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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.
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.
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.
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.
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.
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.
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.
Citrus greening, or huanglongbing (HLB), caused by the phloem-limited bacterium Candidatus Liberibacter asiaticus (CaLas), threatens the global citrus industry. Field observations have demonstrated that some citrus cultivars are more tolerant to the CaLas pathogen than others. ‘Parson Brown’ is an early maturing sweet orange variety that has consistently exhibited minimal leaf and fruit drop in the field compared with the ‘Hamlin’ sweet orange under similar conditions. This study aimed to understand performance of the ‘Parson Brown’ cultivar in several locations across the citrus production regions of Florida. Results indicated that the CaLas bacterial titer in both cultivars were similar with the quantitative polymerase chain reaction cycle threshold values ranging between 24.99 and 28.61 in ‘Hamlin’ and between 25.48 and 30.89 in ‘Parson Brown’. Leaves from the ‘Parson Brown’ trees however demonstrated higher chlorophyll content and total phenolic compounds in most of the locations. We also detected higher relative expression of CsPR1 and CsPR2 transcripts in ‘Parson Brown’ leaves in the first sampling period (March) and the fourth period (November). Additionally, Phloem protein 2 transcripts were downregulated in ‘Parson Brown’ leaves compared with ‘Hamlin’ at all locations. The ‘Hamlin’ juice had higher acidity, whereas ‘Parson Brown’ juice demonstrated a higher Brix to acidity ratio and juice color. The oil content in the juice ranged between 0.020% and 0.042%, and there was variation in the oil content between the locations, which could indicate clonal differences. ‘Parson Brown’ juice however contained higher limonin and nomilin content than ‘Hamlin’ juice in most of the locations. Taken together, the current results confirmed the enhanced tolerance of ‘Parson Brown’ trees to HLB when compared with ‘Hamlin’ in all sampled locations.
Passionfruits (Passiflora sp.) are widely grown throughout tropical regions of the world. Burgeoning new interest in this fruit in both its fresh and processed forms has led to an increase in planting outside of traditional growing zones. Passionfruit production has increased steadily in the United States and its territories since the 2002 US Department of Agriculture Census of Agriculture; however, little is known about how the industry functions across production areas. To assess passionfruit growers’ production practices and support their needs, we conducted a survey during 2021. That survey consisted of 45 questions pertaining to various aspects of passionfruit production, including horticultural practices, pest management, cultivars grown, and industry challenges and needs. The objectives of the survey were to identify where passionfruit is currently grown in the United States, what production practices are being used, and what problems are being encountered so that researchers and extension personnel could provide remedies in the future. Forty-four surveys were complete and allowed for data analyses. Florida had the most responses (21), followed by Puerto Rico (12), California (6), Hawaii, Louisiana, Mississippi, and the Virgin Islands. Most of the passionfruit production in the United States comprises purple passionfruit (Passiflora edulis f. edulis) or intraspecific red types at 68.2%. This value is driven by the high amounts of purple passionfruit and red passionfruit in Florida and other states. In contrast, nearly all farms in Puerto Rico grow yellow passionfruit (P. edulis f. flavicarpa) and fewer purple types. The main obstacle to obtaining optimum production was labor availability. Managing passionfruit, like many other specialty crops, is labor-intensive and includes many activities that require manual labor, such as weeding, training, pruning, pollination, and harvesting. Other obstacles that were noted were weather variability, vine decline, poor pollination, and availability of high-quality cultivars. Diseases, especially fungal diseases, are of particular concern to growers of passionfruit in the United States, although the identification of specific diseases was limited. Online delivery methods of information ranked high on the list of desired products. Online articles, such as those offered by extension services, were the most preferred, followed by webinars, which comprise a more recently developed method of information delivery. Overall, the survey provided baseline information to further develop initiatives to aid passionfruit production within the United States.