Vipasha Verma*, Meenakshi Thakur, Bhavya Bhargava and Gaurav Zinta
Affiliation: CSIR-Institute of Himalayan and Bioresource Technology, Palampur
Corresponding author email: email@example.com
Flowers are the most beautiful creation of God. Their sight is a joyful feeling. The sweet smell of flowers makes the air pleasant to breathe. Flowers add colour to rooms or gardens and mend the quality of life. They are of various colours and hues. Even the same species of flowers exist in several forms and tinges. Cut flowers, namely rose, lily, chrysanthemum, lisianthus, tulip, gerbera, freesia, alstroemeria, carnation and hydrangea, carnations, and chrysanthemums, are commercially important and are sold all over the world.
The global floriculture business is growing at the rate of 6-10 % per annum. India ranks 18th in floriculture trade and has only 0.61 percent share in global floriculture trade (Vahoniya et al., 2018). Development of new varieties, cultivation, marketing and value-added products are essential for the success of floriculture industry. Promoting flowering and flower longevity as well as creating novelty in flower structure, colour range and fragrances are major objectives of ornamental plant breeding.
Various strategies for plant breeding have been employed to improve or enhance colour and shape variation, plant architecture, shelf life, and disease resistance. Multiple cultivars have been developed on the basis of crossbreeding and mutation breeding approaches, which can be applied to a limited number of traits. Transgenic technologies also can enhance ornamental plants by modifying or engineering changes in the plants’ genomes. There are excellent examples of transgenic ornamental plants such as the creation of blue-hued carnations, roses (Katsumoto et al., 2007; Tanaka et al., 2009), and chrysanthemums (Noda et al., 2018) which could not have been produced using conventional breeding methods.
CRISPR Cas9 based Genome editing approaches, allow the development of more precise and efficient tools to induce mutations in plant genes, modifying their expression or silencing them (Hahne et al., 2019). The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) system, which originates from bacteria and archaea (Wiedenheft et al. 2012), is the most widely used genome editing system. Cas9 is a nuclease that can cleave double-stranded DNA. Target DNA specificity is governed by a single-guide RNA (sgRNA) that guides Cas9 to bind to a 20-nucleotide (nt) sequence on the target DNA (referred to as the protospacer). The target DNA requires an additional 3-nt element (protospacer-adjacent motif (PAM)) with the NGG sequence in its downstream to be bound and cleaved by Cas9 (Jinek et al. 2012). DNA cleavage occurs at three base pairs upstream of PAM. The repair of the Cas9-induced DNA double-strand breaks (DSBs) within the protospacer can induce insertion/deletion mutations of variable length. The target specificity of the nuclease is determined directly by a short sequence in the sgRNA.
Therefore, it is necessary only to insert the desired sequence as a DNA oligonucleotide into a vector construct for target site selection. This makes the construction of the CRISPR/Cas9 system easier than other gene editing approaches like ZFNs and TALENs. Moreover, the expression of multiple guide RNAs can be used at once (Mali et al. 2013), which reduces costs and the time needed to generate plants with multiple target mutations. Genome editing methods in plants rely on transgenic technology to incorporate the genome editing system. Just as transgenic plants can inherit a transgene, genome-edited plants can pass on the introduced mutation to its offspring.
Although transgenic technology requires the integration of the transgene to produce the desired traits, genome editing does not require the integration of the transgene into the genome. Therefore, integrated transgenes could be segregated in progeny. Thus, after the transgenes are segregated, genome-edited plants ideally cannot be distinguished from the plants that are mutated via conventional breeding method. Thus, whether such transgene-free genome-edited plants should be treated as transgenic plants under many regulations is a matter of debate. In addition, genome editing is programmable and leads precisely to a specific mutation in the target sequence. Therefore, it is expected to be more effective than conventional mutation breeding to create desired mutant plants.
CRISPR Cas9 mediated genome editing is a great breakthrough for breeding technology that can be adopted in numerous floriculture crops for improving floral attributes (Ma et al. 2016). The possibility to implement such approaches in breeding of ornamental species, however, relies on information about structure and function of plant genomes and genes, and availability of efficient transformation and regeneration protocols. This technology is poised to become a common breeding method for ornamental plants. In addition, novel genome editing technologies are expected to accelerate the speed of breeding programs as the main option for revealing gene function and producing new cultivars in floral crops.
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