Facultative apomixis and chromosome doubling are sources of heterogeneity in citrus rootstock trials: Impact on clementine production and breeding selection

All commercial citrus rootstocks are polyembryonic and propagated by seeds. Although these seeds produce uniform plant material most of the time, zygotic or polyploid citrus seedlings may arise. The aim of this study was to understand how zygotic or tetraploid rootstock could affect a citrus rootstock selection field trial. A trifoliate orange selection field trial, which was planted in 1974 and grafted with clementine, was re-investigated with respect to the presence of rootstocks that were not true-to-type. Among the 288 trees investigated, flow cytometry identified 2.4% of rootstocks as tetraploid and SSR markers indicated that 6.6% were zygotic. Yield data showed that the presence of tetraploid rootstock dramatically decreased (by about 45%) clementine fruit production. However, zygosity did not always affect fruit production and a range of effects were observed, from a slight increase in production to a 24% decrease. Exclusion of non-true-to-type genotypes from the production analysis indicated that the best candidate for rootstock was a clone previously ranked in the middle of the 32 rootstocks under evaluation. However, the presence of zygotic rootstock did not appear to cause any significant differences in fruit quality during the first 5 years of the investigation, which suggests that non-true-to-type plants cannot be identified by fruit quality parameters. This study indicates that tetraploid and zygotic rootstocks have a strong impact on citrus fruit production in orchards, and removal of off-type seedlings is required prior to planting in any agronomic trial.     

单胚槾橘×多胚岩溪晚芦槿柑多胚种子的胚分离培养及后代的叶形态和SSR鉴定

继首次报道单胚清见与多胚桠柑杂交产生高比例多胚种子后,在单胚棱橘（Citrus tardiferax Hort．ex Tan）与多胚岩溪晚芦桠柑（Citrus reticulata Blanco）的杂交组合中,又发现高比例多胚种子,其比例一般为30％左右。为探明多胚来源,对多胚进行了分胚组织培养,获得同一种子来源的多胚...     

韭菜多胚苗及其与无融合生殖关系的研究

以9个韭菜品系为试材, 人工去雄后用二甲基亚砜、激动素和失活韭菜花粉诱导, 获得无融合生殖种子。无融合生殖植株自交后代多胚苗发生频率明显提高, 说明多胚苗与无融合生殖有关。继代选择增大了多胚苗发生频率, 说明多胚苗性状可以遗传。     

Citrus rootstock responses to water stress

Tolerance to drought-stress (DS) of the citrus rootstock Forner–Alcaide no. 5 (FA-5) was tested and compared with that of its parents, Cleopatra mandarin (CM) and Poncirus trifoliata (PT). Nine-month-old seedlings of CM, PT and FA-5 and 15-month-old grafted trees of ‘Valencia’ orange scions on these three rootstocks were cultivated in sand under glasshouse conditions and irrigated with a nutrient solution. Plants were drought-stressed by withholding irrigation until leaves were fully wilted. Survival time of both seedlings and grafted trees under DS was linked to the water extraction rate from the soil, which depended mainly on leaf biomass and on transpiration rate. Seedling responses to DS affecting leaf water relationships and gas exchange parameters varied among genotypes. FA-5 seedlings survived longer than the other seedlings, maintaining the highest levels of water potential, stomatal conductance, transpiration rate and net CO₂ assimilation towards the end of the experiment, when water stress was most severe. Thus, FA-5 was more resistant to DS than its parents (CM and PT). Moreover, rootstock affected the performance of grafted trees under water stress conditions. The higher drought tolerance induced by FA-5 rootstock could be related to the greater osmotic adjustment (OA), which was reflected by smaller reductions in leaf relative water content (RWC) and in higher turgor potentials and leaf gas exchange than the other rootstocks.     

Selection of arbuscular mycorrhizal fungi for citrus growth promotion and Phytophthora suppression

In order to reduce unnecessary amount of P-fertilizer and severity of Phytophthora root rot in citrus orchards, the experiment was set up. Thirteen indigenous arbuscular mycorrhiza (AM) fungi species were isolated from rhizosphere soil of citrus orchards in Thailand and were then propagated into three host plants [sorghum (Sorghum bicolor), maize (Zea mays), and leek (Allium cepa)] by trap culture. We also tested whether indigenous AMF species (13 different species) could colonize into three cultivars of citrus scions and rootstocks (Shogun: Citrus reticulata Blanco cv. Shogun; Tangerine: C. reticulata; and C-35 citrange: Citrus sinensis × Poncirus trifoliata). With root colonization rates, the results indicated that Acaulospora tuberculata and Glomus etunicatum provided the best colonization in all citrus cultivars. We selected, therefore, those AMF species to verify their influences on citrus growth and Phytophthora root rot resistance. Three cultivars of citrus scions and rootstocks, Shogun, Tangerine and C-35 citrange, were inoculated with two effective indigenous AMF species, G. etunicatum or A. tuberculata in order to determine the influences on citrus growth. The plants were investigated to determine the mycorrhizal efficiency index (MEI), AM colonization, P content, and other parameters. Co-inoculation of AMF species (G. etunicatum or A. tuberculata) with Phytophthora nicotianae was also carried out in Shogun scion/C-35 citrange rootstocks. The results of citrus growth revealed that Shogun and Tangerine inoculated with G. etunicatum produced the highest MEI. Tangerine and C-35 citrange amended with fertilizers and G. etunicatum showed the highest P content in leaves. This indicated that G. etunicatum has an influence on citrus growth and P uptake, suggesting it to be the highly effective strain. Shogun scion/C-35 citrange rootstock combinations that were inoculated by both P. nicotianae and different AM fungi (G. etunicatum or A. tuberculata) showed root injury at low level of root rot symptom. However, the part of Shogun scions grafted on rootstocks showed severe symptom of shoot die back in treatment inoculated with P. nicotianae alone, while treatment inoculated with different AM species (G. etunicatum or A. tuberculata) and P. nicotianae rendered lower shoot die back symptoms than that of Phytophthora treatment. The low level of shoot die back symptom was shown at first, then healthy young shoot was restored. Our results indicated the facts that different host plants and different AMF species produced different outcomes of growth and pathogen resistance. The application of both AM isolates, therefore, has an enormous potential to be produced the inoculum for citrus orchards.     

Evaluation of apricot resistance to Plum pox virus (Sharka) in controlled greenhouse and natural field conditions

In this study we compare the evaluation of Plum pox virus (Sharka) resistance of 29 apricot genotypes in controlled greenhouse conditions by grafting onto infected ‘GF305’ peach seedlings growing in pots, and in natural conditions by grafting onto infected 5-year-old apricot trees growing in the orchard. Apricot genotypes evaluated were initially classified into three groups: susceptible to PPV (presence of PPV symptoms and ELISA positive in greenhouse and field assays), resistant (absence of PPV symptoms and ELISA negative in both assays) and undetermined (evaluated differently in both assays). Results showed a similar behavior against Plum pox virus in both assays in 20 out of the 29 apricot genotypes studied (69%). However, in the other nine genotypes results were different (31%). Evaluation in field was more accurate, detecting a higher number of susceptible genotypes, probably due to the higher inoculation pressure in the old trees in comparison with the GF305 rootstocks in pots. However, greenhouse evaluation let to work in controlled isolated conditions with a higher number of genotypes. This situation is greatly required in areas where Sharka is not widely spread. Greenhouse evaluation could be then the first screening method to evaluate Plum pox virus resistance of apricot genotypes, and could be complemented with the evaluation onto infected trees in natural conditions in insect-proof quarantine shelter.     

Chloride and sodium excluding capacities of citrus rootstock germplasm introduced to Australia from the People's Republic of China

The responses to root zone salinity (0, 25 and 50 mM NaCl) by 40 citrus rootstock genotypes introduced from the People's Republic of China, measured as shoot chloride (Cl⁻) and sodium (Na⁺) ion accumulation, growth and dry matter accumulation, were investigated under glasshouse conditions. Two experiments, one using aerated nutrient solutions and the other irrigated sand cultures, were conducted with plants grown from rooted cuttings taken from representative trees of different mandarin (Citrus reticulata Blanco and C. erythrosa Hort. ex Tan.), yuzu (C. junos Sieb. ex Tan.), Ichang papeda (C. ichangensis Swing.), sour orange (C. aurantium L.) and trifoliate orange (Poncirus trifoliata (L.) Raf.) genotypes. Two standard genotypes, viz. Rangpur lime (Citrus x limonia Osbeck.) and William's trifoliate orange were included in the experiments as reference controls.     

椪柑成熟种子胚培养获得四倍体植株

 通过组培方法从374粒　柑种子培养中获得449株小苗。利用流式细胞仪进行染色体倍性分析, 发现其中7株小苗为四倍体植株。采用13对SSR引物分析表明这7株四倍体植株均来自珠心细胞, 为同源四倍体。这些四倍体的获得为下一步培育三倍体无核品种奠定了基础。     

Microsatellite markers reveal low breeding system efficacy and pollen contamination can limit production of full-sib avocado progeny

Phytophthora cinnamomi causes a severe root rot in avocado, Persea americana. Breeding tolerant rootstocks is thought to be the most promising method for phytophthora root rot disease control but breeding avocado is challenging. The avocado flowering syndrome (synchronous protogynous dichogamy), combined with high flowering and low fruit set, render controlled pollination exceedingly difficult. Juxtaposing complementary flowering types of elite parent cultivars (cultivars that produce progeny with tolerance to phytophthora root rot) was performed in an effort to increase the number of full-sib progeny for elite maternal parents and, hypothetically, the number of phytophthora root rot tolerant progeny. Although high outcrossing rates were achieved (estimated ∼93%), the majority of progeny had a non-elite paternal parent (56% of progeny were offtypes) implying maternal trees were pollinated by non-elite distant trees. Among progeny that could be confidently genotyped, a high number of cross types were detected (33). Contrary to our hypothesis, a significant portion of the progeny were the result of crosses between like, and not complementary, flowering types. The spatial distribution of productive trees and grafts helped to explain these data, as productive grafts were directly adjacent to grafts of the same flowering type more often than that of the complementary flowering type. Selfed progeny were significantly less tolerant to phytophthora root rot than outcrossed progeny. Progeny resulting from crosses between an elite maternal parent and non-elite pollen donor (offtypes) were less tolerant than full-sib progeny resulting from crosses between elite parents. Maternal effects may interfere with identifying truly disease tolerant selections. Thus, to reduce maternal effects and non-elite pollen donor contamination, removal of seedling cotyledons before screening for disease tolerance and better isolation of elite parent trees and windbreaks may improve breeding efficacy. This study also demonstrates the usefulness of microsatellite markers in parentage analysis where a high proportion of the putative parents are closely related.     

Distribution of free and glycosylated sterols within Cycas micronesica plants

Flour derived from Cycas micronesica seeds was once the dominant source of starch for Guam's residents. Cycad consumption has been linked to high incidence of human neurodegenerative diseases. We determined the distribution of the sterols stigmasterol and β-sitosterol and their derived glucosides stigmasterol β-d-glucoside and β-sitosterol β-d-glucoside among various plant parts because they have been identified in cycad flour and have been shown to elicit neurodegenerative outcomes. All four compounds were common in seeds, sporophylls, pollen, leaves, stems, and roots. Roots contained the greatest concentration of both free sterols, and photosynthetic leaflet tissue contained the greatest concentration of both steryl glucosides. Concentration within the three stem tissue categories was low compared to other organs. Reproductive sporophyll tissue contained free sterols similar to seeds, but greater concentration of steryl glucosides than seeds. One of the glucosides was absent from pollen. Concentration in young seeds was higher than old seeds as reported earlier, but concentration did not differ among age categories of leaf, sporophyll, or vascular tissue. The profile differences among the various tissues within these organs may help clarify the physiological role of these compounds.     

Relation of carbohydrate reserves with the forthcoming crop,flower formation and photosynthetic rate,in the alternate bearing ‘Salustiana’ sweet orange (Citrus sinensis L.)

The aim of this work was to assess the relation between carbohydrate levels and flower and fruit production, as well as the role of carbohydrates on CO₂ fixation activity, by analysis of leaves, twigs and roots from the alternate bearing ‘Salustiana’ cultivar of sweet orange (Citrus sinensis [L.] Osbeck). A heavy crop load (on year) did not affect photosynthesis activity when compared to non-fruiting trees (off year). Fruiting trees accumulated most of the fixed carbon in mature fruits, whilst no accumulation was observed in roots before harvest. Non-fruiting trees transported part of the fixed carbon to the roots and mobilize it for growth processes and, at the end of the season (December), store it as reserves. Reserve carbohydrates accumulation in leaves started by early December for both tree types, showing the same levels in on and off trees until spring bud sprouting. A heavy flowering after an off year caused the rapid mobilization of the stored reserves, which were exhausted at full bloom. We found no evidence on carbon fixation regulation by either fruit demand or carbohydrate levels in leaves. Carbohydrate reserves played little or no role over fruit set, which actually relied on current photosynthesis.     

Intergeneric hybridizations between Opisthopappus taihangensis and Chrysanthemum lavandulifolium

For the first time, reciprocal intergeneric hybridizations were produced between Opisthopappus taihangensis and Chrysanthemum lavandulifolium without emasculation. About 20% seed set was similarly obtained from reciprocal hybridization. Only 5 and 6 of the viable plants observed from 45 and 78 seedling survived present some conspicuous intermediate characteristics. Phenotypic evaluation among the progenies of the parents and the putative hybrids was performed carefully since an average of 5.3% seed set was produced in the type of self-pollination using pollen from the same flower and >10% seed set was similarly obtained in the types of self-pollination using pollen from different flowers in a plant and flowers in individual plants from different seeds. One individual of each hybrid shared the inflorescence habit with the pollen plant was confirmed further by the sequence of ncpGS. The two hybrids might be used as bridges of breeding of multi-generic hybrids.     

Fruit set and embryo rescue in crosses using parthenocarpic ‘Mopanshi’ persimmon

The ‘Mopanshi’ persimmon is a seedless, astringent parthenocarpic cultivar that does not produce male flowers. After pollination using four non-astringent cultivars (‘Zenjimaru’, ‘Nishimurawase’, ‘Okugosho’ and ‘Hanagosho’), seeds were produced to different degrees. ‘Mopanshi’ fruits pollinated with ‘Zenjimaru’ produced far more seeds than those pollinated with the other three cultivars. The ratio of abnormal seeds obtained from the fruits pollinated with ‘Hanagosho’ was higher than that obtained from the fruits pollinated with the other three cultivars. Most embryos degenerated in the early to late stages of seed development. Immature embryos were cultured in a modified MS medium (half of NO₃ in MS medium + 0.4 μM BA + 0.1 μM IBA) with the greatest success (52–80%) from embryos taken from fruits 60–80 days after pollination. Seedlings failed to initiate radicles so they were transferred to dark culture conditions for 8 days or to a rooting media that contained 3% sucrose and 1% Chinese ink. The seedlings on the medium darkened with ink rooted at greater than 90.83% compared to 75.83% for dark cultured seedlings. This study demonstrated that ‘Mopanshi’ persimmon could be used as a female parent in crosses, but embryos needed to be moved to tissue culture conditions to continue to develop and form plantlets.     

Increased regeneration capacity in spinach lines obtained by in vitro self-fertilisation

Somatic embryos (SEs) were induced from apical sections of the lateral roots of spinach seedlings (1 cm), which were cultivated on solid Murashige and Skoog (MS) medium with 20 μM α-naphthaleneacetic acid and 5 μM gibberellic acid. Apical shoots of the same lines were isolated and cultivated on plant growth regulator-free medium. The regeneration capacities of seedlings randomly chosen from a population were quite low and variable, and only 4 out of 30 lines responded at the frequency of 85–100%, with 6.96–9.96 SEs per explant and up to 347 SEs per seedling over a 12-week period. These SEs were isolated and maintained on medium with 5 μM kinetin. Plants derived from seedlings’ apical shoot and SEs self-fertilised in vitro and set seeds, and these seedlings (S₁) were used to induce regeneration. Similarly, S₂–S₄ seedlings were obtained and the regeneration capacities of 23 S₁, 23 S₂, 17 S₃ and 5 S₄-seedlings were compared to parental lines. Of these, four S₃ and S₄ lines with extremely high regeneration capacities were selected. These lines exhibited 78–139 fold higher embryo-forming capacities than the mother plant, and produced 38.9–68.3 SEs per explant and 1339–2181 SEs per seedling during the same time period. In addition, the process of somatic embryogenesis began 2–4 weeks earlier in these lines, and root explants taken from SE-derived plants of these lines retained high and stable regeneration capacities, and therefore may be ideal material for genetic transformation.     

A new peptide of melon seeds which shows sequence homology with vicilin: Partial characterization and antifungal activity

Plant resistance against pathogens involves a broad variety of proteins, which can be either constitutive or induced. Different types of antimicrobial proteins have been purified from plant seeds, including chitinases, β-1,3-glucanases, defensins, thionins, lipid transfer proteins, lectins and vicilins. The aims of this study were to isolate and characterize defense-related proteins, particularly antimicrobial peptides, present in melon (Cucumis melo L.) seeds. Seed proteins were extracted with phosphate buffer, pH 5.4 and subjected to precipitation with 90% relative ammonium sulfate saturation. The precipitate was re-dissolved in distilled water and heated at 80 °C for 15 min. A DEAE–Sepharose column equilibrated with 0.1 M Tris–HCl pH 8.0 was employed for further purification of the peptides with antifungal properties. Fractions with inhibitory activity were detected in the non-retained basic peak (P1). To test whether P1 peptides were able to permeabilize the yeast plasma membrane, we monitored their effects on glucose-stimulated acidification of the incubation medium by yeast Saccharomyces cerevisiae cells. This fraction strongly inhibited glucose-stimulated acidification of the medium by yeast cells in a dose-dependent manner, and inhibition was up to 60% for all concentrations tested. Effects of the P1 fraction upon the growth of the phytopathogenic fungus Fusarium oxysporum were also observed. Proteins in P1 fraction were also subjected to automated N-terminal amino acid sequencing. Partial N-terminal sequence of a major 8 kDa protein revealed homology with previous reported storage vicilins from different seeds.     

A novel method for Citrus propagation: Seed grafting

Summary

We have developed a novel technique for grafting citrus seeds onto citrus rootstock plants that resulted in successful graft-take with normal vascular connections between the emerging seedling stem tissues and the rootstock plant. The method was found to be suitable for producing grafted plants from seeds of six cultivars and hybrids of Citrus and the citrus relative Murraya paniculata, using four common Citrus rootstocks. Plants produced by this method developed normally and were established in the field more rapidly than those produced by the common practice of grafting the rootstocks with budwood derived from seedlings prepared from seed in soil-based media. Seed grafting is expected to find a range of uses in breeding programmes; for example, by reducing the time required for the evaluation of hybrid seedlings, in cases where the female parent is mono-embryonic, for testing for vertical transmission of pathogens, and for screening for pathogen resistance among hybrid and mutagenised seed sources.     

依据多倍体形态特征快速高效发掘柑橘四倍体

基于柑橘多胚性品种存在珠心细胞自然加倍的特点，建立了基于种子催芽、生长室实生播种、幼苗“观根辨叶看油胞”形态初选和流式细胞仪倍性鉴定的发掘柑橘四倍体的快速方法。采用该方法，分别从无酸甜橙、红江橙、贡柑、年橘、新会柑、滑皮金柑的432、506、1 356、1 870、2 749和685株实生苗中，发掘出疑似四倍体5、6、5、17、45、7株；通过倍性检测，从上述6个品种分别获得四倍体4、5、5、14、39、5株，形态初选准确率分别为80.0%、83.3%、100.0%、82.4%、86.7%和71.4%。与之前的四倍体发掘方法相比，本方法四倍体初选准确率高，并且从种子播种到获得四倍体植株耗时不超过40 d。SSR分子鉴定表明，所鉴定的四倍体均来自其二倍体品种珠心细胞自然加倍形成的双二倍体，为柑橘三倍体无核育种提供了珍贵的四倍体亲本资源。     

Biofertilizers improve plant growth,fruit yield,nutrition, metabolism and rhizosphere enzyme activities of Pomegranate (Punica granatum L.) in Indian Thar Desert

Production of horticultural crops has undergone enormous change in recent years due to development of innovative technologies including integrated nutrient management practices using biofertilizers. The present study represents the positive response of biofertilizers in nursery seedlings followed by their transplantation in harsh field conditions of Indian Thar Desert. Nursery and field experiments were carried out to assess the effectiveness of selected N₂-fixing bacteria and AM fungi alone or in combination, on the growth and biomass production of Punica granatum. In both experiments, the combined treatment of Azotobacter chroococcum and Glomus mosseae was found to be the most effective. Besides enhancing the rhizosphere microbial activity and concentration of various metabolites and nutrients, these bioinoculants helped in better establishment of pomegranate plants under field conditions. A significant improvement in the plant height, plant canopy, pruned material and fruit yield was evident in 5-year-old pomegranate plants in field conditions. In view of the above results, use of biofertilizer technology may be adopted for the establishment and development of other horticultural plant species in arid regions.     

Agrobacterium tumefaciens-mediated genetic transformation and plant regeneration from a complex tetraploid hybrid citrus rootstock

Agrobacterium-mediated genetic transformation of a tetraploid “tetrazyg” citrus rootstock selection ‘Orange #16’ [Nova mandarin (Citrus reticulata Blanco) + Hirado Buntan pummelo (Citrus grandis L. Osbeck)] × [Cleopatra mandarin (C. reticulata Blanco) + Argentine trifoliate orange (Poncirus trifoliata (L.) Raf.)] was performed. Juvenile epicotyl segments were transformed with a construct containing a bifunctional egfp–nptII fusion gene under the control of an enhanced double CaMV 35S promoter. Our protocol resulted in a reasonable transformation efficiency of 18%. Stable integration of the transgene was confirmed by visual observation of EGFP expression, PCR and Southern blot hybridization. The purpose of this work was to investigate the amenability of novel citrus rootstock germplasm being developed for improved tree size control, soil adaptation, and disease resistance, to existing transformation technologies. Seed trees of such transgenic tetraploids also have potential as trap plants containing potent insecticidal transgenes, due to their inedible fruit and inherent crossing barriers with conventional commercial diploid scion cultivars, and could be planted around producing citrus groves.     

Seed inoculation with Azospirillum mitigates NaCl effects on lettuce

Data on the growth-promoting effects of Azospirillum on lettuce exposed to either normal or saline conditions, is scarce. Lactuca sativa L., cv Mantecosa seeds were colonized with A. brasilense Sp245 cells during imbibition. Germination percentages were determined after 7 d treatments with 0, 30, 50 or 80 mol m⁻³ NaCl. In another experiment, seeds germinated in Hoagland were irrigated for 30 d with 0, 30, 50 or 80 mol m⁻³ NaCl supplemented media. Vegetative growth proceeded in a growth chamber with a 13–11 h day–night cycle. Buffer-imbibed seeds were considered non-inoculated controls. Plant samples were taken at 0, 14, 20, and 30 d after the onset of NaCl treatments and dissected in aerial and root portions. The weights of both tissues were measured. Azospirillum-inoculated seeds had significantly higher germination percentages than controls in all treatments. Inoculated dried seeds stored up to 30 d maintained such characteristic in most of the treatments, particularly at 80 mol m⁻³ NaCl. Plants grown from inoculated seeds and irrigated with saline media displayed higher total fresh and dry weights and biomass partition to the aerial portion, than non-inoculated controls. Azospirillum-inoculated lettuce seeds had better germination and vegetative growth than non-inoculated controls after being exposed to NaCl.