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J.P. Syvertsen, J.W Grosser, and L.S. Lee

We grew three diploid (2X) Citrus rootstock seedlings and their autotetraploids (4X) at elevated CO2 to obtain insights into limitations on growth and net gas exchange that have been associated with tetraploidy. Well-nourished Volkamer lemon (Volk), Troyer citrange (Troy), and Cleopatra mandarin (Cleo) were grown in greenhouses at ambient or twice ambient CO2 for 3 months. We measured plant growth, water relations, mineral nutrition, and net gas exchange characteristics of leaves. Overall, tetraploid roots were thicker as 4X had lower root length: dry weight ratio or specific root length (SRL) than 2X roots. Tetraploid plants were smaller and had higher root/shoot ratios, shorter fibrous roots, and lower whole plant transpiration than 2X. Tetraploids also had lower leaf N and P concentrations on a dry weight basis. Since 4X leaves had thicker leaves (more dry weight per area) than 2X leaves, these nutrient differences disappeared when expressed on an leaf area basis. Elevated CO2 increased plant growth but decreased leaf N, P, and K apparently by a growth dilution effect. Elevated CO2 also increased fibrous root thickness, leaf thickness, and net assimilation of CO2 (ACO2) but decreased stomatal conductance and transpiration such that leaf water use efficiency increased. There was no effect of ploidy level on ACO2 but 4X Volk and Troy had lower rates of ACO2 than their diploids at elevated CO2. Hydraulic conductivity of intact root systems (measured in a pressure pot) was correlated to total plant growth but variability obscured effects of CO2 or ploidy on root conductivity. The low SRL of tetraploids were correlated with lower rates of water use and lower leaf nutrient concentrations, which may be operative in determining the growth characteristics associated with tetraploidy.

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J.P. Syvertsen, L.S. Lee, and J.W Grosser

Diploid (2x) and autotetraploid (4x) Citrus L. rootstock cultivars were grown at elevated CO2 to obtain insights into limitations on growth and net gas exchange that have been associated with tetraploidy. Well-nourished 2x and 4x seedlings of `Volkamer' lemon (Volk, C. volkameriana Ten & Pasq.), `Troyer' citrange [Troy, C. sinensis (L.) Osbeck × Poncirus trifoliata (L.) Raf.] and `Cleopatra' mandarin (Cleo, C. reticulata Blanco.), were grown in greenhouses at either ambient or twice ambient CO2 for 4 months. Plant growth, water relations, mineral nutrition, and net gas exchange characteristics of leaves were measured. Most 4x plants were smaller and had lower rates of whole plant transpiration but shorter fibrous roots than 2x plants. Fibrous roots of 4x were thicker than 2x roots as indicated by a lower specific root length (SRL) in 4x than in 2x roots. Root hydraulic conductivity was correlated to total plant growth but there were no effects of CO2 or ploidy on root conductivity. Tetraploid leaves had lower N concentrations than 2x leaves when expressed on a dry weight basis but these differences disappeared when N concentration was expressed on an leaf area basis because 4x leaves had more leaf dry weight per area (LDW/a) than 2x leaves. Plant growth was greater and SRL was lower at elevated CO2 than at ambient CO2. LDW concentrations of N, P, and K were lower at elevated CO2 than at ambient apparently due to a growth dilution effect. LDW/a, net CO2 assimilation (ACO2), and leaf water use efficiency were greater at elevated CO2 than at ambient. Overall, there was no effect of ploidy on ACO2 but 4x Volk and Troy had lower rates of ACO2 than their 2x at elevated CO2. Net gas exchange of tetraploid leaves was less responsive to elevated CO2 than 2x leaves. The low SRL of tetraploids was correlated with low whole plant transpiration rates and low leaf area-based N concentrations, which may be operative in determining the growth characteristics associated with tetraploidy.

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Jude W. Grosser*, J.L. Chandler, and R.M. Goodrich

Sweet orange (Citrus sinensis L. Osbeck) is the most horticulturally important and widely grown Citrus species in Florida and worldwide, and `Valencia' is the most important cultivar for processing. Frozen concentrate orange juice has been the primary product of the Florida and Brazilian industries, but recently there has been a strong shift to not from concentrate (NFC) product in Florida. The higher quality NFC has a greater consumer appeal, and brings a higher market price. The development of higher quality oranges with expanded maturity dates will facilitate this change and should increase the competitive ability of the Florida industry. No true sweet orange cultivars have been developed by conventional breeding due to biological impediments, and alternative methods to obtain genetic variation are being investigated, including studies of somaclonal variation. We have produced nearly 1000 somaclones of `Valencia' sweet orange using organogenesis, somatic embryogenesis, and protoplasts. Following several years of fruit evaluation, early and late maturing high quality somaclones have been identified based on juice analytical data (brix, acid, ratio, juice percentage, juice color, and lbs. solids). These clones have also performed exceptionally in taste panel evaluations comparing them with the traditional mid- and late-season cultivars. Second generation trees of the most promising clones have been propagated for further evaluation, and superior processing clones will be released to the Florida industry in the near future. An overview of this program including pilot plant juice quality data and taste panel results will be presented.

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N. Tusa, J.W. Grosser, and F.G. Gmitter Jr.

Protoplasm culture following the chemical fusion of `Valencia' sweet orange [Citrus sinensis (L.) Osb.] protoplasts, isolated from an embryogenic suspension culture, with `Femminello' lemon [Citrus limon (L.) Burro. f.] leaf protoplasts resulted in the regeneration of an interspecific allotetraploid somatic hybrid plant, two autotetraploid lemon plants, and diploid plants from both parents. The regeneration of plants from lemon leaf protoplasts is an example of protoplast-to-plant regeneration from non-nucellus-derived tissue for Citrus. Regenerated plants were classified according to leaf morphology, chromosome number, and analyses of phosphohexose isomerase (PHI), peroxidase (PER), and 6-phosphoglucose dehydrogenase (PGD) zymograms. The somatic hybrid plant was vigorous, with leaves morphologically intermediate to the parents. The tetraploid lemon plants were similar to diploids, although less vigorous and with thicker leaves. The tetraploid lemon and somatic hybrid plants, if fertile, could be used in interploid sexual crosses to breed triploid seedless lemon cultivars with tolerance of mal secco disease from sweet orange. Further investigation of plant regeneration from leaf protoplasts could increase the number of totipotent Citrus clones amenable to somatic hybridization and genetic transformation experiments.

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J.W. Grosser, J. Jiang, E.S. Louzada, J.L. Chandler, and F.G. Gmitter Jr.

Production of tetraploid somatic hybrids that combine complementary diploid rootstock germplasm via protoplast fusion has become a practical strategy for citrus rootstock improvement, with the overall objective of packaging necessary disease and pest resistance into horticulturally desirable, widely adapted rootstocks. Citrus somatic hybridization techniques have been advanced to the point where numerous somatic hybrid rootstocks can now be produced and propagated for evaluation on a timely basis. Herein we report the production of 11 new somatic hybrid rootstock candidates from 12 different parents, including Milam lemon hybrid (Citrus jambhiri Lush.), Cleopatra mandarin (C. reticulata Blanco), sour orange (C. aurantium L.), `Succari' sweet orange [C. sinensis (L.) Osbeck], `Redblush' grapefruit (C. paradisi Macf.), `Nova' tangelo [C. reticulata × (C. paradisi × C. reticulata)], `Kinkoji' (C. obovoidea Hort. Ex Takahashi), Swingle citrumelo [C. paradisi × Poncirus trifoliata (L.) Raf.], Carrizo citrange (C. sinensis × P. trifoliata), rough lemon 8166 (C. jambhiri), and Palestine sweet lime (C. limettoides Tan.). All hybrids were confirmed by cytological and VNTR-PCR analyses, and have been propagated, budded with a commercial scion, and field-planted for performance evaluation.

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K.C. Taylor, C.D. Chase, L.G. Albrigo, and J. W. Grosser

Citrus blight is an extremely complex decline disorder of unknown etiology, Zinc accumulates in the phloem of the tree 40-50 cm above the bud union 1-3 years prior to visible symptoms of blight (foliage wilt and twig dieback). This is accompanied by Zn deficits in the leaves. A Zn-binding peptide (ZBP) purified from citrus phloem tissue accounts for a symptomatic redistribution of Zn from the canopy to the trunk phloem. ZBP is found in blight and healthy trees and is therefore a normal component of cellular metabolism. To further understand ZBP's role in metabolism two citrus cell culture lines which were selected based on their susceptibility to blight have been characterized as to their growth under Zn treatments as well as Cu and Cd. In addition, their complement of metal-binding constituents is being determined.

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Jude W. Grosser, Frederick G. Gmitter Jr., J.L. Chandler, and Eliezer S. Louzada

Protoplasm culture following polyethylene glycol-induced fusion resulted in the regeneration of tetraploid somatic hybrid plants from the following attempted parental combinations: Cleopatra mandarin (Citrus reticulata Blanco) + Argentine trifoliate orange [Poncirus trifoliata (L.) Raf.]; `Succari' sweet orange [C. sinensis (L.) Osb.] + Argentine trifoliate orange; sour orange (C. aurantium L.) + Flying Dragon trifoliate orange (P. trifolita); sour orange + Rangpur (C. limonia Osb.); and Milam lemon (purported sexual hybrid of C. jambhiri Lush × C. sinensis) + Sun Chu Sha mandarin (C. reticulate Blanco). Protoplasm isolation, fusion, and culture were conducted according to previously published methods. Regenerated plants were classified according to leaf morphology, chromosome number, and peroxidase, phosphoglucomutase, and phosphoglucose isomerase leaf isozyme profiles. All of the somatic hybrid plants were tetraploid, as expected (2n = 4x = 36), and all five selections have been propagated and entered into commercial citrus rootstock trials.

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N. Tusa, J.W. Grosser, F.G. Gmitter Jr., and E.S. Louzada

Allotetraploid somatic hybrid plants of `Hamlin' sweet orange (Citrus sinensis L. Osbeck) + `Femminello' lemon (C. limon L. Burm. f.), and Milam lemon (purported hybrid of C. jambhiri Lush) + `Femminello' lemon were regenerated via somatic embryogenesis following protoplast fusion. `Hamlin' and Milam protoplasts were isolated from undeveloped ovule-derived embryogenic callus cultures and fused using a polyethylene glycol method with seedling leaf-derived protoplasts of `Femminello' lemon. Somatic hybrids were identified on the basis of leaf morphology, root-tip cell chromosome number, and electrophoretic analyses of phosphoglucose isomerase, phosphoglucose mutase, and 6-phosphogluconate dehydrogenase leaf isozymes. The somatic hybrids will be used in interploid crosses with lemon in an effort to generate seedless triploid lemon types with improved tolerance to mal secco disease.

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Jude W. Grosser, Frederick G. Gmitter Jr., E.S. Louzada, and J.L. Chandler

Allotetraploid somatic hybrid plants of `Nova' tangelo [a sexual hybrid of `Clementine mandarin (C. reticulata Blanco) × `Orlando' tangelo (C. reticulata × C. paradisi Macf.)] + `Succari' sweet orange (C. sinensis L. Osbeck), and `Hamlin' sweet orange (C. sinensis L. Osbeck) + `Dancy' tangerine (C. reticulata) were regenerated following protoplast fusion. `Nova' and `Hamlin' protoplasts were isolated from ovule-derived embryogenic callus and suspension cultures, respectively, and fused using a polyethylene glycol method with seedling leaf-derived protoplasts of `Succari' and `Dancy', respectively. Plants were regenerated via somatic embryogenesis, and somatic hybrids were identified on the basis of leaf morphology, root-tip cell chromosome number, and electrophoretic analysis of peroxidase and phosphoglucose mutase isozyme banding patterns. Diploid plants were regenerated from unfused protoplasts of `Hamlin', `Nova', and `Succari'. Tetraploid plants of `Hamlin' and `Succari' were also recovered, apparently resulting from homokaryotic fusions. No `Dancy' plants were recovered. The somatic hybrid and autotetraploid plants can be used for interploid hybridization with selected monoembryonic scions to generate improved seedless triploid tangor/tangelo cultivars. The lack of suitable tetraploid breeding parents has previously inhibited the development of quality seedless cultivars by this method.