Encapsulation for in vitro short-term storage and exchange of ginger (Zingiber officinale Rosc.) germplasm

Synseeds of ginger (Zingiber officinale) were produced using aseptically proliferated 2-week old encapsulating explants (microshoots) upon complexation of 4% sodium alginate prepared in Murashige and Skoog (1962) medium (MS) and 100 mM calcium chloride. Conversion of synseeds into plantlets (conversion) was recorded as 66% and 53% on MS (3% sucrose) and on MS (3% sucrose) + 2.5 mg/l BA media, respectively. However, shoots/synseed were significantly higher on MS (3% sucrose) + 2.5 mg/l BA medium. For short-term storage of germplasm, sucrose-dehydrated synseeds were found better than air-dehydrated or fresh synseeds. Synseeds dehydrated in 0.25 M sucrose liquid medium for 16 h and stored in cryovials (with out medium) at 25 °C for 8 weeks and 12 weeks exhibited 53% and 13% conversion, respectively, on MS (3% sucrose) + 2.5 mg/l BA medium. Plantlets obtained from stored synseeds were hardened, established successfully ex vitro and were morphologically similar to each other as well as their mother plants. This synseed protocol could be useful for short-term storage and exchange of germplasm of ginger between national as well as international laboratories.     

Modeling individual leaf area of ginger (Zingiber officinale Roscoe) using leaf length and width

Leaf area estimation is an important biometrical observation one has to do for comparing plant growth in field and pot experiments. In this study, a leaf area estimation model was developed for ginger (Zingiber officinale Roscoe), using linear measurements of leaf length (L) and maximum width (W). Leaves from five ginger varieties (Varada, Rejatha, Mahima, Maran and Himachal) were used to develop the model in 2006–2007. The actual leaf area (LA) was measured with a leaf area meter (LI-3100, LI-COR, Lincoln, NE, USA) and taken as reference LA. The linear measurements were used to build linear (LA = a + b × L × W) and power models (LA = α × (L × W)^β) for each variety, as the modeling among variety were not different from each other, data for all five varieties have been pooled and compared with earlier models by graphical procedures and statistical criteria such as Mean Square Error (MSE), Root Mean Square Error (RMSE) and Chi-square (χ²). The selected model was validated during 2007–2008. The validation data set was used to produce a validation model for each variety by re-estimating the model parameters to develop the estimation model and the models were compared for consistency. The predicted LA (PLA) was compared with observed LA (OLA) by graphical procedures and lack of agreement was evaluated by calculating the relative bias, estimated by the mean of differences (d) and the standard deviation (SD) of the differences. Normality test was carried out by Spearman's rank correlation coefficient (r_s) and residuals were normally distributed. Finally, the proposed model for leaf area estimation of ginger is LA = −0.0146 + 0.6621 × L × W, R² = 0.997. This model can be reliably used for estimating leaf area of ginger non-destructively. The same equation can be extrapolated to all varieties and land races of ginger as it is vegetatively propagated crop with narrow genetic variability.     

Development of novel chloroplast microsatellite markers for Dendrobium officinale, and cross-amplification in other Dendrobium species (Orchidaceae)

Nine pairs of polymorphic chloroplast microsatellite primers were developed for Dendrobium officinale Kimura et Migo, an endangered herb. Levels of polymorphism were tested across a total of 55 individuals from four natural populations (12–15 individuals per population). Allele numbers varied from two to four per locus, while the number of haplotypes ranged from four to six per population. Transferability of the nine polymorphic chloroplast microsatellite primers was checked on an additional set of 51 Dendrobium individuals (belonging to 17 different species). Three markers could be transferable to all the species tested, while the remaining six markers successfully cross-amplified in most species tested. Moreover, polymorphism of the nine chloroplast microsatellite primers was tested across Dendrobium moniliforme (L.) Sw. and Dendrobium loddigesii Rolfe. All of them were polymorphic in D. moniliforme, while seven of which were polymorphic in D. loddigesii. These polymorphic chloroplast microsatellite primers developed for D. officinale will be a useful tool for the study of genetic diversity, population genetic structure, evolution of D. officinale and establishment of effective conservation strategies.     

Variation in DPPH scavenging activity and major volatile oil components of cassumunar ginger, Zingiber montanum (Koenig), in response to water deficit and light intensity

Cassumunar ginger, Zingiber montanum (Koenig) Link ex Dietr. (Zingiberaceae), is called Phlai in Thailand. The current study investigates the levels of antioxidant activity and major volatile oil components in cassumunar ginger exposed to varying water deficits (0, 30, 60, 90 and 120 days before harvest) and light intensities (100%, 50% and 25% sunlight). A 120-day water deficit resulted in high volatile oil content and increased sabinene content, but DPPH scavenging activity was not affected. The quantity of volatile oil in the cassumunar ginger exposed to a 120-day water deficit was almost twice that of both the control and the sample exposed to a 30-day deficit. The highest terpinen-4-ol content was obtained after a 60-day water deficit, and (E)-1-(3′,4′-dimethoxyphenyl) butadiene content tended to decrease with prolonged water deficiency. The antioxidant activities of cassumunar ginger were 52.98%, 41.75% and 42.93% in plants grown at 100%, 50%, and 25% sunlight, respectively. Light intensity of 50% or 25% increased the biomass of cassumunar ginger but decreased the volatile oil content; antioxidant activity was not significantly affected.     

Genetic diversity analysis in the traditional and improved ginger (Zingiber officinale Rosc.) clones cultivated in North-East India

Identification of clonal or genotypic variations is a prerequisite for ginger (Zingiber officinale Rosc.) improvement programmes. Genetic diversity analysis was carried out in a set of forty-nine ginger clones cultivated in North-East India using random amplified polymorphic DNA (RAPD) markers. The set included clones of released varieties and clones collected from various parts of North East India. Jaccard's genetic similarity, cluster analysis and principal component analysis identified five clusters. Cluster V included four clones traditionally cultivated in the Indian state of Meghalaya known for production of high-quality ginger indicating that the clones were a good candidate for ginger improvement. Specific bands for these clones were also identified. Principal component analysis of the molecular data supported grouping of the clones into six hypothetical populations based on their source or location of collection.     

Optimal pollination environment of tetraploid ginger (Zingiber officinale Roscoe) evaluated by in vitro pollen germination and pollen tube growth in styles

Pollen germination percentages in vitro of a tetraploid ginger (Zingiber officinale Roscoe), ‘4×Sanshu’, tended to be highest at around 20°C. Pollen tube growth in the styles was greatly enhanced at 17°C, i.e., pollen tubes penetrated into the entire stylar length in 66.7% of the styles used. Pollen stored for at least 3 h under 40–80% relative humidity (RH) almost completely lost its germinability, whereas pollen incubated for up to 3 h under 100% RH retained a relatively higher germinability. Moreover, the pollen germinability that was lowered under 40–80% RH did not recover when the pollen was re-humidified for 2 h under 100% RH before inoculation onto a medium. When stigmas were pollinated with fresh pollen at 17°C under 100% RH, pollen tubes penetrated into the entire stylar length in 26.7% of the styles tested. Under 40–80% RH, however, pollen tubes stopped growing at the middle or the base of styles. From these results, I concluded that the optimal pollination environment of the tetraploid ginger was at 17–20°C under 100% RH.     

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.     

Genetic diversity analysis of ginger (Zingiber officinale Rosc.) germplasm based on RAPD and ISSR markers

A global collection of ginger germplasm consisting of 46 accessions were characterized using two types of molecular markers, RAPD (Random Amplified Polymorphic DNA) and ISSR (Inter Simple Sequence Repeats). UPGMA dendrograms constructed based on three similarity coefficients, i.e., Jaccard's, Sorensen–Dice and Simple Matching using the combined RAPD and ISSR markers placed the accessions in four similar clusters in all the three dendrograms revealing the congruence of clustering patterns among the similarity coefficients and a rather less genetic distance among the accessions. Improved varieties/cultivars are grouped together with primitive types. Moreover, in the clustering pattern of the accessions, a geographical bias was also evident implying that germplasm collected from nearby locations especially with vernacular identity may not be genetically distinct. The clustering of the accessions was largely independent of its agronomic features.     

Correlation of saponin content and Fusarium resistance in hybrids from different ploidy levels of Lilium Oriental

Lilium Oriental is of great commercial value, but large losses in production can result from its susceptibility to diseases caused by Fusarium ssp. Here we report a mutant of Lilium Oriental resistant to Fusarium, Cai-74, which was generated from crossing tetraploid (from ‘Star fighter’) and diploid (‘Cor. Amore’ × ‘Acapulco’) plants. Hybrid progeny were screened via inoculating Fusarium oxysporum into tissue cultured plantlets in a greenhouse. Although Cai-74 had a saponin content of 3.81 mg g⁻¹, which is much higher than its parents, the highly Fusarium-resistant wild species Lilium dauricum had the highest content of 4.11 mg g⁻¹ among the tested genotypes. Cai-74 had a 3A karyotype rather than 3B found in normal plants of Lilium Oriental. Taken together, our results suggest that Cai-74 bears a chromosomal variation in its structure, and that the saponin content in the lily scales correlate with the Fusarium resistance of Lilium Oriental. Cai-74 could be used as a gene resource for breeding Fusarium-resistant cultivars of Lilium Oriental.     

Taxonomic revision of Dendrobium moniliforme complex (Orchidaceae)

Taxonomic revision of Dendrobium moniliforme complex is presented. D. moniliforme complex is characterized by the even slim stems, bracts with brownish zone, semi-spherical anther cap and the hairy disc of lip. Dendrobium tosaense, Dendrobium officinale and Dendrobium guangxiense were excluded by having membranous bracts lacking brownish zone, anther cap conical and bifid. Two species are recognized in this complex, i.e., D. moniliforme and Dendrobium wilsonii. D. wilsonii differs from D. moniliforme by having elliptic leaves about 1.3–2 cm wide, dorsal sepal 3.0–4.0 cm long, 0.6–0.9 cm wide, petals elliptic to oblong, 3.0–4.0 cm long, 1.0–1.5 cm wide, lip elliptic to ovate–lanceolate, 2.6–3 cm long, 1.2–1.5 cm wide.     

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.     

Syrian pear (Pyrus syriaca) as a pollinator for European pear (Pyrus communis) cultivars

In Israel four European pear cultivars are grown: ‘Spadona’ is the main cultivar and ‘Coscia’, ‘Gentile’ and ‘Spadochina’ are its pollinators. However, molecular S-genotyping revealed that ‘Spadona’ is semi-compatible with its three pollinators. This explains, at least in part, the relatively low pear yield in Israel. The Syrian pear (Pyrus syriaca) grows wild in Israel and blooms intensively, overlapping the blooming of the cultivated European pears. Cross-fertilization between Syrian pear and ‘Spadona’ was shown to be efficient suggesting that Syrian pear might be a potent pollinator for ‘Spadona’. Twenty-six Syrian pear seedlings, from different sites in north-east Israel were S-genotyped identifying 11 that are fully compatible with the four European pear varieties cultivated in Israel. By this screening, 24 different S-RNases were cloned; ten of them are new, whereas the other fourteen had been identified previously. In addition, seedlings of two wild pear species were also S-genotyped. Two seedlings from Pyrus betulifolia and one from Pyrus korshinskii were found to be genetically compatible with the four European pear cultivars. From these seedlings four S-RNases were cloned, two are new, one had been cloned previously and one was identical to an S-RNase allele cloned from Syrian pear in this work.     

Morphological and cytological studies of diploid and colchicine-induced tetraploid lines of crape myrtle (Lagerstroemia indica L.)

The tips of cotyledon-stage seedlings of three crape myrtle cultivars (“Zi Wei”, “Hong Wei” and “Yin Wei”) were treated with colchicine. Various concentrations of colchicine and different treatment durations were tested. Seedlings of “Zi Wei” treated with 0.5% colchicine for 72 h and seedlings of “Yin Wei” treated with colchicine (0.2% for 96 h, 0.5% for 48 h and 0.8% for 72 h) demonstrated high rates of mutation; “Hong Wei” showed a slightly lower rate. The highest rate of morphological variation was 54.17% and this was achieved when tips were treated with 0.5% colchicine for 72 h. Putative tetraploid plants were identified with morphological and cytological variations, such as larger and thicker leaves, darker green coloration, larger stomata, lower density of stomata across the lower leaf epidermis and increased numbers of chloroplasts per stomata guard cell. Flow cytometric analysis demonstrated that the nuclear DNA content of 15 of these putative tetraploids (of which 7 were “Zi Wei”, 5 were “Yin Wei”, and 3 were “Hong Wei”) was indeed doubled relative to that of control diploid plants. The chromosome number of the tetraploid plants was 2n = 4x = 96, while that of the control diploid plants was 2n = 2x = 48. Ornamental characteristics were also enhanced in the tetraploid plants, with increased diameter of the individual flowers and greater basal length of the petals and claws. Pollen diameter and the size of capsules and seeds were also significantly greater than those of diploid plants.     

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.     

Pollination with laser-irradiated pollens breaks cross-incompatibility between zicaitai (Brassica campestris var. purpurea) and ornamental kale (Brassica oleracea var. acephala) to produce hybrids with the aid of ovule culture

The objective of this study was to produce interspecific hybrids between an Ogura-cytoplasmic male sterile (CMS) line of zicaitai (Brassica campestris var. purupurea, 2n = 20) and cultivars of ornamental kale (Brassica oleracea var. acephala, 2n = 18) to develop a CMS system for hybrid seed production. Pollination with pollen grains of ornamental kales irradiated at a power output of 9.0 mW with a He–Ne laser for 3 min could overcome the cross-incompatibility between the species concerned. Intact hybrids could be efficiently produced from ovules cultured on Murashige and Skoog media supplemented by 0.2 mg l⁻¹ 6-benzyladenine. Chromosome number of hybrids was confirmed to be 2n = 19. Hybrids resembled ornamental kales in leaf morphology and in vernalization response. Pollens of hybrids had a sterile appearance. Moreover the hybridity of the putative hybrids was confirmed by RAPD data on a DNA fragment of 820 bp.     

Ploidy screening of anthurium (Anthurium andreanum Linden ex André) regenerants derived from anther culture

Anther culture was successfully developed in Anthurium andreanum Linden ex André cv. ‘Tropical’, but resulted in variation in morphology and growth response of regenerants. Ploidy levels varied in different morphological variants. Finding a convenient, rapid, reliable, practical and indirect method to screen and determine anthurium ploidy level is important not only for anthurium, but for ornamentals in general. Regenerants derived from anther culture showing three different ploidy levels (haploids, diploids, triploids) were used in this study. Five indirect methods were used to assess chromosome number: chloroplast number in stomatal guard cells (M1), stomatal length and width ratio (M2), stomatal density (M3), ratio of length and width of leaves (M4), and microspore number per anther (M5). These were compared to chromosome counting as the direct and control method (M6). Through simple regression correlation analysis, when compared to M6, M1 was the most convenient and reliable indirect method to determine the ploidy level. This method was highly and significantly positively correlated to anthurium ploidy level (r = 0.945; p < 0.01). This method could also be applied much faster than the conventional chromosome method.     

Identification of seven haplotype-specific SFBs in European pear (Pyrus communis) and their use as molecular markers

The European pear (Pyrus communis) carries the S-RNase-mediated gametophytic self-incompatibility (GSI) system. The S-haplotype is conferred by an S-locus, which contains the style-specific expressed S-RNase, and the pollen-specific expressed F-box genes (SFB). Both the S-RNase and the SFB genes are multi-allelic and each is characteristic of one of the S-haplotypes. Therefore, they are ideal markers for molecular S-genotyping. In this work, for the first time, seven haplotype-specific SFBs were isolated from European pears. Particular primers for each of these SFBs were generated, thus providing an additional tool for S-genotyping of European pear cultivars.     

DNA content,morphometric and molecular marker analyses of Citrus limonimedica, C. limon and C. medica for the determination of their variability and genetic relationships within the genus Citrus

This work investigated the fingerprinting and phenotyping of Citrus germplasm; species selected were of historical importance belonging to Citrus limonimedica Lush. and its supposed ancestors, along with some other species of the Citrus genus. An integrated approach based on the exploitation of nuclear DNA content, morphological traits and molecular markers, such as RAPD fingerprints and ITS-based SNPs, was employed. We studied a core collection of 54 distinct accessions, including 43 genotypes of the Citrus species (18 species or supposed species) and 11 genotypes of the Poncirus genus, which was used as the reference outgroup. Morphological trait analysis and statistical analysis of DNA content and markers were useful for reconstructing a Citrus phylogeny. In particular, our experiments aimed at estimating the genetic variation within and the genetic relatedness among C limon (L.) Burm., C. limonimedica and C. medica L. to shed light on the hybrid origin hypothesis of C. limonimedica. The results of the multidisciplinary analyses allowed us to confirm a remarkable differentiation between Poncirus and Citrus genera and to highlight a close relationship among the three investigated Citrus species but a distinct difference between these three species and other species in the Citrus genus. RAPD fingerprints and ITS polymorphisms enabled us to point out a variation gradient between lemon and citron, with C. limonimedica as a possible intermediate species. Some accessions of C. medica and C. limonimedica that deviate from such a trend suggest recurrent introgression and/or hybridisation with other species of Citrus.     

Pollen biology of ornamental ginger (Hedychium spp. J. Koenig)

An improved in vitro pollen germination assay was developed to assess the viability of stored Hedychium pollen. The effect of polyethylene glycol (PEG) (10, 15, and 20%, w/v) on pollen germination and tube growth was evaluated for Hedychium longicornutum and two commercial Hedychium cultivars, ‘Orange Brush’ and ‘Filigree’. Overall, the inclusion of PEG 4000 in the medium improved both pollen germination and tube growth for the three different genotypes tested and the results varied depending on genotype. In vitro germination was used to assess the viability of Hedychium pollen stored up to two months. Pollen nucleus status was determined for four Hedychium cultivars, ‘Orange Brush’, ‘Anne Bishop’, ‘Filigree’, and ‘Daniel Weeks’. Pollens of ‘Orange Brush’, ‘Anne Bishop’, and ‘Daniel Weeks’ were found to be binucleate but ‘Filigree’ was shown to possess both binucleate and trinucleate pollens. High pollen:ovule ratio values were obtained in several Hedychium taxa. The results obtained on the nuclear pollen status and pollen:ovule ratios will further our understanding of the pollination biology and help clarify the taxonomy and phylogeny of Hedychium species.     

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.