Zygotic embryo explants of grape cultivar AXR#1 were isolated from maw-e seeds and cultured on medium supplemented with naphthoxy acetic acid beta-(NOA) and benzylaminopurine (BA). Embryo explants dedifferentiated to form embryogenic callus. Globular stage embryos were visible in 9-10 months. On transfer 10 a growth regulator free medium supplemented with charcoal these globular embryos underwent further stages of embryo development. In a period of 30-40 days embryogenic tissues turned into clumps of somatic embryos displaying different stages of development Cotyledonary stage embryos were separated and transferred to basal medium. These embryos developed into complete plants. Cold and desiccation treatment of somatic embryos significantly enhanced the rate of plant conversion. Hypocotyl segments of elongated somatic embryos were good source explant for induction of shoot organogenesis. The hypocotyl-length and the proximity to-shoot-apex were found to influence the rate of shoot induction from hypotyl segments. Multiple shoot complexes which formed on hypocotyl segments were separated and individual shoots were grown on a root induction medium resulting in complete plant development. The possibility of both embryogenic and organogenic modes of plant regeneration make somatic embryos a highly versatile explant source for experiments on genetic manipulation.
Don Waneck, H. Mathews, J. Stamp, and R. Bestwick
Abraham Cruz-Mendívil, Javier Rivera-López, Lourdes J. Germán-Báez, Melina López-Meyer, Sergio Hernández-Verdugo, José A. López-Valenzuela, Cuauhtémoc Reyes-Moreno, and Angel Valdez-Ortiz
and Levy, 2002 ; Meissner et al., 1997 ). The generation of transgenic plants involves the development of plant tissue culture and gene transfer procedures, in which the lack of efficient regeneration protocols is the limiting step ( Herrera
Y.D. Park, A.A. Boe, and M.K. Ehlenfeldt
Leaf disks of potato cv. Kennebec and ND 860-2 (North Dakota potato breeding clone) were cultured on Murashige Skoog (MS) medium supplemented with 6 levels of indole acetic acid (IAA) and 7 levels of zeatin riboside (ZR). Shoots were induced at various combinations of hormone levels. The medium containing 3.5 mg/l IAA and 4.0 mg/l ZR produced the most shoots. Rooted plantlets were grown in the greenhouse. The growth of regenerated plants obtained from the MS medium supplemented with 7.0 mg/l IAA and 3.0 mg/l ZR was significantly greater than those grown from nodal explants. In ND 8602, a leaf chimera with chlorophyll deficient (light yellow) sectors was found in plants regenerated from leaf disks (grown on MS medium supplemented with 3.5 mg/l IAA and 3.0 mg/l ZR) but not in plants grown from nodal explants. Phenotypic variability was also observed for tuber number, size and weight.
Violeta Colova-Tsolova, Rachel Gollop, Sharon Farchie, Sylvie Even, Nahman Shahar, and Avi Perl
Embryogenic cell suspension was developed from in vivo anthers of seedless grape cv. Sugarone. Agrobacterium genetic co-transformation was realized with two vectors carried respectively two different reporter genes: hpt and nptII, and three (1+2) agronomicaly beneficial genes encoding for proteins that are involved in fungus disease resistance. The effciency of transformation procedure and integraton of foreign genes was verified by hystochemical assay as a first step after insertion in embryogenic suspension two different constructs with Gus-reporter gene under control of different promotors. PCR assay and Southern blot analysis were used to confirm the co-transformation in regenerated grape plants.
Wendy J. Wagoner, Jill A. Kellogg, Richard K. Bestwick, and James A Stamp
Broccoli and cauliflower are among the most regeneratively intractable genotypes found in the brassicaceae. To develop a method for transfer of the gene encoding S-adenosylmethionine hydrolase (SAMase) into inbred broccoli and cauliflower germplasm, we investigated the morphogenic competence and Agrobacterium susceptibility of a wide range of tissues of varied source. Appropriately controlled expression of the SAMase gene should, theoretically, reduce the plant's capacity for ethylene biosynthesis and extend the post harvest shelf life of the flower head.
Through examination of the in vitro response of a wide range of tissues we identified procedures which support caulogenesis from 100% of explants, each producing more than 30 shoots which readily convert to plantlets. Studies with several wild type and disarmed Agrobacterium strains, and utilization of the binary vector system and appropriate marker and reporter genes, led to the identification of methods for high frequency T-DNA transfer to explant tissues and the flow frequency of transgenic plants containing SAMase gene.
V.M. Gingas and B.D. Stokes
Funded by the Ohio Thomas Edison Program, Ohio Dept. of Development. We thank Hugh Daubeny for supplying plant material and Luann Henry and Amy McMullen for technical assistance. The cost of publishing this paper was defrayed in part by the
H. Lou and S. Kako
The embryogenic capacity of seven cucumber (Cucumis sativus L.) cultivars was examined by tissue culture of cotyledon, young first-leaf, and internode explants. Somatic embryogenesis frequencies differed significantly among the tested cultivars, and `Fushinarimidori' produced the highest number of embryos from either cotyledons or young first leaves. Cotyledon- and first-leaf-derived calluses produced more embryos than calluses from internodes. Somatic embryos were induced from `Aonaga F1' internodes. With relatively high sucrose levels (6% and 9%) in the initiation medium, the frequency of embryogenic callus formation from `Fushinarimidori' cotyledon explants was >90%. The highest yield of somatic embryos occurred in cultures initiated with high sucrose levels (9% or 12%), although 12% sucrose inhibited callus formation and growth. Somatic embryos germinated in a basal liquid medium supplemented with 0.5% activated charcoal, and they developed into well-shaped, healthy plantlets on semisolid medium with 1% sucrose.
Sachiko Matsubara and Hegazi H. Hegazi
Callus initiation and growth and plantlet regeneration were studied using eight cultivars of Raphanus sativus L., including six Japanese radishes, one Chinese and one small `Comet' radish. The basal medium was composed of Murashige and Skoog inorganic salts, 2.0 mg myo-inositol/liter, 0.5 mg each of nicotinic acid and pyridoxine·HCl/liter, and 0.1 mg thiamine·HCl/liter, 30 g sucrose and 2 g Gelrite/liter. High callus yields were obtained on basal medium containing (mg·liter-1) 0.1 2,4-D and 1.0 BA for two Japanese radishes and 0.1 NAA and 1.0 kinetin for `Comet' radish. Shoots were regenerated from callus by subculturing on basal medium containing 0.1 or 1.0 mg BA/liter and then transferring to basal medium. Rooting occurred on basal medium. Although callus was obtained in all eight cultivars, shoots and plantlets were regenerated only from `Moriguchi', `Nerima Shirinaga', and `Comet'. Chemical names used: 2-(l-naphthyl) acetic acid (NAA); N-(phenylmethyl)-lH-purine-6-amine (BA); 2,4-dichlorophenoxy acetic acid (2,4-D); 6-(furfurylamino)purine (kinetin).
Paula P. Chee
A procedure for the regeneration of muskmelon (Cucumis melo L.) cv. Topmark via shoot organogenesis from cotyledon explants is described. The best induction medium for a morphogenic response was MS salts and vitamins medium with BA at 1.0 mg·liter-1. Further vegetative bud development was completed by transferring organogenic tissue to MS medium containing BA at 0.05 mg·liter-1 . The shoots were rooted in MS medium containing NAA at 0.01 mg·liter-1. Morphologically normal plantlets were obtained. Chemical abbreviations used: 6-benzylaminopurine (BA); indoleacetic acid (IAA); naphthaleneacetic acid (NAA).
Masanori Kadota, Dong-Sheng Han, and Yoshiji Niimi
Anthers of six apple [Malus ×domestica (L.) Borkh.], three Japanese pear (Pyrus pyrifolia N.) and two European pear (Pyrus communis L.) scion cultivars were cultured. Callus formation occurred from anthers of all cultivars and androgenic embryogenesis was observed from all except P. pyrifolia `Kosui' and P. communis `La France'. Regeneration of adventitious shoots from anther-derived embryos was shown from all apple cultivars and P. pyrifolia `Shinko'. Many of these shoots did not grow or died on half-strength Murashige and Skoog medium (1962) with 4.4 μm BA and 0.5 μm IBA, whereas several shoots of apple `Starking Delicious' grew to plantlets. Chromosome counts of shoot apical cells of four clones derived from embryos of `Starking Delicious' showed that three clones were diploids and one clone comprised diploid and haploid shoots, suggesting that at least one clone originated from a microspore. Chemical names used: 3-indolyl-butyric acid (IBA); N6-benzyladenine (BA).