Reciprocal differences in DNA sequence and methylation status of the pollen DNA between F<Subscript>1</Subscript> hybrids of <Emphasis Type="Italic">Solanum tuberosum</Emphasis> × <Emphasis Type="Italic">S. demissum</Emphasis>

Reciprocal differences, mostly caused by cytoplasmic effects, are frequently observed in interspecific hybrids. Previously, we found that crosses onto Solanum demissum were much successful with the pollen of interspecific hybrids between S. tuberosum as female and S. demissum as male (TD hybrids) than the reciprocal ones (DT hybrids). To elucidate this reciprocally different crossability, we analyzed the pollen DNA of TD and DT using methylation-sensitive amplified polymorphism (MSAP) analysis. Using 126 primer combinations, MSAP analysis revealed 57 different bands between bulked pollen DNA samples of TD and DT. Individual examination of 16 TD and 9 DT plants disclosed eight bands uniformly different between TD and DT. Their sequencing results revealed two pairs of bands to be identical to each other, resulting in six distinct sequences. As expected, one band shared high homology with chloroplast DNA, and another one with mitochondrial DNA. However, one band that was apparently different at DNA sequence level and maternally transmitted from S. demissum, showed no homology with any known sequence. The remaining three bands were of DNA methylation level differences with no or uncertain homology to known sequences. To our knowledge, this is the first report detecting reciprocal differences in DNA sequence or DNA methylation other than those in cytoplasmic DNA.     

Clonal propagation of triploid <Emphasis Type="Italic">Acorus calamus</Emphasis> Linn. Using dual-phase culture system

Acorus calamus is an important medicinal plant which has been used in Indian traditional medicine since time immemorial. Various bioactive molecules, viz., acorin, α- and β-asarone, asaryldehyde, caryophylene, isoasarone, methylisoeugenol, and safrol have been isolated from this plant. However, the use of this plant for medicinal purpose has been recently banned due to the high toxic property of β-asarone. The triploid Acorus calamus is reported to be low in β-asarone content and thus found to be the ideal raw material for medicinal use. The present investigation represents our finding for successful in vitro clonal propagation of the elite triploid accessions of Acorus calamus for mass propagation. In the dual-phase culture system consisting of agar-solidified Murashige and Skoog medium overlaid by liquid fraction of the same medium, maximum multiple shoot induction was favored by supplementation of α-naphthaleneacetic acid (0.5 mg L⁻¹) and 6-benzylaminopurine (2.0 mg L⁻¹). In vitro rooting of the microshoots was maximum in the medium supplemented with indolebutyric acid at 2.0 mg L⁻¹. The well-rooted microshoots could be successfully hardened and transplanted in the field. This result can be reproduced and is a viable protocol for successful clonal propagation of the seedless triploid Acorus calamus for conservation and sustainable development.     

Foliar application of the exogenous plant hormones at pre-blooming stage improves flowering and fruiting in cashew (<Emphasis Type="Italic">Anacardium occidentale</Emphasis> L.)

The problem of declining tree yield has led to an investigation into the effectiveness of foliar application of exogenous hormones to improve flowering, fruit set, and fruit retention in cashew. Five exogenous hormones, one Gibberellic Acid (GA₃) and four Auxins (IAA, IBA, NAA, and 2,4-D) at seven different rates of application (0 mg L⁻¹, 10 mg L⁻¹, 25 mg L⁻¹, 50 mg L⁻¹, 100 mg L⁻¹, 250 mg L⁻¹, and 500 mg L⁻¹) were tested on six yield-related components of the two Brazilian cashew genotypes. This trial was a factorial split-split-plot design with each treatment replicated five times within a tree and three replications (three trees) per genotype. Responses varied significantly between exogenous hormones, concentrations and genotypes. The cashew plants used showed hormone-specific and optimum concentration response patterns. Of the five exogenous hormones tested, GA₃ was most effective as its application at 50–100 mg L⁻¹ gave five-fold improvements in flowering (precocity and number of hermaphrodite flowers) and fruiting, and about 69% increase in fruit retention ability and 25% in nut size. Panicles treated with GA₃ also produced relatively bigger nuts compared to the untreated. Days to flowering was found to be hormone sensitive, while production of hermaphrodite flowers, fruit set, and nut development tended to be concentration specific. The GA₃ exhibited a broad concentration tolerance among the five exogenous hormones investigated. Our data showed that using GA₃ at 50 mg L⁻¹ will enhance flowering precocity, shorten flowering duration, increase production of hermaphrodite flowers and fruit set significantly, and resultant nuts develop optimally with high percentage retention. Thus, it suggests cashew yield could be increased by exogenous foliar application of GA₃ at 50–100 mg L⁻¹ at pre-blooming stage.     

High frequency plantlet regeneration from nodal segment culture of female <Emphasis Type="Italic">Momordica dioica</Emphasis> (Roxb.)

An in vitro propagation method for female plants of Momordica dioica (Roxb.) has been established. The nodal segments were harvested and the cut ends of the explants were sealed with wax and then surface sterilized and cultured. Bud breaking occurred on Murashige and Skoog’s (MS) agar-gelled medium + 2.0 mg L⁻¹ 6-Benzylaminopurine (BAP) + 0.1 mg L⁻¹ Indole-3 acetic acid (IAA). The cultures were amplified by passages on MS medium supplemented with 1.0 mg L⁻¹ BAP + 0.1 mg L⁻¹ IAA. Further, shoot amplification (29.2 shoots per vessel) was achieved by subculturing of in vitro regenerated shoot clump on MS medium + 0.5 mg L⁻¹ BAP + 0.1 mg L⁻¹ IAA. The micropropagated shoots were subsequently transferred for root formation on half-strength MS medium + 2.0 mg L⁻¹ Indole-3 butyric acid (IBA) with 89% success rate. The in vitro-regenerated shoots were also rooted ex vitro with 34% success. These plantlets were hardened in the greenhouse and transferred to the field. The established protocol is suitable for true to type cloning of mature female plant of M. dioica.     

QTL identification of yield-related traits and their association with flowering and maturity in soybean

Two soybean recombinant inbred line populations, Jinpumkong 2 × SS2-2 (J × S) and Iksannamulkong × SS2-2 (I x S) showed population-specific quantitative trait loci (QTLs) for days to flowering (DF) and days to maturity (DM) and these were closely correlated within population. In the present study, we identified QTLs for six yield-related traits with simple sequence repeat markers, and biological correlations between flowering traits and yield-related traits. The yield-related traits included plant height (PH), node numbers of main stem (NNMS), pod numbers per plant (PNPP), seed numbers per pod (SNPP), 100-seed weight (SW), and seed yield per plant (SYPP). Eighteen QTLs for six yield-related traits were detected on nine chromosomes (Chrs), containing four QTLs for PH, two for NNMS, two for PNPP, three for SNPP, five for SW, and two for SYPP. Two highly significant QTLs for PH and NNMS were identified on Chr 6 (LG C2) in both populations where the major flowering gene, E1, and two DF and DM QTLs were located. One other PNPP QTL was also located on this region, explaining 12.9% of phenotypic variation. Other QTLs for yield-related traits showed population-specificity. Two significant SYPP QTLs potentially related with QTLs for SNPP and PNPP were found on the same loci of Chrs 8 (Satt390) and 10 (Sat_108). Also, highly significant positive phenotypic correlations (P < 0.01) were found between DF with PH, NNMS, PNPP, and SYPP in both populations, while flowering was negatively correlated with SNPP and SW in the J × S (P < 0.05) and I × S (P < 0.01) populations. Similar results were also shown between DM and yield-related traits, except for one SW. These QTLs identified may be useful for marker-assisted selection by soybean breeders.     

Flow cytometric analysis of nuclear DNA content,mitotic chromosome number and protein separation by SDS-PAGE in three accessions of African locust bean (<Emphasis Type="Italic">Parkia biglobosa</Emphasis> Benth.)

Nuclear DNA of three accessions of Parkia biglobosa collected from three locations in northern Cross River State was investigated using a Patec PA II flow cytometer equipped with an argon ion laser (488 nm), and pictures of mitotic chromosomes were taken using a digital micro-camera (Canon) placed on the eye piece of a binocular microscope at 100X oil immersion. Metaphase chromosome counts of 2n = 22 for accessions A and C and 2n = 24 for accession B, were obtained and through flow cytometry, the three accessions were confirmed to be diploids. The nuclear DNA content and genome size for the accessions were 1.5085, 1.489, and 1.5266 pg (737.7054, 728.121, and 746.5074 Mbp) for accessions A, B, and C, respectively. In another experiment, leaf samples from greenhouse-germinated seeds were analyzed for variation in the banding pattern of the protein by SDS-PAGE in the three accessions. Protein was resolved into three banding groups according to their electrophoretic mobility: slow, medium, and fast, clustering between 100–200, 40–70, and 10–25 kDa, respectively. There was 76% similarity in the banding pattern between the accessions.     

Application of DNDC model to estimate N<Subscript>2</Subscript>O emissions from green tea fields in Japan

Heavy doses of N fertilizers are commonly applied to green tea fields in Japan, and cause large amount of nitrate leaching in ground water and emission of ammonia and nitrous oxide (N₂O) to the atmosphere. The Denitrification and Decomposition (DNDC) model was tested against experimental data on N₂O emissions from the tea field in Nishio, Aichi, Japan. There were reasonable agreements between the simulated and measured values of N₂O emissions for this site. The model was then applied for estimating the environmental impacts as affected by farm management practices, climate change, and soil properties. The model results were assessed with respect to major indicators of agro-ecosystems including crop yield, soil organic carbon sequestration, nitrate leaching loss, and N₂O emission. The results indicated that use of compost significantly reduced nitrate leaching and N₂O emissions in comparison with N fertilizer. When soil pH and texture shifted to non-acidic and coarser soil, N₂O emission increased; and a change in temperature and precipitation affected N₂O emission, nitrate leaching, and SOC sequestration. This study thus revealed the biogeochemistry model as a powerful tool in addressing the complex efficacy of the alternative farm management practices in tea fields across various climate and soil conditions.     

Greenhouse evaluation and inheritance analysis of transgenic cotton expressing <Emphasis Type="Italic">PTM3</Emphasis>, a MADS-box gene of Aspen

The PTM3 (MADS-box) gene characterized from flower of Aspen was ectopically expressed in cotton and was found to result in desirable agronomic traits. Among several transgenic lines, the PTM3 cotton event-10 was found to flower significantly earlier than the control and yield better [Ramachandran et al. (2011)]. We report our findings on performance based on greenhouse evaluation and inheritance of PTM3 cotton event-10. The T₁ progeny of event-10 were grown in pots under RBD design. The progeny were confirmed by kanamycin imbibition test, PCR, and GUS assay which showed the segregation of transgene at about 3:1 ratio. GUS assay was performed with pollen from all transgenic plants; plants that expressed gus in all pollen versus those that showed segregation for expression were found to be at a ratio of 1:2. Similar to observations made in the T₀ generation of event-10, the agronomic evaluation of T₁ progeny exhibited, on an average, earliness of 13 days in flowering, 13.5 days in crop maturity, and 22% of yield enhancement. The T₂ progeny of homozygous lines were grown on field soil in the greenhouse under strip trial design to see the effect as observed from potted plants of T₁ progeny. Inheritance of transgene cassette to T₂ progeny was confirmed by the same tests used to analyze T₁ progeny. As exhibited by T₀ and T₁ progenies, the T₂ progeny also showed earliness of 8 days in flowering, 12 days in crop maturity, and 17% of yield enhancement. The data generated from the progeny over two generations confirms that the PTM3 cotton event-10 is superior in agronomic characters as compared to non-transgenic cotton and is of interest for breeding shorter duration varieties with improved yield.     

Varietal difference in physicochemical properties of pigmented rice varieties

The physicochemical properties of pigmented (Midorimochi, Jeokjinjubyeo, and Heukmichalbyeo) and non-pigmented (Hwayoungbyeo) rice varieties were investigated. Starch from non-pigmented rice consisted mainly of large polyhedral granules that were more loosely packed than those of the non-pigmented sample. Pigmented rice showed higher linoleic acid content (36–40%) and hydrolysis rate (63–79%) but lower oleic acid content (35–39%), blue value (0.05–0.17), and viscosity values than the non-pigmented one. Significantly higher mineral content, blue value, and pasting and viscosity values were observed in the Jeokjinjubyeo sample compared to those of the other pigmented rice samples. The Midorimochi variety, on the other hand, exhibited the highest total amino acid (816.82 ng mg⁻¹) and sugar (0.15–3.35 μg g⁻¹) contents and the lowest pasting and viscosity values. No substantial difference in the X-ray diffraction pattern was observed among the samples. Results of this study could serve as baseline information for the quality evaluation of three pigmented rice cultivars and provide a better understanding of their potential uses and food industry applications.     

Estimating canopy cover from color digital camera image of rice field

Canopy cover (CC) is a good predictor variable for plant growth parameters such as leaf area index and aboveground biomass. A nondestructive, low-cost, and convenient method is presented for estimating CC using digital camera image analysis. CC was estimated by the ratio of plant pixels to total pixels of digital camera image of rice field. To determine the criteria for segmenting the rice plant from variable soil background, three mosaic images for rice plant, flooded/bare soil, and algae-infested background were prepared from digital camera images that were taken in various field conditions. An image analysis program was developed in Visual Basic to extract red, green, and blue (RGB) features from the mosaic images, calculate RGB-based color indices, and compute the minimum segmentation error for separating rice plant from background. When judged by the segmentation error, modified excessive green index (MEGI) showed the highest potential for segmenting rice plant from flooded/bare soil background, followed by normalized green (g) and excessive green index (EGI). At the threshold MEGI value of 0.03, the segmentation error was the lowest as 0.13%. Any single index considered was not satisfactory in segmenting rice plant from algae-infested background. However, a discriminant function of 1.2553EGI + 0.01735G − 0.01474B was successful in segmenting rice plant from flooded/bare soil and algaeinfested background with segmentation errors of 0.34 and 1.17%, respectively. CC for four rice varieties from tillering to booting stage was estimated based on the threshold value of MEGI and discriminant function and also manually using commercial software. Both estimates of CC showed good relationship of r² = 0.94, suggesting that a digital camera could be used efficiently for measuring the CC of rice field.