Tolerance of wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) to high soil and solution selenium levels

The fertilisation of wheat crops with Se is a cost-effective method of enhancing the concentration of organic Se in grain, in order to increase the Se intake of animals and humans. It is important to avoid phytotoxicity due to over-application of Se. Studies of phytotoxicity of Se in wheat grown in Australia, where rainfall and grain yield are usually relatively low, have not been reported previously, and overseas studies have had varied results. This study used trials conducted in the field, glasshouse and laboratory to assess Se phytotoxicity in wheat. In field trials that used rates of up to 120 g ha^–1Se as selenate, and in pilot trials that used up to 500 g ha^–1 Se soil-applied or up to 330 g ha^–1 Se foliar-applied, with soils of low S concentrations (2–5 mg kg^–1), no Se toxicity symptoms were observed. In pot trials of four weeks duration, the critical tissue level for Se toxicity was around 325 mg kg^–1 DW, a level attained by addition to the growth medium of 2.6 mg kg^–1 Se as selenate. Solution concentrations above 10 mg L^–1 Se inhibited early root growth of wheat in laboratory studies, with greater inhibition by selenite than selenate. For selenite, Se concentrations around 70 mg L^–1 were required to inhibit germination, while for selenate germination % was unaffected by a solution concentration of 150 mg L^–1 Se. Leaf S concentration and content of wheat increased three-fold with the addition of 1 mg kg^–1 Se as selenate to the growth medium. This effect is probably due to the induction of the S deficiency response of the main sulphate transporter. This study found wheat to be more Se-tolerant than did earlier studies of tobacco, soybeans and rice. We conclude that Se phytotoxicity in wheat will not be observed at the range of Se application rates that would be used to increase grain Se for human consumption (4–200 g ha^–1 Se as selenate, which would result in soil and tissue levels well below those seen in the above studies), even when – as is common in Australia – soil S concentration and grain yield are low.     

Molecular genetic improvement of cereals: transgenic wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Only modest progress has been made in the molecular genetic improvement of wheat following the production of the first transgenic plants in 1992, made possible by the development of efficient, long-term regenerable embryogenic cultures derived from immature embryos and use of the biolistics method for the direct delivery of DNA into regenerable cells. Transgenic lines expressing genes that confer resistance to environmentally friendly non-selective herbicides, and pests and pathogens have been produced, in addition to lines with improved bread-making and nutritional qualities; some of these are ready for commercial production. Reduction of losses caused by weeds, pests and pathogens in such plants not only indirectly increases available arable land and fresh water supplies, but also conserves energy and natural resources. Nevertheless, the work carried out thus far can be considered only the beginning, as many difficult tasks lie ahead and much remains to be done. The challenge now is to produce higher-yielding varieties that are more nutritious, and are resistant or tolerant to a wide variety of biotic as well as abiotic stresses (especially drought, salinity, heavy metal toxicity) that currently cause substantial losses in productivity. How well we will meet this challenge for wheat, and indeed for other cereal and non-cereal crops, will depend largely on establishing collaborative partnerships between breeders, molecular biologists, biotechnologists and industry, and on how effectively they make use of the knowledge and insights gained from basic studies in plant biology and genetics, the sequencing of plant/cereal genomes, the discovery of synteny in cereals, and the availability of DNA-based markers and increasingly detailed chromosomal maps.     

Genotyping of somatic hybrids between <Emphasis Type="Italic">Festuca arundinacea</Emphasis> Schreb. and <Emphasis Type="Italic">Triticum aestivum</Emphasis> L.

In order to genotype hybrid genomes of distant asymmetric somatic hybrids, we synthesized hybrid calli and plants via PEG-mediated protoplast fusion between recipient tall fescue (Festuca. arundinacea Schreb.) and donor wheat (Triticum aestivum L.). Seventeen and 25 putative hybrid clones were produced from the fusion combinations I and II, each with the donor wheat protoplast treated by UV light for 30 s and 1 min, respectively. Isozyme and RAPD profiles confirmed that ten hybrid clones were obtained from combination I and 19 from combination II. Out of the 29 hybrids, 12 regenerated hybrid plants with tall fescue phenotype. Composition and methylation-variation of the nuclear and cytoplasmic genomes of some hybrids, either with or without regenerative ability, were compared by genomic in situ hybridization, restriction fragment length polymorphism, and DNA methylation-sensitive amplification polymorphism. Our results indicated that these selected hybrids all contained introgressed nuclear and cytoplasmic DNA as well as obvious methylation variations compared to both parents. However, there were no differences either in nuclear/cytoplasmic DNA or methylation degree between the regenerable and non-regenerable hybrid clones. We conclude that both regeneration complementation and genetic material balance are crucial for hybrid plant regeneration.     

The endophytic fungi from wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

In order to study the species composition of endophytes from wheat healthy plants in Buenos Aires Province (Argentina) and to determine their infection frequencies from leaves, stems, glumes and grains, wheat plants were collected from five cultivars at five growth stages from crop emergence to harvest. A total of 1,750 plant segments (leaves, stems, glumes and grains) were processed from the five wheat cultivars at five growth stages, and 722 isolates of endophytic fungi recovered were identified as 30 fungal genera. Alternaria alternata, Cladosporium herbarum, Epicoccum nigrum, Cryptococcus sp., Rhodotorula rubra, Penicillium sp. and Fusarium graminearum were the fungi that showed the highest colonization frequency (CF%) in all the tissues and organs analysed. The number of taxa isolated was greater in the leaves than those in the other organs analysed.     

A new PCR-based marker on chromosome 4AL for resistance to pre-harvest sprouting in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Pre-harvest sprouting (PHS) is a complex trait controlled by multiple genes with strong interaction between environment and genotype that makes it difficult to select breeding materials by phenotypic assessment. One of the most important genes for pre-harvest sprouting resistance is consistently identified on the long arm of chromosome 4A. The 4AL PHS tolerance gene has therefore been targeted by Australian white-grained wheat breeders. A new robust PCR marker for the PHS QTL on wheat chromosome 4AL based on candidate genes search was developed in this study. The new marker was mapped on 4AL deletion bin 13-0.59-0.66 using 4AL deletion lines derived from Chinese Spring. This marker is located on 4AL between molecular markers Xbarc170 and Xwg622 in the doubled-haploid wheat population Cranbrook × Halberd. It was mapped between molecular markers Xbarc170 and Xgwm269 that have been previously shown to be closely linked to grain dormancy in the doubled haploid wheat population SW95-50213 × Cunningham and was co-located with Xgwm269 in population Janz × AUS1408. This marker offers an additional efficient tool for marker-assisted selection of dormancy for white-grained wheat breeding. Comparative analysis indicated that the wheat chromosome 4AL QTL for seed dormancy and PHS resistance is homologous with the barley QTL on chromosome 5HL controlling seed dormancy and PHS resistance. This marker will facilitate identification of the gene associated with the 4A QTL that controls a major component of grain dormancy and PHS resistance.     

Identification of SNPs and development of functional markers for LMW-GS genes at <Emphasis Type="Italic">Glu-D3</Emphasis> and <Emphasis Type="Italic">Glu-B3</Emphasis> loci in bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Low-molecular-weight glutenin subunits (LMW-GS) have great effect on wheat processing quality, but were numerous and difficult to dissect by SDS-PAGE. The development of functional markers may be the most effective way for a clear discrimination of different LMW-GS genes. In the present study, three different approaches were used to identify SNPs of different genes at Glu-D3 and Glu-B3 loci in bread wheat for the development of six STS markers (3 for Glu-D3 and 3 for Glu-B3 genes) that were validated with distinguished wheat cultivars. Firstly, seven LMW-GS gene sequences ( AY585350, AY585354, AY585355, AY585356, AY585349, AY585351 and AY585353 ) from Aegilops tauschii, the diploid donor of the D-genome of bread wheat, were chosen to design seven pairs of AS-PCR primers for Glu-D3 genes. By amplifying the corresponding genes from five bread wheat cultivars with different Glu-D3 alleles (a, b, c, d and e) and Ae. tauschii, a primer set, S13F2/S13R1, specific to the gene AY585356, was found to be positive to cultivars with alleles Glu-D3c and d. Nevertheless, the other five pairs of primers designed from AY585350, AY585349, AY585353, AY585354 and AY585355, respectively, did not produce specific PCR products to the cultivars tested. Secondly, all the PCR products from the five primer sets without specific characteristics were sequenced and an SNP from the gene AY585350 was detected in the cultivar Hartog, which resulted in the second STS marker S1F1/S1R3 specific to the allelic variant of AY585350. Thirdly, three Glu-D3 sequences (AB062851, AB062865 and AB062872) and three Glu-B3 sequences (AB062852, AB062853 and AB062860) defined by Ikeda et al. (2002) were chosen to query wheat EST and NR databases, and DNA markers were developed based on the putative SNPs among the sequences. Using this approach, four STS markers were developed and validated with 16-19 bread wheat cultivars. The primer set T1F4/T1R1 was also a Glu-D3 gene-specific marker for AB062872, while T2F2/T2R2, T5F3/T5R1 and T13F4/T13R3 were all Glu-B3 gene specific markers for AB062852, BF293671 and AY831800, respectively. The chromosomal locations of the six markers were verified by amplifying the genomic DNA of Ae. tauschii (DD), T. monococcum (AA) and T. turgidum (AABB) entries, as well as Chinese Spring and its group 1 chromosome nulli-tetrasomic lines. The results are useful to discriminate the corresponding Glu-D3 and Glu-B3 genes in wheat breeding programs.     

An integrative genetic linkage map of winter wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

We constructed a genetic linkage map based on a cross between two Swiss winter wheat (Triticum aestivum L.) varieties, Arina and Forno. Two-hundred and forty F₅ single-seed descent (SSD)-derived lines were analysed with 112 restriction fragment length polymorphism (RFLP) anonymous probes, 18 wheat cDNA clones coding for putative stress or defence-related proteins and 179 simple-sequence repeat (SSR) primer-pairs. The 309 markers revealed 396 segregating loci. Linkage analysis defined 27 linkage groups that could all be assigned to chromosomes or chromosome arms. The resulting genetic map comprises 380 loci and spans 3,086 cM with 1,131 cM for the A genome, 920 cM for the B genome and 1,036 cM for the D genome. Seventeen percent of the loci showed a significant (P < 0.05) deviation from a 1:1 ratio, most of them in favour of the Arina alleles. This map enabled the mapping of QTLs for resistance against several fungal diseases such as Stagonospora glume blotch, leaf rust and Fusarium head blight. It will also be very useful for wheat genetic mapping, as it combines RFLP and SSR markers that were previously located on separate maps. S. Paillard and T. Schnurbusch contributed equally to the work     

Quantitative structure analysis of genetic diversity among spring bread wheats (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) from different geographical regions

Genetic diversity in spring bread wheat (T.␣aestivum L.) was studied in a total of 69 accessions. For this purpose, 52 microsatellite (SSR) markers were used and a total of 406 alleles were detected, of which 182 (44.8%) occurred at a frequency of <5% (rare alleles). The number of alleles per locus ranged from 2 to 14 with an average of 7.81. The largest number of alleles per locus occurred in the B genome (8.65) as␣compared to the A (8.43) and D (5.93) genomes, respectively. The polymorphism index content (PIC) value varied from 0.24 to 0.89 with an average of 0.68. The highest PIC for all accessions was found in the B␣genome (0.71) as compared to the A (0.68) and D␣genomes (0.63). Genetic distance-based method (standard UPGMA clustering) and a model-based method (structure analysis) were used for cluster analysis. The two methods led to analogical results. Analysis of molecular variance (AMOVA) showed that 80.6% of the total variation could be explained by the variance within the geographical groups. In comparison to the diversity detected for all accessions (H _e = 0.68), genetic diversity among European spring bread wheats was H _e = 0.65. A comparatively higher diversity was observed between wheat varieties from Southern European countries (Austria/Switzerland, Portugal/Spain) corresponding to those from other regions.     

Comparative effectiveness of <Emphasis Type="Italic">Pseudomonas</Emphasis> and <Emphasis Type="Italic">Serratia</Emphasis> sp. containing ACC-deaminase for improving growth and yield of wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) under salt-stressed conditions

Ethylene synthesis is accelerated in response to various environmental stresses like salinity. Ten rhizobacterial strains isolated from wheat rhizosphere taken from different salt affected areas were screened for growth promotion of wheat under axenic conditions at 1, 5, 10 and 15 dS m⁻¹. Three strains, i.e., Pseudomonas putida (N21), Pseudomonas aeruginosa (N39) and Serratia proteamaculans (M35) showing promising performance under axenic conditions were selected for a pot trial at 1.63 (original), 5, 10 and 15 dS m⁻¹. Results showed that inoculation was effective even in the presence of higher salinity levels. P. putida was the most efficient strain compared to the other strains and significantly increased the plant height, root length, grain yield, 100-grain weight and straw yield up to 52, 60, 76, 19 and 67%, respectively, over uninoculated control at 15 dS m⁻¹. Similarly, chlorophyll content and K⁺/Na⁺ of leaves also increased by P. putida over control. It is highly likely that under salinity stress, 1-aminocyclopropane-1-carboxylic acid-deaminase activity of these microbial strains might have caused reduction in the synthesis of stress (salt)-induced inhibitory levels of ethylene. The results suggested that these strains could be employed for salinity tolerance in wheat; however, P. putida may have better prospects in stress alleviation/reduction.     

Direct somatic embryogenesis and synthetic seed production from<Emphasis Type="Italic"> Paulownia elongata</Emphasis>

We have developed a reproducible system for efficient direct somatic embryogenesis from leaf and internodal explants of Paulownia elongata. The somatic embryos obtained were subsequently encapsulated as single embryos to produce synthetic seeds. Several plant growth regulators [6-benzylaminopurine, indole-3-acetic acid, -naphthaleneacetic acid, kinetin and thidiazuron (TDZ)] alone or in combination were tested for their capacity to induce somatic embryogenesis. The highest induction frequencies of somatic embryos were obtained on Murashige and Skoog (MS) medium supplemented with 3% sucrose, 0.6% Phytagel, 500 mg l^-1 casein hydrolysate and 10 mg l^-1 TDZ (medium MS10). Somatic embryos were induced from leaf (69.8%) and internode (58.5%) explants on MS10 medium after 7 days. Subsequent withdrawal of TDZ from the induction medium resulted in the maturation and growth of the embryos into plantlets on MS basal media. The maturation frequency of somatic embryos from leaf and internodal explants was 50.8% and 45.8%, respectively. Subculturing of mature embryos led to their germination on the same medium with a germination frequency of 50.1% and 29.8% from leaf and internode explants, respectively. Somatic embryos obtained directly on leaf explants were used for encapsulation in liquid MS medium containing different concentrations of sodium alginate with a 30-min exposure to 50 mM CaCl₂. A 3% sodium alginate concentration provided a uniform encapsulation of the embryos with survival and germination frequencies of 73.7% and 53.3%, respectively. Storage at 4°C for 30 days or 60 days significantly reduced the survival and complete germination frequencies of both encapsulated and non-encapsulated embryos relative to those of non-stored somatic embryos. However, the survival and germination rates of encapsulated embryos increased following storage at 4°C. After 30 days or 60 days of storage, the survival rates of encapsulated embryos were 67.8% and 53.5% and the germination frequencies were 43.2% and 32.4%, respectively. These systems could be useful for the rapid clonal propagation and dissemination of synthetic seed material of Paulownia elongata.Abbreviations BAP 6-Benzylaminopurine - IAA Indole-3-acetic acid - NAA -Naphthaleneacetic acid - TDZ ThidiazuronCommunicated by H. Lörz     

Micropropagation of <Emphasis Type="Italic">Kalopanax pictus</Emphasis> tree via somatic embryogenesis

Summary Kalopanax pictus (Thunb.) Nakai is a tall tree, and its wood has been used in making furniture, while its stem bark is used for medicinal purposes. Here, we report on the micropropagation of Kalopanax pictus via somatic embryogenesis. Embryogenic callus was induced from immature zygotic embryos. The frequency embryogenic callus induction is influenced by days of seed harvest. Callus formation was primarily observed along the radicle tips of zygotic embryos incubated on Murashige and Skoog (MS) medium with 4.4 μM 2,4-dichlorophenoxyacctic acid (2,4-D). Somatic embryogenesis was observed following transfer of embryogenic callus to MS medium lacking 2,4-D. Somatic embryos at the cotyledonary stage were obtained after 6 wk following culture. Frequency of conversion of somatic embryos into plantlets was low (35%) on a hormone-free MS basal medium, but it increased to 61% when the medium was supplemented with 0.05% charcoal. Gibberellic acid (GA₃) treatment markedly enhanced the germination frequency of embryos up to 83%. All plantlets obtained showed 98% survival on moist peat soil (TKS2) artificial soil matrix. About 30 000 Kalopanax pictus plants were propagated via somatic embryogenesis and grown to 3-yr-old plants. These results indicate that production of woody medicinal Kalopanax pictus plantlets through somatic embryogenesis can be practically applicable for propagation.     

Induction of somatic embryogenesis in cultured cells of <Emphasis Type="Italic">Chenopodium quinoa</Emphasis>

A major limiting factor for quinoa cultivation as a grain crop on a large scale are virus diseases, in particularly seed borne diseases. Therefore, a somatic embryogenesis protocol is a necessary tool to produce virus free plants. Somatic embryogenesis offers the possibility of mass production of transgenic plants and therefore can be used easily to study the effect on plants resulting from breeding processes. An in vitro protocol has been developed for somatic embryogensis from calluses and cell cultures of Chenopodium quinoa. Callus was induced from hypocotyl explants within 2 weeks of culture on a modified Murashige and Skoog (MS) medium supplemented with 0.45 M 2,4-D. Calluses were cultured on solid or liquid MS medium and later the development of somatic embryos was observed on both employing the same MS medium without 2,4-D. To our knowledge this is the first report of somatic embryogenesis in Chenopodium quinoa.     

Recovery and characterization of transgenic plants from two spring wheat cultivars with low embryogenesis efficiencies by the bombardment of isolated scutella

Summary Two commercial wheat cultivars with low embryogenesis efficiencies, AC Karma and Hy417, were transformed by the bombardment of isolated scutella with two gene constructs. Three AC Karma plants (433, 436, and 437) carrying plasmid pRC62 containing a gus:npt fusion gene, and one Hy417 plant (438) carrying plasmid pBARGUS containing a bar gene and a gusA gene were recovered and characterized. Presence of transgenes in T0 and T1 plants was confirmed by both PCR and Southern hybridization. Copy number of transgenes varied from one to six in these four plants. The inheritance of transgenes in the progeny was characterized. The gusA gene and its activity in AC Karma plant 436 and bar gene and its activity in Hy417 plant 438 segregated in the selfed T1 progeny in a Mendelian 3:1 ratio, but gusA gene and its activity in AC Karma plants 433 and 437 segregated in selfed T1 progeny in a non-Mendelian 1:1 ratio. The gusA activity in all three AC Karma plants was stably transmitted to selfed T2 or T3 progenies. The levels of gusA and nptII activities in nine T1 plants from AC Karma plant 437 were also determined. A GusA fluorometric assay indicated that gusA activity in the nine T1 plants increased by 2.5–7.2-fold compared with the nontransformed control, while and NptII ELISA assay detected nptII activity only in two of the nine T1 plants, suggesting the nptII gene was silenced in the other seven T1 plants.     

The histological analysis of indirect somatic embryogenesis on <Emphasis Type="Italic">Drosera spathulata</Emphasis> Labill

We studied indirect somatic embryogenesis in the callus tissue of Drosera spathulata Labill. originated from isolated leaves. Callogenesis was induced on MS medium (Murashige and Skoog 1962), supplemented with various concentrations of NAA and BA. Somatic embryos regenerated on half-strength MS medium supplemented with 20 μM of NAA or without growth regulators. The highest efficiency of somatic embryo production was achieved on hormone-free medium. Globular, heart-, torpedo- and cotyledonary-shaped embryos were observed in embryogenic clusters. Histological and scanning electron microscopy analysis verifies somatic embryogenesis. Regenerated plants were transferred to soil and were grown to maturity.     

Plant regeneration by somatic embryogenesis and organogenesis in commercial pineapple (<Emphasis Type="Italic">Ananas comosus</Emphasis> L.)

Summary Axillary and terminal buds from suckers of Ananas comosus cv. Phuket were established on Murashige and Tucker-based (MT) medium with 2.0 mgl⁻¹ (9.8 μM) indolebutyric acid, 2.0 mgl⁻¹ (10.74 μM) naphthaleneacetic acid, and 2.0 mgl⁻¹ (9.29 μM) kinetin, followed by multiplication on Murashige and Skoog-based (MS) medium containing 2.0 mgl⁻¹ (8.87 μM) benzyladenine (BA) to provide a continuous supply of axenic shoots. Leaves, excised from such cultured shoots, produced adventitious shoots from their bases when these explants were cultured on MS medium containing 0.5 mgl⁻¹ (2.26 μM) 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.0 mgl⁻¹ (8.87 μM) BA. Embryogenic callus was produced when leaf explants were cultured on MS medium with 3.0 mgl⁻¹ (12.42 μM) 4-amino-3,5,6-trichloropicolinic acid (picloram). Somatic embryos developed into shoots following transfer of embryogenic tissues to MS medium with 1.0 mgl⁻¹ (4.44 μM) BA. Cell suspensions, initiated by transfer of embryogenic callus to liquid MS medium with 1.0 mgl⁻¹ (4.14 μM) picloram or 1.0 mgl⁻¹ (4.52 μM) 2,4-D, also regenerated shoots by somatic embryogenesis, on transfer of cells to semisolid MS medium with 1.0 mgl⁻¹ (4.44 μM) BA. All regenerated shoots rooted on growth regulator-free MS medium, prior to ex vitro acclimation and transfer to the glasshouse. These studies provide a baseline for propagation, conservation, and genetic manipulation of elite pineapple germplasms.     

Osmotic sucrose enhancement of single-cell embryogenesis and transformation efficiency in <Emphasis Type="Italic">Oncidium</Emphasis>

Traditional breeding processes to genetically modify the long reproductive cycle and slow seed maturation of orchids have limits. We developed a more efficient protocol using particle bombardment to produce transgenic plants of Oncidium Sharry Baby OM8 (Orchidaceae). Pretreating protocorm-like bodies (PLBs) with 0.5 M sucrose for 2 h increased single-cell embryogenesis 3- to 4-fold; however, shoot formation was suppressed. In addition, new PLBs were regenerated from the entire sucrose-pretreated PLBs, whereas in untreated PLBs, this occurred only from the bases. Pretreated PLBs were bombarded with pSPFLP containing genes encoding a sweet pepper ferredoxin-like protein (pflp), hygromycin phosphotransferase (hpt) and -glucuronidase (GUS) driven by the cauliflower mosaic virus 35S promoter. Pretreated PLBs showed a 14.8-fold increase in GUS expression over the untreated PLBs 40days after bombardment. The presence of pflp and hpt transgenes in the 40 putatively stably transformed lines that produced 113 clones was confirmed by PCR analysis. Six lines (eight clones) were positive for both pflp and hpt transgenes. In addition, clones derived from these lines were either all positive or all negative for the two transgenes, which suggests homogeneity in pretreated PLBs with more single-cell embryogenesis. Thus, sucrose pretreatment enhanced the regeneration of PLBs, single-cell embryogenesis and efficiency of transformation.     

Somatic embryogenesis in <Emphasis Type="Italic">Schisandra chinensis</Emphasis> (Turcz.) Baill.

Summary Regeneration of plants via somatic embryogenesis was achieved from zygotic embryo explants isolated from mature seeds of Schisandra chinensis. Merkle and Sommer's medium, fortified with 2,4-dichlorophenoxyacetic acid (2,4-D; 9.04 μM) and zeatin (0.09 μM), was effective for induction of embryogenic callus. The development of a proembryogenic mass and somatic embryos occurred on Murashige and Skoog medium (MS) free of plant growth regulators. The embryogenic callus induced on Merkle and Sommer's medium supplemented with 2,4-D (9.04 μM) and zeatin (0.09 μM) showed development of the maximum number of somatic embryos when transferred to MS medium free of plant growth regulators. The maximum maturation and germination of cotyledonary somatic embryos (46.3%) occurred on MS medium supplemented with 2,4-D (0.45 μM) and N⁶-benzyladenine (1.11 μM). The somatic embryo-derived plants were successfully hardned, with a survival rate of approximately 67%, and established in the field.     

Histological analysis of direct somatic embryogenesis in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> (L.) Heynh

In Arabidopsis the in vitro culture of immature zygotic embryos (IZEs) at a late stage of development, on the solid medium containing synthetic auxin, leads to formation of somatic embryos via direct somatic embryogenesis (DSE). The presented results provide evidence that in IZE cells competent for DSE are located in the protodermis and subprotodermis of the adaxial side of cotyledons and somatic embryos displayed a single- or multicellular origin. Transgenic Arabidopsis lines expressing the GUS reporter gene, driven by the DR5 and LEC2 promoters, were used to analyse the distribution of auxin to mark embryogenic cells in cultured explants and develop somatic embryos. The analysis showed that at the start of the culture auxin was accumulated in all explant tissues, but from the fourth day onwards its location shifted to the protodermis and subprotodermis of the explant cotyledons. In globular somatic embryos auxin was detected in all cells, with a higher concentration in the protodermis, and in the heart stage its activity was mainly displayed in the shoot, root pole and cotyledon primordia. The embryogenic nature of dividing protodermal and subprotodermal cells accumulating auxin was confirmed by high expression of promoter activity of LEC2 in these cells. Analysis of symplasmic tracer (CFDA) distribution indicated symplasmic isolation between tissues engaged in DSE and other parts of an explant. Symplasmic isolation of somatic embryos from the explant was also detected.     

Changes in jasmonates and 12-oxophytodienoic acid contents of <Emphasis Type="Italic">Medicago sativa</Emphasis> L. during somatic embryogenesis

Jasmonic acid (JA), its methyl ester (MeJA) and the biosynthetic precursor 12-oxophytodienoic acid (OPDA) were detected quantitatively during somatic embryogenesis of Medicago sativa L. Using GC-MS analysis, these compounds were found in initial explants, in calli and in somatic embryos in the nanogram range per gram of fresh weight. In distinct stages of somatic embryogenesis, JA and 12-OPDA accumulated preferentially in cotyledonary embryos. Initial explants exhibited about five-fold higher JA content than OPDA content, whereas in other stages OPDA accumulated predominantly. These data suggest that also in embryogenic tissues OPDA and JA may have individual signalling properties.     

Studies on nuclear degeneration during programmed cell death of synergid and antipodal cells in<Emphasis Type="Italic"> Triticum aestivum</Emphasis>

Morphological changes in the nuclear degeneration of the synergid (mainly the synergid that receives the pollen tube) and antipodal cells in Triticum aestivum were studied. Although located in the same embryo sac, and derived from the same megaspore, nuclear degeneration of the synergid and antipodal cells differs greatly. Nuclear degeneration in the synergid is characterized by pycnosis, i.e., total chromatin condensation, nuclear deformation and distinct shrinkage in volume, followed by the formation of an irregular and densely stained mass—the degenerated nucleus—while the nucleolus disappears prior to the degradation of chromatin. In contrast, in the nuclear degeneration of antipodal cells, chromatin is only partly condensed and the nuclear volume changes only slightly after the distinct chromatin condensation. Chromatolysis then occurs, i.e., stainable contents disappear while the nuclear envelope is retained. The nucleoli persist after the disappearance of the chromatin. The possible functions of nuclear degeneration of synergid and antipodal cells are discussed, especially with respect to the guidance of pollen tube growth and the proliferation of free-nuclear endosperm. The degeneration of synergids and antipodal cells in T. aestivum are distinct forms of programmed cell death, regarded as cytoplasmic cell death and nuclear degradation in advance of cell death, respectively.