Improved Agrobacterium-mediated transformation and high efficiency of root formation from hypocotyl meristem of spring Brassica napus ‘Precocity’ cultivar

Abstract

ABSTRACT. Rape seed (Brassica napus L.) is one of the most important oil seed crops in the world. Genetic manipulation of rapeseed requires a suitable tissue culture system and an efficient method for plant regeneration, as well as an efficient transformation procedure. However, development of transgenic B. napus has been problematic, and current studies are limited to cultivated varieties. In this study, we report a protocol for regeneration of transgenic rape after Agrobacterium-mediated transformation of hypocotyls from the spring B. napus 'Precocity' cultivar. We analyzed the effects of plant growth regulators in the medium on regeneration. Additionally, factors affecting the transformation efficiency, including seedling age, Agrobacterium concentration, infection time, and co-cultivation time, were assessed by monitoring GUS expression. Results from these experiments revealed that transformation was optimized when the meristematic parts of the 16841 Transformation and root formation of Brassica napus ©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 14 (4): 16840-16855 (2015) hypocotyls were taken from 8 day-old seedlings, cultured on Murashinge and Skoog basal media containing 0.1 mg/L 1-naphthaleneacetic acid and 2.5 mg/L 6-benzylaminopurine, and incubated in Agrobacterium suspension (OD 600 = 0.5) for 3 to 5 min, followed by 2 days of cocultivation. Integration of T-DNA into the plant genome was confirmed by polymerase chain reaction (PCR), b-glucuronidase histochemical staining, and quantitative real-time PCR. The protocols developed for regeneration, transformation, and rooting described in this study could help to accelerate the development of transgenic spring rape varieties with novel features.

High efficiency transformation of Brassica oleracea var. botrytis plants by Rhizobium rhizogenes

“…The percentage pattern of both, the particular pathways and shoots and/or roots induced, were changed as different concentrations of the particular phytohormones were provided. With the increase in BAP concentration the shoot formation (understood as the total number of shoots without pathway distinction) grew gradually, confirming the rule that a high ratio of cytokinin-to-auxin induced shoot regeneration (Ikeuchi et al 2013; Liu et al 2015). On the contrary, the cauliflower callus seemed to be induced (in hypocotyls of the Pionier cultivar) only in the presence of both regulators and at a high ratio of BAP to NAA, which is not a typically observed relation (it usually takes an equal cytokinin-to-auxin ratio) (Ikeuchi et al 2013).…”

“…As it is known from biotechnological research data, it is not the only species that displays troublesome recalcitrance towards genetic manipulations (e.g. Phaseolus vulgaris ; Brassica napus ) (Hnatuszko-Konka et al 2014; Liu et al 2015). As in the case of other important crops, the establishment of a reliable procedure of transformation would provide the space for their improvement or biopharming.…”

In vitro selection for Fusarium oxysporum f. sp. conglutinans resistance in brassica vegetables

“…The regeneration efficiency dramatically depends on the explant, the genotype variant, the age of the explant due to the physiological status. [12][13][14] In this study, three types of explants, hypocotyl, shoot tip and cotyledon, in two age groups, 6 day and 10 day, taken from seedlings, were examined for the regeneration capacity of selected cultivars of B. oleracea var. capitata.…”

“…capitata. Yet Qamar et al observed 100% callus induction from hypocotyls of cabbage cultured in a medium supplemented with 2,4-D (2.0 mg/L) and BAP (0.25 to 1.0 mg/L) but a considerably higher dosage of cytokinin was essential for shoot induction from the callus to break the strong auxin effect of 2,4-D. 14,15 They achieved 100% of shoot induction by a combination of BAP (4 mg/L), Zeatin (4 mg/L) and IAA (5 mg/L). Munshi et al also observed the maximum percentage of callus induction from cotyledon explants on 2,4-D (1.0 mg/L) and NAA (0.5 mg/L), and shoot induction was only observed from the calli derived from cotyledon explants.…”

“…36 As all reported previous studies emphasized the genotype dependency on in vitro organogenesis of Brassica oleracea, this study likewise clearly observed a similar response. 12,[14][15][16]19,[36][37][38][39] After successful mutation induction and selection, we need to evaluate the resistance capacity of developed resistant plants to a live pathogen in both greenhouse and field conditions, in order to confirm the stability of the mutational resistance developed in the plant. Identification of induced mutational resistance gene or genes through in vitro mutation induction would be of benefit for future studies.…”