In-vitro morphogenesis of corn (Zea mays L.)

The objective of this research was to study the in-vitro morphogenetic pattern of corn (Zea mays L.) shoot tips excised from aseptically-grown seedlings, and of expiants of axillary shoot buds, immature tassels and ears (staminate and pistillate inflorescences) obtained from greenhouse-grown corn plants. The seedling shoot tips and immature ears first regenerated clumps of multiple shoots within four weeks of culture on Murashige and Skoog (MS) basal medium supplemented with 500 mg/L casein hydrolysate (CH) and 9.0 M N⁶-benzyladenine (BA). Multiple shoot clumps were also differentiated from spikelets of immature tassels cultured on MS medium containing 500 mg/L CH, 4.5 M BA and 0.45 M 2,4-dichlorophenoxy acetic acid (2,4-D). All these multiple shoot clumps in turn differentiated clusters of ears after further four subcultures at four-week intervals under light on MS medium supplemented with 500 mg/L CH and 2.25, 4.5, 9.0 or 18 M BA. Axillary shoot buds readily differentiated clusters of ears within four weeks of the initial culture on these media. Secondary and tertiary ear clusters were initiated following subculture of primary ears on MS medium containing 500 mg/L CH and 4.5 or 9.0 M BA. Most of the ear primordia developed into ears with well-developed ovaries and styles on subculture on MS medium containing 500 mg/L CH and 1.0 M BA. Corn kernels were obtained after pollination of in-vitro-formed ears with pollens collected from greenhouse-grown corn. These kernels germinated in vitro and developed into mature corn plants in the greenhouse. Clusters of tassels were also differentiated in darkness from the multiple shoot clumps after six months successive subcultures but the spikelet primordia of tassels failed to develop fully under the in-vitro conditions tested. Somatic embryos arose directly from spikelet primordia of young tassels or ears on MS medium containing 500 mg/L CH and 4.5 M 2,4-D, or indirectly from calli derived from spikelets of young tassels and ears on MS medium containing 500 mg/L CH and 9.0 M 2.4-D.Abbreviations BA N⁶-benzyladenine - CH casein hydrolysate - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (basal medium) Heng Zhong is a Rockefeller Foundation Fellow on leave from the Institute of Botany, Academia Sinica, Beijing, P.R. China. This work was supported by a grant from the MidWest Plant Biotechnology Consortium and U.S.-A.I.D. grant No. DAN-4197-A-00-1126-00 to M.B. Sticklen. Thanks are due to Illinois Foundation Seeds, Champaign, USA for the supply of Honey N Pearl sweetcorn seeds and the Services of Center for Electron Optics, Michigan State University, for the electromicroscopic work as related to this publication.     

Suppression of somatic embryogenesis in Citrus cell cultures by extracellular proteins

Nucellar-derived cell cultures of sour orange (Citrus aurantium L.) proliferate as proembryogenic masses. By a change in the carbon source of the medium from sucrose to glycerol they are induced to undergo synchronous embryogenesis forming embryo initials that develop into globular embryos. The proembryogenic masses released glycoproteins to the medium. Exogenous addition of the glycoproteins to cells in glycerol-containing medium modified the course of embryo development in a dose-dependent manner. Addition of 20 g · ml^–1 of glycoproteins blocked embryogenesis and resulted in an accumulation of embryo initials. When glycoproteins were added to cultures containing advanced globularstage embryos further development was suppressed. The inhibitory component of the glycoproteins was found to be a family of polypeptides with apparent molecular masses of 53–57 kDa. While these proteins normally accumulated only in cultures of proembryogenic masses, they could be induced to accumulate in glycerol-containing medium by the addition of the glycoproteins. Thus, their accumulation was not a direct consequence of the type of growth medium used or the developmental state of the cultures. The results indicate that the 53-to 57 kDa glycoproteins could play a regulatory role in in-vitro embryogenesis in sour orange. The normal progression of embryo development appears to depend, in an obligatory manner, on the absence of these glycosylated extracellular proteins from the medium.Abbreviations kDa kilodalton - PEM proembryogenic masses - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - 2D-PAGE Two-dimensional polyacrylamide gel electrophoresis We thank Dr. S. Satoh (Institute of Biological Sciences, Tsukuba, Japan) for sending protein samples of the purified 57-kDa glycoprotein. This research was supported by a grant from the Charles H. Revson Foundation for Basic Research in the Life Sciences of the Israel Academy of Sciences. R.F. is a recipient of the Jack and Florence Goodman Career Development Chair.     

Enclosure of bacteria by the rough endoplasmic reticulum of shrimp hepatopancreas cells

Summary Zygotic embryos ofArabidopsis thaliana showed three different types of developmental response, when cultured in vitro: (1) normal development, (2) formation of morphogenetic callus, and (3) somatic embryogenesis. Early zygotic embryos were mechanically isolated and inoculated into different volumes of various culture media. It was possible to isolate embryos to the octant stage. Survival and further development in culture were observed in embryos to the early globular stage. Culture success increased with the initial size of the cultivated embryos. Neither the volume of the culture medium nor its composition were found to significantly influence the proportion of embryos developing in vitro. Whereas normal development from stages beyond 35 m diameter was possible without phytohormones, callus formation was frequently observed in the presence of phytohormones, even if used at very low concentrations. Embryos smaller than 35 m formed callus even without added phytohormones, and the proportion of embryos undergoing callus formation decreased with increasing embryo size at the time of culture initiation. Shoot morphogenesis was easily induced in embryo derived callus. Somatic embryogenesis was reliably observed during the culture of embryos from later stages (post heart-shaped) in liquid medium on a shaker.Abbreviations IAA indoleacetic acid - KIN kinetin - NAA -naphthaleneacetic acid     

Induction of somatic embryogenesis and caulogenesis from cotyledon and leaf protoplast-derived colonies of melon (Cucumis melo L.)

Summary A procedure leading to the regeneration of whole plants from protoplasts of melon is described. Protoplasts were isolated from cotyledons and leaves of plants grown in vitro. After 14 days of culture, average viability and division rates were respectively 60% and 30% for the two organs, considering total initial protoplasts plated. The manipulation of the exogenous auxin / cytokinin balance in regeneration media enabled to direct morphogenesis towards somatic embryogenesis (1 mg·l^–1 2,4-dichlorophenoxyacetic acid and 0.1 mg·l^–1 6-benzylaminopurine) or caulogenesis (0.5 mg·l^–1 6-benzylaminopurine and 0.5 mg·l^–1 kinetin). Contrary to division ability, regeneration capacity was genotype-dependent under our conditions, but the two organs expressed similar division and regeneration capacities. Maltose was superior to sucrose for the development of caulogenic nodules into buds. Some plants were transplanted to soil, where they appeared to be fertile and produced seeds.Abbreviations BAP 6-benzylaminopurine - CPW Cell and Protoplast Washing medium - KIN kinetin - MES 2-(N-morpholino) ethanesulfonic acid - MS Murashige and Skoog (1962) - NAA 1 — naphthaleneacetic acid - PAS H (staining), Periodic Acid-Schiff / Hematoxylin - 2,4-D 2,4-dichlorophenoxyacetic acid     

Adventitious shoot induction and somatic embryogenesis with intact seedlings of several hybrid seed geranium (Pelargonium x hortorum Bailey) varieties

Summary Intact seedlings of hybrid seed geranium (Pelargonium x hortorum Bailey) were tested for their ability to produce adventitious shoots and somatic embryos by direct culture of mature seeds on Murashige and Skoog (1962) medium (MS) supplemented with growth regulators BAP, BAP + IAA, or thidiazuron (TDZ). Ten varieties were tested in the presence of different BAP concentrations, four with BAP + IAA, and two with TDZ. Varieties used in this study differed in their response to BAP in the medium. Multiple adventitious shoots were produced by seven of the ten varieties tested. Multiple adventitious shoots were induced at all levels of TDZ in the medium. TDZ also induced callusing from roots and direct embryogenesis from intact hypocotyls. Adventitious shoots were separated, rooted and transferred to soil where they grew as normal healthy plants and flowered.Abbreviations BAP N⁶-benzylaminopurine - IAA indole-3-acetic acid - MS Murashige and Skoog (1962) - TDZ thidiazuron     

Contrasting pattern of somatic and zygotic embryo development in alfalfa (Medicago sativa L.) as revealed by scanning electron microscopy

Scanning electron microscopy has been used to investigate the morphological changes occurring during the development of alfalfa somatic embryos. Embryos were initiated from callus, transferred to suspension culture and matured on solid agar medium. This developmental pattern was compared to that of zygotic embryos developing in ovulo. Somatic embryos begin as distinct pro-embryos within the callus tissue pieces placed in suspension culture. They become globular and heart-shaped while on solid agar medium and then undergo cotyledon elongation and maturation. Somatic embryos develop comparatively slower at early stages of development and faster at the later stages than zygotic embryos. They lack a well-defined suspensor and have a very rough, poorly-differentiated epidermis, the first layer of which is lost after pro-embryo formation. The cotyledons of somatic embryos are multiple and poorlydeveloped; there appears to be a correlation between the amount of surface roughness of the developing embryo and the extent to which polycotyledony occurs.     

First evidence of somatic embryogenesis from needles of 1-year-old Picea abies plants

Summary Somatic embryogenesis was obtained from hypocotyls and cotyledons of one month old plantlets of Picea abies. Embryogenic yield was higher with expiants from somatic embryo-derived plantlets (80 %) than with plantlets issued from zygotic embryos (10 %). This report also describes production of embryogenic calli from needles of 14 month old somatic embryo-derived plants cultivated in greenhouse. The influence of the physiological status and genotype of the mother plant on somatic embryogenic potential is discussed.Abbreviations ABA abscisic acid - (±) ABA racemic ABA - BAP 6-benzylaminopurine - CI callus inducing culture medium - NAA 1-naphtaleneacetic acid     

Somatic embryogenesis in polyembryonic Secale cereale L.

Summary The progeny of polyembryonic Secale cereale L., was used to study the in vitro response of the immature embryos. The formation of embryogenic calli was very high, and this response and its distribution was statistically different to that shown by the normal regenerated plants and the original population. This behaviour seems to be related to a genetic condition which favours the presence of supernumerary embryos, in vivo as well as in vitro.     

Embryogenic cell suspensions of Triticum aestivum x Leymus angustus F1 hybrids: characterization and plant regeneration

Summary Embryogenic cell suspension cultures were established from Triticum aestivum X Leymus angustus F₁ hybrids, using compact nodular calli derived from inflorescence segments. Calli originating from leaf segments did not give rise to stable cell suspensions. Growth measurements of the cell suspensions revealed that they continued rapid growth up to 10 days after subculturing. Flow cytometric studies of the cell cycle over a 7 day culture period showed that the majority of cells were in G₁ phase while the rest were either in S or G₂. During the 7 days of culture, no significant differences in DNA distribution patterns were observed. The cells from suspension cultures produced somatic embryos when they were transferred to different solid media. The embryos germinated and gave rise to plantlets which were successfully rooted and transferred to soil.