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Kathryn Kamo and Bong Hee Han

the transformation of lilies. Agrobacterium -mediated transformation of Lilium longiflorum and Oriental lilies has been demonstrated ( Hoshi et al., 2004 ; Mercuri et al., 2003 ; Ogaki et al., 2008 ). Biolistic-mediated transformation has been

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William B. Miller and Shi Niu

Sucrose is the major form of translocated carbohydrate in most plants. While enzymes of sucrose degradation have been well studied in many agronomic crop sinks, little is known about the physiology of sucrose breakdown in most floral tissues. Invertase and sucrose synthase are accepted as the key enzymes responsible for sucrose breakdown. As the first step in studying sucrose breakdown in Lilium longiflorum, we characterized floral bud invertase enzymes. Three soluble invertases were present in developing buds, and were resolved by DEAE-Sephacel chromatography (Invertases I, II, and III, in order of elution). After further purification, each enzyme was characterized. Each was an acid invertase (pH optima of 4.0 to 5.0). each had Km values for sucrose of 5.0 to 7.0 mM. To determine if the enzymes had tissue-specific localization, anthers were dissected from tepal, pistil, and filament tissues. Invertase I was localized primarily in anthers, with invertases II and III being present in much smaller amounts. Invertases II and III were the major forms in the other floral tissues with essentially no invertase I detectable.

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Pablo A. Marinangeli, Luis F. Hernández, Cecilia P. Pellegrini, and Néstor R. Curvetto

External, middle and inner scales in parent bulbs were studied to evaluate bulblet differentiation in Lilium longiflorum Thunb. during scale propagation at 25 °C. A 13-stage developmental process describes different steps including preprimordial, primordial, and bulblet formation. For all scales, preprimordial and primordial stages occurred within the first 4 days. The differentiation process depended on parent scale position. Most bulblets arising from external scales developed three true scales after 30 days while bulblets from middle scales formed four true scales. Homogeneity in the morphology of the parent scales, only shown in the middle ones, was associated with a rapid change in developmental stage for the population of bulblets. Inner scales showed few bulblets with three and four true scales, the rest remaining at earlier developmental stages. Bulblet production decreased from external to internal scales: 2.6, 2.2, and 1.2 bulblets per scale, respectively, and showed a positive correlation with the scale base width. Maximum scale weight and surface area and maximum bulblet fresh and dry weight occurred in the middle scales. We conclude that middle scales are the ideal starting material for experimental uses involving scaling propagation. For production purposes, the external scales, in addition to the middle scales, must also be included for propagation.

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Pablo Marinangeli, Silvia Delmastro, and Néstor Curvetto

Both dibutyryl-cAMP and forskolin, with and without a pretreatment with traumatic acid, stimulated bulbing in vitro of Lilium longiflorum Thunb. by up to 65% over the controls. Dibutyryl-cAMP, forskolin, their combinations with traumatic acid, and adenosine significantly increased the micropropagation yield. Traumatic acid alone produced similar results on bulblet number per explant, but with increased fresh mass per bulblet. Chemical names used: 9-ß-D-ribofuranosyladenine (adenosine); adenosine 3′, 5′ - cyclic monophosphate (cAMP); dibutyryl adenosine 3′, 5′ - cyclic monophosphate (DB-cAMP); 9-ß-L (+) adenosine or 9-ß-L (+) ribofuranosyladenine; forskolin; 10(E) dodeca-1,12-dicarboxylic acid (traumatic acid).

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Theo J. Blom, M.J. Tsujita, and Glen L. Roberts

Potted plants of Lilium longiflorum Thunb. cvs. `Ace' and `Nellie White' were grown either under an ambient photoperiod (APP) or under an 8-hour photoperiod (8PP) in a greenhouse. The latter photoperiod was achieved by pulling black cloth over the plants at 1615HR and removing the cloth at 0615HR each day, from emergence to flowering. Within each photoperiod, ambient light intensity was reduced by 0, 20, 40 or 60% using various shade cloths permanently suspended above the plants. Heating was set at 20/16C for the dark/light period, respectively. Plant height, determined from the rim of pot to the top of plant, was 25% lower under 8PP compared to APP for both cultivars. Plant height of `Ace' and `Nellie White' increased by 1.5 mm and 2.5 mm, respectively, per 1% light reduction.

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Anil P. Ranwala and William B. Miller

In mature Lilium longiflorum flower buds, anther and stigma had the highest soluble acid invertase activity [3.29 and 2.31 μmol of reducing sugars (RS)/min per gram of fresh weight (FW), respectively] compared to style, ovary, petal, and filament with activities of 1.52, 1.08, 0.99 and 0.98 μmol RS/min per gram of FW, respectively. DEAE-sephacel chromatography revealed that invertase activity in petal, ovary, style, and stigma was composed exclusively of invertase II and III isoforms. Anther invertase was mainly invertase I with small amounts of invertase II and III. Filament tissue mainly had invertase II and III isoforms with a small amount of invertase I. Wall-bound invertases were extracted with 1.0 m NaCl. Anthers had the highest wall-bound invertase activity (4.42 μmol RS/min per gram of FW) followed by stigma (0.42 μmol RS/min per gram of FW). Other tissues had low wall-bound invertase activity (<0.1 μmol RS/min per gram FW). For further purification, the binding of soluble invertases to nine different reactive dyes was investigated. Invertase I was bound to Reactive Green 5, Reactive Green 19, and Reactive Red 120 columns and was eluted with 0.5 m NaCl, resulting in increase in specific activity ≈10-fold with ≈70% recovery. Invertase II and III bound only to Reactive Red 120. Elution with 0.5 m NaCl resulted in an ≈6-fold increase in specific activity.

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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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William B. Miller, P. Allen Hammer, and Terri I. Kirk

Commercial greenhouse operators are increasingly using “negative DIF” temperature regimes to control crop height. A negative DIF exists when greenhouse night temperature is greater than the day temperature. Large negative differences in day and night temperatures strongly suppress stem elongation in many crops. We have explored the effects of negative DIF temperature regimes on leaf, flower, and stem carbohydrate levels in Lilium longiflorum Thunb. `Nellie White'. During two growing seasons, `Nellie White' plants were grown under positive or negative DIF regimes (±5 or 8C) under prevailing daylengths, with temperatures adjusted so that daily temperature averages were equal between regimes. Plants were harvested ≈10 days after visible bud stage and at anthesis. Carbohydrates in stems, leaves, and flowers were analyzed by high-performance liquid chromatography. Compared to plants grown under positive DIF, negative DIF plants showed significantly reduced stem length and leaf and stem dry weights. Negative DIF regimes reduced leaf and stem total soluble carbohydrate (TSC) content by 39% to 46% at visible bud and anthesis, while flower TSC content was reduced by 10% to 13%.

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Wendy S. Higgins and Dennis P. Stimart

Lilium longiflorum Thunb. `Ace' bulblets generated in vitro at 25 or 30C were stored at 4C for O, 1, 2, 4, or 6 weeks after removal from culture and before planting to ascertain the effects of in vitro generation temperature and post-in vitro cold storage duration on bulblet growth responses during 36 weeks of greenhouse growth. Increasing post-in vitro storage duration decreased the number of days to first leaf emergence and percentage of plants producing shoots within 36 weeks, but increased the number of days to shoot emergence and anthesis, leaf number, and flower bud number. The length of time required for bulblet development from planting to shoot emergence was affected by storage duration more than periods from shoot emergence to visible bud and anthesis. It is feasible to produce high-quality L. longiflorum pot plants from in vitro-produced bulblets.

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Andrew A. Waber and Michael R. Evans

Euphorbia pulcherrima `Freedom' (poinsettia) were grown in coir dust, sphagnum peat, and perlite at the following ratios (respectively) 20:0:80, 40:0:60, 60:0:40, 80:0:20, 0:20:80, 0:40:60, 0:60:40, and 0:80:20 (v/v) substrates. Days to anthesis were not significantly different between substrates. Heights were greater for plants produced in 80% coir compared to plants grown in 80% peat. Overall, plants grown in coir-based substrates were taller than plants grown in peat-based substrates. Plants grown in 60% coir had a greater number of lateral shoots, increased shoot fresh weight and increased bract area compared to plants grown in 60% peat. Overall, plants grown in coir-based substrates had greater shoot fresh weights compared to plants grown in peat-based substrates. Lilium longiflorum `Nellie White' (lily) plants were grown in 40:0:20:40, 0:40:20:40, 0:57:14:28, 0:73:9:18 (v/v sphagnum peat: coir dust: loam: perlite) substrates. As the proportion of coir in the substrate increased, height, and shoot and root fresh weights increased. Nodes to flower, days to flower, and number of flowers were not significantly affected by substrate.