Kathryn Kamo

Regeneration of Zea mays L. from Embryogenic Callus

Regeneration of corn (Zea mays L.) from embryogenic callus of the inbred line A188 was investigated. Immature zygotic embryos cultured on N6 medium in the dark at 25 C produced a friable, embryogenic callus. Maturation of somatic embryos was enhanced by transferring the embryogenic callus after 3 wk to medium containing 6% sucrose and lacking 2,4-D. During the next 1-2 wk, as the somatic embryos developed, the cultures were transferred to light. When appropriately cultured, between 80% and 90% of the zygotic embryo explants produced embryogenic callus and then plants. An average of seven to eight plants was produced from the callus per zygotic embryo; 70%-80% of these plants were phenotypically normal when evaluated at a height of 7-10 cm. Of ca. 100 plants grown to maturity, 90% exhibited a normal phenotype.

The establishment of cell suspension cultures ofGladiolus that regenerate plants

SummaryInflorencence stalks from greenhouse-grownGladiolus plants of the cultivars ‘Blue Isle’ and ‘Hunting Song’ cultured on a Murashige and Skoog basal salts medium supplemented with 53.6 μM 1-napthaleneacetic acid formed a compact, not friable type of callus that regenerated plantlets. Cormel slices and intact plantlets of three cultivars (‘Peter Pears’, ‘Rosa Supreme’, ‘Jenny Lee’) propagated through tissue culture formed a friable type of callus when cultured on Murashige and Skoog basal salts medium supplemented with 2,4-dichlorophenoxyacetic acid. This friable callus readily formed a cell suspension when the callus was placed in a liquid medium. Plants were regenerated from two-month-old suspension cell cultures of the commercial cultivar ‘Peter Pears’ after the suspension cells had been cultured on solid medium.

Characterization of Somatic Embryo Development and Plant Regeneration from Friable Maize Callus Cultures

Friable, rapidly growing callus was produced from immature embryos of maize (Zea mays) inbred A188 and hybrids A188 x B73 and B73 x A188. The callus consisted of nodules with active superficial cell layers that produced new nodules and regenerated numerous plantlets by somatic embryogenesis. Early stages of somatic embryo development, observed in paraffin and plastic sections, indicated probable single-cell origin of the somatic embryos. Three genotypes of friable callus had somatic embryos of identical anatomy, although hybrids A188 x B73 and B73 x A188 regenerated more plants than inbred A188 after 6-9 mo in culture. Selection and subculturing of embryogenic regions of the friable callus greatly increased the plant regeneration capacity of the cultures.

Tissue specificity and expression level of gusA under rolD, mannopine synthase and translation elongation factor 1 subunit a promoters in transgenic Gladiolus plants

Abstract Transgenic plants of Gladiolus cv. Jenny Lee were developed that contain the bargusA fusion gene under either the mannopine synthase 2 (mas2), translation elongation factor 1 subunit α (EF-1α), rolD, or the cauliflower mosaic virus 35S (CaMV 35S) promoters. The relative level of gusA expression in leaves of five to ten independently transformed, in-vitro-grown plants representing each promoter was similar for transgenic plants containing the rolD and CaMV 35S promoter and 2.0-fold and 3.3-fold higher than the level for the mas2 and EF-1α promoters, respectively. The maximum level of gusA specific activity by leaves was 135–173 nmol 4-methylumbelliferone (4-MU)/h per milligram protein for plants containing either CaMV 35S or rolD as compared to only 27–38 nmol 4-MU/h per milligram protein for plants with either mas2 or EF-1α. Histochemical staining confirmed the relatively high level of gusA expression throughout the length of the older, 6-cm-long leaves of plants that contained bargusA under rolD, whereas gusA expression was infrequently observed throughout the older leaves of plants containing either the mas2 or EF-1α promoters. In contrast to the older leaves, staining showed that strong gusA expression was frequently observed throughout young leaves of plants with either the mas2, EF-1α, or rolD promoters. Roots of plants with the rolD and EF-1α promoters showed strong gusA expression specifically in 93% and 68%, respectively, of the root tips. Roots of the plants with the mas2 promoter showed strong gusA expression throughout the entire length of the root.

Stable transformation of maize: the impact of feeder cells on protoplast growth and transformation efficiency

SummaryThe importance of cell culture conditions, including the use of feeder cells, on protoplast growth and transformation in maize (Zea mays L.) was investigated. Total GUS activity, measured two days after transformation, was five-fold higher in protoplasts cultured on feeder cells compared to those grown in the absence of feeder cells. Since the specific activity of GUS was only slightly higher in the transformed protoplasts plated over feeder cells, the stimulation in transient gene expression resulted mainly from the improved environment provided by the feeder system. For stable transformation, either PEG treatment or electroporation of protoplasts was used to introduce the neo gene. When PEG was used, over 85% of the putative transformants (resistant to kanamycin) contained the neo gene. The combination of PEG transformation and the optimized cell culture protocol using feeder cells enabled the selection of about 100 stably transformed lines per gFW of cells. Electroporation was less efficient.

Effect of the cauliflower mosaic virus 35S, actin, and ubiquitin promoters on uidA expression from a Bar-uidA fusion gene in transgenic Gladiolus plants.

SummaryTissue-specific patterns and levels of gene expression were characterized in transgenic Gladiolus plants that contained the phosphinothricin acetyltransferase (bar)-β-glucuronidase (uidA) fusion gene under transcriptional control of the promoter from either the cauliflower mosaic virus 35S (CaMV 35S), duplicated CaMV 35S (2×CaMV 35S), rice actin (Act1), or Arabidopsis ubiquitin (UBQ3) promoters. The bar gene confers resistance to phosphinothricin (PPT)-containing herbicides and allowed selection of transgenic cells. The β-glucoronidase gene encoded by the uidA locus of E. coli functioned as a reporter gene. Maximum levels of β-glucuronidase (GUS) activity in leaves were 173, 112, 50, and 10 nmoles 4-methylumbelliferone h−1 mg−1 protein for transgenic plants with the bar-uidA fusion gene under the control of the CaMV 35S, 2×CaMV 35S, UBQ3, and Act1 promoters, respectively. There was frequently considerable variability in GUS activity between the leaves of a single plant, and levels of uidA expression varied between independently transformed plants for each promoter. Callus derived from transgenic plants showed much less variation in GUS expression than leaves. The mean level of GUS activity was significantly higher (over 3×) for transgenic lines of callus containing the CaMV 35S as compared to the UBQ3 promoter, and this confirmed the higher (2×) level of GUS activity in levels of plants with the CaMV 35S promoter as compared to the UBQ3 promoter. Tissue-specific patterns of uidA expression were determined by histochemical staining. Leaves 5–6 cm long from plants with any of the four promoters tested exhibited uidA expression primarily in the vasculature. Under all four promoters uidA was expressed more frequently in root tips as compared to leaves.

Resistance to Cucumber mosaic virus in Gladiolus plants transformed with either a defective replicase or coat protein subgroup II gene from Cucumber mosaic virus.

Transgenic Gladiolus plants that contain either Cucumber mosaic virus (CMV) subgroup I coat protein, CMV subgroup II coat protein, CMV replicase, a combination of the CMV subgroups I and II coat proteins, or a combination of the CMV subgroup II coat protein and replicase genes were developed. These plants were multiplied in vitro and challenged with purified CMV isolated from Gladiolus using a hand-held gene gun. Three out of 19 independently transformed plants expressing the replicase gene under control of the duplicated CaMV 35S promoter were found to be resistant to CMV subgroup I. Three out of 21 independently transformed plants with the CMV subgroup II coat protein gene under control of the Arabidopsis UBQ3 promoter were resistant to CMV subgroup II. Eighteen independently transformed plants with either the CMV subgroup I coat protein or a combination of CMV subgroups I and II coat proteins were challenged and found to be susceptible to both CMV subgroups I or II. Virus resistant plants with the CMV replicase transgene expressed much lower RNA levels than resistant plants expressing the CMV subgroup II coat protein. This work will facilitate the evaluation of virus resistance in transgenic Gladiolus plants to yield improved floral quality and productivity.

Stable transformation of Gladiolus by particle gun bombardment of cormels

Transgenic plants of Gladiolus were produced following particle bombardment of cormel slices. Plant cells were cotransformed with the gene for phosphinothricin acetyltransferase under control of the cauliflower mosaic virus 35S promoter and the uidA gene coding for β-glucuronidase (GUS) under control of the actin promoter isolated from rice. The optimum concentration for the first selection of transformed plants was 8 mg/l phosphinothricin which resulted in 14% of the bombarded cormel slices regenerating plants that were transformed as confirmed by polymerase chain reaction amplification. Polymerase chain reaction amplification, Southern hybridization and histochemical staining for GUS gene expression on plants after two selective screenings with phosphinothricin indicated that regenerated plants were transformed. Histochemical staining for GUS gene expression showed that the actin promoter resulted in GUS gene expression primarily in callus cells and root meristems. Leaves were typically chimeric for GUS gene expression.

Effect of phytohormones on plant regeneration from callus ofGladiolus cultivar “Jenny Lee”

SummaryCallus capable of plant regeneration was initiated at a higher frequency from the basal leaves of in vitro plants (70% explants) as compared to cormel slices (30% explants) when cultured on medium containing various concentrations of auxin. The greatest number of plants were regenerated from 4-mo.-old callus (112 plants/g fresh weight callus) cultured on medium containing 10 mg/liter (53.8µM) 1-napthaleneacetic acid. The addition of 2 mg/liter (9.3µM) kinetin to a Murashige and Skoog’s basal salts regeneration medium resulted in an average two- to three-fold increase in the number of plants regenerated compared to regeneration on medium without hormones. Ten months after callus initiation, all callus maintained on auxin-supplemented media showed a drastic reduction in its capacity to regenerate plants. Ten-month-old callus maintained on dicamba regenerated the greatest number of plants (14 to 23 plants regenerated per gram fresh weight callus) as compared to callus maintained 10 mo. on medium containing 1-napthaleneacetic acid or 2,4-dichlorophenoxyacetic acid. Cormel slices cultured on cytokinin-supplemented media formed small amounts of callus which regenerated up to 19 plants per cormel slice within 1 to 2 mo. after the cormel slice had been placed on either 10 mg/liter (49.2µM) N6-2-isopentenyladenosine or 1 mg/liter (4.4µM) 6-benzylaminopurine.

The 5′UTR-intron of the Gladiolus polyubiquitin promoter GUBQ1 enhances translation efficiency in Gladiolus and Arabidopsis

BackgroundThere are many non-cereal monocots of agronomic, horticultural, and biofuel importance. Successful transformation of these species requires an understanding of factors controlling expression of their genes. Introns have been known to affect both the level and tissue-specific expression of genes in dicots and cereal monocots, but there have been no studies on an intron isolated from a non-cereal monocot. This study characterizes the levels of GUS expression and levels of uidA mRNA that code for β-glucuronidase (GUS) expression in leaves of Gladiolus and Arabidopsis using GUBQ1, a polyubiquitin promoter with a 1.234 kb intron, isolated from the non-cereal monocot Gladiolus, and an intronless version of this promoter.ResultsGladiolus and Arabidopsis were verified by Southern hybridization to be transformed with the uidA gene that was under control of either the GUBQ1 promoter (1.9 kb), a 5′ GUBQ1 promoter missing its 1.234 kb intron (0.68 kb), or the CaMV 35 S promoter. Histochemical staining showed that GUS was expressed throughout leaves and roots of Gladiolus and Arabidopsis with the 1.9 kb GUBQ1 promoter. GUS expression was significantly decreased in Gladiolus and abolished in Arabidopsis when the 5′UTR-intron was absent. In Arabidopsis and Gladiolus, the presence of uidA mRNA was independent of the presence of the 5′UTR-intron. The 5′-UTR intron enhanced translation efficiency for both Gladiolus and Arabidopsis.ConclusionsThe GUBQ1 promoter directs high levels of GUS expression in young leaves of both Gladiolus and Arabidopsis. The 5′UTR-intron from GUBQ1 resulted in a similar pattern of β-glucuronidase translation efficiency for both species even though the intron resulted in different patterns of uidA mRNA accumulation for each species.