Abstract
Onion (Allium cepa L.) is considered the most important Allium vegetable crop grown worldwide. The formation of the onion bulb, which is the most utilised part of the plant, is controlled by both photoperiod and temperature. However, the molecular mechanisms and genes involved in bulbing are not fully elucidated with limitations on onion genome sequencing and genetic transformation. With this, efforts to develop new onion cultivars have been hampered by these major constraints in molecular genetic studies and environmental sensitivities of onion bulb formation and flowering.
In this study, candidate genes previously identified in the Macknight Laboratory which were associated with onion bulb formation were studied. The genetic basis of daylength-dependent onion bulb formation was investigated by sequencing the onion FLAVIN-KELCH REPEAT F-BOX 1 (AcFKF1) gene and the FLOWERING LOCUS T (AcFT1 and AcFT4) genes using next-generation sequencing. Further, a system for the efficient in vitro culture and regeneration of materials which can be used for genetic transformation studies was developed. Several selectable markers were also tested for their use in the efficient selection of transformed cells and subsequent regeneration of onion plants. The use of these high-end tools, such as high-throughput sequencing and recently available tools for genetic transformation were utilised to overcome the difficulties of generating sequence information and functional gene characterisation in onions.
The sequences generated using Illumina MiSeq to sequence a region of AcFKF1 exon 2 showed five polymorphic regions in the gene’s coding sequence across 25 short-day (SD) and 27 long-day (LD) cultivars. These mutations were found between the F-box and the first Kelch-repeat region (269, 280, 294, and 305) and in the first Kelch-repeat (322), which may indicate the difference by which these accessions interact with target proteins that may be involved in onion bulbing and/or flowering. On the other hand, the use of Oxford Nanopore enabled the sequencing of the entire promoter to exon 4 regions of the AcFT1 and AcFT4 genes. The nucleotide sequence alignment of the genes among six SD and six LD onion cultivars showed a high degree of conservation among the samples. However, several mutations were found on the intron 2 of the genes, which may play a role in the transcriptional activation of AcFTs and subsequent bulbing adaptation of onion cultivars.
Further, the study developed a system for the induction of materials for onion genetic transformation, which is essential in the future functional characterisation of these candidate genes. It was found that callus induction of the onion cultivar Pukekohe Long Keeper (PLK) from the shoot tip of mature seeds is best at Murashige-Skoog (MS) medium with 1.0 mg/L 2,4-D, with the highest percentage of callus initiation recorded at 74.11%. For somatic embryogenesis (SEM), the optimum growth and formation of embryos were recorded in SEM 2b at 82.14%. Also, it was shown that increasing the concentration of 2,4-D for both callus induction and somatic embryogenesis results in a significant decrease in callus response which was recorded at concentrations higher than 2.0 mg/L. It was also demonstrated that the presence of 6-benzylaminopurine in shoot induction is essential in the regeneration of onion plants. Lastly, rooting of plantlets can be quickly attained in half-strength MS without plant growth regulators and media additives. The developed protocol would allow the production of embryogenic callus materials for transformation experiments after 16 weeks of callus induction and 35 weeks to generate acclimatised onion plants from somatic embryos of PLK.
The transformation of onion cells was also tested using different selectable markers. It was found that 60 mg/L is the highest concentration that can be used to select callus, while 15 mg/L during shoot regeneration of onion using the antibiotic selection marker hygromycin. The visual reporter of transformation, called RUBY, in onion callus resulted in transient expression of betalain in the onion cells, which were visible after 3-5 days of co-cultivation. Results also showed that one-hour co-cultivation time is best for transformation. A longer co-cultivation duration of two and three hours resulted in callus stress and tissue necrosis after two weeks. However, betalain was only transiently expressed and may be inhibitory to the growth of the callus. Later, callus browning, and shrinkage were evident on the sixth week of selection on 60 mg/L hyg. There were no calli recovered after eight weeks of hygromycin selection for all the treatments.
The same transient expression was also observed in the overexpression of the morphogenic genes, GROWTH-REGULATING FACTOR 4 and GRF-INTERACTING FACTOR 1 (GRF4-GIF1) in the onion callus, with the presence and expression of GIF1 detected from six-week-old callus. A total of 252 shoots were regenerated from experiments with one-hour Agrobacterium cultivation, but subsequent qPCR analyses of 19-week-old shoot forming callus and acclimatised plants showed no GIF1 expression. The transient expression of morphogenic genes encouraged the growth and shoot proliferation of previously tissue culture recalcitrant plants. Further experiments should be conducted to confirm this in onion callus regeneration. Overall, the findings of the study will be beneficial in the discovery of genes controlling bulb formation, which is a valuable resource in the development of onion cultivars.