In Vitro Fertilization with Isolated, Single Gametes Results in Zygotic Embryogenesis and Fertile Maize Plants.

We demonstrate here the possibility of regenerating phenotypically normal, fertile maize plants via in vitro fertilization of isolated, single sperm and egg cells mediated by electrofusion. The technique leads to the highly efficient formation of polar zygotes, globular structures, proembryos, and transition-phase embryos and to the formation of plants from individually cultured fusion products. Regeneration of plants occurs via embryogenesis and occasionally by polyembryony and organogenesis. Flowering plants can be obtained within 100 days of gamete fusion. Regenerated plants were studied by karyological and morphological analyses, and the segregation of kernel color was determined. The hybrid nature of the plants was confirmed.

Karyogamy after Electrofusion of Single Egg and Sperm Cell Protoplasts from Maize: Cytological Evidence and Time Course.

In maize, in vitro fusion of isolated male gametes with isolated egg cell protoplasts can be induced by electric pulses. Until now, karyogamy has not been demonstrated. In this study, we cytologically examined fusion products fixed at different times after electrofusion with phase contrast microscopy and transmission electron microscopy. We obtained a precise timetable from 23 samples studied during the first 3 hr. The sperm nucleus was integrated within the egg cell protoplast, migrated toward the egg cell nucleus, and fused with it within 1 hr, as demonstrated by ultrastructural observations, three-dimensional reconstructions of nuclei, and subsequent nuclear volume estimates. Fusion of nuclei occurred before zygotic mitosis, as is the case in vivo. These findings demonstrate karyogamy during in vitro fertilization of maize.

Endosperm development after fusion of isolated, single maize sperm and central cells in vitro

We demonstrate here the possibility of endosperm development in vitro after the fusion of pairs of an isolated sperm and an isolated central cell of maize. The occurrence of karyogamy and the time course of the fusion of sperm and central cell nuclei are presented. The fusion of the sperm nucleus occurred either with one of the two polar nuclei or with the secondary nucleus and was completed within 2 hr after in vitro cell fusion. The in vitro study of early events after cell and nuclear fusion indicates that the resulting primary endosperm cell develops into a characteristic tissue capable of self-organization apart from the mother tissue. The technology presented here opens the way for new cellular and molecular studies, especially of early events after sperm and central cell fusion. These studies should lead to a better understanding of the processes of double fertilization and endosperm development.

Heterologous expression of genes mediating enhanced fungal resistance in transgenic wheat.

Three cDNAs encoding the antifungal protein Ag-AFP from the fungus Aspergillus giganteus, a barley class II chitinase and a barley type I RIP, all regulated by the constitutive Ubiquitin1 promoter from maize, were expressed in transgenic wheat. In 17 wheat lines, stable integration and inheritance of one of the three transgenes has been demonstrated over four generations. The formation of powdery mildew (Erysiphe graminis f. sp. tritici) or leaf rust (Puccinia recondita f. sp. tritici) colonies was significantly reduced on leaves from afp or chitinase II- but not from rip I-expressing wheat lines compared with non-transgenic controls. The increased resistance of afp and chitinase II lines was dependent on the dose of fungal spores used for inoculation. Heterologous expression of the fungal afp gene and the barley chitinase II gene in wheat demonstrated that colony formation and, thereby, spreading of two important biotrophic fungal diseases is inhibited approximately 40 to 50% at an inoculum density of 80 to 100 spores per cm2.

In vitro development of wheat (Triticum aestivum L.) from zygote to plant via ovule culture.

Ovules of the wheat breeding line Veery #5 were excised and transferred to culture within 24 h after pollination. When ovules were cultured on Phytagel-solidified medium, and the pericarp removed exclusively at the micropylar tip and the abaxial side, zygotes from up to 79.2% of the ovules underwent embryogenesis with the same developmental pattern as found in planta. Embryos from more than 50% of the cultured ovules germinated when transferred to regeneration medium. More than 100 plantlets were randomly chosen for transfer to soil, all of which developed to phenotypically normal and fertile plants. With this system, the entire process of zygotic embryogenesis can be studied using living material. Furthermore, the method could be used as an embryo rescue technique for plant breeding purposes.

Establishment of a highly efficient regeneration system from leaf base segments of oat (Avena sativa L.).

A reliable and efficient protocol for the regeneration of fertile plants derived from leaf base segments of young in-vitro-grown oat seedlings has been developed successfully. Callus induction and shoot regeneration were achieved when the basal region of young seedlings was cultured on auxin-containing medium. Callus induction efficiencies as well as regeneration frequencies were correlated with the developmental stage and the genotype of the explants. In five different genotypes of oat, an average of 25 plants per explant could be produced and for the most responsive genotype more than 50 regenerants per explant could be regenerated reproducibly. This high regeneration potential makes oat leaf bases a very attractive target for transformation.

Efficient in vitro plant regeneration from immature zygotic embryos of pearl millet [Pennisetum glaucum (L.) R. Br.] and Sorghum bicolor (L.) Moench.

We report here an in vitro culture system that provides reliable, highly efficient regeneration from immature embryos of pearl millet [Pennisetum glaucum (L.) R. Br.] and sorghum [Sorghum bicolor (L.) Moench]. Immature embryos were isolated 10-20 days after pollination and cultured on various L3 media. The influence of different parameters during the callus induction phase was examined with respect to the regeneration rate: (1) the concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and various cytokinins; (2) the addition of AgNO3; (3) the use of maltose or sucrose as a carbon source. Modifications in the phytohormones alone resulted in the regeneration of fertile sorghum plants at high efficiency. Significant increases in the regeneration rates of pearl millet genotypes were achieved by the combination of sucrose as a carbon source and silver nitrate as a potential ethylene inhibitor.

Biokinetic behavior of technetium in the red abalone, Haliotis rufescens: a re-assessment.

The biokinetic behavior of 95mTc in the red abalone, Haliotis rufescens, is reviewed in light of recent experiments with other molluscs. Additional experimentation has confirmed that, when uptake is directly from labeled seawater, abalones exhibit concentration factors in excess of 100. Bivalve molluscs under the same experimental conditions have concentration factors that do not exceed 2. However, uptake and loss kinetics in the abalone cannot be described by a single compartment model as had been previously advanced. Assimilation of 95mTc by abalones following a single feeding of labeled macroalga, Nereocystis luetkeana, is approximately 45% and loss kinetics are similar to those observed following direct uptake from seawater.

Site-specific recombination in Zea mays.

The elimination of marker genes after selection is recommended for the commercial use of genetically modified plants. We compared the applicability of the two site-specific recombination systems Cre/lox and Flp/FRT for marker gene elimination in maize plants. The selection marker gene pat surrounded by two identically directed lox or FRT sites was introduced into maize. Sexual crossing with plants harboring the corresponding constitutively expressed recombinase led to the precise and complete excision of the lox-flanked marker gene in the F1 progeny, whereas Flp-mediated recombination of FRT sequences occurred rarely. Further examination of site-specific integration was done by biolistic bombardment of immature embryos harboring only one lox site with a lox.uidA sequence with results indicating directed integration.

In vitro fertilisation of maize by single egg and sperm cell protoplast fusion mediated by high calcium and high pH.

We present evidence for the fusion of isolated single maize egg and sperm cell protoplasts in a mannitol solution (400-430 mosmol/kg H2O) containing 0.05 M CaCl2 at pH 11.0, followed by cell division of the fusion products. These findings allow the performance of in vitro fertilisation of higher plants by combining single gametes as in lower plant and animal systems. Further, our findings open new avenues for investigating the basic mechanisms of adhesion and fusion of higher plant gametes and eventually for examining processes that inhibit polyspermy in higher plants.

Investigations on the transfer of isolated nuclei into plant protoplasts.

Nuclei were isolated from various types of donor protoplasts and were transferred into receptor protoplasts in numerous combinations. Five percent uptake was achieved under conditions which did not interfere with viability and subsequent culture of receptor protoplasts. Methodological investigations on nuclei uptake were carried out with cereal and tobacco protoplasts. To look for biological proof of integration and replication of transferred nuclear genes, two complementing, chlorophyll-deficient, light-sensitive mutants of tobacco were used as sources of nuclei and receptor protoplasts. Ca. 5.5 × 10(7) receptor protoplasts were cultured following transplantation experiments involving these complementing mutants and about 1.8 × 10(7) of the resulting calli were subjected to selective conditions which discriminate against the parental types. No nuclear hybrids were detected, although in control experiments somatic hybrids were obtained by protoplast fusion. Some explanations for failure of nuclear hybrid formation are discussed together with other possible approaches for selective somatic combination of plant cell genophores.

Single androgenetic structures of maize (Zea mays L.) for the initiation of homogeneous cell suspension and protoplast cultures.

Induction of androgenesis in maize leads to heterogeneous callus types in terms of chromosome number and regeneration ability. In order to obtain homogeneous cell suspensions from androgenetic material we initiated maize cultures from single microspore-derived structures. Cultures were established either by transfer of previously selected type-II callus or by transfer of single aggregates directly into suspension medium. During establishment no selection was performed. Independent suspension lines were analysed with respect to their chromosome number, their embryogenic capacity, and their regeneration ability. Cytological analysis revealed that most of the cultures tested showed a constant chromosome number over a period of 18 months, indicating a homogeneous constitution. Within regenerable lines no difference in embryogenic capacity could be observed when smaller (1mm) and larger (4mm) aggregates were compared. Furthermore, their homogeneous character was confirmed by the regeneration of only green or albino plants from the individual lines. Chromosome analysis of regenerants by root tip cytology showed that their numbers corresponded to that of the suspension they were regenerated from. Similar results showing the homogeneous nature of the lines were obtained in protoplast culture and regeneration experiments.

Plant regeneration and initiation of cell suspensions from root-tip derived callus of Oryza sativa L. (rice).

Root-tip derived suspended callus of Oryza sativa cv. Thaipei showed the capacity for plant regeneration via organogenesis. Cell cultures were induced in liquid Murashige-Skoog medium containing 2 mg/l 2.4-dichlorophenoxyacetic acid. Dicamba or Picloram were effective for induction of organogenesis. Shoots and roots differentiated following subculture on medium lacking auxins but containing kinetin. At 1 and 4 mg/l Dicamba and 1 mg/l Picloram normal green plants were regenerated whereas with 7 mg/l Dicamba in the medium only albino plantlets were obtained. Regenerated plantlets were grown to maturity and set seed. Cell suspension cultures, initiated from the root-tip derived calli, provided suitable material for protoplast isolation.

Initiation of morphogenic cell-suspension and protoplast cultures of barley (Hordeum vulgare L.).

Cell-suspension cultures were initiated from embryogenic calli of various barley cultivars. Seven fast-growing suspension lines were obtained from four different cultivars (cvs. Dissa, Emir, Golden Promise and Igri). Two of these cell suspensions showed morphogenic capacity. From a cell suspension of cv. Dissa, albino plantlets were regenerated when aggregates were cultured on solid medium. Aggregates of cv. Igri usually stopped differentiation at the globular stage, but occasionally formed scutellum-like structures. Five suspension lines were used for protoplast isolation and culture. Dividing protoplasts were obtained from all lines, but with cv. Igri a few divisions only and no further development were observed. Protoplasts from the various lines differed in the time of first division (2-14 d), division frequency (0.09-70.9%) and efficiency of colony formation (0.09-7.3%). Protoplasts isolated from the morphogenic cell suspension of cv. Dissa developed compact calli which sporadically regenerated albino plantlets.

A segment of rye chromosome 1 enhances growth and embryogenesis of calli derived from immature embryos of wheat.

The influence of the short arm of rye chromosome 1 (1RS) from Secale cereale var. Imperial on the growth and differentiation of callus cultures from wheat Triticum aestivum var. Chinese Spring immature embryos was analysed. This chromosome arm was found to stimulate both embryogenesis and the rate of growth of calli. Recombinant lines carrying segments of 1RS were used to delineate the regions of 1RS responsible for the tissue culture effects. The enhancement of embryogenesis and the stimulation of growth were shown to be associated with two distinct genetic regions of the chromosome arm; the former is located between the centromere and the Sec 1 locus, while the latter is situated in the immediate vicinity of the Sec 1 locus.

Fertile transgenic wheat from microprojectile bombardment of scutellar tissue.

A reproducible transformation system for hexaploid wheat was developed based on particle bombardment of scutellar tissue of immature embryos. Particle bombardment was carried out using a PDS 1000/He gun. Plant material was bombarded with the plasmid pDB1 containing the beta-glucuronidase gene (uidA) under the control of the actin-1 promoter of rice, and the selectable marker gene bar (phosphinothricin acetyltransferase) under the control of the CaMV 35S promoter. Selection was carried out using the herbicide Basta (Glufosinate-ammonium). From a total number of 1050 bombarded immature embryos, in seven independent transformation experiments, 59 plants could be regenerated. Putative transformants were screened for enzyme activity by the histochemical GUS assay using cut leaf material and by spraying the whole plants with an aqueous solution of the herbicide Basta. Twelve regenerants survived Basta spraying and showed GUS-activity. Southern-blot analysis indicated the presence of introduced foreign genes in the genomic DNA of the transformants and both marker genes were present in all plants analysed. To date, four plants have been grown to maturity and set seed. Histochemically stained pollen grains showed a 1:1 segregation of the uidA gene in all plants tested. A 3:1 segregation of the introduced genes was demonstrated by enzyme activity tests and Southern blot analysis of R1 plants.

Representative cDNA libraries from few plant cells.

A reverse transcriptase/polymerase chain reaction (RT/PCR) method for the construction of representative cDNA libraries originating from few isolated cells is described. Poly(A)+ RNA was extracted from an average of 100 maize cells and reverse transcribed into sscDNA. The sscDNA was dG-tailed at its 3' end and amplified during a two-step PCR reaction. The generated PCR products were analysed and the majority < or = 2 kbp were full-size cDNAs. A fraction of the amplified cDNA from 128 isolated maize egg cells was cloned into the lambda Uni-ZAP XR vector and a primary library of 6.8 x 10(6) p.f.u. was obtained. The average insert size is 860 bp. It was further determined, that 0.31% of the clones hybridized to a cytosolic GAPDH probe. It is thought that, with this method, the first cDNA library of egg cells in higher plants was generated.

Isolation of viable egg cells of rape (Brassica napus L.).

The isolation of viable egg cells of rape (Brassica napus L.) has been achieved from microdissected ovules. The non-gametic cells of the embryo sac, synergids and central cells have also been isolated. Their morphology corresponded to that of these cells in situ, making a discrimination from isolated sporophytic cells possible. Two hours after isolation the egg cells were still viable. Viable egg cells have been reproducibly isolated with a frequency of 25% per dissected ovule.

Plant regeneration from protoplasts of wild barley (Hordeum murinum L.).

We have produced a large number of plants regenerated from protoplasts originally isolated from embryo-derived cell suspensions of wild barley, Hordeum murinum L.. Suspensions initially allowed protoplast isolation and culture 5.5 to 9 months from the date of callus initiation. Colony formation efficiencies ranged from 1.5 to 3.0 % and from 0.1 to 1.4 % for protoplast cultures with and without nurse cells, respectively. Both nurse and non-nurse techniques allowed efficient embryogenesis and plant regeneration. More than 400 shoots/plantlets have been obtained from 6 independent experiments. Over 150 plants have been transferred to the greenhouse. Protoplasts isolated from the youngest suspensions (5.5 months old) gave rise to the largest number of plants. Protoplasts isolated from suspensions as old as 15 months were also regenerable.