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Manjul Dutt and Robert Geneve

Impatiens (Impatiens wallerana Hooker F.) and petunia (Petunia ×hybrida Hort. Vilm.) seeds were imaged using a flat-bed scanner interfaced with a personal computer programmed to capture images every hour. Images were used to measure time to radicle protrusion and seedling growth. Time to radicle protrusion was calculated as time to 50% germination or as actual germination for each seed. Seedling growth after germination was calculated from linear regression of growth over time. Radicle protrusion and seedling growth were evaluated as indicators of seed vigor. Both were good indicators of seed vigor in impatiens seed lots. These measurements of vigor were highly correlated for each impatiens seed lot and for pooled seed lots. However, there was little or no correlation between time to radicle protrusion and seedling growth on an individual seed basis. The relationship between germination speed and seedling growth rate observed in impatiens was confirmed in two petunia seed lots. This study supports the use of time to radicle emergence and seedling growth as good indicators of seed vigor. However, it appears that different aspects of seed vigor may be measured by these indicators because there was no relationship between time to radicle protrusion and seedling growth rate on an individual seed basis.

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Vladimir Orbović, Manjul Dutt and Jude W. Grosser

The effects of fruit age on the seed quality and germination percentage of ‘Duncan’ and ‘Flame’ grapefruit and ‘Hamlin’ sweet orange were investigated. Our results suggested that seed germination varied from 98% to 100% for the two grapefruit cultivars and 85% to 100% for ‘Hamlin’ regardless of time of harvest. Within the first 5 months of the harvest season, chilling of ‘Duncan’ and ‘Hamlin’ seeds at 4 °C for 7 days after fruit sampling resulted in a lower germination percentage only with the ‘Hamlin’ seeds. Seed moisture content of all three cultivars varied slightly through the season and remained steady at 60% and 70% for batches of fresh seeds stored at room temperature or at 4 °C. Our results suggest that high seed viability and germination percentage can allow the use of these seeds for experimentation regardless of the time the fruit were picked during the harvest season.

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Jude W. Grosser, Divya Kainth and Manjul Dutt

Colchicine-induced stable autotetraploid plants were recovered through indirect organogenesis from stem sections of in vitro-grown zygotic seedlings of pink/red-fleshed pummelo (Citrus grandis L. Osbeck) selections cybrid Hirado Buntan (C-HBP) (pink), 5-1-99-3 (dark pink), and HBJL-5 (red), all derived from Hirado Buntan pink pummelo. Multiple shoot induction was achieved through indirect organogenesis from the callus produced from the cut ends of the treated explants. Ploidy levels of regenerated plantlets were determined through flow cytometry at a stage when recovered shoots had one to two expanded leaves. Recovered tetraploids proved to be stable after 2 years in the field. As expected, higher colchicine concentrations and treatment durations decreased the survival rates of the regenerated plantlets. Colchicine concentrations of 0.05% and 0.1% produced the most tetraploids; of the 19 total tetraploids produced, 10 were produced from the treatment with a colchicine concentration of 0.1% and six were produced from treatment with a concentration of 0.05%. After flowering, these stable pink/red-fleshed tetraploid plants generated should be useful as breeding parents in our grapefruit/pummelo improvement program, especially if any show canker tolerance or reduced furanocoumarins. Use of monoembryonic tetraploids in interploid citrus crosses eliminates the need for embryo rescue to recover seedless triploid progeny; this research expands our pool of available high-quality monoembryonic tetraploid parents.

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Manjul Dutt, Daniel Stanton and Jude W. Grosser

Anthocyanins are beneficial bioflavonoids that have numerous roles in human health maintenance, disease prevention, and overall well-being. In addition, anthocyanins are key to the consumer appeal of many ornamental plants. Most citrus (Citrus L.) plants do not produce anthocyanins under warm tropical and subtropical conditions. Anthocyanin pigments, responsible for the “blood” color of blood orange [Citrus sinensis (L.) Osbeck], are produced after exposure to cold conditions during the fruit’s development. The transcription factor Ruby is responsible for the production of anthocyanin in blood orange. Functionally, similar genes exist in other fruit crops such as grape [Vitis vinifera L. (VvmybA1 and VvmybA2)] and apple [Malus ×domestica Borkh (MdMYB10)]. Here, VvmybA1 and Ruby genes were constitutively expressed in ‘Mexican’ lime (Citrus aurantifolia Swingle). This cultivar performs optimally under Florida’s humid subtropical environment and has a short juvenile phase. Constitutive expression of VvmybA1 or Ruby resulted in anthocyanin pigmentation in the leaves, stems, flowers, and fruit. An increased pigmentation of the outer layer(s) of stem tissue was observed in ‘Mexican’ lime overexpressing the VvmybA1, whereas lower anthocyanin levels were observed in plants overexpressing Ruby. Enhanced pigmentation was also observed in the young leaves; however, pigment intensity levels decreased as the leaves matured. Flower color ranged from light pink to fuchsia and the fruit pulp of several ‘Mexican’ lime lines were maroon; similar to a blood orange. The results demonstrate that expression of anthocyanin-related genes can affect temporal pigmentation patterns in citrus. It also opens up the possibility for the development of modified blood orange and other cultivars adapted to the subtropical environment.

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Manjul Dutt, Zhijian T. Li, Sadanand Dhekney and Dennis J. Gray

Genetic transformation of plants necessitates the use of promoters to control transgene expression. Numerous promoters have been isolated from a wide range of organisms for use in plants. However, many of these natural promoters exhibit relatively low activity and/or have limited use. To provide an alternative, we constructed a composite promoter (EP) using a genomic DNA sequence and a 35 bp TATA-containing fragment from the 2S albumin (VvAlb1) gene core promoter of grapevine. The 0.9-kb genomic sequence was identified after TAIL-PCR, based on the presence of several unique cis-acting elements. The sequence showed no homology to any known plant gene, enhancer, and promoter. Two binary vectors, pEP-EGFP/NPT and pEP-GUS, containing a bifunctional EGFP/NPTII fusion gene and a GUS gene, respectively, were constructed to test transcriptional activity of the composite promoter both qualitatively and quantitatively. Transient GFP expression was observed in somatic embryos (SE) of Vitis vinifera `Thompson Seedless' after Agrobacterium-mediated transformation using pEP-EGFP/NPT. Quantitative GUS assay of stably transformed SE containing pEP-GUS indicated that the EP composite promoter was capable of producing GUS activity as high as 12% of that from a doubly enhanced Cauliflower Mosaic Virus 35S promoter or eight times higher than that from a doubly enhanced Cassava Vein Mosaic Virus promoter. In addition, transformation of Arabidopsis with pEP-GUS yielded comparable GUS activity throughout the plant. These data indicate that the EP composite promoter can be used in transformation studies to provide sustained constitutive gene expression in plants.

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Xiuli Shen, Vladimir Orbović, Manjul Dutt, William S. Castle and Frederick G. Gmitter Jr.

An efficient in vitro regeneration system through direct shoot organogenesis was established for Murraya paniculata (L.) Jack (Orange Jessamine). Epicotyls, leaves, roots, and cotyledons from in vitro-germinated seedlings and several plant growth regulators (PGRs) were evaluated for their effects on plant regeneration. Longitudinally cut epicotyl segments were observed to be the optimal explants followed by uncut epicotyls (not longitudinally cut). Roots, leaves, and cotyledons were not suitable as explants as a result of little or no shoot induction. Adventitious shoot induction was enhanced by the addition of 6-benzyladenine (BA). The highest percentage of shoot induction (87%) and the greatest number of shoots per explant (12.7) occurred on Murashige and Skoog (MS) medium supplemented with 15 μM BA from longitudinally cut epicotyls followed by 5.2 shoots per explant from uncut epicotyls. Optimal concentration of gibberellic acid (GA3) for shoot elongation was observed to be 15 μM. Eighty-five percent of the regenerated shoots produced roots with an average of three roots per shoot on MS medium supplemented with 5 μM indole-3-butyric acid (IBA). Our protocol for direct shoot organogenesis can potentially lead to the development of a robust method for production of transgenic plants of M. paniculata through Agrobacterium-mediated genetic transformation.

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Manjul Dutt, Dennis J. Gray, Zhijian T. Li, Sadanand Dhekney and Marilyn M. Van Aman

A major drawback to the use of embryogenic cultures for transformation of grapevine is that their ability to undergo genetic transformation is cultivar-dependent. Also, depending on cultivar, embryogenic cultures are difficult to impossible to maintain over time, reducing their utility for use in genetic transformation. An alternative to the use of embryogenic cultures for transformation of grapevine is the use of micropropagation cultures, which are easier to initiate from a wide range of grapevine cultivars and can be maintained over time without loss of function. Vitis vinifera `Thompson Seedless' was used as a model for genetic transformation using micropropagation cultures. In vitro cultures were initiated from apical meristems of actively growing vines and maintained in C2D medium containing 4 μM of 6-benzylaminopurine (C2D4B). Shoot tips and nodes were collected from proliferating in vitro cultures for transformation studies. A variety of wounding techniques, including nicking, sonication, and fragmenting of meristematic tissues was employed in order to enable Agrobacterium infection. We used a construct containing a bidirectional 35S promoter complex with a marker gene composed of a bifunctional fusion between an enhanced green fluorescent protein (EGFP) gene and a neomycin phosphotransferase (NPTII) gene in one direction and a hybrid lytic peptide gene in the other. Transgenic shoots growing in C2D4B medium containing 200 mg·L-1 each of carbenicillin and cefotaxime and 20 mg·L-1 of kanamycin were selected based on GFP fluorescence. Transgenic shoots were rooted and transferred to a greenhouse. To date, 18 transgenic lines have been generated. Details on the transformation procedure will be discussed.