Investigating the In Vitro Regeneration Potential of Commercial Cultivars of Brassica

Abstract

In vitro regeneration is a pre-requisite for developing transgenic plants through tissue culture-based genetic engineering approaches. Huge variations among different genotypes of the genus Brassica necessitate the identification of a set of regeneration conditions for a genotype, which can be reliably used in transformation experiments. In this study, we evaluated the morphogenesis potential of four commercial cultivars (Faisal canola, Punjab canola, Aari canola, Nifa Gold) and one model, Westar, from four different explants namely cotyledons, hypocotyls, petioles and roots on three different Brassica regeneration protocols, BRP-I, -II and -III. The regeneration efficiency was observed in the range of 6-73%, 4-79.3%, 0-50.6%, and 0-42.6% from cotyledons, petioles, hypocotyls and roots, respectively, whereas, the regeneration response in terms of average shoots per explant was found to be 0.76-10.9, 0.2-3.2, 0-3.4 and 0-2.7 from these explants. Of the commercial varieties tested, almost all varieties showed poorer regeneration than Westar except Aari canola. In comparison to Westar, its regeneration frequency from cotyledons was up to 7.5-fold higher on BRP-I, while it produced up to 21.9-fold more shoots per explant. Our data show that the explant has strong influence on the regeneration response, ranging from 24% to 92%. While the growth of commercial cultivars was least affected by the regeneration conditions provided, the effect on Westar was twice that of the commercial cultivars. After determining the optimal explant type and regeneration conditions, we also determined the minimum kanamycin concentration levels required to selectively inhibit the growth of untransformed cells for these cultivars. Regenerated shoots of Aari canola could be successfully grown to maturity within 16-18 weeks, with no altered phenotype noted and normal seed yields obtained. Therefore, the commercial variety, Aari canola, could be a good candidate for future genetic transformation studies.

In Vitro Regeneration Potential of White Lupin (Lupinus albus) from Cotyledonary Nodes

“…Plant tissue culture system is an efficient in vitro based technique used for genetic modification and plant regeneration, and reduces yield loss [15][16][17]. Development of plants with potential improved traits such as superior quality, high yield, better adaptability, disease tolerance and stress resistance is mainly based on micropropagation technique of tissue culture system [18][19][20]. Relatively very poor progress has been accomplished on plant regeneration system in legumes [21], due to the unsuccessful in vitro generation tissue culture systems.…”

In Vitro Production of Somaclones with Decreased Erucic Acid Content in Indian Mustard [Brassica juncea (Linn.) Czern&Coss]

“…Further, the environmental and nutritional conditions can be controlled uniformly and precisely under tissue culture conditions, and at a given time large number(s) of somatic cell lines could be screened swiftly to regenerate variants. In genus Brassica , the in vitro regeneration capabilities have been genotype-specific [ 31 , 51 ]. Levels of variations have been observed to be dependent on the regeneration potential of the genotypes.…”

Genotype-Independent Transformation and Genome Editing of Brassica napus Using a Novel Explant Material

“…The hypocotyl system has worked well for a limited range of genotypes, with Westar being the most commonly employed genotype ( De Block et al, 1989 ; Cardoza and Stewart, 2003 ). Other researchers have been able to extend hypocotyl transformation to a few other genotypes using a modified version of the protocol with no selection ( Maheshwari et al, 2011 ), it has been challenging to make the hypocotyl system work efficiently for elite commercial canola germplasm representing a broad range of breeding genetics ( Hao et al, 2010 ; Farooq et al, 2019 ).…”

“…While transforming haploid microspores or microspore-derived embryos is attractive because the process produces homozygous double-haploid plants, however the process is lengthy, laborious and genotype-dependent ( Cegielska-Taras et al, 2008 ; Chen and Tulsieram, 2015 ). Leaf tissue, cotyledons, and petioles have also been described in the literature as having regeneration potential ( Akasaka-Kennedy et al, 2005 ; Farooq et al, 2019 ) but these explant types have not been widely adopted for canola transformation. For a comprehensive review of Brassica transformation methodology, please see Rani et al (2013) .…”