Effect of Agrobacterium Infection Time, Co-Cultivation and Cell Density on in vitro Response in Hypocotyl of Eggplant (Solanum melongena L.)

The present study purports to assess the effect of Agrobacterium infection time, co-cultivation and cell density on in vitro response in hypocotyl explants of eggplant (brinjal) cv. Manjarigota. Agrobacterium(OD600 0.3-0.5) infection for 10-15 minutes (24.44±2.34%) was found to be optimum, while, higher or lower infection-time resulted in reduced callus initiation, shoot regeneration and explant survival. Explants with no (only Agrobacterium infection) or short (1 day) co-cultivation, showed reduced callus-initiation response and turned yellow, with no regeneration. Callus-initiation response increased from Day 1 (96.66±03.33%), and reached a maximum on Day 2 and Day 3 (100±00.00%). It decreased on further increase in co-cultivation time. Explants co-cultivated for three days showed highest regeneration response (30.00±02.96%) which thereafter reduced with further increase in co-cultivation time. Explants infected with Agrobacterium culture at 0.05 OD600 showed hardly any regeneration, and turned yellow and necrotic on the selection medium. Highest regeneration response (28.33±02.33%) was obtained in explants infected with 0.1 OD600 culture, and this gradually reduced as celldensity increased (upto 1.0 OD600), becoming zero in explants treated with cultures at 1.5 OD600 or above. Agrobacterium overgrowth was noticed on explants infected with cultures of 0.5 OD600 and above. Exposure of hypocotyl explants to higher cell-density, longer infection-time and prolonged co-cultivation regime resulted in severe necrosis of explants; time taken for development of Agrobacterium overgrowth was less with increase in the level of these factors. Regenerated shoots were healthy, green, elongated and showed root induction on culture medium containing Kanamycin

Factors Affecting in Vitro Shoot Regeneration in Hypocotyls of Brinjal (Solanum melongena L.) in the Early Steps of Agrobacterium-Mediated Transformation

An attempt was made to assess the effect of size, age and position of the explant, pre-culture and high cytokinin concentration in the pre-culture medium on shoot regeneration in brinjal hypocotyls co-cultivated with Agrobacterium. The study was carried out using hypocotyl explants of brinjal cv. Manjarigota, Agrobacterium strain A208 and shoot regeneration medium (full-strength basal MS medium, 2μM BAP + 0.05μM NAA, 3% sucrose and 0.8% agar) containing Cefotaxime (250-500mg l-1) and Kanamycin (100mg l-1). Hypocotyl explants showed callus initiation and shoot regeneration response after 10-12 and 20-22 days of culture, respectively. Five-day-old explants did not survive Agrobacterium infection, and ten-day-old explants showed higher shoot regeneration (29±1.91%) than older explants. Explants of medium size (1cm long; 32±2.62%) from the apical region (38.57±2.61%) showed better shootregeneration ability than explants of any other size or region. A period of four days of pre-culture (33.33±3.76) was optimal best for best shoot-regeneration in hypocotyl explants. No regeneration was seen in hypocotyl explants at shorter or longer pre-culture period. High cytokinin (10μM) in shoot regeneration medium during pre-culture enhanced shoot regeneration response (47.27±2.98%) in explants co-cultivated with Agrobacterium. Effects of various factors documented in this study will be useful in developing an efficient Agrobacterium-mediated transformation protocol in brinjal cv. Manjarigota

Effect of Age and Size of Hypocotyl Explant on in vitro Shoot Regeneration in Eggplant

In the present study, effect of size and age of hypocotyl explant on in vitro organogenetic responses was assessed in eggplant cv. Manjarigota. Size and age did not affect callus-initiation response, but showed marked influence on shoot regeneration response. Hypocotyl explants 1.5cm long showed highest shoot regeneration response (77.4%); either increase or decrease in size resulted in reduced response. Five to 15 day old hypocotyl explants showed direct shoot regeneration from cut ends, whereas 20-30 day old hypocotyl explants showed indirect shoot regeneration from callus produced on cut ends. Five day old explants were most responsive, with highest (91.23%) and thirty day old explants least responsive with reference to shoot regeneration response (20.85%). Shoot regeneration frequency decreased with increasing age, whereas shoot regeneration efficiency increased with increasing age of hypocotyl explants

Effects of Growth Regulators and Explant-Type on Agrobacterium-Mediated Transformation in Brinjal (Solanum melongena L.) cv. Manjarigota

Effects of growth regulators and type of explants on transformation and in vitro morphogenetic responses of brinjal cv. Manjarigota were studied. Both hypocotyl and cotyledonary explants showed marked influence on in vitro morphogenetic responses after Agrobacterium co-cultivation. Hypocotyl explants showed callus initiation and regeneration responses earlier than cotyledonary leaves. Hypocotyl explants were found to be better than cotyledonary leaf explants in regenerating shoots after Agrobacterium co-cultivation. There was delay and reduction in both callus and regeneration responses in Agrobacterium co-cultivated explants. Hypocotyl explants showed the highest regeneration response on MS medium containing 2 μM BAP and 0.05 μM NAA while cotyledonary leaves did not show regeneration response after Agrobacterium co-cultivation. However, they showed green buds on MS medium containing 10 μM BAP and 1 μM NAA, which could not differentiate into shoots. Overall, hypocotyl explants were found better in regenerating shoots after Agrobacterium co-cultivation

Effect of Antibiotics and Gelling Agents in Transformation of Brinjal (Solanum melongena L.) cv. Manjarigota

A study was conducted to find out the effect of antibiotics and gelling agents on Agrobacterium-mediated transformation using hypocotyl explants of brinjal cv. Manjarigota. Hypocotyl explants of brinjal were found to be sensitive even to the lowest level of kanamycin (25 mg/l) tested. Explants that showed increased callus initiation and regeneration response upon cocultivation with Agrobacterium and on kanamycin at 100 mg/l were selected as this indicated a highly effective selection pressure. Cefotaxime did not affect regeneration response and at 500 mg/l, it effectively inhibited Agrobacterium overgrowth completely on Agrobacterium cocultivated hypocotyl explants. There were marked differences in regeneration response in hypocotyl explants cultured on medium solidified with various gelling agents indicating the influence of gelling agent on the activity of kanamycin in culture medium, which indirectly affects selection and recovery of transformants. Antibiotics and gelling agents could therefore affect, directly or indirectly, transformation of brinjal cv. Manjarigota

Percutaneous Device Closure of Congenital Isolated Ventricular Septal Defects:A Single-Center Retrospective Database Study Amongst 412 Cases

To identify suitable cases and reduce failure/complication rates for percutaneous ventricular septal defect (VSD) closure, we aimed to (1) study causes of device failure and (2) compare outcomes with different VSD types and devices in a high-volume single center with limited resources. Retrospective data of 412 elective percutaneous VSD closure of isolated congenital VSDs between 2003 and 2017 were analyzed. Out of 412, 363 were successfully implanted, in 30 device implantation failed, and in 19 the procedure was abandoned. Outcome was assessed using echocardiography, electrocardiography, and catheterization data (before procedure, immediately after and during follow-up). Logistic regression analyses were performed to assess effects of age, VSD type, and device type and size on procedural outcome. Median [interquartile range] age and body surface area were 6.6 [4.1-10.9] years and 0.7 [0.5-1.0] m(2), respectively. Device failure was not associated with age (p = 0.08), type of VSD (p = 0.5), device type (p = 0.2), or device size (p = 0.1). Device failure occurred in 7.6% of patients. As device type is not related to failure rate and device failure and complication risk was not associated with age, it is justifiable to use financially beneficial ductal devices in VSD position and to consider closure of VSD with device in clinically indicated children

Synergistic Use of Hypocotyl Explants and High Bap Preconditioning for Enhanced Transformation Frequency in Brinjal (Solanum melongena L.)

Poor regeneration is one of the limiting factors in the development of transgenic crops since Agrobacterium as a plant pathogen can disturb the fragile in vitro conditions with wounding and infection regimes. We have tried to optimize the transformation system in two important varieties of brinjal after Agrobacterium infection to the explants. The effect of explant was studied and hypocotyls were found to be better than cotyledonary leaves. High BAP during the preconditioning period was found to further enhance the regeneration rate. Therefore, use of hypocotyls and high BAP during preconditioning can improve the regeneration of transformed cells and recovery of transformants in vegetables especially brinjal

Biochemical characterization of Fusarium oxysporum f. sp. cubense isolates from India

The Fusarium wilt caused by Fusarium oxyspoum f. sp. cubense (Foc) is a major biotic constraint for banana production. The characteristics of F. oxyspoum f. sp. cubense isolates were investigated using electrophoretic studies of isozyme and whole-cell protein. The morphological characteristics of the isolates were very similar to each other. All the Foc isolates were pathogenic to banana cultivar 'Nanjangud Rasabale' but they did not induce any disease symptoms on cultivar 'Cavendish'. F. oxyspoum (Isolate 6) did not induce wilt symptoms on either 'Nanjangud' or 'Cavendish' cultivar. Isozyme banding patterns showed 46 scoreable markers and cluster analysis with UPGMA using genetic distance showed that the isolates belonged to three main groups. Group 1 contained isolates 1, 2, 4, 5, 7 and isolate 3 and 6 were placed in group 2 and 3. Results indicated that the estimated intra-specific variation may be more pronounced with isozyme analysis than with protein markers. The level of isozyme variability detected within F. oxysporum f.sp. cubense suggested that it is reliable, efficient and effective in determining genetic relationships among Foc isolates

Identification and genetic diversity analysis of Memecylon species using ISSR, RAPD and Gene-Based DNA barcoding tools

Background: Memecylon species are commonly used in Indian ethnomedical practices. The accurate identification is vital to enhance the drug's efficacy and biosafety. In the present study, PCR based techniques like RAPD, ISSR and DNA barcoding regions, such as 5s, psbA-trnH, rpoC1, ndh and atpF-atpH, were used to authenticate and analyze the diversity of five Memecylon species collected from Western Ghats of India. Results: Phylogenetic analysis clearly distinguished Memecylon malabaricum from Memecylon wightii and Memecylon umbellatum from Memecylon edule and clades formed are in accordance with morphological keys. In the RAPD and ISSR analyses, 27 accessions representing five Memecylon species were distinctly separated into three different clades. M. malabaricum and M. wightii grouped together and M. umbellatum, M. edule and Memecylon talbotianum grouped in the same clade with high Jaccard dissimilarity coefficient and bootstrap support between each node, indicating that these grouped species are phylogenetically similar. Conclusion: Data from the present study reveals that chloroplast psbA-trnH region could be used as a potential candidate region for identifying Memecylon species, and ISSR marker system could be used for estimating genetic diversity since it has high percent polymorphism compared to RAPD marker

High resolution mapping of QTLs for fruit color and firmness in Amrapali/Sensation mango hybrids

IntroductionMango (Mangifera indica L.), acclaimed as the ‘king of fruits’ in the tropical world, has historical, religious, and economic values. It is grown commercially in more than 100 countries, and fresh mango world trade accounts for ~3,200 million US dollars for the year 2020. Mango is widely cultivated in sub-tropical and tropical regions of the world, with India, China, and Thailand being the top three producers. Mango fruit is adored for its taste, color, flavor, and aroma. Fruit color and firmness are important fruit quality traits for consumer acceptance, but their genetics is poorly understood.MethodsFor mapping of fruit color and firmness, mango varieties Amrapali and Sensation, having contrasting fruit quality traits, were crossed for the development of a mapping population. Ninety-two bi-parental progenies obtained from this cross were used for the construction of a high-density linkage map and identification of QTLs. Genotyping was carried out using an 80K SNP chip array.Results and discussionInitially, we constructed two high-density linkage maps based on the segregation of female and male parents. A female map with 3,213 SNPs and male map with 1,781 SNPs were distributed on 20 linkages groups covering map lengths of 2,844.39 and 2,684.22cM, respectively. Finally, the integrated map was constructed comprised of 4,361 SNP markers distributed on 20 linkage groups, which consisted of the chromosome haploid number in Mangifera indica (n =20). The integrated genetic map covered the entire genome of Mangifera indica cv. Dashehari, with a total genetic distance of 2,982.75 cM and an average distance between markers of 0.68 cM. The length of LGs varied from 85.78 to 218.28 cM, with a mean size of 149.14 cM. Phenotyping for fruit color and firmness traits was done for two consecutive seasons. We identified important consistent QTLs for 12 out of 20 traits, with integrated genetic linkages having significant LOD scores in at least one season. Important consistent QTLs for fruit peel color are located at Chr 3 and 18, and firmness on Chr 11 and 20. The QTLs mapped in this study would be useful in the marker-assisted breeding of mango for improved efficiency