplasts were successfully isolated from subcultured, friable, embryogenic pollen calluses and cultured on N₆BM and N₆K media using a feeder layer, obtained from 2-day-old suspension culture. Healthy plants were regenerated from protoplast-derived calluses.     

Cono- and seminiphagous insects of Norway spruce Picea abies (L.) Karst. and their parasitoids in lower and upper montane zone of the Tatra National Park in Poland

Abstract: A study of the insects inhabiting of cones of Norway spruce Picea abies (L.) Karst. in the Tatra National Park was conducted during 1987–92. As a result of breeding and analyses of the cones collected from trees at various altitudes above sea level, 53 746 specimens of cono- and seminiphagous insects were collected. Interior damage to cones and the losses of spruce seeds caused by pests were measured, with regard both to the variable cone crop abundance of spruce and to different altitudes above sea level. The role of parasitoids in limiting the numbers of some cono- and seminiphagous insects was also estimated.     

Analysis of quantitative trait loci which contribute to anther culturability in rice (Oryza sativa L.)

Anther culturability of rice is significantly different between indica and japonica varieties. A doubled haploid (DH) population was established via anther culture of an indica/japonica hybrid on SK₃ medium, which had been shown particularly suitable for anther culture of indica/japonica hybrids. For analyzing the quantitative trait loci (QTLs) responsible for anther culturability, anthers of the DH lines were again cultured with SK₃ medium and parameters for four traits representing the anther culturability were surveyed and analyzed with the molecular map constructed from the same DH population. The parameters for four major traits were as follows: callus induction frequency (CI), green plantlet differentiation frequency (GPD), albino plantlet differentiation frequency (APD), and green plantlet yield frequency (GPY). All four traits displayed continuous distributions among the DH lines. The correlation coefficients between these traits were also tested and showed that there was no relationship between callus induction and green plantlet differentiation frequencies, but both showed strong positive correlation with the frequency of green plantlet yield. For callus induction frequency, five QTLs were identified on chromosomes 6, 7, 8, 10 and 12. Two QTLs for green plantlet differentiation frequency were located on chromosomes 1 and 9. There was a major QTL for albino plantlet differentiation frequency on chromosome 9. No independent QTL was found for green plantlet yield frequency. The results may be useful in the selection of parents with high response to anther culture for rice haploid breeding and in the establishment of permanent DH populations for molecular mapping.     

Chronic environmental pollution alters adenylate levels in needles and fine roots of Scots pine

Contents of ATP, ADP, AMP, inorganic phosphate, and values of ATP/ADP ratio, adenylate energy charge (AEC), phosphorylation potential (PP) and adenylate kinase activity were analysed in needles and fine roots of Scots pine trees grown at the polluted and control (free of acute air pollution) site. Also chemical properties of the soil and mineral elements in needles from both sites were analysed. In comparison with the control, developing needles from the polluted site contained less ATP, the same amount of ADP and more AMP, and had lower values of ATP/ADP, AEC and PP. In one-year-old needles from the polluted site no change or a decrease in ATP was recorded, while ADP decreased, AMP increased, AEC did not change, and ATP/ADP ratio and PP were higher. In fine roots from the polluted site AMP level was higher, while ATP, ADP, ATP/ADP ratio, PP and AEC were lower than in the control.     

The effect of AOA on ethylene and polyamine metabolism during early phases of somatic embryogenesis in Medicago sativa

Changes in the levels of ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC), 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC) and polyamines were simultaneously investigated during the early phases of alfalfa somatic embryogenesis. These included the period of induction and subculture of callus, and 3- and 7-day suspension cultures for the induction of somatic embryogenesis. The polyamines contained in the embryogenic callus were found to include putrescine (Put), spermidine (Spd) and spermine (Spm), but the level of Spm was much less than that of Put and Spd. There was a dramatic increase in MACC after induction of embryogenesis, and ACC levels were lower in somatic embryos than in embryogenic callus. Induction of embryogenesis for 3 days increased the levels of ACC and polyamines to a maximum level, and these then reduced as the embryogenesis proceeded. The ratios of Put/Spd and ACC/MACC were decreased during the induction. This indicated that both high levels of ACC and polyamines might be a prerequisite for early differentiation during the induction of the embryogenesis. Thus, there appears not to be competition between polyamine biosynthesis and ethylene biosynthesis at least during the induction of somatic embryogenesis, because both the polyamines and ACC were simultaneously increased during the induction period. Conversion of ACC into MACC and the maintenance of a relatively high level of polyamines, especially Spd, appear to be important for further development of the embryos.
When aminooxylvinylglycine (AOA) was added at the initiation of the callus subculture, it had no significant effect on the callus growth, the ethylene production and ACC level of the callus. However, AOA increased the numbers of the embryos accompanying an increase in Spd level and S-adenosylmethionine decarboxylase (SAMDC) activity. Thus, the AOA effect could be associated with Spd increase rather than with the effect of ethylene biosynthesis.     

水稻花药培养力的遗传分析及基因定位

在栽培稻的籼粳亚种间,花药培养力存在显著差异,这一差异主要是由遗传因素引起的。以适合籼粳稻杂种花药培养的SK_3培养基,经花药培养获得了一个籼粳交F_1代的加倍单倍体(DH)群体,对该群体的110个株系用同一种培养基进行花药培养,利用该群体构建的分子图谱进行有关水稻花药培养力的数量性状基因座位(QTLs)的分析。结果表明,与水稻花药培养力有关的4个性状在DH群体中均表现为连续分布,愈伤组织诱导率与绿苗分化率之间不存在相关性,而绿苗产率与愈伤组织诱导率和绿苗分化率均显著相关。在第6、7、8、10和12 5条染色体上分别检测到与愈伤组织诱导率有关的5个QTLs,其加性效应均为正。在第1和第9染色体上检测到与绿苗分化率有关的2个QTLs,这两个性状间的QTs不存在连锁。在第9染色体上有一个主效基因与白苗分化率有关,对绿苗产率则没有检测到特有的QTL。     

Nitrogen distribution in young Norway spruce (Picea abies) trees as affected by pedospheric nitrogen supply

In numerous locations in Europe spruce trees are exposed to high loads of nitrogen. The present study was performed to characterize the distribution of nitrogen compounds under these conditions. For this purpose Norway spruce ( Picea abies [L.] Karst.) trees were cultivated under close-to-natural conditions of a forest understory in soil from an apparently nitrogen-limited field site in the Black Forest either with, or without supplementation of nitrogen as ammonium nitrate. After 11 and 20 months, growth, total nitrogen contents of the biomass, and total soluble non-proteinogenic nitrogen compounds (TSNN, i.e. nitrate, ammonium, soluble proteinogenic and non-proteinogenic amino compounds) in needles, xylem sap and phloem exudate were analysed. After 20 months of growth, N-fertilization had slightly enhanced the biomass of current-, but not of 1-year-old shoots. At both harvests, total N-content of 1-year-old needles was increased by N-fertilization, whereas current-year needles were not significantly affected. By contrast, TSNN was elevated by N-fertilization in both current-year and 1-year-old needles. The increase in TSNN was mainly attributed to an accumulation of arginine. Xylem sap analysis showed that the increase in TSNN of the needles was a consequence of enhanced nitrogen assimilation of the roots rather than the shoot. Since also TSNN in phloem exudates was enhanced, it appears that N-fertilization elevates the cycling pool of amino compounds in young Norway spruce trees. However, this pool seems to be subject to metabolic interconversion, since mainly glutamine and aspartate are transported in the xylem from the roots to the shoot, but arginine accumulated in the needles and the phloem.     

Differences in growth characteristics and dynamics of elements absorbed in seedlings of three spruce species raised on serpentine soil in northern Japan

BACKGROUND AND AIMS: Serpentine soils are characterized by the presence of heavy metals (Ni and Cr) and excess Mg; these elements often suppress plant growth. Picea glehnii is nevertheless distributed widely on serpentine soils in northern Japan. Growth characteristics were compared among P. glehnii, Picea jezoensis (distributed in the same region) and Picea abies (planted for timber production), and concentrations of elements in various tissues over time and the amount of ectomycorrhizal infection in short roots were evaluated. METHODS: Seedlings of three spruce species were planted in two types of experimental plots, comprising serpentine soil and brown forest (non-serpentine) soil, and these seedlings were grown for 3 years. Growth, ectomycorrhizal infection of short roots, and elemental composition of tissues were examined. KEY RESULTS: The total dry mass of P. glehnii planted on serpentine soil was almost the same as on brown forest soil, and a large number of needles survived to reach later age classes. By contrast, growth of P. jezoensis and P. abies in serpentine soil was significantly less than in brown forest soil, and needle shedding was accelerated. Moreover, roots of seedlings of P. glehnii on serpentine soil were highly infected with ectomycorrhiza, and the concentration of Ni in needles and roots of P. glehnii was the lowest of the three species. CONCLUSIONS: Picea glehnii has a high ability to maintain a low concentration of Ni, and the ectomycorrhizal infection may have the positive effect of excluding Ni. As a result, P. glehnii is more tolerant than the other spruce species to serpentine soil conditions.     

Protein polymorphism between 2 Picea abies populations revealed by 2-dimensional gel electrophoresis and tandem mass spectrometry

In species with high gene flow and consequent low interpopulation differentiation over wide geographic ranges, differential gene expression along ecological gradients often reveals adaptive significance. We investigated potential differences in protein expression between Picea abies ecotypes adapted to contrasting altitude conditions. Protein expression patterns were compared between needles and roots of 2-month-old P. abies seedlings by means of 2-dimensional electrophoresis. Proteins exhibiting differential expression between the 2 ecotypes were analyzed by tandem mass spectrometry. A total of 19 proteins exhibited qualitative or quantitative polymorphism between the 2 populations. These proteins exhibited organ-specific expression, and the level of interpopulation protein polymorphism was organ dependent. Among differentially expressed proteins, we identified proteins involved in photosynthesis, photorespiration, root tracheary element differentiation, and transmitochondrial membrane transport. Our results show that P. abies seedlings from locally adapted ecotypes exhibit consistent differences in protein expression. The expression polymorphism of some of these proteins has potential adaptive significance.     

Selection of stable mutants from cultured rice anthers treated with ethyl methane sulfonic acid

To increase the frequency of stable mutants from cultured anthers of rice, the effects of EMS treatment on callus induction, plant regeneration and mutant induction were investigated according to the timing of treatment after anther inoculation on the medium. The frequency of callus induction was highest in anthers treated with 0.5% EMS 10 days after culture. Anthers treated directly at the initiation of culture exhibited a very low callus induction level, and the such calluses exhibited a poor plant regeneration capacity. The frequency of regeneration of green plants was significantly decreased by EMS treatments immediately after anther inoculation as compared with control. The frequencies of stable mutants were 20.7% and 12.0% in EMS treatments at 10 and 20 days, but unstable mutants were 43.1% and 52.6%, respectively. A total of 14 stable mutants, semidwarf mutants (4 lines), grain-shape mutants (2 lines) and glabrous mutants (8 lines) were selected from doubled haploid lines of the A₂ generation. The frequencies of callus induction, green plant regeneration and stable mutants were maximal in anthers treated with 0.5% EMS 10 days after culture.     

Ploidy stability of maize anther culture-derived callus lines

Summary Ploidy levels of 26Zea mays L. anther culture-derived callus lines of the F1 hybrids (H99 × Pa91, Pa91 × FR16, and H99 × FR16) were determined at various times after culture initiation using flow cytometry (for 21 lines) or chromosome counting of callus cells or regenerated plants (for the remaining 5 lines). Twenty of the lines remained haploid, whereas 6 were diploid. The results from flow cytometry, after examining the DNA content of 5000 nuclei of each callus line, show that each callus line consisted of homogenous haploid or diploid cells. Thus for diploid callus lines, spontaneous chromosome doubling must have occurred before or in the early stages of androgenesis, before the initiation of callus cultures. These long-term callus cultures (growing for up to 38 mo.) have stably maintained their ploidy levels so it is unlikely that the culture conditions have caused chromosome doubling. The restriction fragment length polymorphism pattern obtained with 52 to 58 markers for each diploid callus line shows that all the diploid lines are homozygous diploid so each originated from a microspore and not from diploid maternal F1 hybrid tissue.     

The early ontogeny of embryoids and callus from pollen and subsequent organogenesis in anther cultures of Datura metel and rice

Summary Haploidy induction through anther culture has been examined in Datura metel and rice with a view to tracing the precise sequence of development of the pollen, either directly or through an intervening callus, into an embryo and seedling. In D. metel, the vegetative cell of the young pollen grain assumes the major role in formation of embryos whereas the generative cell and its few derivatives degenerate. Embryos and seedlings arising directly from pollen without an intervening callus phase always proved to be haploids, whereas those differentiating from pollen-derived callus gave haploid, diploid and even triploid plants. Cytological analysis of callus tissue showed cells of various ploidy levels ranging from haploid to triploid, and in rare instances even with higher chromosome numbers.In rice anther cultures the embryoids arose from an initial callus phase. Of 15 different rice cultivars tried, only four produced a callus, and in only one, was there differentiation of plants, both haploid and diploid ones. Among other species tried, egg plant has also yielded plantlets through a callus phase whereas only callus production has been achieved in jute, tea and petunia. No response has been obtained in wheat, maize, cotton and coconut.Coconut milk (CM) appears to be the most important component of the medium for the initial induction of embryoids and callus in anther cultures of most of the species tried. However, further growth and differentiation of plants may require a simpler medium; in D. metel, continued culture on CM led to dedifferntiation.Dedicated to the memory of the late Dr. J. P. Nitsch.     

Effects of age and site quality on the distribution of biomass in Scots pine (Pinus sylvestris L.)

The distribution of the above-ground and below-ground biomass of Scots pine in southern Finland were investigated in trees of different ages (18–212 years) from two types of growth site. Secondly, some structural regularities were tested for their independence of age and growth site. Trees were sampled from dominant trees which could be expected to have a comparable position in stands of all ages. All stands were on sorted sediments. The biomass of the sample trees (18 trees) was divided into needles, branch sapwood and heartwood, stem sapwood and heartwood, stem bark, stump, large roots (diameter >20 cm), coarse roots (five classes) and fine roots. The amount of sapwood and heartwood was also estimated from the below-ground compartments. Trees on both types of growth site followed the same pattern of development of the relative shares of biomass compartments, although the growth rates were faster on the more fertile site. The relative amount of sapwood peaked after canopy closure, coinciding with the start of considerable heartwood accumulation. The relative amount of needles and fine roots decreased with age. The same was true of branches but to a lesser degree. The relative share of the below-ground section was independent of tree age. Foliage biomass and sapwood cross-sectional area were linearly correlated, but there were differences between the growth sites. Needle biomass was linearly correlated with crown surface area. The fine root to foliage biomass ratio showed an increasing trend with tree age.     

The role of plant hormones in higher plant cellular differentiation. II. Experiments with the vascular cambium, and sclereid and tracheid differentiation in the pine, Pinus contorta

In sterile-cultured explants of stems of the pine Pinus contorta Dougl., fusiform cambial cells differentiated entirely into axial parenchyma cells when exogenous indol-3yl-acetic acid (IAA) was omitted. The normal appearance of the cambial zone was maintained when IAA was included in the medium. The IAA-maintained stability of cambial structure suggests physiological rather than epigenetic control over vascular cambium structure. IAA was essential for the occurrence of callus growth in stem explants. Callus growth was similar in appearance and extent in winter- and summer-explanted material. Tracheids differentiated in explants only when actively differentiating tracheids were already present at the moment of explanting, suggesting the absence of factors necessary for tracheid differentiation in over-wintering tissues. Sclereid differentiation, which normally does not occur in phloem or xylem development in P. contorta, occurred in callus derived from active cambial explants. The sclereids were identical to sclereids which differentiated in pith of intact stems. The possibility that sclereid and tracheid differentiation may be fundamentally similar types of gene expression is discussed. Growth of P. contorta trees in continuous darkness resulted in extensive compression-wood tracheid differentiation in the upright main stem. Normal-wood tracheids differentiated in similar trees grown in light. More tracheids differentiated in light than in darkness. This apparently is the first report of induction of compression-wood tracheid differentiation in the absence of hormone treatment or tilting of trees. Different types and numbers of tracheids differentiated at different position in two-year-old disbudded defoliated stem cuttings of P. contorta in response to apically supplied IAA. No evidence for new tracheid differentiation was seen in control cuttings; however, the results suggest that neither cambial cell division nor tracheid differentiation were actually initiated by IAA. Directed transport of additional regulatory factors toward areas of high IAA concentration is formulated as a hypothesis to explain these observations. Gibberellic acid, (S)-abscisic acid and IAA inhibited tracheid differentiation when individually supplied to basal ends of P. contorta cuttings predisposed to differentiate new tracheids. Experiments with single intact needles on Pinus cembroides var. monophylla cuttings confirmed a previous interpretation that the mature pine needle, rather than the short-shoot apical meristem at its base, promotes tracheid differentiation in the stem.