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.
Jude W. Grosser, Frederick G. Gmitter Jr., J.L. Chandler, and Eliezer S. Louzada
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.
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.
Jude W. Grosser, Frederick G. Gmitter Jr., Franca Sesto, Xiu Xin Deng, and J.L. Chandler
Protoplasm culture following polyethylene glycol (PEG) -induced fusion resulted in the regeneration of somatic hybrid plants from the following six parental combinations: Citrus sinermis (L.) Osbeck cv. Hamlin + Severinia buxifolia (Poir.) Tenore (Chinese box-orange); C. reticulate Blanco cv. Cleopatra + Poncirus trifoliata (L.) Raf. cv. Flying Dragon; C. reticulate cv. Cleopatra + Swingle citrumelo (C. paradisi Macf. × P. trifoliata); C. sinensis cv. Hamlin + C. jambhiri cv. Rough lemon; C. sinensis cv. Valencia + C. jambhiri cv. Rough lemon; and C. paradisi cv. Thompson + `Murcott' tangor (purported hybrid of C. reticulate × C. sinensis). Diploid plants were regenerated from nonfused embryogenic culture-derived protoplasts of `Cleopatra' mandarin and `Hamlin' and `Valencia' sweet orange, and from nonfused leaf-derived protoplasts of Rough lemon and `Mnrcott'. Regenerated plants were classified according to leaf morphology, chromosome number, and isozyme analyses. All of the somatic hybrids reported herein are tetraploid (2n = 4x = 36), with the exception of the `Hamlin' + S. buxifolia hybrid, which was unexpectedly found to have a chromosome number of 2n = 27. These six new somatic hybrids have potential in citrus scion and rootstock improvement for commercial use.
Sudip Kunwar, Jude Grosser, Fred G. Gmitter Jr., William S. Castle, and Ute Albrecht
Most of the commercially important citrus scion cultivars are susceptible to Huanglongbing (HLB), which is the most devastating disease the citrus industry has ever faced. Because the rootstock can influence the performance of the scion in various ways, including disease and pest tolerance, use of superior rootstocks can assist citrus growers with minimizing the negative effects of HLB. The objective of this study was to assess rootstock effects on the horticultural performance and early production potential of ‘Hamlin’ sweet orange (Citrus sinensis) trees in commercial field settings under HLB-endemic conditions. Two field trials were conducted in different locations in Central and Southeast Florida. The trials were established in 2015 and included 32 diverse diploid and tetraploid rootstock cultivars and advanced selections. One trial was performed in Highlands County, FL, on a poorly drained flatwoods-type site. Another trial was performed in Polk County, FL, on a well-drained sandy Central Florida Ridge site. Horticultural traits including tree height, canopy volume, trunk diameter, canopy health, leaf nutrient content, yield, and fruit quality were assessed during the 2018–19 and 2019–20 production years. Significant differences were found among trees on different rootstocks for most of the measured traits, particularly tree vigor and productivity, but rootstock effects also varied by location. Rootstocks that induced large tree sizes, such as the diploid mandarin × trifoliate orange hybrids ‘X-639’, ‘C-54’, ‘C-57’, and ‘C-146’, also induced higher yield, but with lower yield efficiency. Most of the tetraploid rootstocks significantly reduced tree size, among which ‘Changsha+Benton’, ‘Green-3’, ‘Amb+Czo’, ‘UFR-3’, and ‘UFR-5’ induced high yield efficiency. Therefore, these rootstocks have the potential to be used in high-density plantings. However, trees on some of these small size-inducing rootstocks had a higher mortality rate and were more vulnerable to tropical force winds. This study provides important information for the selection of rootstocks with the greatest production potential in an HLB-endemic environment, especially during the early years of production.
Aditi D. Satpute, Chunxian Chen, Fredrick G. Gmitter Jr., Peng Ling, Qibin Yu, Melinda R. Grosser, Jude W. Grosser, and Christine D. Chase
In cybridization, new combinations of nuclear and cytoplasmic genes result in a unique genotype that may bring cellular, physical, physiological, and biochemical changes to the plant. This has been demonstrated in the unexpected cybrids generated from the fusion of citrus (Citrus sp.) protoplasts in two independent experiments. The first experiment was conducted to generate potentially seedless triploids by fusing diploid protoplasts of embryogenic ‘Dancy’ mandarin (Citrus reticulata) suspension culture cells with haploid ‘Ruby Red’ grapefruit (C. paradisi) protoplasts derived from tetrad-stage microspores. After multiple attempts, only one triploid was recovered, but several diploid plants with typical grapefruit morphology were also regenerated. In the second experiment, protoplasts derived from embryogenic ‘Dancy’ mandarin suspension culture were fused with nonembryogenic protoplasts from ‘Duncan’ grapefruit leaves in an effort to produce an allotetraploid somatic hybrid. The fruit from the resulting trees resembled grapefruit in morphology and type, and maintained excellent quality throughout the summer, when commercial grapefruit rapidly loses quality. Fruit on these trees remained firm with exceptional sweetness and good flavor into August, and without seed germination. The regenerants obtained in the protoplast fusion experiments were confirmed as cybrids by genetic marker analyses. The test grapefruit were identical to commercial ‘Ruby Red’ grapefruit at six nuclear simple sequence repeat (SSR) marker loci, but identical to ‘Dancy’ with respect to a mitochondrial intron marker. The plastid genomes of individual trees originated from either fusion partner. In the first experiment, haploid ‘Ruby Red’ protoplast preparations must have also contained contaminant diploid protoplasts. Apart from the value of altered fruit quality attributes in the marketplace, these plants provide an opportunity to understand the contributions of cytoplasmic organelle genetics to important citrus fruit-breeding objectives.
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.
Jude W. Grosser, Hyun Joo An, Milica Calovic, Dong H. Lee, Chunxian Chen, Monica Vasconcellos, and Frederick G. Gmitter Jr
Somatic hybridization through protoplast fusion has proven to be a valuable technique in citrus for producing unique allotetraploid breeding parents that combine elite diploid selections. Many citrus somatic hybrids are now flowering and being used in interploid crosses to generate triploid hybrids that produce seedless fruit, a primary objective of citrus breeding programs. Most of the early somatic hybrids produced for mandarin improvement combined sweet oranges with mandarins, because the performance of sweet oranges in tissue/protoplast culture generally exceeds that of most mandarin selections. However, a high percentage of triploid progeny from interploid crosses using sweet orange + mandarin somatic hybrids as the tetraploid parent produce fruit that are difficult to peel. We report nine new allotetraploid somatic hybrids and five new autotetraploids from somatic fusion experiments involving easy-peel mandarin parents. These tetraploids can be used in interploid crosses to increase the percentage of seedless triploid progeny producing easy-to-peel fruit. Ploidy level of the new tetraploids was determined by flow cytometry and their genetic origin by expressed sequence tag–simple sequence repeat marker analysis.
Rhuanito S. Ferrarezi, Alan L. Wright, Brian J. Boman, Arnold W. Schumann, Fred G. Gmitter, and Jude W. Grosser
Completely enclosed screen houses can physically exclude contact between the asian citrus psyllid [ACP (Diaphorina citri)] and young, healthy citrus (Citrus sp.) trees and prevent huanglongbing (HLB) disease development. The current study investigated the use of antipsyllid screen houses on plant growth and physiological parameters of young ‘Ray Ruby’ grapefruit (Citrus ×paradisi) trees. We tested two coverings [enclosed screen house and open-air (control)] and two planting systems (in-ground and container-grown), with four replications arranged in a split-plot experimental design. Trees grown inside screen houses developed larger canopy surface area, canopy surface area water use efficiency (CWUE), leaf area index (LAI) and LAI water use efficiency (LAIWUE) relative to trees grown in open-air plots (P < 0.01). Leaf water transpiration increased and leaf vapor pressure deficit (VPD) decreased in trees grown inside screen houses compared with trees grown in the open-air plots. CWUE was negatively related to leaf VPD (P < 0.01). Monthly leaf nitrogen concentration was consistently greater in container-grown trees in the open-air compared with trees grown in-ground and inside the screen houses. However, trees grown in-ground and inside the screen houses did not experience any severe leaf N deficiencies and were the largest trees, presenting the highest canopy surface area and LAI at the end of the study. The screen houses described here provided a better growing environment for in-ground grapefruit because the protective structures accelerated young tree growth compared with open-air plantings while protecting trees from HLB infection.
Rhuanito S. Ferrarezi, Alan L. Wright, Brian J. Boman, Arnold W. Schumann, Fred G. Gmitter, and Jude W. Grosser
Huanglongbing (HLB) disease is a threat to most citrus (Citrus sp.) producing areas and is associated with the bacterium Candidatus Liberibacter asiaticus. The disease is transmitted by the vector asian citrus psyllid [ACP (Diaphoria citri)]. Antipsyllid screen houses can potentially reduce and eliminate HLB development in young citrus plantings by excluding the insect vector. These structures are also anticipated to represent a new environmental platform to cultivate high-valued fresh citrus. The purpose of this investigation was to evaluate the effect of screen houses on excluding infective ACP from inoculating grapefruit (Citrus ×paradisi) trees and determine changes on environmental conditions caused by the screen cloth. We tested two coverings [enclosed screen house and open-air (control)] and two planting systems (in-ground and container-grown), with four replications arranged in a split-plot experimental design. Psyllid counting and HLB diagnosis were performed monthly, and the antipsyllid screen excluded the HLB vector from the houses. ACP and HLB-positive trees were found only at the open-air plots. Weather monitoring was performed every 30 minutes from 22 Feb. to 31 July 2014. Solar radiation accumulation averaged 6.7 W·m−2·minute−1 inside the screen houses and 8.6 W·m−2·minute−1 in the open-air. Air temperature was greater inside the screen houses whereas wind gusts were higher in the open-air. Reference evapotranspiration accumulation averaged 3.2 mm·day−1 inside the screen houses and 4.2 mm·day−1 in the open-air. There was no difference in cumulative rainfall between screen houses and open-air. The antipsyllid screen houses reduced solar radiation, maximum wind gust, and reference evapotranspiration (ETo). The environmental conditions inside the protective screen houses are suitable for grapefruit production.