SummaryClustered regularly interspaced short palindromic repeat (CRISPR) and Cas9‐associated protein systems provide a powerful genetic manipulation tool that can drive plant research forward. Nuclease‐dead Cas9 (dCas9) is an enzymatically inactive mutant of Cas9 in which its endonuclease activity is non‐functional. The applications of CRISPR/dCas9 have expanded and diversified in recent years. Originally, dCas9 was used as a CRISPR/Cas9 re‐engineering tool that enables targeted expression of any gene or multiple genes through recruitment of transcriptional effector domains without introducing irreversible DNA‐damaging mutations. Subsequent applications have made use of its ability to recruit modifying enzymes and reporter proteins to DNA target sites. In this paper, the most recent progress in the applications of CRISPR/dCas9 in plants, which include gene activation and repression, epigenome editing, modulation of chromatin topology, live‐cell chromatin imaging and DNA‐free genetic modification, will be reviewed. The associated strategies for exploiting the CRISPR/dCas9 system for crop improvement with a dimer of the future of the CRISPR/dCas9 system in the functional genomics of crops and the development of traits will be briefly discussed.

Knowledge of heat-tolerant/sensitive cultivars based on morpho-physiological indicators and an understanding of the action and interaction of different genes in the molecular network are critical for genetic improvement. To screen these indicators, the physiological performance of two different varieties of white and red cabbages (B. oleracea var. capitate f. alba and f. rubra, respectively) under heat stress (HS) and non-stress (NS) was evaluated. Cultivars that showed considerable cell membrane thermostability and less reduction in chlorophyll content with better head formation were categorized as the heat-tolerant cultivars (HTC), while those with reduction in stomatal conductance, higher reduction incurred in chlorophyll and damage to thylakoid membranes are categorized as the heat-sensitive cultivars (HSC). Expression profiling of key genes in the HS response network, including BoHSP70 (HEAT SHOCK PROTEIN 70), BoSCL13 (SCARECROW-LIKE 13) and BoDPB3-1 (transcriptional regulator DNA POLYMERASE II SUBUNIT B3-1 (DPB3-1))/NUCLEAR FACTOR Y SUBUNIT C10 (NF-YC10), were evaluated in all cultivars under HS compared to NS plants, which showed their potential as molecular indicators to differentiate HTC from HSC. Based on the results, the morphophysiological and molecular indicators are applicable to cabbage cultivars for differentiating HTC from HSC, and potential target genes for genome editing were identified for enhancing food security in the warmer regions of the world.

Several seamless DNA assembly kits based on in vitro homologous recombination activity are commercially available in recent years for efficient and rapid construction of expression vectors, subsequent transformation and investigation of gene functionality. This study was performed to estimate the efficiency of uni seamless cloning system, through cloning of multiple DNA fragments derived from Elaeis guineensis stearoyl-ACP desaturase (SAD) and metallothionein-like protein genes (MET1 and MET2) into a plasmid cloning vector. PCR fragments were assembled based on homologous overlapping regions of 25-30 bp into a pUC19 linearized vector. The successful cloning of the three DNA fragments was validated by blue white screening, colony cracking and colony PCR. The observed overall cloning efficiency for the three assembled DNA fragments of about 2.7 kb in size was above 65%. This system produces seamless junctions, is directional and does not rely on restriction enzyme digestion and any specific site within the DNA fragments.

Development of in vitro plant regeneration method from Brassica explants via organogenesis and somatic embryogenesis is influenced by many factors such as culture environment, culture medium composition, explant sources, and genotypes which are reviewed in this study. An efficient in vitro regeneration system to allow genetic transformation of Brassica is a crucial tool for improving its economical value. Methods to optimize transformation protocols for the efficient introduction of desirable traits, and a comparative analysis of these methods are also reviewed. Hence, binary vectors, selectable marker genes, minimum inhibitory concentration of selection agents, reporter marker genes, preculture media, Agrobacterium concentration and regeneration ability of putative transformants for improvement of Agrobacterium-mediated transformation of Brassica are discussed.

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