Leaflet development, induction time, and medium influence somatic embryogenesis in peanut (Arachis hypogaea L.).

Factors affecting somatic embryogenesis in peanut (Arachis hypogaea L.) using leaflet explants of seedlings obtained from aseptically germinated embryo axes were evaluated. Somatic embryogenesis was influenced by developmental stage, leaflet size, induction medium, and time on induction medium. Leaflets that were 5-7 mm long had a greater embryogenic response than smaller or larger leaflets. Percent embryogenesis and mean number of embryos were related to the developmental stage of germinating seedlings. A greater response was obtained if leaflets were folded and closely appressed. Preselection of leaflets increased percent embryogenesis from 21% up to 67%. As leaflets unfolded, embryogenesis decreased; open leaflets lost the potential for embryogenesis. The optimal induction conditions were a 7-day incubation period on Murashige and Skoog medium with 136 µM 2,4-dichlorophenoxyacetic acid and 0.93 µM kinetin. Somatic embryos germinated to form plants that exhibited a normal morphology.

High frequency somatic embryogenesis in peanut (Arachis hypogaea L.) using mature, dry seed.

Peanut (Arachis hypogaea L.) somatic embryos were produced from the embryo axes of mature, dry seeds of cultivar GK-7. Percent embryogenic explants ranged from 88-100% using 10-40 mg/1 of 2,4-D in the induction medium. Neither 2,4-D concentration nor photoperiod during the induction period had a large effect on percent embryogenesis, mean number of embryos per explant, or embryo morphology. However, embryos obtained from cultures grown in the dark were easier to remove from the explant than those under a 16-h photoperiod. Somatic embryos developed on the epicotyl portion of the embryo axis, primarily on the young, expanding leaves. A survey of 14 genotypes indicated that genotype had a large influence on embryogenic capacity, with all genotypes being embryogenic to some extent. The ability to recover somatic embryos from axes of harvested, stored seeds represents significant advantages for the establishment of peanut embryogenic cultures, including the use of simple sterilization procedures and a constant source of explant tissue.

Influence of photoperiod and medium formulation on peanut somatic embryogenesis.

Somatic embryos were produced from peanut (Arachis hypogaea L.) immature zygotic cotyledons. Comparisons were made of the level of α-naphthaleneacetic acid during induction, nitrogen formulation of the medium, and photoperiod. Over 70% embryogenesis was obtained regardless of NAA level used. Percent embryogenesis and number of embryos were markedly lower in explants induced on NAA compared to 2,4-D. Embryo production was not greatly affected by either the use of Murashige & Skoog versus Finer & Nagasawa salts or light versus dark culture conditions. However, embryo morphology was noticeably affected by photoperiod. Embryos produced under a 16 h photoperiod were tough, woody and difficult to separate for subsequent germination and conversion. Those produced under a 0-h photoperiod were succulent and pliable.

The effect of auxin type and concentration on pecan (Carya illinoinensis) somatic embryo morphology and subsequent conversion into plants.

Embryogenic cultures were initiated from immature pecan zygotic embryos. Explants were induced for one week on Woody Plant Medium with either α-naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid at 2, 6 or 12 mg/l, then subcultured monthly to fresh basal medium. Observations were made on callus production, embryo formation, and embryo morphology. Somatic embryo morphology and overall callus proliferation were affected by auxin type. Callus proliferation was less extensive and more somatic embryos resembling zygotic embryos were obtained from cultures initiated with α-naphthaleneacetic acid than with 2,4-dichlorophenoxyacetic acid. Repetitive somatic embryogenesis was obtained in all auxin treatments. Conversion into plantlets was affected by somatic embryo morphology in that embryos with poorly developed apices exhibited lower percentages of conversion than those with well developed single or multiple apices. Consequently, although more embryos were obtained with 2,4-dichlorophenoxyacetic acid, naphthaleneacetic acid was the superior auxin for production of somatic embryos more likely to convert into plants.

Somatic embryogenesis and plant regeneration from leaflets of peanut, Arachis hypogaea.

Somatic embryos were induced on peanut (Arachis hypogaea) leaflets from aseptically germinated embryo axes. Leaflet size influenced percent somatic embryogenesis; 5-8 mm long cut leaflets were superior to 2-3 mm long uncut leaflets. Maximum embryogenesis of 14.6% was obtained after a 15 d incubation on induction medium (modified MS with B5 vitamins, 30 g/l sucrose, 4 g/l Gel-Gro, 40 mg/l 2,4-D +0.2 mg/l kinetin) followed by transfer to a secondary medium with 5 mg/l 2,4-D+0.2 mg/l kinetin. Primary somatic embryos were fused along the axes with no distinct cotyledons, but secondary embryos had single axes with two cotyledons. Other treatments had lower percent embryogenesis, no secondary embryogenesis, and embryos with single axes with two cotyledons. Some somatic embryos converted into normal plants capable of greenhouse survival.

Protoplast isolation and callus production from leaves of tissue-cultured Vitis spp.

Protoplasts were isolated from mesophyll of axenic cultures of grape, Vitis rotundifolia cv. Summit and V. vinifera cv. Cabernet Sauvignon. Enzymes effective for protoplast isolation were Macerozyme R-10 (0.5% and 0.1%) and Cellulase Onozuka R-10 (1.0% and 0.5%) for V. rotundifolia and V. vinifera, respectively. Polyvinylpyrrolidone and 2-(N-morpholino)ethanesulfonic acid were essential in the isolation media. Protoplasts were purified using flotation/centrifugation. The protoplasts of V. rotundifolia cultured in Gamborg's B5 basal medium with 2.2 µM 6-benzyladenine, 4.5 µM 2,4-dichlorophenoxyacetic acid and 0.4% agarose gave the best plating efficiency of conditions tried in this study. Cell division occurred within 5 to 6 days and visible microcalli developed within one month. After 6 weeks in culture, microcalli transferred to liquid medium exhibited active callus growth. Protoplasts of V. vinifera cultured under these conditions had similar results.

Effects of Quantum Flux Density on Photosynthesis and Chloroplast Ultrastructure in Tissue-Cultured Plantlets and Seedlings of Liquidambar styraciflua L. towards Improved Acclimatization and Field Survival.

Liquidambar styraciflua L. seedlings and tissue-cultured plantlets were grown under high, medium, or low (315, 155, or 50 microeinsteins per square meter per second photosynthetically active radiation) quantum flux densities. Net photosynthesis, chlorophyll content, and chloroplast ultrastructure of leaves differentiated from these conditions were investigated. Seedling photosynthetic rates at light saturation were positively related to light pretreatments, being 6.44, 4.73, and 2.75 milligrams CO(2) per square decimeter per hour for high, medium, and low light, respectively. Cultured plantlets under all light conditions had appreciably higher photosynthetic rates than noncultured seedlings; corresponding rates were 12.14, 13.55, and 11.36 milligrams CO(2) per square decimeter per hour. Chlorophyll in seedlings and plantlets was significantly higher in low light-treated plants. Seedling leaves had chloroplasts with abundant starch regardless of light pretreatment. In high light, starch granules were predominant and associated with disrupted granal structure. Low light seedling chloroplasts had smaller starch grains and well-formed grana. In contrast, tissue culture-differentiated leaves were devoid of starch; grana were well organized in higher quantum flux density treatments, but disorganized at low flux densities.

The lipid solubility of fixative, staining and embedding media, and the introduction of LX-112 and poly/bed-812 as dehydrants for epoxy resin embedment.

The manufacture of Shell, Epon-812 (E-812) resin has recently been discontinued. E-812 and two newly introduced E-812 substitutes, and Ladd-112 (LX-112) and the Polysciences, Poly/bed-812 (PB-812) resins, were studied biochemically and morphologically for their effectiveness as polar dehydrants. Their technical properties as general E-812 replacements were also explored. In the biochemical studies, acetone was more effective in retaining lung phospholipid components than ethanol, the resin dehydration was more effective than either acetone or ethanol. There was no appreciable difference in lipid solubility among the three resins. Acetone and uranyl magnesium acetate each had a loosening effect on previously fixed phospholipids. The PB-812 and E-812 resin dehydrated blocks of dense animal tissues, demonstrated serious technical difficulties during sectioning. The L-112 resin substitute, due to its low viscosity and improved infiltration, was found to be technically as effective a dehydrant as ethanol or acetone. None of the three resins was successful as dehydrating agents for the plant tissue. With organic solvent dehydration, both epoxy resin substitutes demonstrated excellent embedment properties with both animal and plant tissues.

The ultrastructural histochemistry and stereoscanning electron microscopy of the rodent and amphibian surfactant systems.

Ultrastructural histochemical precedures were employed to determine the carbohydrate components and their contributions to the rodent and amphibian surfactant systems. Zirconium stained the rodent (rat) cytoplasm surrounding the multilamellar bodies, the Golgi, and was associated with the membrane structures of the compact lamellae of alveolar multilamellar bodies. In the rodent and amphibian (Rana pipiens), ruthenium red stain was observed within all tubular myelin surfactant matricies. The "gutters," tubular myelin surfactant matrix, and intratubular myelin surfactant matrix materials all demonstrated a positive reaction product. The periodic acid-chromic acid-silver procedure revealed irregular channels extending from the multilamellar bodies to the surface of the rodent great alveolar pneumocyte. The extra-pulmonary and respiratory surfaces in both species were additionally studied by stereoscanning electron microscopy. The respiratory anatomy of the rodent was corroborated. The amphibian lung demonstrated three orders of septa, and in the expired state, tertiary septal pits. The amphibian primary septa were hollow, blind tubules containing respiratory surfaces.

The stereoscanning electron microscopy and ultrastructural histochemistry of the avian and reptilian surfactant systems: Indian dove, desert spiny and Taiwan golden skink lizards.

Lung tissues from the Indian dove, Scardafella inca, desert spiny lizard, Sceloporus magister, and the Taiwan golden skink lizard, Mabuya aurates, were studied by transmission electron microscopy utilizing ruthenium red as a carbohydrate stain and with the so-called lipid-carbohydrate retention procedures to elucidate the morphology of the surfactant systems. Stereoscopic scanning electron microscopic procedures were utilized for a comparative anatomical study of these three species, and the results were compared with the rat and frog in the companion article. The avian lung tissues demonstrated several perculiarities. The ciliated epithelial cells of the bronchus had cytoplasmic ciliated projections between the boundaries of mucus secreting cells. The discrete morphology of the main bronchus, secondary bronchi, parabronchi, and the air capillaries, and their three-dimensional morphologic perspective were elucidated. The skink illustrated an arrangement of primary, secondary, and tertiary septa, with elaborate tertiary septal pits, similar to the amphibian. All septa contained a solid connective tissue core. The desert lizard was similar to the skink except the tertiary septal pits were rudimentary. All three species had a modified great alveolar pneumocyte and a laminated surfactant which included a carbohydrate matrix material between layered phospholipid-based membranes. The ruthenium red additionally stained the homogeneous surface-lining material. A comparative analysis of the surfactant systems of these three species with each other, and with the rodent and amphibian in the companion article, were discussed in terms of phylogenetic origin.