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.

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.

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.

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.

Silencing of HaAce1 gene by host-delivered artificial microRNA disrupts growth and development of Helicoverpa armigera.

The polyphagous insect-pest, Helicoverpa armigera, is a serious threat to a number of economically important crops. Chemical application and/or cultivation of Bt transgenic crops are the two strategies available now for insect-pest management. However, environmental pollution and long-term sustainability are major concerns against these two options. RNAi is now considered as a promising technology to complement Bt to tackle insect-pests menace. In this study, we report host-delivered silencing of HaAce1 gene, encoding the predominant isoform of H. armigera acetylcholinesterase, by an artificial microRNA, HaAce1-amiR1. Arabidopsis pre-miRNA164b was modified by replacing miR164b/miR164b* sequences with HaAce1-amiR1/HaAce1-amiR1* sequences. The recombinant HaAce1-preamiRNA1 was put under the control of CaMV 35S promoter and NOS terminator of plant binary vector pBI121, and the resultant vector cassette was used for tobacco transformation. Two transgenic tobacco lines expressing HaAce1-amiR1 was used for detached leaf insect feeding bioassays. Larval mortality of 25% and adult deformity of 20% were observed in transgenic treated insect group over that control tobacco treated insect group. The reduction in the steady-state level of HaAce1 mRNA was 70-80% in the defective adults compared to control. Our results demonstrate promise for host-delivered amiRNA-mediated silencing of HaAce1 gene for H. armigera management.

CRISPR/Cas9: a promising way to exploit genetic variation in plants.

Creation of variation in existing gene pool of crop plants is the foremost requirement in crop improvement programmes. Genome editing is a tool to produce knock out of target genes either by introduction of insertion or by deletion that disrupts the function of a specific gene. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system is the most recent addition to the toolbox of sequence-specific nucleases that includes ZFNs and TALENs. The CRISPR/Cas9 system allows targeted cleavage of genomic DNA guided by a small noncoding RNA, resulting in gene modifications by both non-homologous end joining and homology-directed repair mechanisms. Here, we present an overview of mechanisms of CRISPR, its potential roles in creating variation in germplasm and applications of this novel interference pathway in crop improvement. The availability of the CRISPR/Cas9 system holds promise in facilitating both forward and reverse genetics and will enhance research in crops that lack genetic resources.