Genetic diversity of bitter gourd (Momordica charantia L.) genotypes revealed by RAPD markers and agronomic traits

Bitter gourd or bitter melon (Momordica charantia L.) is considered as minor cucurbitaceous vegetable in spite of having considerable nutritional and medicinal properties. Although some reports on genetic diversity based on morphological characterization are available, no work has been conducted to estimate genetic diversity using molecular markers in this crop. In the present study, 38 genotypes of M. charantia including few commercially cultivars collected from different parts of India based on agro-ecological zones were analysed for diversity study both at morphological and molecular levels. Genomic DNA was extracted from young healthy leaves following the procedure of Doyle and Doyle [Doyle, J.J., Doyle, J.L., 1990. A rapid DNA isolation procedure from small quantity of fresh leaf material. Phytochem. Bull. 119, 11–15]. Pair-wise comparison of genotypes was calculated as per the procedure of Jaccard [Jaccard, P., 1908. Nouvelles recherches sur la distribution florale. Bull. Soc. Vaud. Sci. Nat. 44, 223–270]. Dendrogram was constructed using the unweighted pair group method with arithmetic averages (UPGMA) and the computation for multivariate analysis was done using the computer programme NTSYS-pc Version 2.0 [Rohlf, F.J., 1998. NTSYS-pc Numerical Taxonomy and Multivariate Analysis System, Version 2.01. Exeter Software, Setauket, NY, USA]. Diversity based on yield related traits and molecular analysis was not in consonance with ecological distribution. Among 116 random decamer primers screened 29 were polymorphic and informative enough to analyse these genotypes. A total of 208 markers generated of which 76 (36.50%) were polymorphic and the number of bands per primer was 7.17 out of them 2.62 were polymorphic. Pair-wise genetic distance (GD) based on molecular analysis ranged from 0.07 to 0.50 suggesting a wide genetic base for the genotypes. The clustering pattern based on yield related traits and molecular variation was different.     

Biocontrol of mildew with Bacillus subtilis in bitter gourd (Momordica charantia L.) seeds during germination

The bitter gourd seed has a thick, hard seed coat. Mildew often occurs during germination and causes uneven and low rates of seed germination. However, the problems caused by mildew can be overcome by treating seeds with hot water, by soaking in water, or by using microorganisms. Seeds of the ‘Ching Pi’ bitter gourd were treated in a water bath at 60 °C for 10 min and then soaked in tap water at 25 °C for 24 h. The resulting germination percentage was 86.7%, and the resulting percentage of mildewed seeds was 10%. The biocontrol potential of three commercially available Bacillus subtilis solutions was examined. For seeds primed with Huodijun B. subtilis solution, the germination percentage was 73.3% and the mildewed percentage 6%. In dual cultures, the antibiotic content in the Huodijun B. subtilis solution was significantly greater than in Yunghsing and Huolibao, the other B. subtilis solutions examined. B. subtilis effectively inhibited the growth of Rhizoctonia solani mycelium and caused abnormal mycelial growth.     

Comparative analysis of genetic diversity in Indian bitter gourd (Momordica charantia L.) using RAPD and ISSR markers for developing crop improvement strategies

A genetic analysis of 38 diverse Indian bitter gourd (Momordicacharantia var. charantia, and var. muricata) accessions was performed using 29 RAPD and 15 ISSR markers. RAPD primers yielded 208 amplicons of which 76 (36.5%) were polymorphic providing an average of 2.6 amplicons per primer. RAPD amplicons per primer ranged from 3 (OPE-19, OPW-09) to 15 (OPW-05), and varied in size from 200 bp to 3000 bp. Fifteen ISSR primers provided a total of 125 bands of which 94 (74.7%) were polymorphic. Polymorphic ISSR markers ranged from 0 (UBC-841) to 12 (UBC-890) providing a mean of 6.3 amplicons per primer that ranged in size from 150 bp to 2700 bp. Nevertheless, the concordance among bitter gourd accession groupings after cluster analysis was relatively high (r = 0.77), indicating that RAPD- and ISSR-based diversity assessments in this germplasm array were generally consistent. The M.charantia var. charantia (domesticated) and var. muricata (wild, free-living) accessions examined were genetically distinct, and these differences provided for the development of strategies for genetic analyses and crop improvement in this species.     

Genetic relatedness (diversity) and cultivar identification by randomly amplified polymorphic DNA (RAPD) markers in teasle gourd (Momordica dioica Roxb.)

Genetic relationship and variation of 29 accessions of teasle gourd (Momordica dioica Roxb.) and 1 accession of Momordica cochinchinensis Spreng. (wild relatives of teasle gourd) were examined by RAPD analysis using 44 dodecamer oligonucleotide primers. A total of 496 fragments were produced by 44 primers of which 95% bands were polymorphic. Using presence or absence of specific RAPD markers or combination of primers, 23 out of 30 accessions were identified. The genetic relatedness or genetic distance based on Nei and Li's genetic similarity varied from 0.86 to 0.65 with an average of 0.74 among 29 M. dioica accessions (when M. cochinchinensis excluded). In the phenetic dendrogram developed from cluster analysis using UPGMA method, M. cochinchinensis was out grouped as single accession, while others showing relatively weak grouping formed four groups. Clustering pattern did not demonstrate any relationship between geographical origin and genetic diversity. A DNA extraction method has been standardized. This is the first report of using RAPD techniques in teasle gourd. It was concluded that RAPD analysis is a useful tool for genotypic identification and estimation of genetic similarity in teasle gourd.     

Rose (Rosa hybrida L.) EST-derived microsatellite markers and their transferability to strawberry (Fragaria spp.)

The ‘Genome database for Rosaceae (GDR)’ provides a large collection of expressed sequence tags (ESTs) harboring simple sequence repeats (SSRs) from several Rosaceae genera, including Rosa (rose). Primer pairs flanking SSR were designed for 312 unique Rosa ESTs based on GDR database. Eight rose (Rosa hybrida L.) genotypes were tested for PCR amplification, and 287 (92%) of the primer pairs generated allele-specific PCR bands that were readily scored. From 183 (63.7%) primer pairs that evidenced polymorphic alleles among the eight rose cultivars, 20 pairs evidencing EST sequence homology to known gene functions and high levels of polymorphism were selected and utilized for DNA fingerprinting and genetic diversity assessments of 47 rose hybrids. A total of 202 polymorphic bands were scored and generated unique fingerprints for each rose hybrid. The Nei–Li genetic similarity coefficients among 1081 pair-wise comparisons of 47 cultivars exhibited a broad range of genetic variations from 0.30 (‘Grand King’ and ‘Carnival’) to 0.99 (‘First Red’ and ‘Red Champ’). UPGMA cluster analysis divided 47 hybrids into five major groups and two sub-groups. The cross-species transferability of 273 EST-SSR primer pairs was evaluated using four genotypes of the strawberry, a genus member of the Rosaceae family. PCRs on the DNA samples of strawberry were successful for 165 primer pairs; among these, 123 pairs amplified 243 polymorphic bands. As surrogates of the marker transfer, the phenetic relationship among the four strawberry genotypes was evaluated. Genetic similarity coefficients varied from 0.78 (‘Maehyang’ and ‘Janghyee’) to 0.64 (‘Janghyee’ and ‘Pragana’). The results of cluster analysis showed that the three octaploid strawberry cultivars were quite similar, whereas the diploid ‘Pragana’ was related distantly at the genomic DNA level. The EST-SSR markers developed in the present study can be efficiently utilized for genetic diversity studies in Rosaceae.     

African spider flower (Cleome gynandra L./Gynandropsis gynandra (L.) Briq.) as a red spider mite (Tetranychus urticae Koch) repellent in cut-flower rose (Rosa hybrida L.) cultivation

Companion planting of Cleome gynandra, of Kenyan origin, in beds of cut-flower roses reduces significantly red spider mite (Tetranychus urticae Koch) infestation without any detrimental effect on productivity or flower quality. The level of reduction is dependent upon the density of the C. gynandra plants with 15 plants in a 1.8 m² bed (8.3 plants m²) being the most effective, planted either around the bed perimeter or within the rows of roses. The relatively high density of C. gynandra plants required may limit the direct application of this technology in export-focused, greenhouse rose production yet may be of significant value as a supplement to other mite-control strategies. The potential benefits of such companion planting for growers of field roses and those involved in some domestic markets are also evident. Research into the nature and extraction of the active, volatile mite-repellant components of C. gynandra is indicated.     

Antioxidant phytochemicals and gingerol content in diploid and tetraploid clones of ginger (Zingiber officinale Roscoe)

Fresh rhizomes of 18 diploid and tetraploid genotypes of Indian ginger, including commercial cultivars and experimental genotypes, were assayed for gingerol content and antioxidant activity. The tetraploid ginger type was derived from the respective diploid ginger by shoot tip culture. [6]-Gingerol was identified as the major pungent phenolic compound in all samples (mean 1354.78 ± 470 μg/g), while [8]- (mean 180.94 ± 45 μg/g) and [10]-gingerol (mean 234 ± 79 μg/g) occurred in lower concentrations. The total gingerol content of the tetraploid type was much higher than that of the respective diploid type and especially the [10]-gingerol. Both diploid and tetraploid types of the cv. Meghalaya Local contained the highest level of gingerol. At both ploidy levels, the genotypes showed significant differences in phenols and antioxidant activities. Correlation of total phenolics with different antioxidant assay at both diploid and tetraploid level was linear and strong to moderate. For genotypes at both ploidy levels, antioxidant activity (BCO) was determined especially by [6]-gingerol while [8]- and [10]-gingerol did not affect BCO. These results demonstrated that in the tetraploid types of ginger, the gingerol content and antioxidant activity were higher than in the diploid ones.     

Early detection of graft incompatibility in apricot (Prunus armeniaca L.) using phenol analyses

The study investigated the role of phenols in apricot graft incompatibility. Assays of phloem with cambium from 1-year-old apricot trees of cultivars Marlen, Leskora and Betinka which were grafted on the rootstocks of different genetic origin: M-LE-1, Lesiberian, MY-KL-A, Tetra, Penta, Green Gage, Julior, MRS 2/5 and Isthara were analysed with HPLC (together 23 scion/stock combinations). The phloroglucinol, catechin, p-coumaric acid and further non-identified phenols with the retention time 23–25 and 30 min were determined. The content of individual phenol compounds was related to specific cultivar/rootstock combination. The minimum number of statistical significant differences in the phenol content between tissues above and below graft union was established in homospecific combinations (P. armeniaca/P. armeniaca). Cultivars Marlen, Leskora and Betinka differ in the degree of compatibility or incompatibility with rootstocks. The pattern of non-identified phenol 23 in different graft combinations is similar to catechin and p-coumaric acid.     

Towards crop improvement in bell pepper (Capsicum annuum L.): Transgenics (uid A::hpt II) by a tissue-culture-independent Agrobacterium-mediated in planta approach

Agrobacterium tumefaciens-mediated transformation has been one of the methods used to generate transgenic plants in bell pepper. An alternate transformation method that avoids/minimizes tissue culture would be beneficial for the improvement of bell pepper due to its recalcitrant nature. In this report, transgenic bell pepper plants have been developed by a tissue-culture-independent A. tumefaciens-mediated in planta transformation procedure. In the present study, two open pollinated varieties viz., Arka Gaurav and Arka Mohini were used for transformation. Agrobacterium strain EHA105 harboring the binary vector pCAMBIA1301 that carries the genes for β-glucuronidase (uid A) and hygromycin phosphotransferase II (hpt II) was used for transformation. GUS histochemical analysis of T₀ and T₁ plants at various stages of growth followed by molecular analysis using PCR, Southern analysis and RT-PCR allowed selection of transgenics. The method resulted in 17.8% and 11.4% of the T₀ plants in Arka Gaurav and Arka Mohini being selected as chimeric and 35.0% and 29.7%, respectively, were identified as stable transformants in the T₁ generation based on PCR analysis.     

A strain of Bacillus subtilis stimulates sunflower growth (Helianthus annuus L.) temporarily

Preliminary studies showed that a Bacillus subtilis strain stimulates plant growth. We investigated how inoculating seeds of a sunflower cultivar (Helianthus annuus L.) with this strain stimulated plant growth, soil properties and emissions of greenhouse gasses, i.e. carbon dioxide (CO₂) and nitrous oxide (N₂O), when cultivated in a greenhouse. Unfertilized sunflowers or fertilized with urea served as controls. After one month, root length and fresh and dry root weight of the sunflower was significantly higher in the bacteria amended plant than in the urea and unfertilized plants. However, at harvest, no positive effect was observed. The number of seeds per plant and seed weight was not significantly different between the treatments, but total plant N was significantly higher in urea-amended plants than in unfertilized plants. The CO₂ production rate was not affected by treatment, but the N₂O emission rate was significantly higher in soil amended with urea plus bacteria soil compared to the unfertilized treatments. It was found that the B. subtilis strain used in this study had a positive, but only temporarily effect on growth of the sunflower cultivar used.     

Phenotypic variability and genetic structure in plum (Prunus domestica L.), cherry plum (P. cerasifera Ehrh.) and sloe (P. spinosa L.)

In the present study, phenotypic variability of 80 plum (Prunus domestica L.) varieties maintained in the French National Plum Collection was evaluated with 19 quantitative traits. In addition, genetic diversity and genetic structure was studied in three plum species (P. domestica L., Prunus cerasifera Ehrh. and Prunus spinosa L.) using chloroplast DNA (cpDNA) markers and five single sequence repeat (SSR) loci. Based on phenotypic traits, some varieties, such as mirabelle plums, grouped together. Bayesian structure analysis was used to identify different genetic groups, whereby damson plums were clearly distinguished from greengage plums. When examining the three species together, a higher level of cpDNA allelic richness was found in P. cerasifera and in P. spinosa than in P. domestica where only five cpDNA haplotypes were detected in the national plum collection, with one main haplotype that accounted for 80% of the varieties studied. P. domestica cpDNA haplotypes tended to group together with P. cerasifera haplotypes whereas most of P. spinosa haplotypes formed a separate cluster. SSR markers were somewhat able to distinguish the three species. These results provide some clues as to the origin of plum and the various plum varieties. Our results also provide useful information for the management of plum genetic resources.     

A SCAR marker linked to the N gene for resistance to root knot nematodes (Meloidogyne spp.) in pepper (Capsicum annuum L.)

The root-knot nematodes (Meloidogyne spp.) are important nematode pests and cause serious diseases in pepper in the world. No molecular markers linked to the nematodes resistance N gene have been reported. In this paper, ‘Carolina Wonder’ (Capsicum annuum L.), a sweet pepper line resistant to root-knot nematode with N gene, ‘20080-5-29’ (C. annuum L.), an inbred line susceptible to root-knot nematode with good horticultural characteristics, and their F₂ progeny with 320 individuals were used as materials. Evaluation of resistance and susceptibility of parental lines, F₁ and F₂ progeny inoculated with root-knot nematodes (Meloidogyne incognita) were carried out. ‘Bulked segregant analysis’ method was used to search for polymorphic markers from 512 pairs of AFLP primers. Based on the assessment of resistance and susceptibility and polymorphism of the AFLP marker in F₂ population, the genetic linkage distance between the AFLP marker and the N gene was estimated. One AFLP marker E39/M41-339 was obtained and transferred to a SCAR marker amplifying a 315 bp DNA fragment linked to the N resistant allele and a 331 bp fragment linked to the N⁺ susceptible allele. The distance between the molecular marker and the nematodes resistance N gene is 6.3 cM. This research delivered a valuable tool for the marker assisted selection of nematodes resistance in pepper.     

Phytochemical and antioxidant properties of selected fig (Ficus carica L.) accessions from the eastern Mediterranean region of Turkey

Fig has been a typical fruit component of the health-promoting Mediterranean diet for a very long time. Phytochemical characters and antioxidant capacity of green-, yellow-, brown-, purple-, and black-fruited fig (Ficus carica L.) accessions were investigated. In this study, total phenolics (TP), total anthocyanins (TA), fructose (FRUC), glucose (GLUC), sucrose (SUC), and variables (such as L*, a*, C*, and hue°) describing fruit skin colors were examined. Also, the antioxidant capacity (TAC) of fig fruits was determined by the ferric reducing antioxidant power (FRAP) assay. Antioxidant capacity was significantly correlated with the polyphenol and anthocyanin (r = 0.74 and 0.63, respectively) contents of fruits. Black fig accessions had the highest TAC (range of 7.9–16.1, mean 12.4 Fe²⁺ mmol/kg FW), TA (range of 32.3–356.0, mean 128.4 μg cy-3-rutinoside/g FW), and TP content (range of 69.1–220.0, mean 118.9 mg GAE/100 g FW). These black-fruited accessions had 2-fold greater TAC, 15-fold greater TA, and 2.5-fold greater TP than green and yellow fig accessions. However, the FRUC, GLUC, and SUC content of brown and purple fig accessions were higher than those of other color groups. The predominant sugars present were fructose (∼56%) and glucose (∼43%), as determined by HPLC.     

Somatic embryogenesis and plant regeneration in Opuntia ficus-indica (L.) Mill. (Cactaceae)

We present here a method for the regeneration of the prickly-pear (Opuntia ficus-indica (L.) Mill.) through somatic embryogenesis (SE). Direct SE was induced by cultivating shoot apices devoid of leaf primordia on semisolid Murashige and Skoog (MS) basal medium supplemented with 4-amino 3,5,6-trichloropicolinic acid (picloram) at 4 mg l⁻¹. Somatic embryogenesis was influenced by the type, age, physiological and developmental stage of the explants, and also by light conditions, wounding, and the sucrose concentration in the induction medium. A histological analysis confirmed SE by revealing the presence of a closed vascular system in the developing embryos and the absence of a vascular connection between the somatic embryos and the explant.     

The strawberry tree (Arbutus unedo L.) selection in Turkey

These days, the strawberry tree (Arbutus unedo L.), which is a comparatively under-utilized fruit tree species, has attracted attention because of many different possible commercial usages such as for processed and fresh fruit production and ornamental, the pharmaceutical and chemical industrial applications. They are disappearing from the natural flora for many reasons such as their being cut down for fire-wood and structuring on natural vegetation. A selection program was carried out with the aim of selecting strawberry tree genotypes of high quality from the native strawberry tree population grown in the Central Black Sea Region of Turkey. A total of 51 genotypes were evaluated in the program, in 2003 and 2004. The aims of the selection program were focused on fruit pomological characteristics such as fruit weight, total soluble solid, acidity, fruit taste, stoniness, juiciness, roughness and appearance. The data regarding pomological characteristics was evaluated by the Weighted Rankit method. The highest overall score for the characteristics tested was from genotype 57A01 followed by 57A15, 57GÇ08, 57A22 and 57A07, respectively. Their fruit weight was 11.08, 6.17, 6.30, 6.95 and 8.06 g, respectively. TSS contents and acidity of them varied from 21.4 to 30.0% and 0.80 to 1.59%, respectively. Phenological characteristics such as first bloom, fruit set and harvest date and some other fruit, flower and leaf characteristics of selected A. unedo genotypes were also determined. Vitamin C content of the samples varied from 97.83 to 280.00 mg/100 g. Finally, five superior strawberry tree genotypes were selected from the samples we studied for extensive cultivation and thus they were also saved from extinction.     

The conjunctive use of compost tea and inorganic fertiliser on the growth,yield and terpenoid content of Centella asiatica (L.) urban

The conjunctive use of a compost tea (CT) and an inorganic fertiliser (NPK) on the growth, yield and terpenoid content of Centella asiatica (L.) urban was evaluated. CT and NPK applied at half the recommended concentration resulted in the significant enhancement of vegetative growth, yield and antioxidant content. The synergistic effect of CT₅₀ and NPK₅₀ was highly pronounced on the bioactive components Asiatic acid, madecassoside and asiaticoside. The distribution of the total antioxidants was highest in roots (80%), followed by leaves (66%) and petioles (54%). There was a strong positive correlation (r = 0.990) between fertility and total antioxidant content, suggesting an enhanced synthesis of bioactive components resulting from the treatment. This response by C. asiatica to the integrated experimental treatment suggests a viable option for the commercial cultivation of the herb. Augmenting the vegetative biomass production and bioactive components will increase the availability of this medicinal herb for the treatment of various human ailments.     

Chemical compositions and insecticidal effects of essential oils isolated from Achillea gypsicola, Satureja hortensis, Origanum acutidens and Hypericum scabrum against broadbean weevil (Bruchus dentipes)

The essential oils of aerial parts of Achillea gypsicola Hub.-Mor., Hypericum scabrum L., Satureja hortensis L., and Origanum acutidens (Hand.-Mazz.) Letswaart were analyzed in this study by GC and GC–MS and their oils were tested for toxicity against broadbean weevil (Bruchus dentipes). A. gypsicola oil contained camphor (40.17%), 1,8-cineole (22.01%), piperitone (11.29%), borneol (9.50%) and α-terpineol (1.56%) as major components. A total of 74 components were identified by GC–MS in H. scabrum oil, including α-pinene (9.26%), terpinen-4-ol (5.12%), camphor (5.94%), δ-cadinene (4.52%), pulegone (4.45%), γ-muurolene (4.12%), pinocarvone (3.97%) and β-caryophyllene (3.42%) as predominant components. The essential oils of O. acutidens and S. hortensis were characterized by high contents of carvacrol (86.99% and 55.74%), γ-terpinene (0.71% and 20.94%), p-cymene (1.95% and 12.30%), α-terpinene (0.13% and 2.04%) and β-caryophyllene (1.30% and 1.08%). All of the essential oils were toxic to adults of B. dentipes and insect mortality increased with increasing concentration of each oil. The oils (20 μl dose) brought about 100% mortality in 36 h. Although desirable insecticidal activities against the pest were achieved with the oils from all four plant species, S. hortensis and O. acutidens oils were more effective, particularly after 6 h of treatment. The current results concluded that the essential oils, in particular O. acutidens and S. hortensis oils, may be used as potential botanical insecticides against B. dentipes.     

Identification of S-alleles in pear (Pyrus communis L.) cv. ‘Rocha’ and other European cultivars

In this work we report the cloning and identification of S-RNase alleles responsible for gametophytic self-incompatibility (GSI) of ‘Rocha’ pear and of 13 other European pear cultivars that might be used as its pollinators. Partial sequences of S-RNase alleles were amplified by PCR with specific primers hybridising in conserved regions of previously identified S-RNase alleles of Pyrus communis, cloned and sequenced and the S-genotype of eight pear cultivars was fully determined. Three cultivars (‘General Léclerc’ (SqSl), ‘Tosca’ (SbSl) and ‘Alexandrine Douillard’ (SbSk)) shared no S-alleles with ‘Rocha’ (SaSe) and shall be totally compatible with this cultivar. None of the cultivars analysed showed an identical amplification pattern to the one observed in ‘Rocha’, so the other cultivars shall be at least semi-compatible. One new allele was identified in P. communis cv. ‘Beurré d’Avril’ (designated as St). The determination of both S-RNase alleles of cvs ‘Rocha’, ‘Beurré Precoce Morettini’ (SeSk) and ‘Tosca’ and the identification of one S-RNase allele in cvs ‘Carapinheira’ (Sb), ‘Amêndoa’ (Se), ‘Pérola’ (Sk) and ‘Beurré d’Avril’ (St) are important contributions for the effort recently developed worldwide to establish groups of sexual compatibility among European pears.