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Jude W. Grosser, Victor Medina-Urrutia, Govindarajulu Ananthakrishnan and Patricia Serrano

Sour orange has been a premier citrus rootstock worldwide due to its ability to perform on challenging soils and to produce and hold high-quality fruit. However, increasingly widespread quick-decline isolates of citrus tristeza virus (CTV) have destroyed entire industries on sour orange in some countries, and are in the process of destroying millions of trees on sour orange in Florida. CTV also threatens other citrus locations planted heavy to sour orange, including Texas and Mexico. An acceptable alternative rootstock to replace sour orange is in high demand but has yet to be developed. Molecular analyses have recently determined that sour orange is probably a hybrid of pummelo and mandarin. We report the production of 12 new mandarin + pummelo somatic hybrids produced by protoplast fusion from selected superior mandarin and pummelo parents, in efforts to develop a suitable replacement sour-orange-like rootstock that is resistant to CTV-induced quick decline. Somatic hybrids from all 12 parental combinations were confirmed by a combination of leaf morphology, flow cytometry, and RAPD analyses (for nuclear hybridity). These new mandarin + pummelo somatic hybrids are being propagated by rooted cuttings as necessary to conduct quick-decline resistance assays and to assess horticultural performance in replicated field trials.

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Victor Medina-Urrutia, Karla Fabiola, Lopez Madera, Patricia Serrano, G. Ananthakrishnan, Jude W. Grosser and Wenwu Guo

No presently available rootstock combines all the available rootstock attributes necessary for efficient long-term citriculture (production and harvesting) of Mexican limes and other commercially important scions. In the present study, somatic hybridization techniques were used to combine the widely adapted Amblycarpa mandarin (also known as Nasnaran mandarin) with six different trifoliate/trifoliate hybrid selections: Benton, Carrizo, and C-35 citranges; Flying Dragon and Rubidoux trifoliate oranges; and a somatic hybrid of sour orange + Flying Dragon. The ultimate goal of this research is to generate polyploid somatic hybrids that express the complementary horticultural and disease resistance attributes of the corresponding parents, and have direct potential as improved tree-size controlling rootstocks. Somatic hybrids from all six parental combinations were confirmed by a combination of leaf morphology, flow cytometry, and randomly amplified polymorphic DNA (RAPD) (for nuclear hybridity) and cleaved amplified polymorphic sequence (CAPS) analyses (for mtDNA and cpDNA). This is the first report of citrus somatic hybridization using Amblycarpa mandarin. Unexpected hexaploid somatic hybrid plants were recovered from the fusion of Amblycarpa mandarin + C-35 citrange. Hexaploid hybrids should be very dwarfing and may have potential for producing potted ornamental citrus. Resulting somatic hybrid plants from all six combinations have been propagated by tissue culture and/or rooted cuttings and are being prepared for commercial field evaluation for their potential as improved rootstocks for Mexican lime and other important scions.

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Jude Grosser, Milicia Calovic, Patricia Serrano, Fred Gmitter Jr. and J. L. Chandler

The international fresh citrus market now demands high-quality, seedless fruit that must also be easy to peel for consumer convenience, especially when considering new mandarin varieties. High quality varieties that historically perform well in Florida are generally seedy. Florida is therefore losing market-share to `Clementine' and other seedless varieties produced in Mediterranean climates, including Spain, Morocco, and California. In our ongoing program, somatic hybridization and cybridization via protoplast fusion are now playing a key role in strategies to develop competitive seedless mandarin hybrids adapted to Florida. Somatic hybridization is being used to combine elite diploid parents to produce high quality allotetraploid breeding parents that can be used in interploid crosses to generate seedless triploids. Several thousand triploid mandarin hybrids have been produced under the direction of F.G. Gmitter, Jr. Some of our allotetraploid somatic hybrids are producing fruit with direct cultivar potential, i.e., 'Valencia' sweet orange + `Murcott' tangor. New somatic hybrids produced in our program will be discussed, including `Page' tangor + `Dancy' mandarin, `Page' tangor + `Kinnow' mandarin, and `Hamlin' sweet orange + LB8-9 tangelo. Somatic cybridization is being used to transfer CMS (cytoplasmic male sterility) from the seedless `Satsuma' mandarin to other seedy varieties via mtDNA transfer, in efforts to make them seedless. New somatic cybrids produced in our program that contain the `Satsuma' CMS include `Murcott' tangor and `Kinnow' mandarin. Details of these results and other progress will be discussed.

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Chunxian Chen, Jude W. Grosser, Milica Ćalović, Patricia Serrano, Gemma Pasquali, Julie Gmitter and Fred G. Gmitter Jr

Somatic hybridization is a powerful tool for the genetic improvement of citrus rootstocks, and it is part of an efficient in vitro-based breeding system described here. An essential component of the system is the requirement of confirming tetraploidy and the combination of the two donor genomes. Expressed sequence tag–simple sequence repeat (EST-SSR) markers provide a means to accomplish both of these objectives, and their application to a population of pummelo [Citrus grandis (L.) Osbeck] + mandarin (C. reticulata Blanco) somatic hybrids developed for the specific purpose of providing alternative rootstocks for sour orange (Citrus aurantium L.) is detailed. Nineteen new somatic hybrids were produced from various mandarin and pummelo parents, and their ploidy level and the complementation of their nuclear genomes were confirmed using four EST-SSR markers. These markers were selected from markers previously mapped in sweet orange [C. sinensis (L.) Osbeck] and trifoliate orange [Poncirus trifoliata (L.) Raf.] and prescreened for suitable allelic polymorphism within the mandarin and pummelo lines used. After polymerase chain reaction amplification of sequences from the parents and putative hybrids, the products were separated on a genetic sequencer and visualized electronically. Additionally, EST-SSR markers identified the unexpected zygotic origin of a presumed nucellar embryogenic callus line. Integration of EST-SSR techniques for high-throughput genotyping with previously developed approaches to somatic hybrid creation increases substantially the effectiveness and efficiency of this in vitro-based breeding system for citrus rootstock improvement.