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Lignin is one of the main components of plant cell walls, which provides mechanical support for plants and also contributes to resisting against plant pathogenic fungi. In the fruit industry, the lignin content can affect the quality of fruit. The biosynthesis of lignin involves a variety of enzymes, of which caffeoyl-CoA 3-O-methyltransferase (CCoAOMT) is a class of methyltransferases that plays an essential role in lignin biosynthesis. Studies have been conducted on the CCoAOMT gene family in several species, including arabidopsis (Arabidopsis thaliana), black poplar (Populus nigra), and cotton (Gossypium hirsutum). Still, there is relatively little research on this gene family in the Rosaceae. In this study, we used bioinformatics to identify and characterize the CCoAOMT gene family in apple (Malus domestica), chinese white pear (Pyrus bretschneideri), and peach (Prunus persica). In total, 35 CCoAOMT genes were identified in the three Rosaceae species: 8 from chinese white pear, 12 from apple, and 15 from peach. By using structure analysis and collinearity analysis, we found 12 conserved motifs and 12 pairs of CCoAOMT genes with collinearity. In the phylogenetic tree, the gene family was mainly divided into two groups. The genes had different expression patterns during the growth and development stage of fruit, a finding that is consistent with the pattern of lignin accumulation. This study will be beneficial for further study of CCoAOMT genes.

Background Pears and apples are both perennial deciduous trees of the Rosaceae family, and both are important economic fruit trees worldwide. The emergence of many varieties in the market has been mostly domesticated from wild to cultivated and regulated by the differential expression of genes. However, the molecular process and pathways underlying this phenomenon remain unclear. Four typical wild and cultivar pear and apple trees at three developmental stages were used in our study to investigate the molecular process at the transcriptome level. Result Physiological observations indicated the obvious differences of size, weight, sugar acid content and peel color in wild and cultivar fruit among each developmental stage. Using next-generation sequencing based RNA-seq expression profiling technology, we produced a transcriptome in procession of a large fraction of annotated pear and apple genes, and provided a molecular basis underlying the phenomenon of wild and cultivar fruit tree differences. 5921 and 5744 differential expression genes were identified in pear and apple at three developmental stages respectively. We performed temporal and spatial differential gene expression profiling in developing fruits. Several key pathways such as signal transduction, photosynthesis, translation and many metabolisms were identified as involved in the differentiation of wild and cultivar fruits. Conclusion In this study, we reported on the next-generation sequencing study of the temporal and spatial mRNA expression profiling of pear and apple fruit trees. Also, we demonstrated that the integrated analysis of pear and apple transcriptome, which strongly revealed the consistent process of domestication in Rosaceae fruit trees. The results will be great influence to the improvement of cultivar species and the utilization of wild resources.

Strawberry powdery mildew, caused by Podosphaeraaphanis is a major fungal disease that affects strawberry yield and quality. In the model plant species Arabidopsis and the crop plants barley, tomato and pea, the Mildew resistance locus O (MLO) proteins have been found to be required for powdery mildew susceptibility. The present study, based on the sequence of a wild plum (Prunus americana) MLO protein, identified 16 MLO genes within the genome of woodland strawberry, Fragaria vesca and examined their expression pattern in response to powdery mildew infection in three diploid strawberry cultivars. Phylogenetic analysis showed that the FvMLO genes can be classified into six clades. Four FvMLO genes were grouped into clade III, which comprises MLO genes from Arabidopsis, tomato and grapevine that mediate powdery mildew susceptibility. A RNA-seq analysis of two diploid strawberry cultivars, F. vescassp. vesca accession Hawaii 4 (HW) and F. vesca f. semperflorens line “Yellow Wonder 5AF7” (YW) at 1 d (1 DAI) and 8 d (8 DAI) after infection showed the expression of 12 out of the 16 FvMLO genes. The comparison of Fragments Per Kilobase of transcript per Million mapped reads (FPKM values) detected by RNA-seq and expression values of qRT-PCR for FvMLO genes showed substantial agreement. The FvMLO3 gene, which was grouped in clade III and orthologous to the Arabidopsis,tomato and grapevine genes, was highly expressed in YW compared to other FvMLO genes across varieties. The results showed that FvMLO genes can be used as potential candidates to engineer powdery mildew resistance in strawberry based on MLO suppression or genome editing.

The development of a regeneration system from cotton stomatal guard cells directly on epidermal strips is described. The most important factors affecting embryogenic callus initiation in both of the varieties tested (Coker 312 and 315) were the source of the epidermal tissue, including plant age (4–5 months old), the developmental stage of the flower (opening flower stage) from which bracts were obtained, the composition of the culture medium and light irradiance. The flower developmental stage was critical for callus formation, which was observed only from bracts obtained from opening flowers. In addition, epidermal strips excised from the bract basal region were more responsive in culture than those obtained from the top region. Improved callus initiation was obtained on epidermal strips which had their cuticle in contact with the culture medium. Light irradiance was a limiting factor for embryogenic callus formation, which was observed only in calluses cultured under the lower light irradiance (15.8 μmol m–2 s–1). Somatic embryogenesis was observed on callus cultures subcultured consecutively to a culture medium containing naphthalene acetic acid (10.7 μM) and isopentenyladenine (4.9 μM). Histodifferentiation of somatic embryos was improved on a medium containing naphthaleneacetic acid (8.1 μM)+isopentenyladenine (2.5 μM) and abscisic acid (0.19–0.38 μM). Somatic embryo germination and plantlet development were obtained using established protocols with few modifications. On average, one fully developed plant was obtained from the culture of circa 100 epidermal strips in both cultivars.

We investigated how arbuscular mycorrhizal symbiosis can alter trace element uptake, distribution and toxicity in plants by examining some of the molecular mechanisms behind Populus trichocarpa tolerance to Cd and Zn, and the effects of AMF in metal homeostasis. Plants were grown under Cd and Zn contamination, with and without Rhizophagus irregularis inoculation. We determined organ metal concentrations, the expression of genes involved in trace element homeostasis, and the function of metallothionein PtMT2b by heterologous expression in yeast. P. trichocarpa was highly tolerant to both elements, with AMF increasing Zn accumulation. AMF altered the partitioning of Cd, but maintained the same patterns for Zn, indicating that despite being geochemically similar and carried mostly by the same transporters, the nutrient metal (Zn) is handled differently from the non-essential metal (Cd). High Zn and Cd down-regulated PtHMA4 (roots), and up-regulated PtZIP1 (leaves), suggesting their involvement in transporting both metals in poplar. PtMT2b was highly up-regulated in mycorrhizal roots and enhanced Cd tolerance in transformed yeast. R. irregularis reduced Cd transfer to poplar shoots, but did not affect Zn partitioning. The gene expression patterns observed offer a glimpse into the mechanisms behind trace element uptake/translocation dynamic in poplars, influenced by AMF symbiosis.

Seed oils provide a renewable source of food, biofuel and industrial raw materials that is important for humans. Although many genes and pathways for acyl-lipid metabolism have been identified, little is known about whether there is a specific mechanism for high-oil content in high-oil plants. Based on the distinct differences in seed oil content between four high-oil dicots (20~50%) and three low-oil grasses (

Starch is the most widespread and abundant storage carbohydrate in crops and its production is critical to both crop yield and quality. In regard to the starch content in the seeds of crop plants, there is a distinct difference between grasses (Poaceae) and dicots. However, few studies have described the evolutionary pattern of genes in the starch biosynthetic pathway in these two groups of plants. In this study, therefore, an attempt was made to compare evolutionary rate, gene duplication, and selective pattern of the key genes involved in this pathway between the two groups, using five grasses and five dicots as materials. The results showed 1) distinct differences in patterns of gene duplication and loss between grasses and dicots; duplication in grasses mainly occurred before the divergence of grasses, whereas duplication mostly occurred in individual species within the dicots; there is less gene loss in grasses than in dicots, 2) a considerably higher evolutionary rate in grasses than in dicots in most gene families analyzed, and 3) evidence of a different selective pattern between grasses and dicots; positive selection may have occurred asymmetrically in grasses in some gene families, for example, ADP-glucose pyrophosphorylase small subunit. Therefore, we deduced that gene duplication contributes to, and a higher evolutionary rate is associated with, the higher starch content in grasses. In addition, two novel aspects of the evolution of the starch biosynthetic pathway were observed.

The release of genetically modified plants is governed by regulations that aim to provide an assessment of potential impact on the environment. One of the most important components of this risk assessment is an evaluation of the probability of gene flow. In this review, we provide an overview of the current literature on gene flow from transgenic plants, providing a framework of issues for those considering the release of a transgenic plant into the environment. For some plants gene flow from transgenic crops is well documented, and this information is discussed in detail in this review. Mechanisms of gene flow vary from plant species to plant species and range from the possibility of asexual propagation, short- or long-distance pollen dispersal mediated by insects or wind and seed dispersal. Volunteer populations of transgenic plants may occur where seed is inadvertently spread during harvest or commercial distribution. If there are wild populations related to the transgenic crop then hybridization and e...

Background: Podosphaera aphanis, the causal agent of strawberry powdery mildew causes significant economic loss worldwide. Methods: We used the diploid strawberry species Fragaria vesca as a model to study plant pathogen interactions. RNA-seq was employed to generate a transcriptome dataset from two accessions, F. vesca ssp. vesca Hawaii 4 (HW) and F. vesca f. semperflorens Yellow Wonder 5AF7 (YW) at 1 d (1 DAI) and 8 d (8 DAI) after infection. Results: Of the total reads identified about 999 million (92%) mapped to the F. vesca genome. These transcripts were derived from a total of 23,470 and 23,464 genes in HW and YW, respectively from the three time points (control, 1 and 8 DAI). Analysis identified 1,567, 1,846 and 1,145 up-regulated genes between control and 1 DAI, control and 8 DAI, and 1 and 8 DAI, respectively in HW. Similarly, 1,336, 1,619 and 968 genes were up-regulated in YW. Also 646, 1,098 and 624 down-regulated genes were identified in HW, while 571, 754 and 627 genes were down-regulated in YW between all three time points, respectively. Conclusion: Investigation of differentially expressed genes (log2 fold changes �5) between control and 1 DAI in both HW and YW identified a large number of genes related to secondary metabolism, signal transduction; transcriptional regulation and disease resistance were highly expressed. These included flavonoid 3´-monooxygenase, peroxidase 15, glucan endo-1,3-β-glucosidase 2, receptor-like kinases, transcription factors, germin-like proteins, F-box proteins, NB-ARC and NBS-LRR proteins. This is the first application of RNA-seq to any pathogen interaction in strawberry

We have compiled two comprehensive gene expression profiles from mature leaf and immature seed tissue of rice (Oryza sativa ssp. japonica cultivar Nipponbare) using Serial Analysis of Gene Expression (SAGE) technology. Analysis revealed a total of 50 519 SAGE tags, corresponding to 15 131 unique transcripts. Of these, the large majority (approximately 70%) occur only once in both libraries. Unexpectedly, the most abundant transcript (approximately 3% of the total) in the leaf library was derived from a type 3 metallothionein gene. The overall frequency profiles of the abundant tag species from both tissues differ greatly and reveal seed tissue as exhibiting a non-typical pattern of gene expression characterized by an over abundance of a small number of transcripts coding for storage proteins. A high proportion ( approximately 80%) of the abundant tags (> or = 9) matched entries in our reference rice EST database, with many fewer matches for low abundant tags. Singleton transcripts that are common to both tissues were collated to generate a summary of low abundant transcripts that are expressed constitutively in rice tissues. Finally and most surprisingly, a significant number of tags were found to code for antisense transcripts, a finding that suggests a novel mechanism of gene regulation, and may have implications for the use of antisense constructs in transgenic technology.

A field trial was carried out with oil seed rape plants that had been genetically modified to contain genes coding for marker characters (β-glucuronidase, kanamycin resistance and asulam resistance). The aim of the experiment was to examine a method of studying gene dispersal in an agricultural environment. The central area of the field plot comprised 150 genetically modified plants and 450 plants not expressing the marker genes. These were surrounded by additional non-expressing plants. The plants were allowed to freely cross pollinate and a sample of the resultant progeny from non-expressing plants were screened for expression of the marker characters and for the presence of the introduced genes (by PCR). Limited gene dispersal was detected and the frequency of modified seedlings amongst the progeny of a plant appeared to have been strongly influenced by the genotype of the immediately adjacent plants.

A simple and inexpensive system for the generation of fertile, transgenic maize plants has been developed. Cells from embryogenic maize suspension cultures were transformed using silicon carbide whiskers to deliver plasmid DNA carrying the bacterial bar and uidA (gus) genes. Transformed cells were selected on medium containing the herbicide bialaphos. Integration of the bar gene and activity of the enzyme phosphinothricin acetyl transferase (PAT) were confirmed in all bialaphos-resistant callus lines analysed. Fertile transgenic maize plants were regenerated. Herbicide spraying of progeny plants revealed that the bar gene was transmitted in a Mendelian fashion.

The first haploid angiosperm, a dwarf form of cotton with half the normal chromosome complement, was discovered in 1920, and in the ninety years since then such plants have been identified in many other species. They can occur either spontaneously or can be induced by modified pollination methods in vivo, or by in vitro culture of immature male or female gametophytes. Haploids represent an immediate, one-stage route to homozygous diploids and thence to F(1) hybrid production. The commercial exploitation of heterosis in such F(1) hybrids leads to the development of hybrid seed companies and subsequently to the GM revolution in agriculture. This review describes the range of techniques available for the isolation or induction of haploids and discusses their value in a range of areas, from fundamental research on mutant isolation and transformation, through to applied aspects of quantitative genetics and plant breeding. It will also focus on how molecular methods have been used recently to explore some of the underlying aspects of this fascinating developmental phenomenon.

A period of four days preincubation at 25 °C on a medium containing mannitol was found to be superior to those pretreatments requiring incubation at 4 °C. In addition, the yield of green plants was improved by orienting anthers 'flat' on the medium during mannitol preincubation, and reducing the number of anthers cultured per dish.

Introduction Historical Outline Classification of Globulins 11S Globulins: Legumins 7S Globulins: Vicilins Selection and Breeding Physiology and Function Deposition During Seed Development Protein Cleavage During Germination Globulins and Plant Defense Three-dimensional Structure of Globulins Crystallization and Three-dimensional Resolution Structure and Allergenicity of Globulins Evolution Evolution of Variation Within the Globulin Gene Family Similarity of Globulins to Other Desiccation-associated Proteins Structural Evolution Discovery of the Cupin Superfamily Prokaryotic Ancestors of Seed Globulins Over-expression and Modification of Globulins Expression in Bacteria Expression in Yeast Expression in Plants Other Genetic Methods for Manipulation of Globulins Recombinant Systems and Processing Quality Feeding Trials with Recombinant Globulins Production of Pharmaceutically Active Peptides in Glycinins Patents Chronological Summary of Patents Outlook and Perspectives Keywords: cupin; allergy; epitope; germin; sucrose binding protein; glycinin; canavalin; convicilin; glutelin; dioxygenase; overexpression; transgenic; peanut; bean; pea; wheat; rice; germination

An optimized protocol has been developed for the efficient and rapid genetic modification of sugar beet (Beta vulgaris L). A polyethylene glycol- mediated DNA transformation technique could be applied to protoplast populations enriched specifically for a single totipotent cell type derived from stomatal guard cells, to achieve high transformation frequencies. Bialaphos resistance, conferred by the pat gene, produced a highly efficient selection system. The majority of plants were obtained within 8 to 9 weeks and were appropriate for plant breeding purposes. All were resistant to glufosinate-ammonium-based herbicides. Detailed genomic characterization has verified transgene integration, and progeny analysis showed Mendelian inheritance.

It has been successfully demonstrated, using epidermis explants of sugar beet (Beta vulgaris L.), that stomatal guard cells retain full totipotent capacity. Despite having one of the highest degrees of morphological adaptation and a unique physiological specialization, it is possible to induce a re-expression of full (embryogenic) genetic potential in these cells in situ by reversing their highly differentiated nature to produce regenerated plants via a callus stage. The importance of these findings both to stomatal research and to our understanding of cytodifferentiation in plants is discussed.

Oxalic acid is thought to have a primary role in the pathogenicity of several plant pathogens, notably Sclerotinia sclerotiorum. A gene coding for the enzyme oxalate oxidase was isolated from barley roots and introduced into oilseed rape as a means of degrading oxalic acid in vivo. This report describes the production of several transgenic plants of oilseed rape and the characterisation of these plants by Southern, Western and enzyme activity assays. Plants were shown to contain an active oxalate oxidase enzyme and were tolerant of exogenously supplied oxalic acid.

We review here the most recently developed technique for maize transformation which involves the vortexing of silicon carbide whiskers with maize cells in the presence of plasmid DNA. Fertile transgenic plants have been regenerated following whisker-mediated transformation which is compared with the alternatives described to date, namely protoplast uptake, particle bombardment and electroporation of intact tissue.

Two inbred cultivars of Nicotiana tabacum (tobacco), ‘Samsun’ and ‘Xanthi’, were transformed with the plasmid pBI 121 using Bin 19 in Agrobacterium tumefaciens. The plasmid carries the nptII gene conferring kanamycin resistance and the uidA gene encoding β-glucuronidase (GUS). Progeny carrying the genes in the homozygous condition were identified and selfed over several generations. One line homozygous for the introduced genes and one untransformed control from each cultivar were then selected and crossed reciprocally to give four families per cultivar. Seeds from each family were grown in a replicated field trial and all plants scored for a range of morphological and agronomic characters. In addition, leaf samples were taken and GUS activity measured. In the ‘Samsun’ material, which contained one copy of the introduced gene at a single locus and showed high levels of GUS expression, the transformed homozygote showed twice the level of GUS activity as the hemizygotes, wheareas in the ‘Xanthi’ line, which had a lower level of GUS, the hemizygotes showed the same level of GUS activity as the transformed homozygote. The agronomic data showed differences between the families, but the source of such differences could not be ascribed unambiguously. The results are discussed in the light of related information on gene expression and field performance from other transgenic material.

As Haldane stated in 1937 “Every species is at the mercy of its pollen grains”, and although this motto is undoubtedly true in evolutionary terms, it is only relatively recently that we have been able to disrupt at will this critical phase of plant development.

A comparison of four volumes of culture vessel atmosphere revealed that this variable greatly influenced the induction and growth of pollen embryos from anthers of Nicotiana tabacum. The optimum frequency of anthers producing embryos and plantlets was found with a culture atmosphere of 15 ml per anther, whereas the optimum number of plantlets was found with 5-5 ml per anther. A small volume (0-5 ml per anther) almost completely suppressed embryo induction. Removal of specific components of the culture atmosphere (ethylene, carbon dioxide, oxygen) influenced the response of the anthers but did not produce a satisfactory explanation of the inhibition of pollen embryogenesis by the small culture atmosphere volume. In particular, the influence of ethylene absorption on embryo induction and growth depended both on the culture atmosphere volume and on the stage of development of the pollen at the start of culture. Using anthers containing pollen at a stage after the first pollen grain mitosis, ethylene absorption was found to increase the survival of induced embryos. Treatment of anthers for 3 d with silver nitrate, a known antagonist of ethylene action, was not an efficient means of increasing the yield of pollen plantlets.

Excised leaf discs of three Brassica species, B. oleracea, B. napus, and B. campestris were induced to produce adventitious buds and subsequently entire plants by culture on media with specific combinations of 6-benzylaminopurine (BAP) and cr-naphthylacetic acid (NAA). Each species required a particular hormone concentration for optimum growth and differentiation: B. oleracea, BAP 10 mg-l and NAA 1 mg-l; B. napus, BAP 10 mg-l and NAA 10 mg-l; B. campestris, BAP 1 mg-l and NAA 10 mg-l. In a more detailed study on one of these species, namely B. oleracea, the relative influence of other media components such as amino acids and other organic additives was examined. It was also found that the source and size of the explant greatly affected the growth response, as did the size of the culture vessel. The regenerated plants dislayed a range of ploidy as well as phenotypic abnormalities. Findings are discussed in relation to results from other tissue culture systems.

Three cultivars of spring rape (Brassica napus ssp. oleifera), Tower, Willi and Duplo, were used for a study of induction and growth of 'microspore-derived' embryos, Buds, 2.0 mm in length, containing uninucleate microspores were harvested and stored for 14 d at 4 °C in darkness. Anthers were then removed and cultured on a liquid medium based upon that of Murashige and Skoog and containing 8% sucrose, 0.5 mg l-1 naphthylacetic acid and 0-05 mg l-1 benzylaminopurine. Cultures were pre-incubated at 35 °C for 0-3 d and then incubated at 30 °C. After a total of 42 d incubation, cultures were scored for the presence of macroscopic embryos (1-2 mm in length) and for the presence of anthers containing abortive embryoids which had not developed further. The cultivars differed greatly in terms both of the frequency of anthers showing induced embryoids and of the final yield of embryos. Tower showed the highest frequency of induction (maximum 38% of cultured anthers with induced embryoids) whereas the highest yield (equivalent to 1 • 1 embryo per cultured anther) was obtained from anthers of the cv. Duplo after a 3 d treatment at 35 °C. Yields from the other cultivars were much lower and were relatively unaffected by the 35 °C treatment.

Plant tissue culture technology is of increasing interest to plant breeders. As part of a continuing investigation into breeding methods with spring barley two studies were conducted to assess the field performance of the progenies of material regenerated in tissue culture. The first study involved two spring barley cultivars, 'Golden Promise' and 'Mazurka' and compared lines produced from immature embryo (IE) derived callus with those from embryos developed by the Hordeum bulbosum (Hb) technique of chromosome elimination. In general the mean values for the seven characters scored were lower for the IE than the Hb material. In the second study F1 hybrid material ('Golden Promise'x'Mazurka') was used and doubled haploid lines produced by the H. bulbosum and microspore culture (M) techniques were compared with single seed descent (SSD) material. Analysis of these F∝ samples indicated that the mean values for the M lines were significantly lower than those of the Hb and SSD lines. Furthermore, data from the M lines showed significant evidence of variation created during the culture phase. The implications of these findings for barley breeding are discussed.

Isolated root cultures which could be maintained over several months by serial subculture were established from Brassica oleracea var. italica cv. Green Comet Ft. A modified White's medium was found to be the best of several salt compositions tested. The effects on isolated root growth of the following were also examined; nutritional components, culture vessel type and closure, pH of the medium and auxin type and concentration. Using a medium devised for Green Comet, root cultures were established from six other B. oleracea , B. napus and B. campestris cultivars. It was possible to regenerate shoots from segments of cultured roots by incubation on agar-solidified media containing cytokinin and auxin. The effects on regeneration of various auxins and cytokinins were investigated; the combination of Picloram with KN gave the highest frequency of shoot formation. It was demonstrated that isolated roots retained their regenerative ability over a period of 5 months in culture.

The influence of the growth environment of the parent plant, the age of the parent plant, and the stage of pollen development on embryo induction and growth in cultured anthers of N. tabacum has been investigated. The medium used used and the incubation conditions were identical for all anthers cultured, so that differences in response could be attributed to the state of the anther (wall plus pollen) at the moment of culture. By compating the responses of anthers taken from six groups of plants grown in different environments, it was found that differences in photoperiod or light intensity produced a fivefold variation in the number of plantlets produced per anther. Plant age was of secondary importance, althouugh some plants showed up to a sevenfold decline in the number of plantlets produced the 1st to the 100th bud. Pollen stage, the least important of the factors investigated, revealed an optimum in response at the pollen grain mitosis, with a much lower response from older pollen. The possible physiiological basis of these results, together with the connection between floral induction and embryo growth, are discussed.

The effect of four preculture variables on microspore embryo induction and growth were examined: (1) the source of the bud selected for culture (apical or axillary inflorescence; (2) the method of harvest (single harvest of whole inflorescence or sequential harvest of individual buds; (3) the length of the bud (2, 3 or 4 mm); and (4) the application of a 4 °C pretreatment to the bud after harvest. Microscopic and macroscopic analysis of every anther used for culture permitted an assessment of the following parameters: (1) the percentage of induced buds; (2) the number of induced anthers per induced bud; (3) the number of productive buds (with macroscopic embryos) as a percentage of the induced buds; (4) the degree of induction per induced anther (an estimate of the number of microspores in which initial embryogenic divisions had commenced); and (5) embryoid survival (the number of embryos as a proportion of the degree of induction). The product of parameters 1 and 2 gave the number of induced anthers and all five parameters were components of the final yield — the number of embryos produced per bud cultured. It was found that the maximum number of induced buds (67-0 per cent) occurred with 2 mm sequentially harvested non-pretreated buds. Overall, the values decreased with increasing bud length and were lower for pretreated and axillary buds. In contrast, the two other estimates of induction - number of induced anthers per induced bud and degree of induction per induced anther — both had maximum values from 3 mm sequentially harvested, pretreated buds from apical inflorescences. The highest final yield of embryos per cultured bud (44-9) was found with 2 mm non-pretreated buds taken from a single harvest of the apical inflorescence. The study therefore confirmed that the different components of the final embryo yield are differentially affected by the four preculture variables tested. These variables must be controlled if reproducible results are to be achieved.

Ultrastructural features of embryogenic pollen, in Datura innoxia are described, just prior to, during, and after completion of the first division of the presumptive vegetative cell. In anther cultures initiated towards the end of the microspore phase and incubated at 28 °C in darkness, the spores divide within 24 h and show features consistent with those of dividing spores in vivo. Cytokinesis is also normal in most of the spores and the gametophytic cell-plate curves round the presumptive generative nucleus in the usual highly ordered way. Further differentiation of the 2 gametophytic cells does not take place and the pollen either switches to embryogenesis or degenerates. After 48–72 h, the remaining viable pollen shows the vegetative cell in division. The cell, which has a large vacuole and thin layer of parietal cytoplasm carried over from the microspore, divides consistently in a plane parallel to the microspore division. The dividing wall follows a less-ordered course than the gametophytic wall and usually traverses the vacuole, small portions of which are incorporated into the daughter cell adjacent to the generative cell. The only structural changes in the vegetative cell associated with the change in programme appear to be an increase in electron density of both plastids and mitochondria and deposition of an electron-dense material (possibly lipid) on the tonoplast. The generative cell is attached to the intine when the vegetative cell divides. Ribosomal density increases in the generative cell and exceeds that in the vegetative cell. A thin electron-dense layer also appears in the generativecell wall. It is concluded that embryogenesis commences as soon as the 2 gametophytic cells are laid down. Gene activity associated with postmitotic synthesis of RNA and protein in the vegetative cell is switched off. The data are discussed in relation to the first division of the embryogenic vegetative cell in Nicotiana tabacum.

In ultrathin sections of 7- and 8-d-old anther cultures of Nicotiana tabacum examined in the electron microscope, embryogenic pollen was distinguished by the presence of zones in the cytoplasm of the vegetative cell consisting of multivesiculate bodies resembling lysosomes. These zones increased with time, and in 12-d-old samples (in which divisions of the vegetative cell were about to commence), were almost devoid of contents. Ribosomes appeared to be virtually eliminated from the cell and many other organelles degraded; the few that remained, mainly plastids, were clustered around and in close contact with the vegetative nucleus. Plastid contents also declined with time, but mitochondrial structure changed little. It is concluded that the gametophytic cytoplasm is destroyed before the first sporophytic divisions of the vegetative cell. In the same sections, other structurally viable pollen which lacked lysigenous zones was characterized by dense vegetative cytoplasm containing numerous small vacuoles. Mitochondrial and plastid structure differed from that of the embryogenic grains, and in some instances lacked clear structural definition. Extensive stacks of rough e.r., which traversed the cytoplasm of the vegetative cell, were interpreted as assembly sites for excess gametophytic protein and RNA synthesised during the culture period.

The inheritance of the ability of barley anthers to produce microspore-derived callus in vitro was investigated. The genotypes selected were the two spring cultivars 'Dissa' (D) and 'Sabarlis' (S), the two F1 hybrids (DxS, SxD), the two backcross generations [Dx(DxS), Sx(DxS)], and an F2 generation derived from DxS. From a number of individuals of each generation, the first five spikes were harvested sequentially and after pre-treatment the anthers were removed and placed in culture. Cultures were scored for microspore callus production and plantlet differentiation. Although 'Dissa' gave a significantly higher level of callus production than 'Sabarlis', the overall frequencies of green and albino plant production were higher from 'Sabarlis'. There was no significant difference between reciprocal F1 hybrids. Analysis of variance revealed significant differences in response between the spikes sampled from the plants. This was the major source of variation in the experiment. Spike to spike variation also appeared to be a heritable character.

This study was conducted with Lolium temulentum, Festuca pratensis, and the two hybrids L. multiflorum x F. pratensis 'Elmet' and L. perenne x F. pratensis 'Prior'. In a comparison of various durations (7-42 d) of pretreatment at 4 or 7 °C the highest yield of microspore-derived callus of L. temulentum was obtained after pretreatment of spikes at 7 °C for 28 d, conditions which also proved optimal for panicle pretreatment with F. pratensis. For 'Elmet', durations of 21-42 d were optimal, and for 'Prior' the responses tended to decline with increasing duration. In L. temulentum addition of charcoal (1-2 g l-1) to medium containing 2,4-D and KN was beneficial to callus production but inhibited subsequent differentiation. Its beneficial effect was mainly due to its ability to remove supplied hormones from the medium. Addition of 2,4-D (0.1-2.0 mg l-1), NAA (1-10 mg l-1) or IAA (1-10 mg l-1) to charcoal-free medium inhibited callus production. The optimum incubation temperature for callus production was 25 °C but 20 °C allowed superior callus differentiation. Illumination during pretreatment was beneficial but during culture was inhibitory. In F. pratensis, charcoal inhibited growth of calluses. In 'Prior', callus yields varied significantly from plant to plant Only albino plants were regenerated from L. temulentum and F. pratensis but both green and albino pollen plants were regenerated from 'Elmet' and 'Prior'.

Anthers of the Solanum tuberosum genotype H3703 were cultured on medium containing equimolar concentrations of sucrose or maltose. It was found that significantly more pollen embryos became plants after culture on maltose and hence the yield of plants per 100 anthers cultured increased significantly. Mechanisms by which carbohydrate source may influence response to anther culture are discussed.

The calabrese cultivar Brassica oleracea var. italica cv. Green Comet was used in a study of the effects of exogenous hormones on the growth and differentiation of seedling organs in vitro. Four types of explants were tested: hypocotyl segments, root segments, primary leaf discs and cotyledon discs. These explants were incubated on media containing factorial combinations of BAP x IBA, BAP x NAA, KN x IBA and KN x NAA(all at 0, 0.1, 1.0 and 10.0mgl-1). Hypocotyls were the most regenerative explants; shoot production was favoured by cytokinin:auxin ratios greater than one and was decreased by IBA at 10 mg I"1 when callus was produced. Shoot formation from root explants occurred either in the absence of hormones or with low concentrations; no shoot was produced when any hormone was present at lOmgl-1. In contrast, shoot production from primary leaf discs was favoured by high concentrations of both auxin and cytokinin with the combination of BAP and IBA the most effective. Shoot production from cotyledon discs was sporadic with no consistent response on any auxin/cytokinin combination. After further experiments on the optimization of hormone concentration, the following combinations were chosen as allowing reliable regeneration:0.1 mg l-1 BAP+0.lmgl-1 IBA for hypocotyl segments, 0.075 mg l-1 K.N + 0.025 mgl-1 IBA for root segments, and 5.0 mg l-1 BAP+5.0 mg l-1 IBA for leaf discs.

This study concerns anther culture and the production of microspore-derived calluses and plants of the opium poppy (Papaver somniferum L.). It was confirmed that growth regulators were necessary for microspore callus production. Cold treatment (7 d at 7°C) of the buds prior to culture lead to a twofold increase in the frequency of responsive anthers and in the number of calluses per 100 anthers plated. Callus was produced from cultured anthers of several genotypes, covering a wide genetic background. Step by step removal of growth regulators from the culture medium promoted organogenesis and plant regeneration. Most regenerated plants were diploid. The overall process of microspore embryogenesis closely resembled that described in previous reports on somatic callus production and plant regeneration from poppy hypocotyls in vitro.

Barley anthers from cold pretreated spikes produced no or few calluses when plated with both loculi in contact with the medium (‘flat’). When anthers were plated with only one loculus in contact with the medium (‘up’), a high proportion of the anthers produced calluses. The top loculus of the ‘up’ anthers was most productive. ‘Flat’ anthers, when compared with ‘up’ anthers, were not only slower to produce multicellular pollen grains (MCPs) and microcalluses, but also produced fewer of them and ceased production earlier. The MCPs and microcalluses in ‘flat’ anthers grew more slowly and few developed beyond the 30 cell stage. These results establish the importance of anther orientation for barley anther culture.

During the microspore division in Datura innoxia, the mitotic spindle is oriented in planes both perpendicular (PE) and oblique (OB) to the spore wall against which the nucleus is situated. However, irrespective of polarity, the usual type of hemispherical wall is laid down at cytokinesis and isolates the generative cell from the rest of the pollen grain (type A). In PE spores the vegetative nucleus initially occupies a central position in the pollen grain, whereas in OB spores the vegetative nucleus lies at the periphery of the grain close to the generative cell. In anther cultures initiated just before the microspore division is due to take place, no marked change can be observed in either orientation or symmetry of the mitotic spindle when the spores divide. In some, however, cytokinesis is disrupted and deposition of the hemispherical wall arrested. In the absence of a complete wall, differentiation of the generative cell cannot take place and binucleate pollen grains are formed having 2 vegetative-type nuclei (type B). The 2 nuclei in the B pollens are always situated against the pollen-grain wall, suggesting that the disruption phenomenon is related to the OB spores. The incomplete wall always makes contact with the intine on the intine-side of the spindle. Wall material may be represented merely as short stubs projecting out from the intine into the cytoplasm, in which event the 2 nuclei lie close to each other and are separated by only a narrow zone of cytoplasm. In other grains the wall is partially developed between the nuclei and terminates at varying distances from the tonoplast; in these, the nuclei are separated by a wider zone of cytoplasm. The significance of these binucleate grains in pollen embryogenesis is discussed.

In a study of anther culture in Sorghum bicolor it was found that culture incubation temperature had a significant effect on yields of microspore-derived callus. A temperature of 33° C was superior to 25°, 28° or 37° C. With fresh, unpretreated material, callus was produced only from anthers incubated at 33° C. Panicle pretreatment at 7°, 14°, 20°, 25° or 28° C either had no effect or significantly decreased callus yield. Comparison of culture media containing a range of concentrations of 2,4-dichlorophenoxyacetic acid (0–3.0 mg l-1) showed that callus yield increased significantly with concentration up to 2.5 mg l-1 and then declined slightly. Although 1174 calluses were produced, subsequent differentiation was poor and only four albino plants were regenerated.