A droplet vitrification cryopreservation protocol for conservation of hops (Humulus lupulus) genetic resources.

Hops (Humulus lupulus L.) is essentially used in the brewing industry as it contributes to flavor, and aroma of beer. However, the genetic diversity of hops is increasingly threatened by diseases, environmental changes, and urbanization. Cryopreservation has emerged as a pivotal strategy for safeguarding and maintaining the genetic diversity of hops. The present work presents a comprehensive study on the cryopreservation of hops, focusing on the development and optimization of a droplet vitrification based cryopreservation protocol. Shoot tips excised from one month old in vitro cultures were precultured on 0.3 M sucrose, dehydrated in a loading solution followed by treatment with PVS2 solution for different durations. Significant effect of PVS2 dehydration was observed on post-thaw survival and regeneration after cryoconservation with maximum 50% post-thaw regeneration observed in shoot tips dehydrated in PVS2 for 30 min. Genetic fidelity of the regenerated plants was confirmed using 30 ISSR markers. Reproducibility of the developed protocol was tested on seven other accessions and post thaw regeneration ranging from 43 to 70% was observed across the accessions. The present study reports a highly efficient protocol for conservation of hops germplasm. The results indicate that droplet vitrification can be used as a reliable and sustainable approach for hop genetic preservation, with high survival rates and minimal genetic alterations observed in cryopreserved samples. To the best of our knowledge, this is the first report on DV based cryopreservation of hops germplasm.

Comparative analysis of herbivory responsive miRNAs to delineate pod borer (Helicoverpa armigera) resistance mechanisms in Cajanus cajan and its wild relative Cajanus scarabaeoides.

KEY MESSAGE: Comparative analysis of herbivory responsive miRNAs between pod borer susceptible C. cajan and its resistant Crop Wild Relative (CWR) C. scarabaeoides revealed miRNA-based regulation of defense genes and plant-insect interactions. Gram pod borer (Helicoverpa armigera) is one of most devastating pests of pigeon pea (Cajanus cajan) worldwide, responsible for huge losses in crop productivity. The lack of genes conferring resistance to pod borer in pigeon pea has proven to be a bottleneck for its improvement. One of its CWR, C. scarabaeoides has demonstrated resistance to this pest and can be exploited for developing pest resistant crop varieties. Differences in expression patterns of herbivory responsive microRNAs in the susceptible C. cajan and resistant C. scarabaeoides after different time duration of pod borer infestation (2 h, 8 h and 18 h) were identified, characterized and functionally validated to understand their role in insect defense response. A total of 462 conserved and 449 novel miRNAs and 273 conserved and 185 novel miRNAs, were identified in C. cajan and C. scarabaeoides, respectively. Among the identified miRNAs, 65, 68 and 65 miRNAs were found to be differentially expressing between the C. scarabaeoides and C. cajan libraries 2 h, 8 h and 18 h post infestation, respectively. These miRNAs were found to target genes involved in a number of pathways contributing to defense and acquired resistance in C. scarabaeoides against pod borer, indicating miRNA-based regulation of defense pathways. Expression patterns of eight of these miRNAs were validated by qRT-PCR. This study provides novel insights into the miRNA-mediated plant-insect interactions and the mechanisms of regulatory pathways involved in insect defense. These findings can be utilized for further exploring the mechanism of herbivore defense in plant systems.

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.

Loop-Mediated Isothermal Amplification (LAMP): A Rapid and Sensitive Tool for Quality Assessment of Meat Products.

Loop-mediated isothermal amplification (LAMP) is a novel method that amplifies target nucleic acids under isothermal conditions. It is a rapid, specific, and sensitive method, which does not require costly thermal cyclers for the detection of nucleic acids. Thus, it is suitable for on-site detection assays under low-resource settings. It can also be integrated on compact lab-on-a-chip devices for the development of micro-total analysis systems. This review discusses LAMP-based methods, as well as LAMP-based centrifugal, microfluidic, and other fluid-handling devices, which have been developed for the assessment of meat quality parameters that are related to the presence or absence of nucleic acids, for example, animal species identification and microbiological quality. Advances in improving the rapidity, specificity, and sensitivity of LAMP techniques for the assessment of these meat quality parameters are also discussed in this review.

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.

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.

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.

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.

Development of protein fortified mango based ready-to-serve beverage.

Fruit drinks contain negligible amount of protein as nutritional component. Fortification of fruit drinks with protein is a challenge due to protein stability in acidic and ionic environment. Mango ready-to-serve (RTS) beverage was fortified with modified whey protein and its rheological properties were studied. Whey protein was hydrolysed with papain to improve its stability in acidic medium. The water holding capacity of whey protein increased about two times after hydrolysis. Hydrolysed and native whey protein was used at 2, 3 and 4% levels for fortification of mango based RTS beverage. Addition of hydrolysed whey protein at all the three levels did not significantly change the flow behaviour of the beverage. Native whey protein fortification resulted in precipitation; however, addition of hydrolysed whey protein led to stable beverage formulation at all the three levels. Hydrolysed whey protein imparted slight bitter taste to the RTS beverage, which was masked by ß-cyclodextrin @ 0.15% of total protein. The mango RTS beverage with 3.0% hydrolysed whey protein was found acceptable with good sensory appeal and stability during thermal processing as well storage in glass bottles.

Histological and molecular insights in to in vitro regeneration pattern of Xanthosoma sagittifolium.

A study on the effect of various phytohormonal combinations on in vitro propagation of Cocoyam [Xanthosoma sagittifolium (L.) Schott] was conducted to develop an improved and efficient in vitro regeneration protocol for its mass multiplication. Histological analysis to understand the in vitro regeneration pattern and genetic fidelity assessment of regenerated plants were also carried out. Single shoots excised from in vitro established cultures of X. sagittifolium were used as explants. Among the 32 different phytohormonal combinations tested, indirect organogenesis with intervening callus phase was observed on majority of the media combinations. Meristematic clump formation was optimally achieved on all the tested media combinations with maximum 43.54 ± 0.51 shoot primordia on MS medium containing 0.2 mg/L BAP + 0.1 mg/L NAA followed by 36.44 ± 0.76 shoot primordia on MS medium having 2.5 mg/L TDZ. Micro-morphological analysis of different morphogenetic structures revealed that the regeneration of cocoyam is well executed via meristematic nodules, shoot primordia formation that may evolve in to proper shoots. Adventitious shoots (> 2 cm) were successfully (100.00 ± 0.00%) rooted on the half-strength MS medium containing IBA (0.05-1.0 mg/L) and IAA (0.05-0.5 mg/L). The number of roots ranged from 0.78 ± 0.31 on the control half-strength MS medium to 13.94 ± 0.46 on half-strength MS supplemented with 1.0 mg/L IBA. Considering somaclonal variations as a potential restriction to in vitro multiplication of plants, genetic stability was assessed using 40 ISSR primers. The PCR amplification profiles obtained from all the tested propagules (calli, meristematic clumps, regenerated plantlets) were similar to the mother plants indicating the homogeneity of the individuals raised through the regeneration protocol reported here.

Identification of dynamic microRNA associated with systemic defence against Helicoverpa armigera infestation in Cajanus scarabaeoides.

BACKGROUND: Helicoverpa armigera is a major insect pest of several crop plants, including pigeonpea. Resistant gene sources are not available in the cultivated gene pool, but resistance has been observed in its crop wild relative, Cajanus scarabaeoides. Gene regulatory mechanisms governing the systemic immune response of this plant to pod borer infestation have not yet been deciphered. MicroRNA (miRNA) profiles of H. armigera-infested and undamaged adjacent leaves of C. scarabaeoides were compared to gain an insight into the plant-insect interactions and to identify dynamic miRNA molecules potentially acting as mediators of systemic defence responses. RESULTS: A total of 211 conserved, temporally dynamic miRNA were identified in the unfed adjacent leaves, out of which 98 were found to be differentially expressed in comparison to control leaves. On further analysis, most of the miRNA detected in the adjacent leaves was found to target genes involved in the defence pathways and plant immune response. An overlap of the differentially expressing miRNAs was observed between insect-fed and adjacent unfed leaves, indicating the transmission of signal from the site of infestation to the undamaged parts of the plant, indicative of induction of a systemic defence response. CONCLUSION: The miRNA response in the unfed leaves had the signatures of induced changes in metabolism and signal transduction for induction of defence pathway genes. This study reveals the participation of miRNAs in imparting pod borer resistance and mounting a systemic defence response against pod borer infestation in C. scarabaeoides. © 2022 Society of Chemical Industry.

Development of a new set of genic SSR markers in the genus Gentiana: in silico mining, characterization and validation.

Gentiana is an important genus of around 360 medicinally important species, majority of which are not well characterized. Despite its importance, very few genomic resources are available for Gentiana L. Till date, the number of informative and robust simple sequence repeat (SSR)-based markers is limited and very few efforts have been made for their development. A set of robust, freely accessible and informative SSR markers for Gentiana is a pre-requisite for any molecular systematic as well as improvement studies in this group of pharmacologically valuable plants. In view of the importance of these plants, Expressed Sequence Tag (EST) sequences of 18 Gentiana species were surveyed for the development of a large set of non-redundant SSR markers. A total of 5808 perfect SSR with an average length of 17 bp and relative abundance of 214 loci/Mb were identified in the analysed 47,487 EST sequences using Krait software. Mapping of the ESTs resulted in gene ontology annotations of 49.14% of the sequences. Based on these perfect SSRs, 2902 primer pairs were designed, and 60 markers were randomly selected and validated on a set of Gentiana kurroo Royle accessions. Among the screened markers, 39 (65%) were found to be cross-species transferable. This is the first report of the largest set of functional, novel genic SSR markers in Gentiana, which will be a valuable resource for future characterization, genotype identification, conservation and genomic studies in the various species of this group of important medicinal plants. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02969-4.

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.

Temporal and spatial expression analysis of gamma kafirin promoter from Sorghum (Sorghum bicolor L. moench) var. M 35-1.

Different cis acting elements of gamma kafirin gene from Sorghum bicolor var. M 35-1 were amplified and cloned using different combination of the primers. The amplified promoter was replaced with CaMV35S promoter of vector pCMBIA-1304 and resultant vector contained beta-glucuronidase (gus) gene under the control of amplified gamma-kafirin promoter. The resulting fusants were then transformed in to different explants of sorghum via particle bombardment. The regulation of uid gene expression was analyzed to find out the minimum required 5' regulatory sequence and cis acting elements for the efficient expression. However no gus expression was detected in leaves of micropropagated plants, scutellum and calli at any stage of growth. The expression of gus, with pKaf gus-P4 gene construct, was detected in immature embryos and endosperm 20 days after pollination (DAP). The result suggest that at least three motifs (two GCN4 and one prolamin box) besides TATA and CATC boxes are required for the efficient expression of the kafirin gene of sorghum. The study shows that PCR based isolation of different motifs and regions can be used as an alternate to deletion analysis for observing the role of various motifs and their importance in the gene expression and regulation.