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Neil L. Heckman, Garald L. Horst and Roch E. Gaussoin

Buffalograss [Buchloë dactyloides (Nutt.) Engelm.] is a warm-season perennial grass native to the North American Great Plains region and has been used as a low-maintenance turfgrass. Turf-type buffalograsses are available and are commonly used on nonirrigated land. Our objectives were to determine the deepest planting depth of burrs that would allow acceptable emergence, and to evaluate planting depth effects on buffalograss seedling morphology. Two greenhouse experiments were conducted in Fall 2000. Experimental design was a randomized complete block with 4 replications and a 3 (cultivar) × 6 (planting depth) factorial treatment arrangement. Results showed that buffalograss emergence decreased as planting depth increased. All cultivars had <10% total emergence at planting depths >50 mm. Emergence rate indices were greatest when planting depth was 13 mm and were significantly lower at planting depths of 51 and 76 mm. Average coleoptile length was 11 mm. Coleoptile length was similar between all planting depths except for the 13 mm depth which resulted in 9-mm-long coleoptile. Subcoleoptile internode length increased with planting depth up to 38 mm. Planting depths deeper than 38 mm did not significantly increase subcoleoptile internode length.

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Shanqiang Ke, Chiwon W. Lee and Zong-Ming Cheng

Coleoptile tissues excised from young seedlings of `Touchdown' Kentucky bluegrass (Poa pratensis L.) were bombarded with the disarmed Agrobacterium tumefaciens strain EHA 101 carrying rolC (from A. rhizogenes), NPT II and GUS genes. These tissues were then cultured on Murashige and Skoog (MS) medium containing 0.2 mg·L–1 picloram, 0.01 mg·L–1 naphthaleneacetic acid (NAA), 150 mg·L–1 kanamycin, and 50 m acetosyringone. Calli formed on this medium within 2 weeks. The regenerated plants from these calli were analyzed for the presence of the GUS and rolC genes by histochemical GUS assay, PCR, and Southern hybridization. Only 3.7% of the regenerants were transformed when determined by the GUS assay. A similar frequency of transformation in the regenerated plants was obtained after bombarding the coleoptile tissues with the DNA isolated from the pGA-GUSGF-rolC plasmid. Most of the putative transformants were either albinos or variegated plants that are composed of both albino and green tissues.

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Albert Liptay, John L. Barron, Tom Jewett and Ian van Wesenbeeck

Growth of corn seedlings during the coleoptile stage was measured using optical flow. The measurement system was comprised of a digital camera, computer, and related software and measured growth in a continuous, noncontact manner. The use of optical flow to measure shoot elongation, i.e., image motion of the elongating seedling, was most easily computed when there were large spatiotemporal variations of the motion of the corn seedling against the background. The sensitivity of the measurement technique was in the micron per second range. Seedling growth did not occur in a smooth even manner, rather, growth was a series of varying bursts or waves of expansion that appeared to be affected by the physical growth or development of the leaves. Spectral analysis techniques were applied to extract the underlying signal from the observed time series of seedling growth rate and angle.

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Sheila A. Blackman and Eric E. Roos

The low quality of some seed lots received by germplasm repositories such as the National Seed Storage Laboratory can thwart efforts to regenerate seed for storage. This germplasm is in danger of irretrievable loss. The aim of this work is to promote the germination, and hence regeneration, of such low quality seeds through sterile culture of the isolated embryos. Hybrid (B73×LH51) maize seeds were aged 5 y at 32°C and 0.037 g H2O g-1 dry wt. Vigor - but not viability -declined under these conditions. The effects of four factors on growth and germination were systematically examined. These were: seed pretreatments; antibiotics and fungicides; nutrients; and growth substances. Amongst the pretreatments, none surpassed partial hydration of seeds for 24 hr to 0.55 g H2O g-1 dry wt at 25°C prior to embryo dissection. Thiram (2.4 mg mL-1) and kanamycin (50 ug ml1) effectively controlled bacterial and fungal growth with no deleterious effects on growth during culture of the isolated embryos. Exogenous sucrose (optimum 5 % wt/vol) significantly stimulated radicle growth in both deteriorated and non-deteriorated embryos. No other organic or inorganic nutrient stimulated growth. Naphthalene acetic acid did not affect growth while kinetin reduced radicle growth and stimulated coleoptile growth. Gibberellic acid (GA3 at 10-5M) significantly stimulated radicle growth in deteriorated embryos, whereas it promoted coleoptile growth in both deteriorated and non-deteriorated embryos. These data suggest GA or a GA-stimulated process may limit the growth of aged embryos.

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G.D. Leroux, J. Douheret, M. Lanouette and M. Martel

With growing public concern about environmental quality, farmers must turn to new plant protection alternatives that minimize the use of agrochemicals. Flaming has been practiced for several years as a means of weed control in noncropped areas (railroad, ditches, etc.), but its selectivity toward crops has yet to be defined. Experiments were conducted in the ICG-Propane laboratory at Laval Univ. to determine the temperature needed to kill weeds and the temperature that corn could tolerate. Four weed species were studied: Amaranthus retroflexus, Brassica kaber, Chenopodium album, and Setaria viridis and each species was tested at three growth stages: 0–2, 4–6, and >8 leaves. Corn tolerance was tested at four growth stages: coleoptile, 0–2, 4–6, >8 leaves. All plants were grown in the green-house and were submitted to different combinations of operation speeds and of propane pressures, giving 10 temperature intensities ranging from 110 to 390C. The response of each species was evaluated by measuring its height and dry biomass 2 weeks after treatment. The threshold temperature for corn was below 200C; above this temperature, significant corn injury occurred at all growth stages tested. The corn growth stages most tolerant to heat were coleoptile and >8 leaves. While the most sensitive was 4–6 leaves. All weeds tested were sensitive to heat at 0–2 leaf stage. Amaranthus retroflexus and Chenopodium album were controlled until six leaves with temperatures that were not harmful to corn. Weeds with more than eight leaves needed higher temperature, and control rarely reached 60%. Flaming could be a selective method of weed control if operated at a temperature of 170C. Selectivity can be increased by creating a growth differential between corn and weeds.

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Shanqiang Ke, Chiwon W. Lee and Murray E. Duysen

The effects of the expression of the rolC gene on protein accumulation in the chloroplasts of transgenic Kentucky bluegrass (Poa pratensis L.) were investigated. Coleoptile tissues excised from 3-day dark-grown seedlings were bombarded with tungsten particles coated with DNA of the engineered plasmid, pGA-GUSGF, containing the npt II, gus, and rolC genes. The tissues were cultured on callus induction medium, which consists of MS salts supplemented with 0.2 mg/L picloram, 0.01 mg/L naphthaleneacetic acid (NAA) 250 mg/L kanamycin, and 100 mM acetosyringone. The putative transformants were either albinos or variegated plants composed of white and green sections. These albino plants had little or no stroma-based 56-kDa and 14-kDa subunits of the suspected Rubisco proteins, which are expressed in response to genes in the nucleus and plastid, respectively. The albino plants also lacked the 110-kDa and 57–58-kDa, and 43, 47-kDa polypeptides in PS I, coupling factor, and PS II in thylakoid membranes, respectively. These proteins involved in photosynthesis are translated from plastidbased genes. No light-harvesting complex proteins (LHC) were observed in these albino plants. LHC genes are encoded in the nucleus. The thylakoid membrane proteins in the chloroplasts of the rolC transgenic variegated plants contained these proteins. Our data suggest that the nucleus and plastid gene products for plastid development are concomitantly impaired by expression of genes in the transgenic plants.

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Anil P. Ranwala and William B. Miller

Three soluble invertase isoforms from Lilium longiflorum flower buds that had been separated by DEAE-Sephacel chromatography were purified to near homogeneity by further chromatography on hydroxylapetite, Con-A sepharose, phenyl agarose, and Sephacryl S-200 gel filtration. Nondenaturing polyacrylamide gel electrophoresis (PAGE) gave a single band in all three invertases that corresponded to a band of invertase activity in a duplicate gel. The SDS-PAGE of the purified invertase I resulted in a single band with apparent relative molecular mass of 78 kDa. Invertase II and III were resolved to a similar polypeptide pattern by SDS-PAGE with three bands of 54, 52, and 24 kDa. Antiserum of tomato acid invertase cross-reacted with all three invertase protein bands. Antiserum of wheat coleoptile acid invertase cross-reacted only with 54 and 52 kDa bands of invertase II and III but did not recognize invertase I protein. Con-A peroxidase was bound to invertase I protein and all three protein bands of invertase II and III, suggesting that all proteins were glycosylated. Invertase I protein could be completely deglycosylated by incubating with peptide-N-glycosidase F to result in a peptide of 75 kDa. Invertase II and III were partially deglycosylated by peptide-N-glycosidase F resulting proteins bands of 53, 51, 50, and 22 kDa.

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Shanqiang Ke and Chiwon W. Lee

Coleoptile tissues from dark-germinated seedlings of Kentucky bluegrass (Poa pratensis L.) cv. Touchdown were excised and cultured on MS medium supplemented with 1.5-2.5 mg/liter picloram plus 0.2 mg/liter benzyladenine (BA) or with 4 mg/liter 2,4-D. Embryogenic calli were formed on media containing 1.5 mg/liter picloram plus 2.5 mg/liter 2,4-D in the dark. When these embryogenic calli were subcultured on MS medium containing either 0.15-0.3 mg/liter picloram or 0.2-0.5 mg/liter 2,4-D in a 16-h day/8-h night photoperiod, 10.5% of the cultures regenerated shoots. Pretreatment of cultures in the dark for 2 weeks prior to light exposure slightly increased the plant regeneration efficiency to 15.5%. Pigmentation of the regenerants varied with a ratio of 8.5 completely green: 2.5 green plus albino: 1 completely albino plants. The shoots were multiplied in the medium containing 0.5 mg/liter BA plus either 0.2 mg/liter picloram or 0.1 mg/liter indoleacetic acid (IAA). Over 90% cultures in the shoot proliferation medium produced roots after 4 weeks.

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Xiao-Fang Huang, Binh Nguyen-Quoc and Serge Yelle

Sucrose synthase (SS) is one of the key enzymes in plant carbohydrate metabolism. In maize, this enzyme is encoded by two genes, Sh1 and Sus1. We have isolated and determined the 5'-upstream sequence of maize Sus1 gene and compared it with the corresponding sequence in Sh1 gene. Sequence analysis revealed that there was a weak homology between the two promoters and no common sequence elements were found. To understand the differential regulation of the expression of the two genes, we constructed chimeric GUS fusions using the two promoters of SS genes. By using the biolistic system, we delivered these constructs into various plant tissues, and their transient expression was studied. Our results showed that the two promoters of SS genes directed tissue-specific expression in the same way that the two genes are expressed in vivo. The effectiveness of the expression of the constructs was recorded by counting the total blue expression units (blue spots) per shot and by fluorometric assays. High levels of GUS activity were detected in the immature embryos, young coleoptiles, and heterotrophic young leaves bombarded with the Sus–GUS construct. More than 100 expression units were observed in these tissues. Compared with the transient expression of the 35S promoter in the same tissue, Sus promoter activity was twice as high. Strong Sus–GUS expression was also detected in the aleurone cells of developing kernels. In contrast, the Sh-GUS construct was expressed only in the endosperm with an activity twice as high as that of Sus–GUS and 35S–GUS in the same tissue. The results will be discussed in terms of the physiological roles of the two SS isozymes in plant tissues.

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Li Xu, Suzhen Huang, Yulin Han and Haiyan Yuan

(arrows). ( F ) Coleoptile developed within another week (arrows). ( G ) Leaf emerged from coleoptile (arrows). ( H ) Regenerated plantlets with well-developed leaves and roots. ( I ) Accilimatized plantlets in the greenhouse. MS = Murashige and Skoog’s; 2