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.     

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.     

Antioxidant response of bitter gourd (Momordica charantia L.) seedlings to interactive effect of dimethoate and UV-B irradiation

Bitter gourd (Momordica charantia L.) seedlings treated with elevated concentrations of dimethoate (100 and 200 ppm) and fixed ultraviolet-B (0.4 Wm⁻²/30 min) irradiation showed stunted growth and less photosynthetic pigments chlorophylls (Chl) content. The synergistic effects of both the stresses were more pronounced than the individual effect. However, dimethoate at low concentration (50 ppm) stimulated growth and pigmentation but with UV-B it showed slight inhibition. Reactive oxygen species (ROS) accumulated considerably in leaves due to UV-B and high concentrations of dimethoate. Combined exposure further increased the ROS leading to lipid peroxidation and electrolyte leakage. Both the stresses alone and together also caused the increase activity of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD). High concentration of dimethoate and UV-B accelerated the accumulation of ROS particularly H₂O₂ in leaves, causing heavy damage to photosynthetic pigments and growth of bitter gourd seedlings. Simultaneous exposure of UV-B and dimethoate inhibit the growth, photosynthetic pigment and enhanced the accumulation of ROS more severely than the individual exposure. Interestingly, low concentration (50 ppm) of dimethoate significantly reduced the effects of UV-B. The results suggested synergistic effect of dimethoate and UV-B on plant growth as a function of decreased photosynthetic pigments despite increase in the activities of the antioxidant enzyme.     

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.     

Dormancy and germination of Areca triandra seeds

The dormancy mechanisms of Areca triandra Roxb. Ex Buch-Ham seeds were studied by treating the intact or mechanically scarified seeds with scarification, chemical soaking and stratification. The results indicate that the seeds have exogenous and endogenous dormancy. The exogenous dormancy is imposed by the pericarp and it is the major limiting factor for germination. It can be broken by mechanical scarification, but not by chemical scarification in 98% H₂SO₄ for 30 min. Chemical treatments (soaking for 24 h in 100–200 mg/L GA₃, 0.2% KNO₃ and 0.1–0.3% NaNO₂, and for 12 h in 10% H₂O₂ or 20 min or 12 h in 15% H₂O₂) and stratifications, especially, cold stratifications for 30–120 days, broke the endogenous dormancy and significantly hastened germination of mechanically scarified seeds, although they did not increase the germination percentages.     

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.     

Symbiotic seed germination of Grammatophyllum speciosum Blume and Dendrobium draconis Rchb. f., native orchids of Thailand

In vitro symbiotic seed germination is an important tool not only for the study of orchid-fungus specificity but also for the production of mycobiont-infected healthy seedlings that could be valuable for both horticultural and conservation purposes. The current study compared effectiveness of eight putative orchid mycorrhizal fungi obtained from mature orchids in the genera Paphiopedilum, Cymbidium and Dendrobium, in promoting in vitro seed germination and protocorm development of Grammatophyllum speciosum Blume and Dendrobium draconis Rchb. f., native Thai orchids. The developmental stages of seeds and protocorms cultured on Murashige and Skoog (MS) medium, oat meal agar (OMA), or OMA inoculated with one of the eight fungal isolates were evaluated weekly. Two isolates of Epulorhiza repens (Bernard) Moore (=anamorphic species of Tulasnella calospora (Boud.) Juel), Da-KP-0-1 and Pv-PC-1-1, were found to be the most effective fungi in promoting protocorm development of G. speciosum. At week 13, protocorms co-cultured with either one of these two fungal isolates, on the average, were significantly more advanced than those sown on OMA. Protocorms co-cultured with isolate Pv-PC-1-1 were also significantly more advanced than those cultured on MS medium. For D. draconis seed germination, three fungal isolates of different anamorphic species of Tulasnella, C1-DT-TC-1, Pv-PC-1-1, and C3-DT-TC-2, were found to be the most effective fungi in promoting protocorm development. However, none of these fungal isolates outperformed MS medium. Additionally, the compatibility between the fungal isolates tested and the two orchid species was discussed.     

Responses of non-primed or primed seeds of ‘Marketmore 76’ cucumber (Cucumis sativus L.) slurry coated with Trichoderma species to planting in growth media infested with Pythium aphanidermatum

Aqueous slurries of commercial preparations of Trichoderma harzianum Rifai strain KRL-AG2 G41 (Th), T. virens Strain G-41 (Tv), or their combination (ThTv, at half rates each of the single application rate) were applied to ‘Marktetmore 76’ cucumber seeds (Cucumis sativus L.) that were non-primed or primed for 3 days at 25 °C either osmotically (−2.5 MPa from 0.337 molal Ca(NO₃)₂) or osmomatrically (−1.0 MPa from 0.135 molal Ca(NO₃)₂ plus −1.5 MPa from 50% water in exfoliated grade 5 vermiculite). Slurries were applied to seeds (1 mg per seed) either before or after priming. Seeds were sown in soilless, peat-based media with or without inoculation with Pythium aphanidermatum (Pa). Protection against damping-off caused by high pressures of Pa (16% emergence in non-coated, non-primed seeds) was increased by slurry coating Th on non-primed (76.4% emergence) or on osmotically primed seeds, with coating either before or after priming having no effect on efficacy (average 62.6% emergence). Slurry coating Th on osmomatrically primed seeds failed to increase final emergence percentage (FEP). Colony forming units per three seeds (CFU, all 10³) was 2.8 for non-primed seeds, and 3.2 and 2.6, respectively, when osmotically and osmomatrically primed seeds were coated after priming. In a second study with lower disease pressure (58.1 FEP from non-coated, non-primed seeds), slurry coating of non-primed or osmotically primed seeds with Th, Tv or ThTv reduced percentage damping-off and increased FEP. The combination coating eliminated damping-off only in non-primed seeds, and tended to reduce percentage damping-off in osmotically or osmomatrically primed seeds compared to coating with Th or Tv alone. In a third study using only non-primed seeds, slurry coatings with mefenoxam fungicide, Th, Tv, or ThTv decreased total damping-off to 2.6%, 7.4%, 2.0%, and 0%, respectively, from the 30.1% occurring in non-coated seeds. Th, Tv or ThTv applied to growth media at the same rate as the seed coating (1 mg per seed) were generally as effective as the seed coatings, and only the ThTv growth medium application eliminated damping-off. A fourth experiment revealed that Th, Tv or ThTv remained viable on non-primed seeds for up to 4 weeks (the longest storage duration) at 21 or 4 °C, but 21 °C storage resulted in faster seed germination by week 3 and higher CFU per three seeds by week 4. In summary, coating of non-primed seeds with Th, Tv or ThTv was more effective than coating primed seeds in reducing percentage damping-off. While priming treatments generally led to faster seedling emergence and greater seedling shoot fresh weight than was achieved with non-primed seeds, only for non-primed seeds was damping-off eliminated by the ThTv seed coating or growth medium application.     

Interspecific variations in seed germination of Corylopsis

This study was initiated to investigate the differences in germination percentages and rates between Corylopsis coreana Uyeki and Corylopsis sinensis var. calvescens Rehder & E.H. Wilson following a warm stratification (WS) and cold stratification (CS), and to study the effect of different WS temperatures interacting with different durations of CS. Warm stratification at 10 °C, 15 °C, 20 °C, and 25 °C was given for 1 month (1 M 10 °C, 15 °C, 20 °C, and 25 °C WS) followed by 0 M, 1 M, 2 M, and 3 M of CS at 5 °C (0 M, 1 M, 2 M, 3 M CS) and seeds were germinated in an air conditioned greenhouse maintained at 18.5 °C/18 °C. On average, less than 1% of C. coreana seeds germinated when sown without any WS and CS or with 1 M 15 °C, 20 °C, and 25 °C WS without CS treatment. However, 26% C. coreana seeds germinated after 1 M 10 °C WS without any CS treatment. Germination was not affected by WS temperatures when followed by 2 M 5 °C CS. It is concluded that C. coreana exhibited low seed germination at 10 °C and that this temperature could be considered the upper limit of CS for C. coreana. Only 2 M CS was required for more than 90% seeds to germinate. However, C. sinensis var. calvescens required longer than 3 M CS for more than 29% seeds to germinate. This clearly shows that there is an interspecific variation in optimum dormancy-breaking requirements.     

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.     

Chitosan specificity for the in vitro seed germination of two Dendrobium orchids (Asparagales: Orchidaceae)

The effects of different types of chitosan on seed germination and protocorm development were determined for two orchid species, Dendrobium bigibbum var. compactum and Dendrobium formosum. Six chitosan types derived from polymer or oligomer chitosan each with 70, 80 or 90% levels of deacetylation (P70, P80, P90, O70, O80 and O90, respectively), were evaluated as direct medium supplements at 0, 10, 20, 40 or 80 mg/L in modified VW medium by following seed germination and protocorm growth for 12 weeks. Chitosan of all six tested types and four concentrations were found to significantly enhance the proportion of D. formosum seeds that germinated, when compared to these germinated without chitosan. In contrast, chitosan caused no enhanced germination rate was noted for D. bigibbum var. compactum with all tested chitosans and doses tested. However, almost all types of chitosan at 10 mg/L, except O90, were able to significantly improve the growth of D. bigibbum var. compactum protocorms, whilst 10 or 20 mg/L of P70 chitosan was the best formula to enhance the growth of D. formosum protocorms. It is concluded that chitosan responses in seed germination and protocorm development were somewhat species and developmental stage dependent. Therefore, the appropriate chitosan application for each plant species should be evaluated first before use.     

Ovules that failed to form seeds in zinnia (Zinnia violacea Cav.)

The structure of ovules that failed to form seeds after compatible pollination in double-flowered zinnias (Zinnia violacea Cav.) was investigated by observing histological sections, and comparing the normal and abnormal development of the seeds. In an embryo sac of zinnia at anthesis, a large nucleus of fused polar nuclei was clearly recognized. Success in double fertilization was determined by the disappearance of this nucleus. In normally developing seeds, the cellular endosperm became vague at 5–6 days after pollination (DAP) and the developing embryos occupied the entire portion of the seeds up to 10 DAP. A number of ovules failed in fertilization for the lack of an embryo sac. In most of the aborted seeds, the embryo sacs degenerated, whereas the aborted embryos were still alive in a small number of aborted seeds. A small percentage of the aborted seeds exhibited an aborted embryo and an unfertilized fused nucleus of two polar nuclei or ‘single fertilization’. The three major problems suggested for the failure in seed formation in pollinated florets of zinnia included failure in fertilization, ovules lacking embryo sacs, and abortion of developing seeds.     

Alteration of tomato fruit quality by root inoculation with plant growth-promoting rhizobacteria (PGPR): Bacillus subtilis BEB-13bs

In this study the effect of inoculation of tomato (Lycopersicon esculentum Mill.) roots with plant growth-promoting rhizobacteria (PGPR) on yield and fruit quality was evaluated. The control treatment was non-inoculated (CTL) and the PGPR treatment was inoculated with Bacillus subtilis BEB-lSbs (BS13). Yield per plant and marketable yield, as well as fruit weight and length were increased by the BS13 treatment when compared to the CTL treatment. Texture of red fruits was also enhanced by the BS13 treatment compared to that in the CTL treatment. These results demonstrated that PGPR have positive effects on tomato fruit quality attributes, particularly on size and texture.     

Callus induction and plant regeneration from cotyledonary explants of ash gourd (Benincasa hispida L.)

A protocol for the production of complete plantlets through multiple shoots from the cotyledon-derived calli of ash gourd (Benincasa hispida L.) is described. The embryos were excised from mature seeds and cultured on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurin (BAP, 1–5 μM). After 10 days the well-developed green cotyledons from the growing embryos were isolated and cultured on MS medium fortified with 2,4-D (1–6 μM). The cultured cotyledons gave rise to luxuriantly growing calli after 6 weeks. These calli were subcultured on MS medium supplemented with various concentrations of BAP (1–6 μM) alone or in combination with naphthalene acetic acid (NAA, 0.2 and 0.5 μM) for regeneration. The regenerated shoots were multiplied and rooted on quarter strength MS medium supplemented with indole-3-butyric acid or NAA (1–5 μM). The rooted shoots were transplanted to soil with 90% success.     

In vitro germination of walnut (Juglans regia L.) embryos

The present studies were undertaken with a view to standardize the medium and culture conditions for embryo culture of five cultivars of walnut viz., ACO 38853, Netar Akhrot, Gobind, Solding Selection and Blackmore. Embryos from mature fruits were aseptically excised and cultured on MS medium supplemented with different combinations of BAP, kinetin and GA₃. Best performing medium was MS with 0.5 mg l⁻¹ kinetin, 0.5 mg l⁻¹ BAP and 2 mg l⁻¹ GA₃ yielding 66.6% germination in Netar Akhrot after 12 days of culturing. Percent germination of excised embryos was higher when GA₃ and cold treatments were simultaneously applied as compared to those when applied separately. Netar Akhrot was found to be the best responding cultivar, which had a range of 25–66.6% embryo germination under different culture conditions. Plantlets with shoots and roots have been obtained in Netar Akhrot and ACO38853 and are transferred to soil after hardening.     

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.     

In vitro asymbiotic seed germination and seedling growth of Ansellia africana Lindl.

The effects of sodium hypochlorite treatment and growth medium on in vitro seed germination and seedling growth of the leopard orchid (Ansellia africana Lindl.) were investigated. Forty minutes treatment of seeds with 1.75% sodium hypochlorite stimulated germination (70.6%) and seedling development when measured at Stage 5 (emergence of first leaf) on P668 medium after 8 weeks of culture. The growth medium played a significant role in determining the germination response of A. africana seeds. Dark pretreatment of seed cultures significantly enhanced the germination percentage and the growth of rhizoids on the protocorms. Leaf growth in terms of length was substantially enhanced on P668 medium. It was significantly reduced in modified Knudson C medium after 16 weeks of culture. Seedlings developed on P668 medium showed a significantly better growth performance in relation to leaf length, leaf number, root length, fresh and dry weights per plant in vermiculite after 12 weeks of ex vitro growth in a mist house with 90% relative humidity. Further studies developing a suitable method for in vitro symbiotic germination could assist in reintroduction of this threatened orchid species into the wild.     

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.