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Kathryn C. Taylor, Danielle R. Ellis, and Luciano V. Paiva

Zinc in xylem and phloem of the citrus rootstock, rough lemon [Citrus jambhiri (L.)] was associated with a Zn-binding protein, designated citrus vascular Zn-binding protein (CVZBP). The apparent molecular mass of the CVZBP was 19.5 kDa after nondenaturing size exclusion chromatography and 21.8 kDa after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Ion exchange chromatography demonstrated that CVZBP was anionic, requiring 0.43 n NaCl for elution from quaternary aminoethyl Sepharose. Antiserum to the protein cross-reacted more with total protein extracts from leaf midveins than with total protein from the rest of the leaf lamina, further suggesting a vascular location of the Zn-binding protein. Quantitative analysis indicated that ≈2 to 3 mol of Zn were associated with 1 mol of native protein. Binding studies with the partially purified CVZBP demonstrated a capacity to bind several divalent cations: Cd, Ni, Pb, and Zn. Reaction with Ellman's reagent suggested that the protein has significant sulfhydryl group content that may be involved in metal binding. N-terminal sequencing demonstrates identity with papaya latex trypsin inhibitor, sporamin, or other Kunitz soybean proteinase inhibitors.

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Danielle R. Ellis and Kathryn C. Taylor

A partial cDNA (cvzbp-1) was cloned based on the N-terminal sequence of a citrus (Citrus L.) vascular Zn-binding protein (CVZBP) previously isolated from vascular tissue (Taylor et al., 2002). CVZBP has homology to the Kunitz soybean proteinase inhibitor (KSPI) family. Recombinant protein produced using the cDNA clone inhibited the cysteine proteinase, papain. Metal binding capacity has not been reported for any other member of this family. CVZBP was present in leaves, stems, and roots but not seeds of all citrus species examined. However, CVZBP was present in germinating seeds after the cotyledons had turned green. Within four hrs after wounding, CVZBP was undetectable in the wounded leaf and adjacent leaves. It has been suggested that many members of the KSPI family serve a function in defense. However, the expression of the CVZBP is in direct contrast with those of KSPI members that were implicated in defense response. Though systemically regulated during wounding, we suggest that CVZBP is not a defense protein but rather may function in vascular development.

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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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Zhiyong Hu, Qing Liu, Meilian Tan, Hualin Yi, and Xiuxin Deng

from the cross between the diploid polyembryonic tangerine BDZ ( Citrus reticulata cv. Huanongbendizao) and the allotetraploid somatic hybrid HR [Hamlin sweet orange ( Citrus sinensis ) + rough lemon ( Citrus jambhiri )] ( Deng et al., 1996 ). Recently

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John D. Lea-Cox and I.E. Smith

Pine bark and peat-based substrates have been shown to have low-phosphorus (P) fixation capacity and high leach-potential, similar to that occurring in high-organic soils lacking in inorganic colloids. A long-term greenhouse experiment was conducted where three rootstock species of varying growth rate, Citrus jambhiri Lush.(RL), Citrus reshni Hort. ex Tan. (CM), and Poncirus trifoliata L. × Citrus sinensis L. (Osbeck) (CC), were grown in 3-L containers in composted pine bark, amended with three forms of P. Two slowly soluble forms (Calmafos and MagAmp) and soluble single superphosphate were incorporated at 0 (control), 200, 400, and 800 g P/m3, in a completely randomized block design (n = six plants). A split fertigation treatment of P at 50 mg·L–1 vs. No P was superimposed on the design (n = 3). Despite significant (P > 0.01) differences in P availability in the substrate after 380 days, particularly between liquid P (μ = 65 mg·L–1) vs. no liquid P (μ = 15 mg·L–1), differences in leaf analysis of seedlings after 235 days showed little significance (2.2 vs. 2.7 mg·g–1). To avoid excessive leaching of P from pine bark substrates, it therefore appears that slow-release forms of P are adequate to maintain relatively high growth rates of citrus stock without supplemental P fertigation.

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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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Muhammad Mumtaz Khan*, Muhammad Azam Khan, Muhammad Asif Ali, and Hasnain Raza

Six-week-old rough lemon (Citrus jambhiri L.) seedlings uniform in size were transplanted from nursery to pots filled with peat, spent compost of mushroom and leaf manure used at different proportions with soil, sand and farm yard manure and grown in green house environment. Initial physical and chemical analysis of media indicated that electric conductivity (EC), total porosity, bulk density, moisture percentage, available nitrogen, phosphorus and potassium are more suitable for citrus plant growth and development than other media of different compositions. Peat + sand (1:1) had pH 6.7 which is optimum for growth of citrus nursery. After every four weeks plant length, stem diameter, number of leaves and leaf area were measured. Leaf analysis for N, P, K and mortality percentage was measured at the end of the experiment. Peat + sand (1:1) produced highest percentage of transplant success, plant height, stem diameter, and number of leaves as compared to all other treatments tested. At initial stage peat + sand (1:1) gave the highest results in relation to leaf area, but at the end of experiment it was observed that treatment with silt + spent compost (button) + spent compost (oyster) (1:1:1), produced maximum leaf area with lush green leaves however, mortality rate was very high. This study suggests that peat + sand (1:1) may serve as a standard medium for the container grown citrus nursery.

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Pedro Gonzalez, James P. Syvertsen, and Ed Etxeberria

. At the whole plant level, different citrus rootstock species can differ in their allocations of Na + between roots and shoots ( Garcia-Sanchez and Syvertsen, 2006 , 2009 ). Although the common citrus rootstock rough lemon (RL) ( Citrus jambhiri

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Ed Stover, Robert G. Shatters Jr., Barrett Gruber, Prem Kumar, and Gloria A. Moore

graft transmission. Trees of rough lemon ( Citrus jambhiri , nucellar seedlings) and citron ( Citus medica , cultivar Mhac Nao from rooted cuttings) were graft inoculated on 20 Oct. 2010 with CLas using infected ‘Valencia’ ( Citrus sinensis ) budwood at

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Abigail J. Walter, YongPing Duan, and David G. Hall

adult ACP sampled for this study had spent their entire life on the infected host plant. Samples of 21, 20, and 30 ACP were taken during Jan. 2011 from colonies reared in cages containing infected Citrus jambhiri Lush. (rough lemon), both infected C