Assessment of the Variations in Selected Industrially Desirable Morphological and Biochemical Traits of Eleven Citrus Species in Sri Lanka

AbstractThe fruits with nutraceutical and medicinal values are having a greater significance in modern food industry. Out of many fruit tree bearing genera, the genus Citrus is extremely important in this regard. It contains well known species such as orange, lime, mandarin, lemon and many other underutilized species. The nutritive value of Citrus is given by many bioactive compounds present in the fruits such as alkaloids, tannins, phenols and saponins. In Sri Lanka, the available Citrus germplasm is not characterized in detail. Especially underutilized Citrus species are yet to receive a proper taxonomic identity. Therefore in the present study we report some of the important morphological and biochemical traits of eleven Citrus species. The fruit size traits; weight, height and width were very highly variable among the studied genotypes and positively correlated to each other (P<0.05). However, juice volume was not highly correlated with the other fruit size traits. The juice volume per unit fruit weight can be given as a better indicator for the selection of these species for industrial applications. Pummelo, a popular edible Citrus fruit, showed the lowest volume to weight ratio of 0.06. The size of the endocarp cells (i.e. juice sacks) was also very highly variable among genotypes / species studied. The brix value, a parameter of total soluble solids, was highest in Sidaran fruits (8.07%) and lowest in cooking types such as lemon and lime (3.27% and 3.76% respectively). Similarly the ascorbic acid concentration was also lowest in lime and lemon (1.9 and 1.5mM respectively). The sweet orange cultivar Sisila (5.9mM) and Pummelo (6.9mM) had the highest ascorbic acid concentrations. The results reported here are very useful to select these genotypes / species for potential industrial applications, future genetic and breeding studies and to further characterize at molecular level to establish an accurate taxonomic scheme for Sri Lankan Citrus germplasm which will be the next focus of our group

Morphological and Bitterness Level Characterization of Phyllanthus emblica Drupes Reveals Higher Diversity

Phyllanthus emblica L. (V. Nelli) is an important constituent of indigenous medicine and acommercially important fresh fruit species. The diversity of the P. emblica germplasm in SriLanka has not been studied in detail according to our knowledge. Therefore, the present studywas conducted to characterize the drupes of P. emblica gremplasm in Sri Lanka.A total of 475 P. emblica drupes harvested from 66 trees in selected areas of Anuradhapura,Kandy and Kurunegala districts of Sri Lanka. For each drupe, seven morphometric data;drupe height, width, weigh, mesocarp thickness, stone width, height and weight weremeasured. Also 61 drupes were used for the quantitative analysis of the epicarp colour ofdrupes using a colourimeter. An organoleptic property; bitterness was analysed in relation todrupe size by having a taste panel of independent human subjects. The data were analysedusing parametric and non parametric statistical methods.The cluster analysis based on drupe size traits clearly characterized the P. emblica germplasmin to four clusters. The means of drupe weight (Cluster 1; 5.98 g, Cluster 2; 3.59 g, Cluster 3;2.13 g, Cluster 4; 14.21 g) were significantly different (P&lt;0.05) among the four clusters.There were significant differences in colour metrics between small and large drupe groups.The results indicated that the smaller drupes (chroma; 21.64, hue angle; -10.86) are lighter,yellowish, sharper and less colourful (i.e. dull) than that of the larger drupes (chroma; 12.66,hue angle; -21.11). The sensory data analysis revealed that generally smaller drupes arebitterer (57% of drupes awfully bitter) in contrast to larger drupes (47% of the drupes mildlybitter) but there is no guarantee that small drupes are always bitter. The Crarmer’s Vcoefficient (0.47) indicated that the association between drupe size and the bitterness has noperfect linear relationship (Crarmer’s V coefficient is 1.00 for such a linear perfectrelationship). The drupe traits were not different with respect to their tree locations or theclimate and the environmental factors contributed less than other factors such as genetics.The P. emblica germplasm has a higher diversity in Sri Lanka and should be conserved inorder to maintain the genetic stochasticity. It sets the foundation to implement conservationmeasures and this diversity structure can be further verified by molecular portrayal which iscurrently undertaken by our research group. The proposed characterization can be used as aguideline to define a working level core-collection that will be useful in taking decisions forgermplasm conservation and in planning crop improvement programs of P. emblica.

Morphological and Microsatellite Marker Analysis of Fruit Size and Shape in Selected Accessions and Commercial Cultivars of Capsicum Species in Sri Lanka

Chili pepper (Capsicum spp.)is a vegetable crop with medical and ornamental uses. In Sri Lanka, 56% of dried chili is imported annually making it a huge burden to the economy. To improve the chili pepper industry, breeding could be suggested as one of the strategies. With the advancement of Molecular Biology,breeding is now practiced with marker assisted selection. According to the present knowledge, no such breeding programs are taking place in Sri Lanka. Therefore the objectives of the present study were to characterize the morphology and validate fruit size and shape linked microsatellite markers using an ex situ Capsicum collection in comparison to the commercial cultivars. The accessions and cultivars were established in a greenhouse at Peradeniya, Sri Lanka. Leaf and fruit morphological data were recorded at flowering and fruit maturity respectively. Five microsatellite markers (HpmsE045, CAeMS010, GPMS178, CAMS451 and CAMS493) were used to genotype all 49 accessions using PCR and bands were size separated using polyacrylamide gel electrophoresis. According to the fruit shape, six groups were identified namely Triangular, Elongated long, Elongated short, Spherical sharp-end, Spherical blunt-end and Rectangular. The plant height had a negative correlation with the number of fruits of the plant at first harvest. Number of seeds per fruit and the fruit diameter were significantly associated with the fruit weight (P<0.05). Microsatellite marker analysis revealed 44 alleles explaining very high level of genetic diversity (in the range of 62% to 87%). Out of the 44 alleles, 15 alleles were found to be significantly affecting on fruit size traits and 23 alleles were associated with fruit shape. This validated marker information could be used to plan future breeding programs and genetic studies for chili pepper.KEYWORDS: QTL, genetic diversity, morphological diversity, chili pepper, Capsicum

Identification of a robust protocol for isolating PCR amenable DNA from caryopses to employ DNA fingerprinting to claim the plant breeders’ rights and varietal ownership of the exporting rice bulks

Rice Research and Development Institute (RRDI) at Bathalagoda and its’ substations are solely conducting rice breeding in Sri Lanka. Recently, RRDI has identified five exportable rice varieties/lines. As rice export in Sri Lanka is undertaken by the private sector, the RRDI does not receive any foreign revenues or credit for breeding rice genotypes. Therefore, RRDI currently is in need of a reliable protocol to claim the Plant Breeders’ Rights (PBR) of these exportable genotypes. The first step of establishing such a protocol based on DNA fingerprinting is depending on the ability to extract good quality DNA from rice caryopses. Therefore, the present study compared the suitability of commonly available DNA extraction methods to purify DNA from rice caryopsis. Three rice genotypes; Bg 250 and At 353 (varieties for local consumption) and Bw-Bs-1-2-31 (with export potential) were selected. Three parboiled rice samples each from long- and short-grain groups were also selected. The DNA extraction, quantification and purity assessment were carried out using four methods viz., CTAB, modified CTAB, Promega kit and Qiagen kit. The extracted DNA was subjected to simplex and multiplex PCR using four rice specific DNA markers. Although positive PCR bands were obtained for all four methods, the modified CTAB method yielded the significantly highest DNA yield (6196.67μg/ml) compared to other methods. Thus, RRDI can employ modified CTAB method in the protocol to claim its PBR and rice varietal ownerships

Molecular mechanisms of reversing neural degeneration by retinoic acid, a major derivative of vitamin A

For the past hundred years since the discovery of vitamin A, the field of research associated with retinoids has been well evolved due to advances in molecular biology, chemistry, biochemistry and medicine. Vitamin A is an essential component in diet. Major derivatives of vitamin A such as retinol, retinaldehydes and retinoic acid are collectively considered as retinoids. Deficit of Vitamin A has a direct correlation with disease development in our body emphasizing the importance of retinol. Retinoic Acid is proclaimed as powerful mitogen acting in our body since the developmental embryonic stages where it is associated with many organ systems in body carrying out different functionalities. Retinoids carry out many functions of nervous systems. This review is mainly focused on contribution of Retinoic acid towards reversing the process of neural degeneration in pathological conditions achieved by neurodegenerative diseases and tumor inducing situations. Retinoids can act on neurons carrying morphogenesis of nerves by promoting stem cell differentiation. Activation of phospholipase A2 pathway can promote neural differentiation. Also by inactivating human Inhibitor of DNA binding 2 gene, retinols can suppress stem cell proliferation and initiate morphogenesis. This gene inactivation can be also used as a therapy to prevent malignant cell proliferation leading to tumors. Anti amyloidogenic activity is discussed in this review locates a significant importance in reversing neural degenerating process in diseased conditions. Moreover, Reactive Oxygen Species accumulation can be suppressed by Retinoic Acid where it can promote cell survival during pathological constrains. The revision of literature is carried out in depth revealing Retinoic Acid related putative drug target mechanisms, where we can use Retinoic Acid as a novel therapeutic drug in neurodegenerative diseases

Real time PCR for the rapid identification and drug susceptibility of Mycobacteria present in Bronchial washings

Abstract Background Mycobacteria have a spectrum of virulence and different susceptibilities to antibiotics. Distinguishing mycobacterial species is vital as patients with non-tuberculous mycobacterial (NTM) infections present clinical features that are similar to those of patients with tuberculosis. Thus, rapid differentiation of Mycobacterium tuberculosis complex from NTM is critical to administer appropriate treatment. Hence the aim of the study was to rapid identification of mycobacterial species present in bronchial washings using multiplex real time Polymerase Chain Reaction (PCR) and to determine the drug susceptibility in identified mycobacterial species. Methods Sputum smear negative bronchoscopy specimens (n = 150) were collected for a period of one year, from patients attending the General Hospital Kandy, Sri Lanka. The specimens were processed with modified Petroff’s method and were cultured on Löwenstein– Jensen medium. DNA, extracted from the mycobacterial isolates were subjected to a SYBR green mediated real time multiplex, PCR assay with primers specific for the M. tuberculosis complex, M. avium complex, M. chelonae-M.abscessus group and M. fortuitum group. DNA sequencing was performed for the species confirmation, by targeting the 16S rRNA gene and the drug susceptibility testing was performed for the molecularly identified isolates of M. tuberculosis and NTM. Results The optimized SYBR Green mediated multiplex real-time PCR assay was able to identify the presence of genus Mycobacterium in 25 out of 26 AFB positive isolates, two M. tuberculosis complex, three M. avium complex and two isolates belonging to M. chelonae-M. abscessus group. DNA sequencing confirmed the presence of M. tuberculosis, M. chelonae-M. abscessus, M. intracellulare, M. avium, Rhodococcus sp. and M. celatum. Remaining isolates were identified as Mycobacterium sp. All the NTM isolates were sensitive to amikacin and seven were resistant to ciproflaxacin. Twenty two of the NTM isolates and the isolate Rhodococcus was resistant to clarithromycin. The two isolates of M. tuberculosis were sensitive to all first line anti tuberculosis drugs. Conclusion The optimized SYBR Green mediated multiplex real time PCR assay could be an effective tool for the rapid differentiation of pathogenic M. tuberculosis complex from the opportunistic nontuberculous mycobacteria and also it confirmed the presence of NTM in 15.3 % of the study population

Characterization and Selection of Phosphorus Deficiency Tolerant Rice Genotypes in Sri Lanka

Phosphorus (P) deficiency in soil is a major constrain for rice production. An important set of rice genotypes (landraces, old improved and new improved varieties) were screened for P deficiency tolerance in two major cropping seasons of Sri Lanka, in 2012. The Ultisol soil, which was collected from a plot cultivated with rice without fertilizer application for past 40 years (P0) at the Rice Research and Development Institute (RRDI), Bathalagoda, Sri Lanka, was used as the potting medium for greenhouse trials. Two field trials were conducted in the same plots at RRDI. Both P0 and P30 (30 mg/kg P2O5) conditions were used in the two greenhouse trials. At the early vegetative (three weeks after transplanting), late vegetative (six weeks after transplanting) and flowering stages, plant height and number of tillers per plant were recorded. At the flowering stage, shoots were harvested and shoot dry weight, shoot P concentration, shoot P uptake and P utilization efficiency were measured. All data were statistically analyzed using analysis of variance, regression and cluster procedures. The measured parameters were significantly different between P0 and P30 conditions (P < 0.05). Higher shoot dry weight was reported by the rice genotypes H4 and Marss under P0 conditions. The regression analysis between shoot dry weight and P utilization efficiency revealed that the studied rice genotypes could be categorized to three P deficiency tolerance classes. A total of 13 genotypes could be considered as highly tolerant and 4 genotypes as sensitive for P deficiency. These results could be used to select parental genotypes for breeding and genetic studies and also to select interesting varieties or landraces for organic rice production