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  • Author or Editor: Jude Grosser x
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Citrus protoplast technology has advanced to where several practical applications in variety improvement and plant pathology are routine. We will report on progress in the following areas: somaclonal variation—`Valencia` and `Hamlin' sweet orange protoclones have been selected for improved juice color, higher soluble solids, seedlessness, and altered maturity dates; somatic hybridization for scion improvement—allotetraploid breeding parents have been created from numerous combinations of elite parental material, and are now being used as pollen parents in interploid crosses to produce seedless triploid varieties; somatic hybridization for rootstock improvement—numerous somatic hybrids combining complementary rootstock germplasm are under commercial evaluation and several look promising for wide adaptation, improved disease resistance, and tree size control; transformation—an alternative protoplast-based transformation that utilizes EGFP for selection has been developed; virus resistance assays—a protoplast-based assay is being used to screen varieties and candidate sequences for resistance to citrus tristeza virus at the cell level, saving time and greenhouse space.

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The genetic engineering of horticultural crops to improve disease/insect resistance, cultivar quality, or other characteristics has become a primary area of focus for many research programs. The technique is attractive because a single beneficial trait can be added to an already successful cultivar without otherwise altering cultivar integrity. However, little information has been available regarding the performance of such transgenic plants in the field, particularly regarding woody perennial crops. The purpose of this colloquium is to provide the latest available information regarding the performance of transgenic plants in the field, covering a wide range of crops including vegetables, woody fruit trees, woody nut trees, and forest trees. Focus will be on the long-term expression of transgenes and promoter efficiency. The information provided should be particularly useful to researchers who are currently designing or performing experiments to improve horticultural crops by genetic engineering.

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Abstract

Young Swingle citrumelo [Citrus paradisi Macf. × Poncirus trifoliata (L.) Raf.] seedlings were sectioned into 0.5-cm and 1.0-cm nodal and internodal segments and plated on an agar-solidified basal medium containing different levels of coumarin as the only growth regulatory substance. The frequency of whole-plant recovery from stem segments was positively correlated with the concentration of coumarin in the medium. Recovery of whole plants from 0.5-cm segments required coumarin, which induced both root and shoot formation. An optimum concentration of coumarin (90–150 μm), increased the number of whole plants recovered per seedling ≈5-fold. Resulting plants were acclimated to soil efficiently and rapidly in humidity tents that received a continuous supply of moist air. This procedure could be used commercially to increase the availability of Swingle citrumelo rootstock.

Open Access

In Florida, pesticides, nutritional and growth regulators are often sprayed in tank mixes to reduce sprayer use. Many individual spray components are phytotoxic and result in spray burns in combination or if applied with adjuvants. The toxicity level of standard spray materials is not known and new product testing for phytotoxicity is not routine. Three tests were developed to allow testing of cellular and whole fruit susceptibility to spray chemicals. Cell suspension cultures initiated from `nucellar derived' embryonic callus of `Hamlin' sweet orange were grown in log phase for 2 weeks with various levels of test chemicals. Fresh weight increase was measured. Peel disks of orange or grapefruit were grown for 4 weeks on solid media. Color changes and callus growth were used to evaluate phytotoxicity. Dilute sprays and droplet applications to on-tree-fruit were used to evaluate individual and combinations of chemicals with and without spray adjuvants. The 3 tests combined effectively demonstrated levels of phytotoxicity and are useful for testing new citrus production chemicals.

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Huanglongbing {HLB [Candidatus Liberibacter asiaticus (C Las)]} has been one of the biggest challenges in citrus (Citrus sp.) production in Florida and wherever it is present. HLB-affected trees show significant shoot and root dieback, fruit drop, and reduction in yield. Currently, there is no cure for HLB, and there is no commercial HLB-resistant germplasm. Nonetheless, intensive nutrient management has been promising for citrus growers. The nutrient requirement of HLB-affected trees seems to be greater than that of healthy citrus trees. By understanding the nutrient uptake potential of rootstocks, fertilizer programs can be customized accordingly to enhance the performance of a rootstock in existing groves. Moreover, a reduction in the application of nutrients is possible by planting rootstocks with a high nutrient absorption capacity. Use of rootstocks with good nutrient uptake efficiency can take some burden off the growers who are intensively managing HLB-affected citrus groves. Therefore, the objective of this study was to evaluate and understand the nutrient uptake potential of the citrus rootstocks. To achieve this objective, a 100% hydroponic greenhouse study was conducted with six rootstocks with a range of tolerance to HLB. Several physiological and molecular tools were applied to evaluate the rootstocks for their nutrient uptake potential. A+Volk × O-19 (HLB-tolerant) rootstock had greater nutrient uptake efficiency, whereas US-896 (HLB-susceptible) had lesser nutrient uptake efficiency. Swingle, one of the most popular pre-HLB rootstocks, had poor zinc uptake and the least expression of ZINC TRANSPORTER, suggesting that zinc applications should be emphasized in Swingle plantings. US-896 rootstock expressed the least level of nutrient transporter genes, such as IRON TRANSPORTER. UFR-4 (a good performer under HLB conditions) had a large root biomass, but the uptake efficiency for nutrients was poor, suggesting that the nutrient uptake potential is a complex process that is not solely dependent on root biomass. This study is unique because it is one of the first citrus studies to report nutrient uptake efficiency and the potential of rootstocks. The information presented can be used to improve performance or select better-performing rootstocks under HLB conditions.

Open Access

Interploid hybridization was conducted using `Key' lime [Citrus aurantifolia (Cristm.) Swing.], `Lakeland' limequat hybrid [C. aurantifolia × Fortunella japonica (Thumb.) Swing.], Palestine sweet lime (C. limettioides Tan.), `Etrog' citron (C. medica L.), and seven lemon [C. limon (L.) Burm. F.] varieties as female progenitors and five allotetraploid somatic hybrids {`Hamlin' sweet orange [C. sinensis (L.) Osbeck] × `Femminello' lemon (C. limon)]; `Key' lime × `Valencia' sweet orange (C. sinensis); `Valencia' sweet orange × rough lemon (C. jambhiri Lush); Milam lemon (purported C. jambhiri hybrid) × `Femminello' lemon (C. limon); and `Valencia' sweet orange × `Femminello' lemon} and two autotetraploids [`Giant Key' lime (C. aurantifolia) and `Femminello' lemon] as pollen progenitors. A few tetraploid × diploid crosses were also performed. Thirty-five parental cross combinations were accomplished in 2000, 2001, and 2002. The breeding targets were seedlessness, cold-tolerance, and disease resistance. Triploid hybrids were recovered through embryo culture. Generation of triploid citrus hybrids was affected by several factors including sexual compatibility, cross direction, embryo developmental stage, pollen viability, as well as horticultural practices and climatic conditions. Efficiency of triploid hybrid production was higher in diploid × tetraploid crosses than the reciprocal. Many more triploid hybrids were generated from lemon seed progenitors compared to the other acid citrus fruit progenitors. `Todo el Año', `Lisbon', and `Limonero Fino 49' showed the highest sexual compatibility. Embryo germination rate and normal plant recovery were also higher in lemons as compared to the other seed progenitors. Low winter temperatures might have affected the hybrid production efficiency from tropical acid fruit progenitors. A total of 650 hybrids (mostly triploid) were transferred to soil. The novel genetic combinations of these progenies should be valuable for the genetic improvement of acid citrus fruit (lemons and limes).

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Although no longer as glamorous as it was a few decades past, the routine application of embryo rescue techniques, leading to plant recovery, is a valuable tool for citrus cultivar improvement. Embryo rescue approaches can be used to generate useful variation or to capture various kinds of spontaneous genetic variation. Embryo rescue, by in vitro culture of undeveloped, and presumably unfertilized, ovules in colchicine-supplemented media is a practical method of producing tetraploid clones, which are used then in crosses with diploids to produce seedless triploid hybrids. This same approach, i.e., in vitro culture of undeveloped ovules, is also used to recover plants from chimeric sectored fruit exhibiting economically important mutations for fruit characteristics, and for producing potentially variant somaclones. Seedlessness is an important objective for fresh citrus fruit cultivar improvement, and triploidy following 2x × 4x hybridizations is one approach being exploited for this objective. When monoembryonic diploid seed parents are crossed with tetraploid pollen parents, however, normal seed development is not usually possible. Embryos must be excised from abortive seeds fairly early in development and cultured appropriately to ensure the recovery of sufficient numbers of 3x offspring from these crosses, to increase the likelihood of identifying superior seedless hybrids. These applications will be described in some detail, and progress toward breeding objectives are highlighted.

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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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