Regeneration and characterization of somatic hybrids combining sweet orange and mandarin/mandarin hybrid cultivars for citrus scion improvement

Protoplast fusion between sweet orange and mandarin/mandarin hybrids scion cultivars was performed following the model ??diploid embryogenic callus protoplast?+?diploid mesophyll-derived protoplast??. Protoplasts were isolated from embryogenic calli of ??Pera?? and ??Westin?? sweet orange cultivars (Citrus sinensis) and from young leaves of ??Fremont??, Nules??, and ??Thomas?? mandarins (C. reticulata), and ??Nova?? tangelo [C. reticulata?×?(C. paradisi?×?C. reticulata)]. The regenerated plants were characterized based on their leaf morphology (thickness), ploidy level, and simple sequence repeat (SSR) molecular markers. Plants were successfully generated only when ??Pera?? sweet orange was used as the embryogenic parent. Fifteen plants were regenerated being 7 tetraploid and 8 diploid. Based on SSR molecular markers analyses all 7 tetraploid regenerated plants revealed to be allotetraploids (somatic hybrids), including 2 from the combination of ??Pera?? sweet orange?+???Fremont?? mandarin, 3 ??Pera?? sweet orange?+???Nules?? mandarin, and 2 ??Pera?? sweet orange?+???Nova?? tangelo, and all the diploid regenerated plants showed the ??Pera?? sweet orange marker profile. Somatic hybrids were inoculated with Alternaria alternata and no disease symptoms were detected 96?h post-inoculation. This hybrid material has the potential to be used as a tetraploid parent in interploid crosses for citrus scion breeding.     

Fatty acids of triglycerides and sterol esters from duncan grapefruit,dancy mandarin and their tangelo hybrids

The fatty acid composition of the triglycerides and sterol esters of juice sac lipids were determined in Duncan grapefruit, Dancy mandarin and three of their tangelo hybrids. The fatty acid profiles of Orlando and Minneola tangelos resembled those of the Dancy mandarin parent more than those of the grapefruit. The profiles of Seminole tangelo were different from those of both parents and of its sister hybrids.     

Production of additional allotetraploid somatic hybrids combining mandarings and sweet orange with pre-selected pummelos as potential candidates to replace sour orange rootstock

Summary Sour orange (Citrus aurantium L.) rootstock has historically been a widely utilized eitrus rootstock throughout the world due to its wide soil adaptability and superior horticultural performance. However, quick-decline isolates of citrus tristeza virus (CTV) have demolished entire industries of sour orange rootstock in some countries, including Brazil and Venezuela. CTV is presently destroying millions of trees of sour orange rootstock in Florida and threatens the citrus industries of Texas and Mexico, where sour orange is the predominant rootstock. Efforts to replace sour orange rootstock are combining traditional breeding and biotechnology approaches, including somatic hybridization and transformation. Molecular techniques have confirmed that sour orange is probably a hybrid of mandarin and pummelo. A major focus of our program continues to be the somatic hybridization of superior mandarins with pre-selected pummelo parents. Here, we report the regeneration of allotetraploid somatic hybrid plants from seven new mandarin+pummelo combinations and one new sweet orange+pummelo combination. All new somatic hybrids were confirmed by leaf morphology, ploidy analysis via flow cytometry, and random amplified polymorphic DNA analysis to show nuclear contributions from both parents in corresponding hybrids. These new somatic hybrids are being propagated by tissue culture and/or rooted cuttings for further evaluation of disease resistance and horticultural performance in field trials.     

A Study on Glutamate Oxaloacetate Transaminase Isozymes of Citrus Germplasm Resources

The glutamate oxaloacetate transaminase isozymes (GOT) extracted from 125 biotypes of Citrus and its relatives, Fortunella, Poncirus and Microcitrus were analyzed using polyacrylamide gel electrophoresis to investigate the taxonomic relationships among citrus plants. Besides all the isozymes reported before, two new bands were detected and designated their putative alleles B and C in GOT-l. Among pummelo cultivars wide variations were found. Most of the mandarins were identical, having SS for GOI-1 and MM for GOT-2 except for Zou-Pi-Gan and Yao-Gan, which both had FS at GOT-1 and might be hybrids. This finding suggests that all of the mandarins may have originated from a common ancestor. Sour orange biotypes showed a considerable variation in GOT isozymes. Most of the sour oranges in China were assumed to be hybrids between pummelo and mandarin based on GOT isozyme patterns, but two biotypes, Xiao-Hong-Cheng and Zhu-lan, had FS at GOT-1 and MB at GOT-2, which strongly suggests that they be hybrids of pummelo and Ichang papeda because B allele of GOT-2 occurs only in Ichang papeda and its close relatives Yuzu and Ichang lemon. From this study Yuzu is assumed to be derived from hybridizationof Ichan, papeda and mandarin.     

Current Distribution of Huanglongbing (citrus greening disease) in India as Diagnosed by Real‐Time PCR

The widespread occurrence of Huanglongbing (HLB) was recorded in sixteen citrus growing states of India using the real‐time quantitative PCR and the derived threshold cycle (Ct) value. All the commercially important citrus varieties of mandarin, sweet orange, lime and lemon, pummelo and Satkara were infected with ‘Candidatus Liberibacter asiaticus’, the bacterium associated with HLB. Ct values positive for HLB were found in all the states except Arunachal Pradesh. The primer–probe combination HLBas‐HLBr‐HLBp was found specific to Ca. L. asiaticus and do not exhibit any cross‐reactivity with other pathogenic residents of citrus.     

A comparison of citrus blossom volatiles

The objective of this study was to identify the major volatiles and their relative concentrations in intact grapefruit, sweet orange, sour orange, mandarin, lemon, lime and pummelo blossoms. Volatiles from freshly picked blossoms were collected and concentrated using static headspace solid-phase microextraction and then separated and identified using GC–MS. Seventy volatiles were detected, 66 identified, of which 29 were identified for the first time in citrus blossoms. Major volatiles consisted of linalool, β-myrcene, α-myrcene, limonene, (E)-ocimene, methyl anthranilate and indole. In terms of total volatiles: pummelo  grapefruit ≈ sweet orange > sour orange ≈ mandarin ≈ lemon–lime > Volkamer lemon > Kaffir lime. Principal component analysis of blossom volatiles demonstrated that there were three widely separated, tightly clustered groups which consisted of mandarin, lemon–lime and pummelo. Other cultivars of possible mixed parentage produced non-overlapping values within the boundaries of these three clustered groups. The first two Eigenvectors explained 83% of the total variance. Linalool, limonene and myrcene had the highest loading values. Those cultivars requiring insect pollination such as pummelo produced highest levels of total volatiles as well as highest levels of known honeybee stimulants such as 1-hexanol and linalool.     

Citrus somatic hybridization with potential for improved blight and CTV resistance

Summary The production of five new somatic hybrids with potential for improved disease resistance is reported herein. Protoplast isolation, fusion, and plant regeneration was achieved from Caipira sweet orange (Citrus sinensis L. Osbeck) as an embryogenic parental source and Volkamer lemon (C. volkameriana Pasquale), Cleopatra mandarin (C. reticulata Blanco), and Rough lemon (C. jambhiri Lushington) as non-embryogenic parental sources. Fusion involving Cleopatra mandarin and Rangpur lime (C. limonia L. Osbeck) as embryogenic parental sources with Sour orange (C. aurantium L.) also resulted in somatic hybrid plants. Somatic hybridization was confirmed by leaf morphology evaluation, chromosome counting, and randomly amplified polymorphic DNA (RAPD) analyses. Somatic hybrids may combine complementary characteristics from both parental sources and have potential for tolerance to blight and citrus tristeza virus (CTV).     

The introduction of cultivated citrus to Europe via Northern Africa and the Iberian Peninsula

The circumstances concerning the diffusion of the main cultivated citrus from their places of origin in Asia are studied here, showing that the citron (’Citrus medica L.) was the only one knew in Ancient times in Europe, while the lemon (C. limon [L.] Osbeck), lime (C. aurantiifolia [Christm.] Swingle), pomelo (C. maxima [Burm.] Merr.) and sour orange (C. x aurantium L.) were introduced to Europe by the Muslims via the Iberian Peninsula and Sicily, and that the grapefruit (C. paradisi Macfad.), mandarin (C. reticulata Blanco) and sweet orange (C. x aurantium L.) arrived to the West between the fifteenth and nineteenth centuries as a result of the trade with the British and Portuguese colonies.     

Genetic diversity of hop stunt viroid from symptomatic and asymptomatic citrus trees in Iran

Hop stunt viroid as the causal agent of cachexia disease has detected from citrus trees in different areas in Iran. Although cachexia has not been reported as a decline disease for citrus trees, it can impair crop quality and reduce plant yields. This study was undertaken to molecularly detect HSVd among different commercial citrus cultivars and determine genetic diversity of this viroid in Mazandaran province of Iran. Sampling was performed from symptomatic and symptomless citrus cultivars in Mazandaran province. HSVd specific primers were used for molecular detection. SSCP and sequencing were applied to assay HSVd genetic diversity. Results showed the detection of HSVd in all symptomatic Satsuma (25 out of 25), Clementine (25 out of 25), sweet lime (20 out of 20) and sweet orange cv. Valencia (7 out of 7), as well as, 31% (14 out of 22), 100% (12 out of 12) and 33% (5 out of 15) of page mandarin, lemon and grapefruit trees, respectively. 10 different HSVd genomes were identified by sequencing the SSCP profiles among which HSVd‐IR1 had the most frequency.     

Production and molecular characterization of two new Citrus somatic hybrids for scion improvement

Somatic hybridization by protoplast fusion from cell suspension cultures and leaf parent has been a well-established technique holding great potential for citrus variety improvement. In this study, somatic hybrid plants were regenerated from the following two fusion combinations: ‘Murcott’ tangor (Citrus reticulata Blanco × C. sinensis (L.) Osbeck) + Hirado Buntan Pink pummelo (HBP) (C. grandis (L.) Osbeck) and ‘Bingtang’ orange (C. sinensis (L.) Osbeck) + Calamondin (C. microcarpa Bunge). Somatic hybrids were selected at an early stage based on their higher capacity for embryogenesis comparing to non-hybrid cells. Flow cytometry analysis showed that all plants from pre-selected lines of the two combinations were tetraploid. SSR analysis confirmed their hybrid nature, with nuclear DNA from both fusion parents, and absence of parental specific bands was also detected. Cytoplasmic compositions of the recovered plants were further revealed by CAPS and cpSSR analysis. The allotetraploid somatic hybrids from the ‘Murcott’ tangor + HBP combination will be applied to develop triploid seedless cultivars by interploid crossing with diploid seedy citrus cultivars, and those from ‘Bingtang’ orange + Calamondin could be valuable for Asiatic citrus canker-tolerant and ornamental citrus breeding.     

不同柑桔品种上桔全爪螨的生长和种群动态差异

通过田间调查、室内盆栽苗接螨、离体叶片饲养等方法,考察了14个柑桔品种上桔全爪螨种群动态,组建了其实验种群生命表,结果表明：该螨在各柑桔品种上的种群密度有明显差异,平均密度以北碚447、台湾晚白柚和垫江白柚上较高,柑新生系最低。雌成螨寿命在台湾晚白柚上最长,垫江白柚次之,柑新生系最短;产卵量以取食台湾晚白柚时最多,垫江白柚、五步红心柚次之,以柑新生系最少;在垫江白柚上的内禀增长率（r_m）最大,沙田柚、台湾晚白柚次之,金弹最小。根据桔全爪螨实验种群的参数判断,金弹和柑新生系属抗螨品种,台湾晚白柚、垫江白柚和北碚447是感螨品种。从品种归类上来看,柚类品种和甜橙中的北碚447感螨程度较高,金柑属的金弹和宽皮柑桔类的柑新生系感螨程度较低。甜橙类与宽皮柑桔类比较,前者感螨程度较高,后者相对较低,但宽皮柑桔类的早津感螨程度相对较高。     

Performance of Cryptolaemus montrouzieri feeding on Pulvinaria aurantii ovisacs on citrus plants

We studied the performance of the coccinellid Cryptolaemus montrouzieri on citrus plant species infested with orange pulvinaria scale, Pulvinaria aurantii, under controlled conditions in the laboratory. The experimental host plants were blood orange, grapefruit, lemon, pummelo and kumquat. All life history parameters indicated best performance of C. montrouzieri, when reared on P. aurantii ovisacs on the leaves of grapefruit. Pummelo, blood orange, lemon or kumquat were less suitable.     

Host-Specific Effects of Toxin from the Rough Lemon Pathotype of Alternaria alternata on Mitochondria

Host-specific toxin from the rough lemon (Citrus jambhiri Lush) pathotype of Alternaria alternata (ACR toxin) was tested for effects on mitochondria isolated from several citrus species. The toxin caused uncoupling of oxidative phosphorylation and changes in membrane potential in mitochondria from leaves of the susceptible host (rough lemon); the effects differed from those of carbonylcyanide-m-chlorophenylhydrazone, a typical protonophore. ACR toxin also inhibited malate oxidation, apparently because of lack of NAD⁺ in the matrix. In contrast, the toxin had no effect on mitochondria from citrus species (Dancy tangerine and Emperor mandarin [Citrus reticulata Blanco], and grapefruit [Citrus paradisi Macf.]) that are not hosts of the fungus. The effects of the toxin on mitochondria from rough lemon are similar to the effects of a host-specific toxin from Helminthosporium maydis (HMT) on mitochondria from T-cytoplasm maize. Both ACR and HMT toxins are highly selective for the respective host plants. HMT toxin and methomyl had no effect (toxic or protective) on the activity of ACR toxin against mitochondria from rough lemon.     

Non-random inheritance of mitochondrial genomes in Citrus hybrids produced by protoplast fusion

Somatic hybridization offers the possibility of manipulating chloroplast and mitochondrial genomes and evaluating their role on cultivar qualities in citrus. Numerous associations between Willow-leaf mandarin (Citrus deliciosa Ten.), as embryogenic parent, and sweet orange cv. Valencia (Citrus sinensis (L.) Osb.), as mesophyll parent, and between Willow-leaf mandarin (embryogenic parent) and grapefruit cv. Duncan (Citrus paradisi Macf.) (mesophyll parent) were obtained by the fusion of protoplasts induced by polyethylene glycol. Regenerated plants were characterized by flow cytometry and nuclear and mitochondrial DNA restriction fragment length polymorphism (RFLP). All plants were diploid. Diploid plants with the nuclear RFLP patterns of mandarin or sweet orange were identified in the progeny between these two parents, while only grapefruit nuclear types were found in the mandarin + grapefruit progeny. The diploid plants with the nuclear profile of the mesophyll parent originated systematically from cells formed through spontaneous association of the nuclear genome of the mesophyll parent and the mitochondrial genome of the embryogenic parent. These plants are assumed to be alloplasmic hybrids or cybrids. They were viable and have been propagated for field testing.     

Detection and Inoculation of Peanut Witches' Broom Phytoplasma (16SrII‐A) and Periwinkle Leaf Yellowing Phytoplasma (16SrI‐B) in Citrus Cultivars in Taiwan

To clarify the phytoplasma associated with Huanglongbing (HLB), a detection survey of phytoplasma in field citrus trees was performed using the standardized nested PCR assay with primer set P1/16S‐Sr and R16F2n/R16R2. The HLB‐diseased citrus trees with typical HLB symptoms showed a high detection of 89.7% (322/359) of HLB‐Las, while a low detection of phytoplasma at 1.1% (4/359) was examined in an HLB‐affected Wentan pummelo (Citrus grandis) tree (1/63) and Tahiti lime (C. latifolia) trees (3/53) that were co‐infected with HLB‐Las. The phytoplasma alone was also detected in a healthy Wentan pummelo tree (1/60) at a low incidence total of 0.3% (1/347). Healthy citrus plants were inoculated with the citrus phytoplasma (WP‐DL) by graft inoculation with phytoplasma‐infected pummelo scions. Positive detections of phytoplasma were monitored only in the Wentan pummelo plant 4 months and 3.5 years after inoculation, and no symptoms developed. The citrus phytoplasma infected and persistently survived in a low titre and at a very uneven distribution in citrus plants. Peanut witches' broom (PnWB) phytoplasma (16SrII‐A) and periwinkle leaf yellowing (PLY) phytoplasma belonging to the aster yellows group (16SrI‐B) maintained in periwinkle plants were inoculated into healthy citrus plants by dodder transmission. The PnWB phytoplasma showed infection through positive detection of the nested PCR assay in citrus plants and persistently survived without symptom expression up to 4 years after inoculation. Positive detections of the phytoplasma were found in a low titre and several incidences in the other inoculated citrus plants including Ponkan mandarin, Liucheng sweet orange, Eureka lemon and Hirami lemon. None of the phytoplasma‐infected citrus plants developed symptoms. Furthermore, artificial inoculation of PLY phytoplasma (16SrI‐B) into the healthy citrus plants demonstrated no infection. The citrus symptomless phytoplasma was identified to belong to the PnWB phytoplasma group (16SrII‐A).     

Improved Transformation Efficiency in Citrus by Plasmolysis Treatment

Procedures for high efficiency production of transgenic citrus plants using an Agrobacterium tumefaciens system with plasmolysis treatment were developed. Longitudinally cut epicotyl segments of eight different citrus species [’Milam’ Rough lemon (Citrus jambhiri Lush), ‘Volkamer’ lemon (Citrus volkameriana L), Rangpur lime (Citrus limonia L), ‘Hamlin’ sweet orange (Citrus sinensis L Osbeck), ‘Duncan’ grapefruit (’Citrus paradisi’ Macf), Sour orange (Citrus aurantium L), ‘Cleopatra’ mandarin (Citrus reticulata Blanco) and Carrizo citrange (Citrus sinensis L Osbeck x Poncirus trifoliata L Raf) ] were plasmolyzed in different concentrations of sucrose and maltose [0, 3, 6, 8, 9, 10, 12 % (w/v) ] prior to Agrobacterium inoculation. Plasmolyzed epicotyl explants were cocultivated with either the hypervirulent Agrobacterium tumefaciens strain, the EHA-101 (harboring a binary vector pGA482GG) or Agl-1 (carrying pCAMBIA1303 vector). Both binary vectors contained neomycin phosphotransferase II (NPT II) and β-glucuronidase (GUS) genes. The binary vector, pCAMBIA1303 also contained a fused mGFP5 gene at the 3’ end of GUS gene as a reporter. Epicotyl explants of Rangpur lime, Rough and ‘Volkamer’ lemons plasmolyzed in 9–12 % maltose showed transient GUS gene expression comprising up to 95 % of the cut surface of explants, while Carrizo citrange showed 80 % expression when they were plasmolyzed in 6–10 % sucrose. On the other hand, epicotyl explants of ‘Hamlin’ sweet orange, Grapefruit, Sour orange and ‘Cleopatra’ mandarin showed transient GUS expession in 80–90 % of explants with 6–10 % sucrose. Basal portions of the regenerated putative transgenic shoots harvested from the cut surface of epicotyl explants within 2–3 months, were assayed for GUS, and apical portions were shoot-tip grafted in vivo for the production of whole plants. The transformation efficiencies in different species obtained are the highest so far reported for citrus.     

Influence of the Sting Nematode, Belonolaimus longicaudalus,on Young Citrus Trees

The sting nematode, Belonolaimus longicaudatus, was associated with poor growth of citrus in a central Florida nursery. Foliage of trees was sparse and chlorotic. Affected rootstocks included Changsha and Cleopatra mandarin orange; Flying Dragon, Rubidoux, and Jacobsen trifoliate orange; Macrophylla and Milam lemon; Palestine sweet lime; sour orange; and the hybrids - Carrizo, Morton, and Rusk citrange and Swingle citrumelo. Root symptoms included apical swelling, development of swollen terminals containing 3-5 apical meristems and hyperplastic tissue, coarse roots, and a reduction in the number of fibrous roots. Population densities as high as 392 sting nematodes per liter soil were detected, with 80% of the population occurring in the top 30 cm of soil; however, nematodes were detected to 107 cm deep. Although an ectoparasite, the nematode was closely associated with citrus root systems and was transported with bare root nursery stock. Disinfestation was accomplished by hot water treatment (49 C for 5 minutes).     

Delay expression of limonoid UDP-glucosyltransferase makes delayed bitterness in citrus

Genes encoding limonoid UDP-glucosyltransferase from albedo of six Citrus species with different levels of delayed bitterness are isolated and cloned in vector pTZ57R/T. Our results indicate that gene sequence of sweet lime (with intense juice delayed bitterness) have complete identity with Satsuma mandarin (without distinctive juice delayed bitterness). Also gene sequence of Marsh seedless grapefruit, local orange and Thompson navel orange (with mild juice delayed bitterness) have very similarity with Satsuma mandarin. On the other hand, this gene started to express 60, 120, and 210 days after full blooming in albedo of Satsuma mandarin, sweet oranges and sour orange, and both grapefruit and sweet lime, respectively. Expression pattern of limonoid glucosyltransferase gene in leaves was quite different with albedo. Thus, we supposed the delayed bitterness in this species was related to delay in expression of limonoid glucosyltransferase gene in albedo and lower limonoid glucoside accumulation in fruits.     

Formation of Adventitious Buds in Decapitated Citrus Seedlings and the Effect of Some Growth Regulators

Young seedlings of various citrus species give rise, after decapitation,to adventitious buds at the cut end of the epicotyl. With suchtreatment, more buds formed on seedlings of sweet orange, sourorange, and grapefruit than of lemon, mandarin, and calamondin.Anatomical observations at the cut stem end show formation ofbuds occurring from tissues close to the cambium. Growth regulatorsapplied at the cut epicotyl end modified the patterns of budformation. 6-Benzylaminopurine (BAP) promoted bud formation;indole-3-butyric acid (IBA), abscisic acid (ABA), and gibberellicacid (GA₃) inhibited it. BAP reversed the inhibitory effectof GA₃. Gibberellic acid caused a typical elongation of thesprout axis. The use of decapitated seedlings as a tool forstudying stem differentiation in citrus is suggested.