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Veli Erdogan and David C. Smith

`Barcelona' hazelnut (C. avellana L.) shoots were girdled and stool layered in a factorial design with three tissue removal (leaf, bud and meristem removal) and two hormone (with or without 750 ppm IBA) treatments. Percent rooting, rooting grade (0 to 5), shoot length, shoot diameter, and total number of buds were determined. Average percent rooting was >90% for all treatments. Girdling alone gave as high percent rooting as hormone application. The main effect of IBA was on root quality rather than percent rooting. About 75% of the hormone-treated rooted layers could be directly planted (grades 3 to 5), compared to 44% for the control, but shoot length, shoot diameter and total number of buds decreased with IBA application. Bud removal did not affect average percent rooting while meristem removal reduced it slightly. The percentage of layers having grades 3 to 5 was lower for the meristem and bud removal treatments than for leaf removal. Our results support the adoption of stool layerage with girdling and IBA application for the production of strong, well-rooted trees suitable for planting directly in the orchard.

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Peter M. Hirst and David C. Ferree

In each of 3 years, vegetative spurs were sampled from l-year-old wood of `Starkspur Supreme Delicious' apple trees (Malus domestica Borkh.) growing on B.9, M.26 EMLA, M.7 EMLA, P.18, and seedling rootstocks. Mineral concentrations of spur leaves and bud apical meristems were determined, and related to spur bud development. The spur leaf P concentration decreased during the growing season each year, hut was unaffected by rootstock. Spur leaves of trees on B.9 rootstock had 30% higher Ca concentrations than trees on M.26 EMLA or seedling rootstocks. In each year, trees growing on M.26 EMLA rootstocks had the highest leaf Mg concentrations. Mineral concentrations were generally unrelated to spur leaf number, leaf area, leaf dry weight, or specific leaf weight. Phosphorus concentrations in spur bud apical meristems declined during two of the three growing seasons of the study and were unaffected by rootstock. Bud P concentration was weakly negatively related to bud diameter and bud appendage number in one year of the study. More vigorous spurs (as indicated by higher spur leaf number, leaf area, and leaf dry weight) had higher bud K levels during each year. No relationships between bud development and either spur leaf mineral concentration or bud apical meristem mineral levels were evident, suggesting that a direct role of mineral nutrition influenced by rootstock at the site of flower formation was unlikely.

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Ana Cristina M. Brasileiro, Francisco J. Lima Aragão, Sílvia Rossi, Diva Maria A. Dusi, Leila M. Gomes Barros, and Elíbio L. Rech

To develop an efficient protocol for Agrobacterium-mediated transformation of common bean (Phaseolus vulgaris L.) and tepary bean (P. acutifolius A. Gray), we have tested the susceptibility of six genotypes to eight Agrobacterium tumefaciens and two A. rhizogenes strains. The virulence of the Agrobacterium strains was shown to be genotype dependent. In general, the tumors observed on common bean cultivars were larger than those observed on tepary bean cultivars. The A. tumefaciens AT8196 and Ach5 strains and the A. rhizogenes 8196 strain induced the best responses in all genotypes tested. Polymerase chain reaction (PCR) analysis confirmed the presence of T-DNA in tumors derived from inoculation with three A. tumefaciens strains in common beans. Apical meristems of P. vulgaris cv. Jalo were bombarded with tungsten microprojectiles and then inoculated with an A. tumefaciens wild-type strain (Ach5). One month later, the explants showed a high frequency of tumor formation (50% to 70%). Similarly, when bombarded meristems were inoculated with an A. tumefaciens disarmed strain (LBA4404/p35SGUSINT), 44% of them showed substantial sectors of GUS activity, suggesting the expression of introduced gene. The bombardment/Agrobacterium system appears to be a promising method to stably transform bean through the regeneration of plants directly from transformed apical meristems.

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Denise V. Duclos* and Thomas Björkman

Cauliflower (Brassica oleracea var. italica) and Broccoli (Brassica oleracea var. botrytis) differ mainly in the stage of reproductive arrest. Cauliflower curd is an inflorescence meristem, while broccoli arrests just before anthesis. Arabidopsis studies led to the hypothesis that a mutant BoCAL allele arrested cauliflower earlier. Later, a mutant in BoAP1 was found to have similar effects. These partially redundant genes, and several identified since, are present in multiple copies in B. oleracea. Understanding their role in the arrest requires quantification of transcript abundance analysis by real-time PCR. Designing selective PCR primers is a critical first step in the process. Designs were based on alignment among the genes of interest (MADS-box genes BoCAL, BoAP1, FUL, and the non MADS-box genes LFY and TFL1) and their paralogs. The high sequence similarity (some over 95%) makes the target transcripts difficult to distinguish. Therefore, primers were designed mostly for targets in the 3'UTR region in order to gain specificity. Short amplicons, 68bp to 200bp, were required for the high PCR efficiency required to quantify these low-abundance transcripts. Primers were evaluated by conventional RT-PCR and real-time PCR. By altering temperature, Total RNA was isolated from plants that were arrested at three developmental stages, inflorescence meristem (cauliflower), floral meristem (intermediate), and floral bud (broccoli) by varying temperature. RT-PCR products were single bands of the expected size, despite the high homology between genes under study. Real-time melting curve analysis (fluorescence derivative vs. melting temperature) corroborated the presence of a single amplicon. The identity of products was confirmed by sequencing and restriction enzyme digestion.

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Adriana Cibele de Mesquita Dantas, Adriano Nunes Nesi, Lilia Bender Machado, Janny Haerter, and Gerson Renan de Luces Fortes

The culture of meristems, shoot tips, and axillary buds leads to the method of in vitro multiplication that is easily used and safe to obtain uniform copies with no undesirable variations. This work aimed to propagate five in vitro pear cultivars: Housui, Carrick, Nijisseiki, Packham's Triumph, and Red Bartlett. The work was carried out in the Tissue Culture Laboratory at Embrapa Temperate Climate. The plants were sprayed with benomyl (1.0 mg./L) and agrimicine (2.4 mg/L) in the fields, 2 weeks before the shoots were collected. The shoots were then cut with two buds with no leaves and desinfested with alcohol 70% for 10 s and 1% sodium hypochloride for 20 min, 50 explants, 25 buds, and 25 meristems, were then transferred to test tubes containing MS salts and vitamins, myo-inositol (100.0 mg/L), sucrose (30.0 g/L), agar (6.0 g/L), added to in mg/L: BAP (1.0), GA3 (0.1), and NAA (0.01). Three pear cultivars were used for in vitro multiplication (`Nijisseiki', `Red Bartlett', and `Housui') by using the same basal salt with N reduced to strength, added to (in mg/L): BAP (1.6), NAA (0.16). The material was kept in growth room under 16-h photoperiod, 25 ± 2 °C and 19 μMol·m-2·s-1 of flux radiation. The in vitro contaminations were mainly due to bacteria derived from the bud material (71.5%). Higher oxidation for meristem material was observed for `Carrick' and `Packham`s Triumph'. `Red Bartlett' showed the best results for all the variable studied, although all cultivars in general presented low response.

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Mark H. Brand, Yiqin Ruan, and Richard Kiyomoto

To characterize the in vitro behavior of Rhododendron `Montego' with tissue proliferation (TP) to cytokinin and auxin, comparisons were made of normal [TP(–)], dwarf TP [TP(+) dwarf], and long TP [TP(+) long] shoot cultures. On basal medium TP(–) and TP(+), long shoots failed to multiply and had a low relative growth rate (RGR) of 0.1, whereas TP(+) dwarf shoots produced 31.8 shoots per tip, with most shoots being <5 mm long, and RGR was 0.3. Addition of 15 μm 2iP to basal medium induced the production of more than six shoots per TP(–) tip and doubled their RGR; TP(+) long shoots produced 16.8 shoots, most <5 mm long, and had an RGR of 0.3; TP(+) dwarf shoots produced only 16% as many shoots as on basal medium, but still exhibited an increase in RGR. Leaves from TP(–) and TP(+) sources failed to produce shoots on basal medium, but 74% of TP(–) leaves formed shoots when cultured on 1 μm IBA and 30 μm 2iP. TP(+) leaves were able to form shoot meristems on media containing only 5 μm 2iP (26% of explants), but these meristems failed to elongate into shoots. Calli from TP(–) leaves, TP(+) leaves, and TP(+) tumors grown on medium containing 10 μm NAA and 15 μm 2iP had higher RGRs than the same calli on basal medium during the first 8 weeks of culture. Over time, RGR decreased in both TP(–) and TP(+) leaf calli, but increased in TP(+) tumor callus. The increased RGR resulted from differentiation of shoot meristems on 85% of the calli between week 4 and week 8. Our results suggest that TP(+) tissues have altered hormone metabolism or sensitivity that leads to dramatic differences in in vitro behavior and probably contributes to tissue proliferation observed in whole plants. Chemical names used: 6-(γ,γ-dimethylallylamino) purine (2iP); indole-3-butyric acid (IBA); α-naphthaleneacetic acid (NAA).

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Samuel Salazar-García, Elizabeth M. Lord, and Carol J. Lovatt

Inflorescence and flower development of the `Hass' avocado (Persea americana Mill.) were investigated at the macro- and microscopic level with three objectives: 1) to determine the time of transition from vegetative to reproductive growth; 2) to develop a visual scale correlating external inflorescence and flower development with the time and pattern of organogenesis; and 3) to quantify the effect of high (“on”) and low (“off”) yields on the flowering process. Apical buds (or expanding inflorescences) borne on summer shoots were collected weekly from July to August during an “on” and “off” crop year. Collected samples were externally described and microscopically analyzed. The transition from vegetative to reproductive condition probably occurred from the end of July through August (end of shoot expansion). During this transition the primary axis meristem changed shape from convex to flat to convex. These events were followed by the initiation of additional bracts and their associated secondary axis inflorescence meristems. A period of dormancy was not a prerequisite for inflorescence development. Continued production of secondary axis inflorescence meristems was observed from August to October, followed by anthesis seven months later. In all, eleven visual stages of bud development were distinguished and correlated with organogenesis to create a scale that can be used to predict specific stages of inflorescence and flower development. Inflorescence development was correlated with minimum temperature ≤15 °C, whereas yield had little effect on the timing of developmental events of individual inflorescence buds. However, the high yield of the “on” year reduced inflorescence number and increased the number of vegetative shoots. No determinate inflorescences were produced during the “on” year. For the “off” year, 3% and 42% of shoots produced determinate and indeterminate inflorescences, respectively.

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James A. Bryan and John R. Seiler

Foliar application of the synthetic growth regulator BA was evaluated for increasing the duration and extent of Fraser fir [Abies fraseri (Pursh.) Poir.] seedling growth. Aqueous solutions of 0, 222, or 444 μm BA (0, 50, or 100 ppm) were sprayed on the shoots of Fraser fir seedlings biweekly from 18 until 38 weeks after planting. Foliar sprays of 444 μm BA increased seedling height 19%, increased shoot weight 57%, reduced root weight 22%, and increased total weight 27%. Apical meristem activity was stimulated and the long periods of dormancy typical of Fraser fir seedlings were avoided. Chemical name used: 6-benzylaminopurine (BA).

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Marietta Loehrlein and Richard Craig

Nine cultivars of Pelargonium × domesticum representing three germplasm sources were evaluated for the effect of daily light integral on floral initiation. Plants were grown at four daily light integrals: 5, 10, 15, or 20 mols/day for a 16-h photoperiod in environmental growth chambers at constant 15.5 °C. Meristems were examined at 50-mol intervals (0 to 350 mols) for morphological changes associated with floral initiation. Two phenotypes were identified, cultivars with an association between floral initiation and irradiance and those with association between floral initiation and chronological time. Genotypic variation was observed among the cultivars of each phenotype.

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Marietta M. Loehrlein and Richard Craig

Nine cultivars of Pelargonium ×domesticum L.H. Bailey were evaluated for the effect of daily light integral on floral initiation. Plants were grown at four daily light integrals: 5, 10, 15, or 20 mol·m-2·d-1 for a 16-hour photoperiod in environmental growth chambers at constant 15.5 °C. Meristems were examined at 50 mol·m-2 intervals for morphological changes associated with floral initiation. Two phenotypes were identified, cultivars with an association between floral initiation and cumulative irradiance and those with association between floral initiation and chronological time. Genotypic variation was observed among the irradiance-associated phenotypes.