Regeneration and Characterization of Plants Derived from Asymmetric Protoplast Fusion in Citrus

RFLP (restriction fragment length polymorphism) was employed to analyze cytoplasmic genome of diploid somatic hybrid plant, morphologically similar to rough lemon which was leaf parent, that was produced via protoplast fusion between rough lemon (Citrus jambhiri Lush) and Hamlin sweet orange (C. sinensis Osb.), the embryogenic parent. Three enzyme-mitocondrial probe combinations and one enzyme-chloroplast probe combination demonstrated that the plant had identical band profiles to Hamlin sweet orange as far as mtDNA and cpDNA were concerned.     

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     

哈姆林甜橙与粗柠檬体细胞杂种的育性

对异源四倍体柑桔体细胞杂种“哈姆林甜橙+粗柠檬”及其亲本的花粉活力、花器官发育、花器官形态发生与花粉母细胞减数分裂四分体阶段进行了观测和统计.结果发现“哈姆林甜橙+粗柠檬”的花粉染色活力、萌发率、每花药中花粉粒数均居于其双亲之间,花器官发育及其形态发生具有双亲的特点.但小花粉及花粉母细胞减数分裂过程中形成的不正常四分体比率远远高于其双亲.以体细胞杂种“哈姆林甜橙+粗柠檬”为花粉亲本,与二倍体单胚类型宜昌橙与华农本地早的有性后代杂交,获得了110棵有性后代植株,其中三倍体82棵,二倍体和其它倍性的植株28棵.     

Photosynthetic responses of citrus trees to soil flooding

Continuous soil flooding reduced leaf photosynthetic rate, stomatal conductance to water vapor, chlorophyll concentration and activity of ribulose bisphosphate carboxylase-oxygenase (Rubisco, EC 4.1.1.39) of sweet orange [ Citrus sinensis (L.) Osbeck cv. Hamlin] trees, grafted onto rough lemon (RL; C. jambhiri Lush.) and sour orange (SO; C. aurantium L.) rootstocks. After 24 days of waterlogging, trees showed senescence, wilting and abscission of leaves, and these symptoms were more evident with flooded Hamlin/SO than Hamlin/RL. Reduction of leaf photosynthetic rate at day 24 was ca 94%, of stomatal conductance, 71%, of chlorophyll, 38% and of Rubisco, 62% for flooded Hamlin/SO, compared with 22, 5, 18 and 33%, respectively, for flooded Hamlin/RL. For both Hamlin/RL and Hamlin/SO, leaf photosynthetic rate and stomatal conductance to water vapor were closely correlated (r²= 0.87). Leaf internal CO₂ concentration of flooded trees, however, was not decreased by reduced stomatal conductance. Dark respiration rates of fibrous roots of flooded trees were greatly reduced, but not in leaf tissues. Total nonstructural carbohydrate concentrations were higher in leaves (50 and 80% increases for Hamlin/SO and Hamlin/RL, respectively), but drastically reduced in roots (60 and 45% reductions for Hamlin/SO and Hamlin/RL, respectively), as a result of flooding. The data indicate that Hamlin grafted onto RL rootstocks was more tolerant to soil flooding than Hamlin grafted onto SO rootstocks.     

RAPD-based genetic analysis of offsprings from the sexual cross using allotetraploid citrus somatic hybrid as pollen parent

Thirty-one polymorphic decamer primers were selected to genotype 92 progenies from the cross between Yiben No.4, a monoembryonic diploid F₁ hybrid of Citrus reticulata Blanco cv Huanongbendizao tangerine and C. ichangensis Swingle, and [Hamlin sweet orange + Rough lemon], an allotetraploid somatic hybrid of Citrus sinensis Osbeck cv. Hamlin and C. jambhiri Lush cv. Rough Lemon. χ² (Chi-square) analysis of RAPD markers in the progenies indicated they were randomly transmitted from the four donor parents, without significant difference between the diploids and triploids. However, these progenies were clustered into three major groups using dendrogram constructed by UPGMA, skewed to three parents in certain degrees, 15 (13 triploids and 2 diploids) to Hamlin, 16 (9 and 7) to Yiben No. 4, and 61 (57 and 4) to [Hamlin sweet orange + Rough Lemon] from which genomic contribution was predominant in progenies, respectively.     

RAPD-based genetic analysis of offsprings from the sexual cross using allotetraploid citrus somatic hybrid as pollen parent

Thirty-one polymorphic decamer primers were selected to genotype 92 progenies from the cross between Yiben No.4, a monoembryonic diploid F1 hybrid of Citrus reticulata Blanco cv Huanongbendizao tangerine and C. ichangensis Swingle, and [Hamlin sweet orange + Rough lemon], an allotetraploid somatic hybrid of Citrus sinensis Osbeck cv. Hamlin and C. jambhiri Lush cv. Rough Lemon. χ² (Chi-square) analysis of RAPD markers in the progenies indicated they were randomly transmitted from the four donor parents, without significant difference between the diploids and triploids. However, these progenies were clustered into three major groups using dendrogram constructed by UPGMA, skewed to three parents in certain degrees, 15 (13 triploids and 2 diploids) to Hamlin, 16 (9 and 7) to Yiben No. 4, and 61 (57 and 4) to [Hamlin sweet orange + Rough Lemon] from which genomic contribution was predominant in progenies, respectively     

Growth responses and vascular plugging of citrus inoculated with rhizobacteria and xylem-resident bacteria

Summary Bacteria, isolated from roots (xylem tissue) of healthy and Young Tree Decline (YTD, Blight)-affected citrus trees, and also from nursery seedlings, were screened for potential pathogenicity by the tobacco hypersensitive reaction (HR). A majority (>75%) of the HR positive strains were classified as nonfluorescent pseudomonads. These HR positive strains were subsequently inoculated into rough lemon (Citrus jambhiri Lush.) and sweet orange (C. sinsensis Osbeck) seedlings or into Valencia sweet orange budded on rough lemon root-stock. Many of the HR positive pseudomonads reduced fresh weights (up to 94%) of roots and shoots and some reduced xylem water conductance and caused scion dieback. There was no evidence of necrosis or root rot in inoculated roots. A few HR negative Pseudomonas and Enterobacter strains significantly, but less severely, inhibited (to 43%) root growth of sweet orange seedlings. HR negative mutants derived from HR positive strains were considerably less inhibitory. Postinoculation stresses (dark and cold) markedly decreased susceptibility of seedlings to bacterial-induced inhibition. Evidence of cultivar-specific effects was obtained in comparable inoculations of rough lemon and sweet orange seedlings. Soil application of a fluorescent pseudomonad, which alone was growth stimulatory, intensified inhibitory effects of nonfluorescent, growth inhibitory, psuedomonads. This study demonstrates that many rhizobacteria isolated from xylem tissue of roots have detrimental effects on citrus.     

柑桔细胞电融合再生两个种间体细胞杂种

朋娜脐橙(Citrussinensis Osbeck)胚性细胞悬浮系原生质体分别与粗柠檬(C.jambhiri Lush)、枸头橙(C.aurantium)叶肉原生质体经电场诱导而融合。经培养,两组合均获得再生植株。对朋娜脐橙+粗柠檬的再生胚状体进行染色体计数,随机取样的52个胚状体中,26个为四倍体,另外26个为二倍体;对74棵再生植株进行染色体计数,由此说明都为四倍体;表明体细胞杂种在植株再生过程中具有明显的竞争优势。朋娜脐橙+枸头橙再生的14棵植株都为四倍体。对朋娜脐橙+粗柠檬部分植株进行POX同工酶和RAPD分析,表明所有检测植株都为杂种。朋娜脐橙+枸头橙再生植株经RAPD分析,表明也为杂种。     

Automated Needle‐free Injection Method for Delivery of Bacterial Suspensions into Citrus Leaf Tissues

A prototype needle‐free device was evaluated for delivery of Xanthomonas citri subsp. citri bacteria into the leaves of cultivars susceptible and resistant to citrus canker. The device delivered a precisely controlled volume of bacterial suspension through infiltration of stomata by injection with pressurized gas. The device produced a uniform inoculation of bacteria into the leaves as measured by the volume of infiltration and diameter of the infiltrated area. No damage to the leaves was observed after inoculation with the automated device, even though a higher number of canker lesions developed compared to a hand‐held needleless syringe injection method. The level of practice needed for operation of the automated device was minimal compared to considerable skill required to perform the hand‐held injection. Results from inoculations with the automated device are in accord with the results with the hand‐held syringe method that demonstrated kumquats are highly resistant to citrus canker while rough lemon and ‘Hamlin’ sweet orange are susceptible.