Anther-derived microspore embryogenesis in pepper hybrids orobelle and Bomby.

BACKGROUND: Traditional breeding methods have long been employed worldwide for the evaluation and development of pepper cultivars. However, these methods necessitate multiple generations of screening, line development, evaluation, recognition, and crossing to obtain highly homozygous lines. In contrast, in vitro anther-derived microspore culture represents a rapid method to generate homozygous lines within a single generation. In the present study, we have optimized a protocol for microspore embryogenesis from anther cultures of pepper hybrids Orobelle and Bomby. RESULTS: We achieved early and successful embryo formation from both genotypes by subjecting the buds to a cold pretreatment at 4 °C for 4 days. Our optimized culture medium, comprised of MS medium supplemented with 4 mg/L NAA, 1 mg/L BAP, 0.25% activated charcoal, 2.6 g/L gelrite, 30 g/L sucrose, and 15 mg/L silver nitrate, exhibited the highest efficiency in embryo formation (1.85% and 1.46%) for Orobelle and Bomby, respectively. Furthermore, successful plant regeneration from the anther derived microspore embryos was accomplished using half-strength MS medium fortified with 2% sucrose and 0.1 mg/L 6-benzylaminopurine (BA), solidified with 2.6 g/L gelrite. The ploidy status of the microspore-derived plantlets was analyzed using flow cytometry technique. Notably, the haploid plants exhibited distinct characteristics such as reduced plant height, leaf length, leaf width, and shorter internode length when compared to their diploid counterparts derived from seeds. CONCLUSION: Our findings highlight the potential of anther culture and microspore embryogenesis as an advanced method for accelerating pepper breeding programs, enabling the rapid production of superior homozygous lines.

Commercial suitability and characterization of newly developed Erucastrum canariense (Can) sterile cytoplasm based cytoplasmic male sterile (CMS) lines in Indian cauliflower.

The study presents the first to characterize novel Erucastrum canarianse Webb and Berthel (or Can) sterile cytoplasm-based CMS lines in Indian cauliflower (Brassica oleracea var. botrytis L.) and investigating their commercial suitability. Eleven Can-based CMS lines were examined for 12 agro-morphological and yield traits,18 floral traits, four seed yield traits together with three each of the Ogura (source: wild Japanese Radish) and Tour (Source: Brassica tournefortii) cytoplasms. All of the recorded floral and seed traits showed significant (P > 0.05) differences between the CMS lines of each group. Agro-morphological and yield traits in CMS lines and their maintainers, however, were non-significantly different. All the Can- and Ogura-based CMS lines showed flowering and appropriate seed formation by natural cross-pollination. Only two Tour cytoplasm-based CMS lines, Tour (DC-41-5) and Tour (DC-67), produced the smallest malformed flowers and stigma. The highest seed yield per plant in CMS lines was in Ogu (DC-98-4) and the lowest in Tour (DC-67). P14 and P15, two polymorphic mtDNA markers, were discovered for the Can CMS system for early detection. Five primers (ITS5a-ITS4, atpF-atpH, P16, rbeL and trnL), along with their maintainers, were sequenced and aligned to detect nucleotide changes including as additions and or deletions at different positions. The newly introduced E. canariense sterile cytoplasm-based CMS system in cauliflower is the subject of the first comprehensive report, which emphasises their potential as a further stable and reliable genetic mechanism for hybrid breeding.

Correlation of stages of microsporogenesis with bud and anther morphology in pepper genotypes through DAPI staining with different levels of mordant in cytological fixative.

The haploid and doubled haploid plants serve as valuable tools for breeders due to their ability to expedite the mapping of genes of agronomic importance, as well as accelerate the breeding cycle for generation of novel hybrids and improved homogenous varieties. Successful anther/microspore culture largely depends on the use of microspores at appropriate developmental stages at the time of culture, which can be specific for each plant species and genotype. In the present study, we described the visible morphological characteristics of flower buds and anthers at different developmental stages to identify the optimal microspore stage within the anther/buds of two pepper hybrids, Indra and Lakshmi. This information enabled us to predict the suitable microspore stage for successful haploid production. To enhance the visualization of nuclei in the pepper microspores, different concentrations of FeCl3 were employed as a mordant to Carnoy's fixative I, followed by DAPI staining. A clear and distinct nucleus was observed using DAPI staining procedures in the pepper microspores when fixed in Carnoy's solution containing ferric chloride (40-90 µl) as mordant. The use of mordant thus facilitated the efficient cytological analysis of the pepper microspores. Present results indicate that, to achieve efficient haploid production, flower buds with an average length of 4.4 to 5.02 mm for the hybrid Indra and 5.15 to 5.40 mm for the hybrid Lakshmi should be utilized. Additionally, these buds should have a calyx covering approximately 80-90% of the total bud length. We observed that in such buds, microspores are in the late-uninucleate and early binucleate stage which has been reported to be the most conducive stage for androgenesis induction in pepper.

Genetic Diversity and Population Structure Analyses in Bitter Gourd (Momordica charantia L.) Based on Agro-Morphological and Microsatellite Markers.

Bitter gourd (Momordica charantia L.) is an important vine crop of the Cucurbitaceae family and is well known for its high nutritional and medicinal values. However, the genetic variation remains largely unknown. Herein, 96 diverse bitter gourd genotypes were undertaken for diversity analysis using 10 quantitative traits, and 82 simple sequence repeat (SSR) markers. Out of 82 SSRs, 33 were polymorphic and the mean polymorphism information content (PIC) value was 0.38. Marker, JY-003 revealed a maximum (0.81) PIC value and, the number of alleles per locus ranged from 2 to 7 (average 3.46). The value of gene diversity showed the presence of a significant level of polymorphism among these genotypes. The unweighted pair group method (UPGMA) cluster analysis grouped the genotypes into two major clusters of which Cluster I comprised mostly small and medium-fruited genotypes of both M. charantia var. charantia and M. charantia var. muricata, whereas Cluster II included mostly long and extra-long fruited genotypes. Furthermore, these genotypes were divided into six distinct groups based on population structure analysis. The diversity analysis based on 10 quantitative traits revealed that earliness and high-yielding ability were exhibited by the predominantly gynoecious line DBGS-21-06 followed by DBGS-48-00. The principal component analysis (PCA) revealed that the first two components exhibited more than 50% of the total genetic variation. The present study deciphered a higher magnitude of agro-morphological and genetic diversity in 96 bitter gourd genotypes. Therefore, trait-specific genotypes identified in this study could be utilized in breeding programmes directed towards the development of improved cultivars and hybrids of bitter gourd.

Genetic analysis of heat tolerance in hot pepper: insights from comprehensive phenotyping and QTL mapping.

High temperatures present a formidable challenge to the cultivation of hot pepper, profoundly impacting not only vegetative growth but also leading to flower and fruit abscission, thereby causing a significant reduction in yield. To unravel the intricate genetic mechanisms governing heat tolerance in hot pepper, an F2 population was developed through the crossing of two distinct genotypes exhibiting contrasting heat tolerance characteristics: DLS-161-1 (heat tolerant) and DChBL-240 (heat susceptible). The F2 population, along with the parental lines, was subjected to comprehensive phenotyping encompassing diverse morphological, physiological, and biochemical heat-related traits under high temperature conditions (with maximum temperature ranging from 31 to 46.5°C and minimum temperature from 15.4 to 30.5°C). Leveraging the Illumina Nova Seq-6000 platform, Double digest restriction-site associated DNA sequencing (ddRAD-seq) was employed to generate 67.215 Gb data, with subsequent alignment of 218.93 million processed reads against the reference genome of Capsicum annuum. Subsequent variant calling and ordering resulted in 5806 polymorphic SNP markers grouped into 12 LGs. Further QTL analysis identified 64 QTLs with LOD values ranging from 2.517 to 11.170 and explained phenotypic variance ranging from 4.05 to 19.39%. Among them, 21 QTLs, explaining more than 10% phenotypic variance, were identified as major QTLs controlling 9 morphological, 3 physiological, and 2 biochemical traits. Interestingly, several QTLs governing distinct parameters were found to be colocalized, suggesting either a profound correlation between the QTLs regulating these traits or their significant genomic proximity. In addition to the QTLs, we also identified 368380 SSR loci within the identified QTL regions, dinucleotides being the most abundant type (211,381). These findings provide valuable insights into the genetics of heat tolerance in hot peppers. The identified QTLs and SSR markers offer opportunities to develop heat-tolerant varieties, ensuring better crop performance under high-temperature conditions.

Molecular marker-based characterization of cytoplasm and restorer of male sterility (Ms) locus in commercially grown onions in India.

BACKGROUND: The cytoplasmic-genic male-sterility system has been extensively employed for the production of onion hybrids. Molecular marker-assisted characterization of the cytotypes and genotyping at the restorer-of male-fertility (Ms) locus is important for the accelerated breeding of onion hybrids. Indian onion breeding has focussed more on open-pollinated varieties than hybrids. To accelerate the breeding efforts, marker-assisted selection (MAS) plays a pivotal role. METHODS AND RESULTS: This study aimed to characterize the Indian breeding lines, varieties, hybrids, and exotic accessions for cytotype and Ms locus. For cytoplasm, cytotype markers, accD, and MKFR and for Ms locus identification, PCR markers AcPMS1 and AcSKP1 were employed. Bulk strategy to identify cytoplasm and Ms locus was tested. Sequencing of PCR products amplified by accD was also tried. Both the accD and MKFR were synonymous in cytoplasm identification except in T821 where T cytoplasm was identified. AcPMS1 was more reliable than AcSKP1 for Ms locus identification. Sequencing proved that N and T cytoplasm are identical. Bulking strategy can be used for cytotype identification but not for Ms locus. CONCLUSIONS: Indian onions have a predominance of normal (N) cytoplasm and homozygous recessive (msms) locus. This might be beneficial for hybrid development. S cytoplasm was identified in exotic varieties. For the first time, T cytoplasm has been reported from India. These findings will assist Indian onion breeders to develop MAS strategies for accelerating hybrid development programs. And for the release of onion hybrids with high productivity and uniformity.

Morphological and molecular changes on cytoplasmic male sterility (CMS) introgression in Asiatic carrot (Daucus carota L.).

MAIN CONCLUSION: 'Petaloid' cytoplasmic male sterility is commonly used as a stable genetic mechanism in carrot hybrid breeding. Its introgression in tropical carrot showed morphometric changes and molecular markers were identified for detection at early stage. Cytoplasmic male sterility (CMS) is the only genetic mechanism in carrot for commercial exploitation of heterosis and production of low cost affordable hybrid seeds. The 'petaloid' CMS system is stable and commonly used in hybrid breeding in temperate carrot but there is no information available on existence of natural CMS system in tropical Asiatic carrot. Therefore, the present study was aimed to investigate morphometric traits and organizational features of cytoplasmic atp9 gene sequences in newly converted CMS lines (BC4-7) of tropical carrot. The CMS lines had root traits at par with fertile counterparts while floral traits had variation. Petal colour and length, petaloids colour and shape and style length showed differences among the CMS lines and with their maintainers. Molecular markers are effective to establish male sterility at genetic level, for this, six fixed and stable CMS lines were screened with seven novel primer combinations. Out of which five pairs produced clearly distinguishable bands in CMS lines and their fertile counterparts. The study confirmed that the region between 3' end of atp9-1/atp9-3 gene and 5' end of region of homology to Arabidopsis thaliana mtDNA is ideal for developing the trait specific markers. These new CMS lines have potential to use in hybrid development and molecular markers will be useful to confirm male sterility to rogue out fertile plants.

Genetics and Expression Analysis of Anthocyanin Accumulation in Curd Portion of Sicilian Purple to Facilitate Biofortification of Indian Cauliflower.

The present study was undertaken to know the genetics of purple color of cauliflower curds using a Sicilian purple 'PC-1' and a white curding mid-late group genotype of Indian cauliflower. For this, a cross was attempted between 'DC-466' (white curd) and 'PC-1' (purple curd) and observed intermediate level of purple pigmentation on curds in F1 plants. Segregation of F2 population (173) revealed that the purple color of the curd was governed by a single gene dominant over white, but the expression of trait was incomplete. It was substantiated by segregation of plants of BC1 and F2:3(intermediate) generations into 1(white):1(intermediate) and 1(white):2(intermediate):1(intense), respectively. The F2, B1, and B2 generations segregated into purple(intermediate to intense): white curding plants in the ratio of 126: 47, 26:24, and 40:0, respectively fitting well with the Mendelian ratio of single gene for purple curds. However, purple pigmentation on curds ranged from very light to intense, which corroborated with the wide range of anthocyanin content in F2 (3.81-48.21 mg/100 g fw). Out of three molecular markers from high resolution map of Pr gene in purple color cauliflower 'Graffiti', only BoMYB3 marker could distinguish purple and white curding parents but did not show co-segregation while investigated in F2 population. Expression of BoMYB1 gene was up regulated in both the purple curd genotypes 'PC-1' and 'Graffiti' in comparison to white curded 'DC-466', while BoMYB2 gene was slightly upregulated in 'PC-1' but down regulated in 'Graffiti'. Occurrence of 'broccoli type' F2 individuals and their genetic stability in F2:3 support the intermediate position of 'Sicilian purple' between broccoli (Calabrese) and cauliflower. There was not any correlation between curd coloration and pigmentation on apical leaf and stem portion, indicating difference of expression in 'PC-1' than 'Graffiti'. The information obtained is useful for breeding anthocyanin rich attractive purple curding 'specialty cauliflower' for better consumer health and growers' earnings.

DNA barcoding for specific and sensitive detection of Cuminum cyminum adulteration in Bunium persicum.

BACKGROUND: Bunium persicum commonly called as Kala zeera, a very high value herbaceous spice used for medicinal purposes is often adulterated with Cuminum cyminum or Safed zeera, a closely related species. Lack of distinctive morphological features makes the identification of genuine kala zeera from its adulterant difficult, the problem is even exaggerated in case of powdered material. METHODOLOGY: Genomic DNA was extracted from all the plant materials by using CTAB-SDS method (Möller et al., 1992) with slight modifications. On the basis of reproducibility and high amplification ability, four universal barcoding loci viz. ITS2, rbcL-a, mat K and psbA-trnH and a specific locus Cum were used in the present study. The amplified PCR products were sequenced bidirectionally and assembled to obtain contigs. The sequences thus obtained were aligned using MUSCLE algorithm (Edgar, 2004) and information pertaining to conserved/ variable/ parsimony informative sites, number of transitions, transversions and Indels was obtained after analyzing the sequences. RESULTS AND CONCLUSION: Among the tested barcoding loci, psbA-trnH has proven to be best barcode in authentication of kala zeera as its amplification and sequencing success was high and it showed the presence of polymorphic sites to detect interspecific variation. This barcode could differentiate between safed zeera and kala zeera in a single reaction, simultaneously.

Conservation and Dispersion of Genes Conferring Resistance to Tomato Begomoviruses between Tomato and Pepper Genomes.

In the present climate change scenario, controlling plant disease through exploitation of host plant resistance could contribute toward the sustainable crop production and global food security. In this respect, the identification of new sources of resistance and utilization of genetic diversity within the species may help in the generation of cultivars with improved disease resistance. Begomoviruses namely, Tomato yellow leaf curl virus (TYLCV) and Chilli leaf curl virus (ChLCV) are known to cause major yield losses in several economically important crop plants of the family Solanaceae. Though co-occurrence, association and synergistic interactions among these viruses in the host plants is reported, whether orthologous genetic loci in related host plants could be responsible for conferring resistance to these viruses has not been investigated yet. Several loci including Ty1, Ty2, Ty3, Ty4, and ty5 have been reported to confer resistance to leaf curl viruses in tomato. Here, we examined the pepper orthologous markers, corresponding to these QTL regions, for polymorphism between ChLCV susceptible and resistant genotypes of pepper. Further, to examine if the polymorphic markers are segregating with the disease resistance, Bulk Segregant Analysis (BSA) was performed on F2 population derived from crosses between resistant and susceptible lines. However, none of the markers showed polymorphism in BSA suggesting that the tested markers are not linked to genes/QTLs responsible for conferring resistance to ChLCV in the selected genotypes. In silico analysis was performed to study the synteny and collinearity of genes located within these QTL regions in tomato and pepper genomes, which revealed that more than 60% genes located in Ty2 and Ty4, 13.71% genes in Ty1, 23.07% in Ty3, and 44.77% genes located within ty5 QTL region in tomato are conserved in pepper genome. However, despite such a high conservation in gene content, the linkage relationship in these regions seems to be greatly affected by gross rearrangements in both the species.

Food adulteration: Sources, health risks, and detection methods.

Adulteration in food has been a concern since the beginning of civilization, as it not only decreases the quality of food products but also results in a number of ill effects on health. Authentic testing of food and adulterant detection of various food products is required for value assessment and to assure consumer protection against fraudulent activities. Through this review we intend to compile different types of adulterations made in different food items, the health risks imposed by these adulterants and detection methods available for them. Concerns about food safety and regulation have ensured the development of various techniques like physical, biochemical/immunological and molecular techniques, for adulterant detection in food. Molecular methods are more preferable when it comes to detection of biological adulterants in food, although physical and biochemical techniques are preferable for detection of other adulterants in food.

Validation of PCR based detection system for aflatoxin producing molds.

Aflatoxins are polyketide secondary metabolites that are produced by certain fungal species in the Aspergillus section Flavi, particularly Aspergillus flavus and Aspergillus parasiticus which contaminate human food as well as animal feed. These are among the most carcinogenic substances known. Due to the toxic and carcinogenic properties of aflatoxins, there is a need to develop reliable methods to detect the presence of aflatoxigenic Aspergilli in contaminated food and feed. Not all Aspergillus strains are able to produce aflatoxins. It requires a detection methodology which can specifically distinguish between the aflatoxin producing and nonproducing strains of Aspergillus. Present communication reports validation of a PCR based detection system based on three genes viz., nor-1, apa-2 and omt-1 involved in aflatoxin biosynthesis, that can specifically distinguish the two aflatoxin producing species viz. Aspergillus flavus ,and Aspergillus parasiticus from non-producers i.e., A. niger, A. fumigates and A. oryzae.

Non-dairy Based Probiotics: A Healthy Treat for Intestine.

Dairy-based fermented products and yoghurts have been utilized as potential probiotic products since ancient times. However, recent upsurge in interest of consumers towards dairy alternatives has opened up new vistas for non-dairy probiotic research and development. Various matrices and substrates such as cereals, fruit juices, or mixture thereof are being utilized for delivering these beneficial microorganisms. Each matrix offers some advantages over the other. Vast knowledge available on a number of conventional fermented foods can also be utilized for future research in this area. The present review provides an insight on the recent research/developments in the field of non-dairy probiotic foods with particular reference to the foods consumed conventionally, in addition to their commercial availability and a way forward.

Utilization of Food Processing By-products as Dietary, Functional, and Novel Fiber: A Review.

Fast growing food processing industry in most countries across the world, generates huge quantity of by-products, including pomace, hull, husk, pods, peel, shells, seeds, stems, stalks, bran, washings, pulp refuse, press cakes, etc., which have less use and create considerable environmental pollution. With growing interest in health promoting functional foods, the demand of natural bioactives has increased and exploration for new sources is on the way. Many of the food processing industrial by-products are rich sources of dietary, functional, and novel fibers. These by-products can be directly (or after certain modifications for isolation or purification of fiber) used for the manufacture of various foods, i.e. bread, buns, cake, pasta, noodles, biscuit, ice creams, yogurts, cheese, beverages, milk shakes, instant breakfasts, ice tea, juices, sports drinks, wine, powdered drink, fermented milk products, meat products and meat analogues, synthetic meat, etc. A comprehensive literature survey has been carried on this topic to give an overview in the field dietary fiber from food by-products. In this article, the developments in the definition of fiber, fiber classification, potential sources of dietary fibers in food processing by-products, their uses, functional properties, caloric content, energy values and the labelling regulations have been discussed.

Molecular Detection of Foodborne Pathogens: A Rapid and Accurate Answer to Food Safety.

Food safety is a global health concern. For the prevention and recognition of problems related to health and safety, detection of foodborne pathogen is of utmost importance at all levels of food production chain. For several decades, a lot of research has been targeted at the development of rapid methodology as reducing the time needed to complete pathogen detection tests has been the primary goal of food microbiologists. With the result, food microbiology laboratories now have a wide array of detection methods and automated technologies such as enzyme immunoassay, polymerase chain reaction, and microarrays, which can cut test times considerably. Nucleic acid amplification strategies and advances in amplicon detection methodologies have been the key factors in the progress of molecular microbiology. A comprehensive literature survey has been carried out to give an overview in the field of foodborne pathogen detection. In this paper, we describe the conventional methods, as well as recent developments in food pathogen detection, identification, and quantification, with a major emphasis on molecular detection methods.

In vitro regeneration in olive (Olea europaea L.) cv, 'Frontio' from nodal segments.

An efficient and reproducible protocol for plantlet regeneration from nodal segments of Olive cv 'Frontio' has been developed. Media and explants browning due to exudation of phenolics from the explants were controlled by fortification of the medium with 100 mg/L ascorbic acid. Best establishment of olive explants was observed on half-strength MS salts fortified with 2.0 mg/L 6-benzylaminopurine (BAP), which resulted in 56.2% of bud break and 93.7% survival whereas, a combination of full strength MS medium with 1.0 mg/L each of 3-indole-butyric-acid (IBA) and kinetin was found to be the best for shoot multiplication, in terms of number of shoots (3.6 shoots/explant) and shoot length (2.2 cm). The in vitro shoots were rooted on half-strength MS medium fortified with 0.2 mg/L IBA and 0.2 mg/L alpha-naphthalene acetic acid (NAA) with 1.5 g/L activated charcoal, which supported optimum rooting (60%), with an average of 2-3 roots/shoot, about 2.4 cm length were produced on four weeks of culture.

Alginate-encapsulation of shoot tips of jojoba [Simmondsia chinensis (Link) Schneider] for germplasm exchange and distribution.

Shoot tips excised from in vitro proliferated shoots derived from nodal explants of jojoba [Simmondsia chinensis (Link) Schneider] were encapsulated in calcium alginate beads for germplasm exchange and distribution. A gelling matrix of 3 % sodium alginate and 100 mM calcium chloride was found most suitable for formation of ideal calcium alginate beads. Best response for shoot sprouting from encapsulated shoot tips was recorded on 0.8 % agar-solidified full-strength MS medium. Rooting was induced upon transfer of sprouted shoots to 0.8 % agar-solidified MS medium containing 1 mg l(-1) IBA. About 70 % of encapsulated shoot tips were rooted and converted into plantlets. Plants regenerated from encapsulated shoot tips were acclimatized successfully. The present encapsulation approach could also be applied as an alternative method of propagation of desirable elite genotype of jojoba.

Use of meristem tip culture to eliminate carnation latent virus from carnation plants.

A successful protocol for meristem tip culture to eliminate carnation latent virus from carnation cv. scania has been described . The virus was found to be mechanically transmissible to Chenopodium quinoa, C. amaranticolor, Dianthus barbatus and Saponaria vaccaria. Murashige and Skoog'smedium (MS) supplemented with NAA (1.0 microM) and Kn (20.0 microM) proved best for meristem establishment and microshoots were rooted in MS medium supplemented with IBA (5.0 microM). Meristems measuring 0.1 and-0.2 mm yielded virus free plants and larger meristems were not effective.