Loren C. Stephens

Transformation of Solanum brevidens using Agrobacterium tumefaciens

Leaf pieces of in vitro-cultured plantlets of the wild potato species Solanum brevidens Phil. were cocultivated with Agrobacterium tumefaciens that contained nptII and uidA genes on the disarmed plasmid pBI121. Independent transgenic shoots were regenerated from solidified and liquid medium that contained 50 mg l−1 kanamycin. Two Agrobacterium strains were investigated for transformation efficiency. GV2260, which contained p35SGUSINT, resulted in a 11% transformation frequency, compared with 1% using LBA4404. Transformation rates were 7% in liquid culture and 3% on solidified medium. All kanamycinresistant, putatively transformed plantlets were confirmed positive by histochemical GUS assays. GUS activity in 22 independently transformed plants was quantified by fluorometric assay. Southern analysis of randomly selected transgenic plants showed that each transgenic plant contained at least one copy of the uidA gene.

Influences of medium consistency and shoot density on in vitro shoot proliferation of Populus alba × P. grandidentata

The in vitro shoot proliferation of Populus alba × P. grandidentata was affected by the medium consistency and shoot density, but not by three sizes of vessels. After 4 weeks of culture, the fresh weight and number of shoots per explant on liquid medium were significantly greater than those on agar-solidified medium. In particular, 3.2 shoots, 7 mm or longer per explant, were produced on liquid medium compared with 1.6 shoots per explant or agar-solidified medium. The fresh weight per explant after 4 weeks of culture on liquid medium and agar-solidified medium were 0.68 and 0.25 g, respectively. Increasing the number of shoots per vessel slowed the growth of the explants as measured by fresh weight and the number of shoots produced. There was little difference in the number of shoots produced between vessels with 1 or 2 shoots per vessel, but there were many fewer shoots produced when 3 shoots were placed in each vessel.

Effect of maternal genotype on development ofPhaseolus vulgaris L. ×P. lunatus L. interspecific hybrid embryos

SummaryCertainPhaseolus vulgaris L. ×P. lunatus L. crosses were performed to study the effect of maternal heterozygosity on development and growth of the interspecific hybrid embryos. Interspecific embryos had a much slower growth rate in vitro compared with embryos derived from self-pollination ofP. vulgaris parents. Thus, interspecific embryos could be identified by growth rate in vitro. TheP. vulgaris maternal genotype affected both the number and size of 15-day-old interspecific embryos. Specifically, ‘76 Spartan Arrow’ produced significantly more interspecific embryos than did ‘Great Northern’ as the seed parent, while maternal intraspecific hybrids produced smaller embryos than did maternal pure lines. There were no reciprocal differences between hybrid maternal parents for embryo number or size. Embryo size at excision and final size after culturing were closely correlated (r2=+0.93). The crossP. vulgaris ‘76 Spartan Arrow’ ×P. lunatus P.I. 214170 produced both the largest mean size at excision and the fastest growth in culture, indicating that specific combining ability affected both characteristics.

Growth regulators affect in vitro propagation of two interspecific Impatients hybrids

Abstract Two Impatiens L. interspecific hybrids were propagated in vitro by culturing shoot tips on agarsolidified MS revised medium containing one of 3 cytokinins (BA, 2iP, kinetin) or a cytokinin plus NAA. BA was most effective for stimulating shoot multiplication on ‘T63-1’, a Java (J) × New Guinea (NG) interspecific hybrid for which 9.3 shoots per explant were obtained at 10 μM. For ‘Star Fire’, a Celebes (C × NG interspecific hybrid, the most shoots per explant occurred on medium with 2iP at 40–60 μM. Multiple shoot production was strongly inhibited on explants of both genotypes by the use of 80 μM of both BA and 2iP. Shoot elongation also was inhibited as cytokinin concentration increased. The fewest but longest shoots of both genotypes were stimulated with kinetin. In the presence of NAA, shoot multiplication was promoted on explants of ‘T63-1’ but inhibited on explants of ‘Star Fire’.

Plants regenerated from embryo cultures of an apomictic clone of Kentucky bluegrass (Poa pratensis L. 'Baron') are not apomictic in origin

Plants were regenerated from tissue cultures of embryos dissected from seeds that were harvested from a self-pollinated clonal selection of Kentucky bluegrass (Poa pratensis L.) ‘Baron’, an apomictic cultivar. Plants were regenerated from 35 embryo-derived callus cultures of the 3280 embryos that were plated. Flow-cytometric (FCM) and RAPD-marker analyses were performed to determine if regenerants were or were not apomictic in origin. Fifteen regenerants with a 3c DNA content were classified as arising from 2n + n aberrant embryos, which was a higher frequency than expected, based on a chi-square analysis. Of the remaining 20 regenerants with a 2c DNA content, a chi-square test showed that all could have arisen from n + n sexually-derived embryos, based on the observed segregation of n + n regenerants, which fit the expected 3:1 ratio of dominant:recessive RAPD-marker phenotypes. The apparent lack of regenerants of apomictic origin, and implications for genetic transformation and breeding of Kentucky bluegrass are discussed.

Ethylene and Ethane from Poa pratensis Callus and from Leaf Blades of Regenerated and Seed-Derived Plants Inoculated with Bipolaris sorokiniana*

Summary Ethylene and ethane production from Poa pratensis callus, from intact leaf blades of plants regenerated from callus, and from intact leaf blades of plants from seed inoculated with Bipolaris sorokiniana was determined during pathogenesis. Ethylene increased from callus and leaf blades in response to inoculation. Ethylene increased progressively from 24 to 96 h after inoculation of callus, whereas, ethylene from inoculated leaf blades peaked at 48 h and then declined through 96 h. The magnitude of ethylene production from leaf blades of plants regenerated from callus was greater than that produced from leaf blades of plants from seed. Ethane from inoculated callus increased sharply at 72 and 96 h after inoculation, whereas that from inoculated leaf blades of plants regenerated from callus and from seed declined at 72 and 96 h. The ethane generated from inoculated callus and leaf blades establishes the gas as a product of pathogenesis in this host-pathogen interaction. The decline in ethylene and ethane in leaf blades at 96 h is believed due to a loss of cell and tissue integrity as pathogenesis progressed and symptoms were expressed by the host. Differences in ethylene production between leaf blades of plants from callus and seed, implications of ethane production during pathogenesis, and the potential use of callus culture for pathogenesis research are addressed in the discussion.

Induction of cathepsin D inhibitor gene expression in response to methyl jasmonate

Summary Two plant species, potato ( Solanum tuberosum cv. Superior) and a nontuberizing potato species ( Solanum brevidens ), were tested for methyl jasmonate induction of the potato cathepsin D inhibitor at the RNA level using a leaf-petiole cutting system. Both species had previously shown accumulation of cathepsin D inhibitor transcripts in leaves in response to wounding. Methyl jasmonate, a hormone-like, lipidderived molecule, is considered to be a secondary messenger in the wound-signal transduction pathway and is a potent inducer of proteinase inhibitor II. A dose-response test was performed for methyl jasmonate induction of cathepsin D inhibitor in the leaves of leaf-petiole cuttings. Transcript induction levels were directly related to the concentration of methyl jasmonate. Cathepsin D inhibitor transcripts could be detected in the Solanum species after only 6 h of methyl jasmonate treatment. Immunoblot analysis showed cathepsin D inhibitor proteins accumulated in «Superior» leaves after methyl jasmonate treatment.

Agrobacterium T-DNA mutation causes the loss of GUS expression in transgenic tobacco

SummaryA new freezer stock of an Agrobacterium tumefaciens clone of LBA4404/pBI121, designated 8999, was found to contain a mutation in the T-DNA region. GUS activity in Agrobacterium 8999 was reduced to levels in negative controls of LBA4404. Additionally, GUS activity in T1 seedlings from tobacco plants transformed with 8999 was reduced to that of untransformed plants. Southern and northern blotting showed that Agrobacterium clone 8999 transferred its T-DNA into the plant, that the correct sizes of 35S promoter and GUS coding region were integrated into the plants genome in the correct orientation, but that no transcript was detectable after 24 h. Genomic DNA from a T1 seedling from 8999 transformation, digested with HpaII and MspI, indicated no methylation in the promoter region. We conclude from this data that Agrobacterium 8999 has a stable mutation that reduces expression at the mRNA level and is responsible for the lack of GUS expression in plants transformed with this Agrobacterium clone. Therefore, unselected genes within the T-DNA region may suffer mutations in Agrobacterium.

Expression of a chimeric proteinase inhibitor II-GUS gene in transgenic Solanum brevidens plants

Summary A wild nontuberizing potato species Solanum brevidens Phil, was transformed with Agrobacterium tume-faciens strain GV2260 containing chimeric neomycin phosphotransferase and potato proteinase inhibitor II ( Pis 4) promoter-GUS ( uid A) genes on the disarmed plasmid pRT210. Several independent transgenic plantlets were regenerated on kanamycin-containing medium. Analysis of polymerase-chain-reaction products followed by Southern blot hybridization confirmed the presence of uid A genes in three of the transformants. Transgenic plants were tested for methyl jasmonate, sucrose, and wound induction of the pinII-GUS genes at the RNA and protein levels using leaf-petiole cuttings and whole plants. Northern blot analysis showed that the Pis4-uidA transgene can be induced by methyl jasmonate and sucrose. Methyl jasmonate-induced GUS activity in transgenic plants was up to fifteen times higher than basal levels after 12-h incubation and up to twenty-three times higher than basal levels after 24 h. Wounding of transgenic S. brevidens leaves on whole plants resulted in a substantial induction of GUS activity both locally and systemically.

Carbohydrate and nitrogen sources affect respectively in vitro germination of immature ovules and early seedling growth of Impatiens platypetala Lindl.

In vitro germination of 20-day old immature ovules of Impatiens platypetala Lindl. was inhibited at concentrations as low as 50 mM sucrose or mannitol and 100 mM glucose. Younger ovules (12, 14, and 16 days old) were similarly inhibited at 100 mM sucrose.Inorganic nitrogen concentration did not affect germination regardless of ovule age, but seedling fresh weight was significantly less and abnormal development of seedlings was significantly increased by total inorganic nitrogen concentrations higher or lower than 30 mM (at a ratio of 20: 10 mM NO3-: NH4+) in the culture medium.