In vitro propagation of some olive (Olea europaea sativa L.) cultivars with different root-ability,and medium development using analytical data from developing shoots and embryos

Short- and long-term objectives for research on tissue culture of the olive are described. Sterile shoots were obtained from single-node woody explants or buds of 3 olive cultivars (‘Frantoio’, ‘Dolce Agogia’ and ‘Moraiolo’) with different root-ability, collected from shoots having different degrees of juvenility (suckers, vigorous nonfruit-bearing and fruit-bearing shoots, which are easy, medium and difficult to root, respectively).Because many of the media tested did not give a satisfactory growth rate and good quality shoots, a new medium was formulated by comparing data from analysis of the main mineral elements found in the apical shoots (4–5 mm long) and in mature embryos in olive and almond. Olive tissues were characterized by a high content of Ca, Mg, S, Cu and Zn compared to almond, which is easy to propagate on MS medium. In this newly derived medium, characterized by a high content of these elements, multiplication rate (number of nodes formed per explant) was about 9× in 40 days. The shoots grew more rapidly and were more tender than when grown in other media. Washing of the explants in water or GSH (reduced glutathione) solution, before sub-culturing, improved quality and growth rate of the shoots.Explants, with 2 or 3 nodes, rooted easily in half-strength MS, in Bourgin and Nitsch, or in half Knop macro and Heller microelements, agar media, with 1 mg 1^?1 NAA and 2% sucrose. Rooting was not affected by the different degrees of juvenility of the original explants used.Hardening-off was achieved by growing plants in a 1:1 mixture of perlite and peat-moss in a transparent plastic chamber with saturated circulating air for 1 month. GA₃ sprayed on the leaves was found to be beneficial in stimulating growth resumption of plantlets.     

一个辣椒肌醇半乳糖苷合成酶同源基因的全长cDNA克隆与低温表达分析

采用RT-PCR结合RACE技术,从低温处理的辣椒(Capsicum annuum L.)品种“苏长红”叶片中克隆得到一个肌醇半乳糖苷合成酶同源基因（GS）的cDNA序列(GenBank登录号：EF566470), 其全长1 444 bp, 推断其编码336个氨基酸。序列分析表明该基因与其他高等植物的GS基因具有较高的同源性。利用RT-PCR技术分析辣椒受低温胁迫后该基因的表达情况,发现该基因常温下在辣椒各组织中均无表达,经4 ℃或10 ℃处理6 h后在叶片中开始表达,而茎和根系中无论低温处理与否均不表达。Southern杂交结果表明辣椒基因组中还存在其他GS同源基因。     

Cloning,molecular analysis,and developmental expression of 3 oleosin cDNA isoforms in coconut (Cocos nucifera L.)

Coconut stores its lipid reserves in the endosperm, specifically in oil bodies that contain proteins called oleosins which stabilise their structure. This study reports the complete cDNA sequences of 3 genes for 3 isoforms of oleosin termed CnOLE500a, CnOLE500c, and CnOLE300a with 396, 375, and 381 nucleotides, respectively. Their predicted amino acid sequences were 131, 124, and 126 residues in length, respectively, with molecular weights of 13,787, 12,982, and 12,988 Da, respectively. The complete CnOLE500a cDNA sequence had 83.1% similarity with that of CnOLE500c, while CnOLE300a cDNA had only 50.7% and 46.5% similarity with CnOLE500a and CnOLE500c, respectively. None of the 3 coconut oleosins had the 18 amino acid insertion characteristics of H-class oleosins, thus they belonged to the L-class of oleosins. However, phylogenetic analysis showed that CnOLE300a was more related to H-class oleosins from dicots. All 3 isoforms were highly expressed at all stages of coconut endosperm development. CnOLE500c exhibited 6% higher expression than CnOLE500a and 15% higher than CnOLE300a at all stages of coconut endosperm development. However, oleosin proteins were barely detectable in the solid endosperm of coconut at 5–6 months, but this increased 20-fold at 6–7 months, and increased by a further 2- and 12-fold, at 7–8 and 8–9 months, respectively.     

Microsatellite markers based assessment of genetic diversity in Iranian olive (Olea europaea L.) collections

To study the genetic variation in Iranian olive collections and some foreign olive cultivars, 47 accessions of 18 local cultivars from 6 olive collections of Iran (Roudbar, Zanjan, Ahvaz, Dezful, Kazeroon and Shiraz), were analyzed along with 30 imported cultivars using 16 microsatellite primer pairs. All the used microsatellite loci revealed polymorphism in the studied genotypes, except GAPU14 and GAPU113 markers. Fourteen microsatellite primers amplified 126 polymorphic alleles in the 87 selected olive accessions. The average number of alleles per locus was 9, ranging from 3 to 14. Polymorphic information content (PIC) was 0.85. The genetic similarity based on Jaccard coefficient ranged from 0.15 to 1. The genetic relationships among accessions were investigated using cluster analysis and principal component analysis (PCA). Most of the accessions with the same name were grouped together; some exceptions were also observed. As expected, close relationship was observed among accessions within same cultivar. Most of the Iranian olive accessions were clustered to a main distinct group. Two-dimensional scatter plot of principal component analysis revealed a clear separation of most of the Iranian olives from Syrian and other introduced cultivars. These suggest that Iranian cultivars have different origin related to West Mediterranean basin cultivars and have evolved independently from the others. Between and within Iranian and foreign cultivars (cultivars including three or more accessions) genetic diversity was analyzed using analysis of molecular variance (AMOVA). AMOVA revealed higher within cultivar genetic variation (62.76%) as compare to that between cultivar variations (37.24%). The intra- and inter-cultivar variance tested by permutation test showed significant genetic variation at both levels. The high level within cultivar genetic variance could be due to mislabeling and presence of homonyms in cultivars produced by vegetative propagation from original plants.     

Biochemical changes involved in self-incompatibility in two cultivars of olive (Olea europaea L.) during flower development

Biochemical analyses were carried out to characterise the basis of self-incompatibility in the olive (Olea europaea L.) cultivars ‘Amygdalolia’ and ‘Konservalia’. Multiple interactions between cells of different types, origin, and function occur in the pistil. Endogenous factors play important roles in ovary and fruit development before (Stage 1) and during pollination (Stage 2), and after fertilisation (Stage 3). Changes in carbohydrate, protein, H₂O_2, and calcium ion concentrations, and in peroxidase, catalase, superoxide dismutase, and ascorbate peroxidase activities in pistil tissue before and during pollination, and after fertilisation were investigated. In both cultivars, H₂O₂ concentrations were significantly higher in pistil tissue before pollination, after which they started to decrease in Stage 2 and continued non-significantly in Stage 3. Peroxidase, catalase, superoxide dismutase, and ascorbate peroxidase activities were lower at Stage 1 and Stage 2, whereas these enzyme activities increased at Stage 3 in both cultivars. The lowest concentration of calcium ions was observed at Stage 1, whereas at Stage 2, calcium ion concentrations increased and reached their highest level, then decreased at Stage 3. Calcium ion concentrations in ‘Konservalia’ were higher than in ‘Amygdalolia’. In ‘Konservalia’, the highest protein concentration was observed at Stage 2. No significant differences were found in carbohydrate concentrations between the two cultivars. The biological significance of the presence of these products may differ between Stage 1, when they have a defence function, Stage 2 when there are interactions between pollen and pistil, and Stage 3 after fertilisation. This study provides support for the hypothesis that there is a correlation between self-incompatibility, stress-related enzyme activities, and calcium ion concentrations in the pistils of olive.     

白菜S位点糖蛋白基因的克隆与表达分析

 以白菜自交不亲和系为材料,采用RT-PCR技术,用特异引物对SLG基因进行克隆,获得SLG基因cDNA序列长度为1 324 bp,命名为BcSLG。序列分析表明：所获得的白菜BcSLG基因cDNA序列包含一完整的编码框,编码432个氨基酸,含有12个保守的半胱氨酸残基和7个N-糖基化位点。序列比对和系统进化分析表明：BcSLG与其它植物的SLG基因氨基酸序列具有较高的同源性,与大白菜和甘蓝亲缘关系最近。荧光定量PCR分析表明：BcSLG基因在自交不亲和系的柱头中表达量最高,其次是花蕾,叶片中表达最低, 在自交亲和系的柱头、花蕾和叶片中相对表达较低。     

Cloning and expression analysis of three genes encoding ubiquitins in papaya (Carica papaya L.)

Papaya (Carica papaya L.) fruit have a short shelf-life due to rapid ripening induced by ethylene, which usually results in a high percentage of product loss. However, little is known about the genetic mechanism of ripening and the attributes of fruit quality. Ubiquitin (UBQ) proteins have received increasing attention because they play important roles in response to ripening and in regulating certain developmental processes in plants. In the present study, three genes encoding UBQ proteins, CpUBI1, CpUBI2, and CpUBI3, were isolated from papaya fruit. The lengths of the cDNAs of CpUBI1, CpUBI2, and CpUBI3 were 1,485 bp, 1,642 bp, and 529 bp, encoding 306, 308, and 156 predicted amino acids, respectively. Sequence analysis demonstrated that the CpUBI1 and CpUBI2 proteins contained four consecutive structural domains of the UBQ superfamily, while CpUBI3 contained a ribosomal domain structure of the S27a superfamily. RT-qPCR analysis demonstrated that ethephon treatment increased CpUBI gene expression significantly, compared to 1-methylcyclopropene (1-MCP) treatment and the untreated controls. Levels of CpUBI1 and CpUBI2 gene expression were significantly higher than CpUBI3. These results suggested that CpUBI1, CpUBI2, and CpUBI3 might have different roles during papaya fruit ripening and softening.     

Effects of water deficit on the vegetative response,yield and oil quality of olive trees (Olea europaea L., cv Coratina) grown under intensive cultivation

An experiment was carried out in a young high-density olive grove (556 plants ha⁻¹—Olea europaea L., cv Coratina) located in Southern Italy to evaluate the effect of different soil water availability on the vegetative and productive performances of olive trees also looking into the quality of the resulting oils. Trials were carried out over a 3-year period on trees subjected to irrigation and grown under rainfed conditions. Vegetative tree response, as shoot elongation and trunk diameter, was evaluated. Yield per plant, fruit characteristics, oil quality indices (free fatty acid content, peroxide value, UV adsorption at 232 and 270 nm, total phenols, α-tocopherol content) were determined for both irrigated and non-irrigated treatments in the ‘on’ years 1997 and 1999 (6th and 8th year after planting, respectively).     

Molecular characterisation of coconut (Cocos nucifera L.) varieties using ISSR and SSR markers

The present study was carried out to standardise a DNA isolation protocol for coconut and to characterize five coconut varieties using 18 inter-simple sequence repeat (ISSR) and 14 simple sequence repeat (SSR) markers. DNA was extracted from tender young leaf samples collected from the fronds of five different trees of each coconut variety. A protocol using 0.095 g ml^?1 glucose, 0.025 g ml^?1 polyvinylpyrrolidone, 0.0045 g ml^?1 sodium bisulphite, 0.0055 g ml^?1 sodium dodecyl sulphate, and 50 µl ml^?1 sarcosine produced good quality DNA. The average polymorphism percentages revealed using ISSR or SSR markers between the five varieties were 31.9% or 92.9%, respectively. Using ISSR markers, the overall similarity between all five varieties ranged from 0.657 to 0.775, whereas it was 0.037–0.304 using SSR markers. The levels of polymorphism detected using ISSR markers among the five samples each of ‘Banawali’, ‘Gangabondum Green Dwarf’, ‘Pratap’, ‘Konkan Bhatye Coconut Hybrid-I’, and ‘East Coast Tall’ were 23.2%, 24.2%, 25.6%, 27.1%, and 21.2%, respectively. The levels of polymorphism detected using SSR markers among the five samples of the same five varieties were 85.7%, 86.9%, 85.7%, 100%, and 92.9%, respectively. This study indicated that genetic variation existed both between and within samples of each of the five varieties of coconut. SSR markers were superior to ISSR markers. The extent of genetic variation obtained within a variety was not expected, so it is essential to maintain seed purity via artificial pollination.     

菜薹花芽分化及BrcuFLC基因的克隆与表达

通过制作石蜡切片研究了菜薹(Brassica campestris L. ssp. chinensis (L.) Makino var. utilis Tsen et Lee)早熟品种‘油青49’和晚熟品种‘油青甜菜心80天’的花芽分化过程,结果表明,当展开2~3片真叶时花芽分化开始启动。用已报道的拟南芥Flowering locus C (FLC) 基因和FRIGIDA (FRI) 基因的保守区域设计引物,通过RT-PCR的方法从两个菜薹品种中均克隆得到了两个决定开花的关键基因,并命名为BrcuFLC (GenBank登录号为EF138603)和BrcuFRI (GenBank登录号为EU700362)。半定量式RT-PCR表达分析表明, BrcuFLC基因在早、晚熟菜薹品种的不同发育时期表达存在差异,表达量随真叶数增加而逐步减少,但在晚熟品种中BrcuFLC表达量降低幅度小;BrcuFRI则在早、晚熟品种的所有阶段表达都较低。BrcuFLC在菜薹不同部位表达的情况不同,在茎、叶中的表达强,花次之,根中表达较弱;而BrcuFRI在早、晚熟品种根中的表达量明显高于其它3个部位。     

Assessment of genetic purity of tomato (Lycopersicon esculentum L.) hybrid using molecular markers

Three DNA molecular marker systems, RAPD, ISSR and SSR, were used to test seed genetic purity of two commercial hybrid tomato (Lycopersicon esculentum L.) cultivars ‘Hezuo 903’ and ‘Sufen No. 8’. Genomic DNA from the two F₁ hybrid cultivars and their corresponding parental lines was screened with 218 RAPD decamer primers, 54 ISSR primers and 49 SSR primers. Among the 321 primers, 4 primers for ‘Hezuo 903’ and 3 for ‘Sufen No. 8’, which could produce both female and male parent-specific markers, were selected for testing the genetic purity. A total of 210 hybrid individuals of each cultivar were analyzed using the identified primers. The combined results of the marker analysis showed that eight of the 210 F₁ plants in ‘Hezuo 903’ and 13 of 210 in ‘Sufen No. 8’ were false hybrids, and the overall genetic purity of the two F₁ hybrid seed lots was 96.2 and 93.8%, respectively. This study showed that RAPD and SSR markers could provide a practical and efficient tool in quality control of the tomato commercial hybrid seeds.     

Assessment of genetic diversity among mango (Mangifera indica L.) genotypes using RAPD markers

Knowledge about the extent of genetic diversity/relatedness in mango germplasm is vital for developing coherent strategies for future gains in productivity. The genetic diversity/relatedness among mango cultivars/genotypes developed in Pakistan has not been investigated previously. We have assessed the genetic diversity among 25 mango genotypes/cultivars using randomly amplified polymorphic DNA (RAPD). Sixty random ten-mer primers were surveyed, out of which 45 yielded amplicons in all the genotypes. Genetic similarity between genotypes/cultivars was in the range of 64–89% with an average of 74%. Similarly, the genetic relatedness among all variants derived from a mango cultivar Chaunsa was in the range of 81.18–88.63%. These coefficients were utilized to construct a dendrogram using the unweighted pair group of arithmetic means (UPGMA). The genotypes were grouped into three (A, B, C) clusters. Generally, genotypes originating from Pakistan were grouped in cluster ‘A’ while cluster ‘B’ primarily composed of southern India as well as Florida cultivars. Kensington Pride was the most distantly related genotype which grouped with Maya and Yakta, forming a distinct cluster ‘C’.     

Analysis of blueberry (Vaccinium corymbosum L.) fruit water dynamics during growth using an ecophysiological model

Blueberry (Vaccinium corymbosum L.) fruits exhibit three growth stages associated with distinct biological processes. During these periods, water and carbon accumulate in the fruit, determining quality traits such as fruit size and sugar concentration. We adapted the Fishman-Génard model to blueberry and used it to analyse the effect of fruit load on fruit fresh mass and water dynamics, based on empirical observations of dry mass and sugar content performed throughout the fruit growth period. Different fruit load treatments were imposed during two seasons on ‘Brigitta’ blueberry plants growing under different culture systems. Increasing fruit load significantly reduced the fresh mass of the fruits at harvest, but did not affect sugar concentration, which was simulated and validated with a mean error of 7% for fresh mass and 15% for sugar concentration for the tested conditions. The most sensitive model parameters were those related to cell wall extensibility and sugar uptake. The simulations indicated that larger fresh mass of the fruit was mainly caused by increases in water fluxes rather than pressure differences. The model implementation provides the first estimates of a set of parameters which govern blueberry fruit water dynamics from fruit set to harvest.     

Optimisation of a highly efficient shoot regeneration system using leaf explants of Chinese jujube (Ziziphus jujuba Mill.) by response surface methodology

Chinese jujube (Ziziphus jujuba Mill.) is a major fruit crop in Asia. In this study, response surface methodology (RSM) was successfully employed to establish a highly efficient in vitro propagation and regeneration system for the ‘Teapot’ jujube via shoot organogenesis. Among the tested factors, gibberellic acid (GA₃) concentration showed the most significant positive effect. The pre-culture darkness timing and medium were also important factors for highly efficient shoot regeneration of the ‘Teapot’ jujube. The highest regeneration (> 75%) was achieved by 1 week in darkness and culture on wood plant medium (WPM) containing 0.25 mg·L^?1 GA₃, 0.5 mg·L^?1 6-benzylaminopurine (BAP) and 0.1 mg·L^?1 3-indoleacetic acid (IAA). In vitro-derived shoots rooted very well in the modified ¹/₂ Murashige and Skoog (MS) medium containing 0.4 mg·L^?1 3-indolebutyric acid (IBA), resulting in a 100% rooting rate. These findings suggest that the RSM can be employed to optimise the protocols needed for successful in vitro plant regeneration of jujube cultivars, with potential applications in plant genetic transformation practices, polyploidy induction and germplasm preservation.     

Characterization of Tunisian pomegranate (Punica granatum L.) cultivars using amplified fragment length polymorphism analysis

The amplified fragment length polymorphism (AFLP) analysis of DNA was used to characterize 34 pomegranate cultivars. By using a combination of six primers, a total of 327 markers were scored with a mean of 57.5. The high percentage of polymorphic bands (ppb) of 94.7 and the resolving power (R_p) collective rate value of 129.14 were scored. Data proved that the tested primers were informative to discriminate among cultivars and to survey the genetic diversity in this fruit crop. It has been assumed that the local pomegranate germplasm is characterized by a typically continuous genetic diversity. The derived dendrogram proved that cultivars are clustered independently from their geographical origin and their denomination. In addition, AFLP permitted the generation of a nearly unlimited number of molecular markers that are reliable in differentiating the cultivars and/or the polyclonal varieties.     

Cryopreservation of coriander (Coriandrum sativum L.) somatic embryos using sucrose preculture and air desiccation

An efficient protocol for cryopreservation of somatic embryos of Coriandrum sativum, an important spice and medicinal herb, was developed. The successful cryopreservation procedure utilized embryo clumps (ECs) comprised of 3–4 somatic embryos at the globular or heart-shape stage. These ECs were precultured for 3 days on medium supplemented with 100 g/L sucrose, desiccated under the current of sterile air for 100 min, then sealed in cryovials and plunged directly into liquid nitrogen. Preliminary incubation on sucrose-enriched medium (100 g/L) improved both desiccation- and cryo-tolerance of ECs compared to medium with normal sucrose content (30 g/L) and enhanced embryo formation after cryopreservation. The regrowth after cryopreservation and average number of new embryos developed from cryopreserved ECs were retained at the level of the untreated control (98% and 13 embryos per clump, respectively). Both normal and abnormal plants were produced from control and cryopreserved cultures, indicating that appearance of abnormalities was not related to cryopreservation. The regenerants with normal phenotype showed the same peaks of relative DNA content regardless of cryopreservation. The results suggest that simple desiccation method is effective for cryopreservation of coriander somatic embryos with subsequent regeneration.     

Genetic diversity and population genetic structure of pomegranate (Punica granatum L.) in Iran using AFLP markers

Pomegranate (Punica granatum L.) is one of the oldest known edible fruits. It is native to Iran and spread from Iran to other areas. In this study amplified fragment length polymorphism (AFLP) was used to detect intra- and inter-population genetic diversity of pomegranate. A group of 67 accessions belonged to 4 populations from Iran was studied using eight primer combinations. A total of 221 scorable bands were amplified, of which, 118 (54.13%) were polymorphic. Resolving power (Rp) ranged from 5.70 to 9.21, and the average of polymorphism information content (PIC) per primer pair was 0.40. According to Nei's gene diversity and allelic statistics, Isfahan population had a highest genetic diversity (H = 0.3646, I = 0.5327, N_e = 1.6467). Coefficient of gene differentiation between populations (GST) was 0.124, indicated that mainly proportion of genetic variation (87.6%), was within populations and the remaining (12.4%) of the variation was among populations that, also supported by analysis of molecular variance (AMOVA). The gene flow (Nm) varied from 0.969 to 10.404 between pair-wise populations and was 3.504 among all of the populations. The Jaccard similarity coefficient between individuals ranged from 0.26 to 0.88. The UPGMA dendrogram clustered all 67 accessions into 6 groups. In some cases accessions from same region were grouped together but in most cases, there was gene exchange. To study the genetic relationships among populations, a principal coordinate analysis (PCoA) based on Nei's genetic distances was performed. Results of this study showed that AFLP marker can be a useful tool for investigating the genetic diversity of pomegranate genotypes.