Finger printing of acid lime varieties and clones having varied resistance to bacterial canker,using RAPD marker

Citrus is an important fruit crop having divergent genetic variation within the species. The germplasm identification and characterisation is an important link between the conservation and utilisation of genetic resources. Conventionally, variety/clone identification has relied on morphological characters such as growth habit, leaf, floral and fruit characters etc. Investigation through RAPD (random amplified polymorphic DNA) markers was carried out for determination of genetic variation among 12 acid lime clones having varied resistance to bacterial canker disease. DNA was extracted from the leaf of 12 acid lime clones and was subjected to PCR using 20 random primers (nine from OPM and 11 from OPA series) which yielded a total of 127 distinct DNA fragments, out of which 103 were polymorphic. Genetic similarity was evaluated based on the presence or absence of bands. The bands obtained were polymorphic, with sizes ranging from 750 bp to 2.5 kb. Cluster analysis using the similarity coefficient showed that the Balaji, RHRL-124 and PKM-1 formed one cluster and the remaining clones formed a second cluster, which in turn were divided into TAL 94-9, TAL 94-10, TAL 94-11 and TAL 94-12 which formed the first subcluster; the Nalgonda selection and local acid lime formed a second subcluster; TAL 94-8, RHRL-49 and RHRL-122 did not resemble any other clones. Among the 12 acid lime clones, Balaji, RHRL-124, RHRL-122 and PKM-1 were found to be moderately resistant to bacterial canker. Correlation of RAPD data with canker disease incidence in the moderately resistant acid lime clones viz., Balaji, RHRL-124 and PKM-1 were formed as one cluster, and all susceptible clones formed as a second cluster viz., except TAL-94-9, RHRL-122, which were found to be moderately resistant and did not form a cluster with any other acid lime clone.     

Protoplast-fusion-mediated transfer of organelles from Microcitrus into Citrus and regeneration of novel alloplasmic trees

Summary Iodoacetate-treated Citrus protoplasts from embryogenic nucellar calli of Sour orange (C. aurantium) or from Rough lemon (C. jambhiri) were fused with -irradiated protoplasts from a related genus, Microcitrus. The fused protoplasts were cultured to obtain colonies and micro-calli. Micro-calli derived from these two fusion combinations were isolated, propagated and differentiated into embryos, which subsequently regenerated trees having the morphology of Sour orange or Rough lemon. These intergeneric fusions resulted in mitochondria with novel DNA, indicating recombination between the chondriomes of Citrus and Microcitrus. Chloroplast DNA analyses of fusion-derived embryos indicated that they contained the chloroplasts of either fusion-partner or a mix of these chloroplasts. Later plastome analyses of leaves from fully differentiated plants showed that cybrids having Rough lemon morphology had either Rough lemon or Microcitrus chloroplast DNA, indicating complete sorting out of chloroplasts. Likewise, sorting out of Microcitrus chloroplasts was detected in a cybrid plant having Sour orange morphology.Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, 50250 Israel. No. 2663-E, 1989 series     

柑桔原生质体融合再生叶肉亲本型植株的遗传分析

叶肉亲本（粗柠檬）型植株叶形指数、气孔特征与亲本粗柠檬无异,与同组合体细胞杂种差异显著。染色体计数为二倍体（２ｎ＝２ｘ＝１８）。过氧化物酶（ＰＯＸ）、多酚氧化酶（ＰＰＯ）、谷草转氨酶（ＧＯＴ）同工酶图谱与粗柠檬一致。ＲＡＰＤ分析表明,在具多态性的５４个随机引物中,大多数引物（５２个）上的图谱与粗柠檬相同。但ＯＰＷ－１２上,叶肉亲本型植株含有哈姆林甜检的特征谱带。ＯＰＶ－０４扩增产物显示,叶肉亲本型     

Soil temperature and flooding effects on two species of citrus

Summary Rough lemon (Citrus jambhiri Lush.) and sour orange (C. aurantium L.) seedlings were grown at constant soil temperatures of 16, 24, and 33 C for 3 months. Shoot and root growth of rough lemon was greatest at 33 C while growth of sour orange was greatest at 24 C. There were no significant effects of soil temperature on shoot: root ratio, leaf water potential or stomatal conductance. The hydraulic conductivity of intact root systems of both species was highest when seedlings were grown at 16 C. Thus, acclimation through greater root conductivity at low soil temperature may have compensated for decreased root growth at 16 C and negated effects of soil temperature on plant water relations. Half the plants growing at each soil temperature were subsequently flooded. Within 1 week, the soil redox potential (Eh) dropped below zero mV, reaching a minimum Eh of –250mV after 3 weeks of flooded conditions. Flooded plants exhibited lower root conductivity, a cessation of shoot growth, lower leaf water potentials, lower stomatal conductances, and visual sloughing of fibrous roots. Decreases in root conductivity in response to flooding were large enough to account for the observed decreases in stomatal conductance.Florida Agricultural Experiment Stations Journal Series No. 4080.     

Citrus replant problem in Iraq II. Possible role of allelopathy

The role of allelopathy in citrus replant problems was investigated in Iraq. The failure of citrus seedlings to grow normally in old citrus orchards was not caused by differences between old and non-citrus soils in electrical conductivity, pH, organic matter, soil texture and those minerals tested. Extracts of soil collected from old citrus orchards significantly reduced the growth of sour orange seedlings. Extracts and decaying sour orange roots reduced the growth of sour orange seedlings as did extracts of non-senescent sour orange leaves and decaying senescent leaves. Thus it appears that allelopathy is at least partly involved in the citrus replant problem.     

Isozymes variability among <Emphasis Type="Italic">Fusarium udum</Emphasis> resistant cultivars of pigeonpea (<Emphasis Type="Italic">Cajanus cajan</Emphasis> (L.) (Millsp)

The present study was carried out to select the different pigeonpea cultivars for resistance against wilt caused by Fusarium udum and to assess the genetic variability among the resistant and susceptible cultivars. These cultivars were screened by root dip inoculation and classified into resistant (ICP 8863 and 9145), moderately resistant (ICP 11681 and Selection-1), susceptible (ICP 7118, TRG-1 and LRG-30) and highly susceptible cultivars (ICP-2376 and LRG-41). The peroxidase activity (PEO) in both leaf and root tissues of four pigeonpea cultivars (ICP 8863, Selection-1, ICP 2376 and LRG-30) were determined at 1^st, 4^th and 7^th day after inoculation (DAI) in healthy and F. udum infected tissues. Higher PEO activity in both leaf and root was observed and at 4^th DAI in susceptible cultivars. In native-PAGE analysis of isozymes, the induction of specific leaf peroxidase band (Em=0.17) and two root peroxidase bands (Em=0.24 and 0.55) were observed in ICP 8863 after inoculation. Significant differences were observed in the leaf phosphatase and esterase banding profiles of all the cultivars. The presence of leaf phosphatase band at Em of 0.04 was observed only in ICP 8863 and 11681. The leaf esterase band (Em=0.3) was well expressed in ICP 8863 when compared to other cultivars. The significance of peroxidase in plant defense mechanism and utility of biochemical markers in breeding programmes are discussed. Part of M.Sc. (Ag) thesis of the first author and approved by the Acharya N.G. Ranga Agricultural University during March 2002.     

Aconitase activity and expression during the development of lemon fruit

Citrus fruits are characterized by the accumulation of high levels of citric acid in the juice sac cells and a decline in acid level toward maturation. It has been suggested that changes in mitochondrial aconitase (EC 4.2.1.3) activity affect fruit acidity. Recently, a cytosolic aconitase (cyt-Aco) homologous to mammalian iron-regulated proteins was identified in plants, leading us to re-evaluate the role of aconitase in acid accumulation. Aconitase activity was studied in 2 contrasting citrus varieties, sweet lime ( Citrus limettioides Tan., low acid) and sour lemon ( Citrus limon var. Eureka, high acid). Two aconitase isozymes were detected. One declined early in sour lemon fruit development, but was constant throughout sweet lime fruit development. Its reduction in sour lemon was associated with a decrease in aconitase activity in the mitochondrial fraction. Another isozyme was detected in sour lemon toward maturation, and was associated with an increase in aconitase activity in the soluble fraction, suggesting a cytosolic localization. The cyt-Aco was cloned from lemon juice sac cells, but in contrast to the changes in isozyme activity, its expression was constant during fruit development. We present a model, which suggests that reduction of the mitochondrial aconitase activity plays a role in acid accumulation, while an increase in the cyt-Aco activity reduces acid level toward fruit maturation.     

Influence of paclobutrazol in the soil on growth,nutrient elements in the leaves,and flood/freeze tolerance of citrus rootstock seedlings

Paclobutrazol [(2RS,3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)pentan-3ol] was applied to soil at 0, 100, or 250 mg/3.78-liter pot containing seedlings of Swingle citrumelo, Carrizo citrange, Cleopatra mandarin, sour orange, rough lemon, and Sun Chu Sha. All cultivars were sensitive to paclobutrazol, which caused a proliferation of shorter/thicker roots, and top growth showed shorter internodes and lower dry weight. Induced changes resulted in greater root/shoot ratios, and paclobutrazol treatments showed higher concentrations of nitrogen, calcium, boron, iron, and manganese in the leaves of different cultivars. Paclobutrazol-treated seedlings did not show a greater ability to tolerate flooded soil for 60 continuous days under greenhouse conditions nor survive-6.7°C controlled freeze tests. Paclobutrazol is a potentially useful plant growth regulator to dwarf citrus, but it apparently is not a strong candidate for increasing flooding and freezing tolerance in citrus rootstock seedlings.Abbreviations PPFD photosynthetic photon flux density - ANOVA analysis of variance     

Molecular characterization of the nuclear and cytoplasmic genomes of intergeneric diploid plants from cell fusion between Microcitrus papuana and Rough lemon

Simple-sequence-repeat (SSR) and PCR-RFLP were employed to characterize the nuclear and cytoplasmic genomes of intergeneric diploid plants derived from symmetric fusion between Microcitrus papuana Swingle and Rough lemon ( Citrus jambhiri Lush). Three out of five SSR primers distinguished the fusion parents from each other and the regenerated plants showed band profiles completely identical to the leaf parent, Rough lemon. Amplified products from the intergenic regions of cpDNA between trnD -trnT were digested with HaeIII and MspI, and those between trnH -trnK were digested with HinfI, and both the regenerated plants and Rough lemon shared the same band patterns, which were different from the embryogenic parent, M. papuana. With mtDNA, only 2 out of 12 primer pair/restriction enzyme combinations ( nad4 ex 1–2/ TaqI and nad4 ex 1–2/ HindIII) revealed polymorphisms between the fusion parents. With the former combination the regenerated plants showed the same fragment distribution as that of the embryogenic parent, M. papuana, whereas with the latter, a novel band absent in the fusion parents was detected in all of the regenerated plants, suggesting a possible rearrangement. The present research indicates that the plants analyzed were putative cybrids containing nuclear DNA and cpDNA from Rough lemon and mtDNA from M. papuana. Presumed mechanisms leading to the regeneration of diploid hybrid plants following symmetric fusion are discussed herein.     

澳洲指橘与柑橘属间原生质体电融合再生二倍体体细胞杂种

电场诱导粗柠檬（CitrusjambhiriLush,2n＝2x＝18）叶肉原生质体与澳洲指橘（MicrocitruspapuanaSwingle,2n＝2x＝18）悬浮系原生质体融合,融合产物培养后再生出丛芽,经试管嫁接得到完整植株。再生植株的细胞学检查表明它们具有18条染色体,为二倍体;植株的叶片形态与叶肉亲本（粗柠檬）一样;用6个10-mer随机引物分析再生植株的杂种特性：在4个引物（OPA－07、OPAN－07、OPE－05和OPA－08）的扩增带型图中,再生植株的带型与粗柠檬完全一样,澳洲指橘的特征带未在植株中出现;在引物OPS-13和引物OPA－04的扩增带型图中,再生植株都具有澳洲指橘的特征带。细胞学和RAPD分析的结果表明,通过对称融合得到了澳洲指橘与粗柠檬的属间二倍体体细胞杂种植株。这是柑橘属间对称融合再生二倍体叶肉亲本类型植株的首例报道。     

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).     

Regeneration of Diploid Intergeneric Somatic Hybrid Plants Between Microcitrus a Electrofusion

Leaf-derived protoplasts of Rough lemon ( Citrus jambhiri Lush, 2n = 2x = 18) were electrofused with embryogenic suspension protoplasts of its relative, Microcitrus papuana 5wingle (2n = 2x = 18), with an intention of creating novel germplasm. Six plants were regenerated following protoplasts fusion. Cytological examination demonstrated that they were diploids with 18 chromosomes (2n = 2x = 18). RAPD (random amplified polymorphic DNA) analyses with six arbitrary 10-mer primers showed that the regenerated plants had identical band pattems to those of Rough lemon for primers OPA-07, OPAN-07, OPE-05 and OPA-08, whereas for the other two primers, OPA-04 and OPS-13, bands specific to M. papuana could be detected in the regenerated plants. Cytological and RAPD analysis revealed that the regenerated plants were diploid somatic hybrids between M. papuana and Rough lemon. The putative hybrids were morphologically similar to Rough lemon. This is the first report on production of diploid somatic hybrid plants between citrus with its related genus via symmetric fusion.     

The movement and distribution of citrus tristeza virus and citrus exocortis viroid in citrus seedlings1

The systemic movement of citrus tristeza virus (CTV) in sour orange (Citrus aurantium) seedlings and of citrus exocortis viroid (CEVd) in Etrog citron (C. medica) seedlings was studied. The movement of the two pathogens was analysed by detection in sections of roots and stems at different time intervals. Both pathogens were detected initially in the basal parts and the roots and subsequently spread to the shoot. CTV and CEVd moved in young citrus seedlings at similar rates. The findings are consistent with long distance phloem transport of the virus and the viroid. The practical implications of the pattern of systemic movement for diagnosis of infected trees are discussed.     

Alteration of Bark Proteins Associated with Citrus Tristeza Virus (CTV) Infection on Susceptible Citrus Species and Scion-Rootstock Combinations

The effect of citrus tristeza virus (CTV) on bark protems of susceptible citrus species and scion-rootstock combinations was studied by polyaerylamide gel electrophoresis (PAGE). Protein pattern of sour orange bark from CTV-infected trees on this rootstock showed reduced intensity in a protein band, about 20,000 daltons molecular weight, as compared with similar CTV-free trees. This protein modification appears specifically associated with decline induced by tristeza since it was observed on trees of different ages and scion-rootstock combinations, grown in various locations and infected with several CTV isolates, but not on trees exhibiting decline from other causes. The observed protein alteration was localized in the ribosomic fraction. No protein alteration, associated with CTV infection could be found on lemon bark, although this citrus species also behaves as a CTV-susceptible rootstock. Electrophoretic profiles obtained from CTV infected Mexican lime and Etrog citron seedlings also showed reduced intensity in a protein band with the same electrophoretic mobility as the tnodified band of sour orange.     

Reaction of Citrus Rootstocks to Meloidogyne javanica

The response of Citrus spp. and related rootstocks to a population of Meloidogyne javanica was evaluated in a screenhouse experiment. Palestine and Rangpur lime, rough lemon, sour orange, Sexton and Thentriton tangelo, and Volkamer lemon were not infected by M. javanica. Galls and tip swellings were observed on the roots of Poncirus triloliata and Troyer citrange. There was no evidence of nematode development. Symptoms induced by the nematode were stelar division, syncytia formation in the vascular tissues, and necrotic cells.     

Growth and root hydraulic conductivity of several citrus rootstocks under salt and polyethylene glycol stresses

The water relations responses to salt of several important citrus rootstocks such as Swingle citrumelo, sour orange, and Milam lemon have not been studied in detail before. Studies were set up to compare growth and root hydraulic properties of these rootstocks to other citrus rootstocks by exposing them to NaCl and polyethylene glycol (PEG) stresses. Seedlings of 7 citrus rootstocks were irrigated for 5 months with nutrient solutions containing NaCl or PEG that had been adjusted to osmotic potentials of -0.10, -0.20 or -0.35 MPa. The 7 rootstocks studied were sour orange (Citrus aurantium), Cleopatra mandarin (Citrus reticulata Blanco), Swingle citrumelo (C. paradisi x P. trifoliata), Carrizo citrange (C. sinensis x P. trifoliata), rough lemon (Citrus jambhiri Lush), Milam lemon (C. jambhiri hybrid), and trifoliate orange (Poncirus trifoliata [L.] Raf.). In both shoot and root growth, Cleopatra mandarin and sour orange were the least sensitive to salt, Milam and trifoliate orange were the most sensitive, and rough lemon, Swingle, and Carrizo were intermediate in sensitivity. Even though the roots were exposed to solutions of equal osmotic potentials, plant growth and root conductivity were reduced more by the PEG treatments than the corresponding NaCl treatments. At -0.10 and -0.20 MPa, shoot and root dry weights were reduced 16 to 55% by NaCl and 24 to 68% by PEG. Shoot root ratio was lowered at the higher concentrations, particularly by PEG. There was a major decrease in root conductivity caused by NaCl at -0.10 MPa (19 to 30% in sour orange and Cleopatra mandarin and 78 to 85% in trifoliate orange and Milam). Conductivity decreased more at -0.20 and -0.35 MPa, but not proportionally as much as at -0.10 MPa. Root weight per unit length increased at the higher salt levels, particularly in trifoliate orange. Water flow rate through root systems followed the same trend as root conductivity; salt affected sour orange and Cleopatra mandarin the least and trifoliate orange and Milam the most. However, reductions in fibrous root length by salt treatment differed. Root lengths of Swingle and Carrizo were least affected by salt while sour orange. Milam, and rough lemon were the most affected. Hence, even though sour orange and Cleopatra mandarin were more tolerant than the other rootstocks in terms of water flow rate or root conductivity, these 2 rootstocks showed a proportionally greater decrease in root length than Carrizo, Swingle, or trifoliate orange.     

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.