In vitro adventitious shoot regeneration and acclimatisation of Brassica oleracea subsp. italica cv. Green Marvel

Cotyledonary explants of Brassica oleracea subsp. italica (broccoli) cv. Green Marvel were cultured on Murashige and Skoog (MS) medium containing different combinations of the growth regulators 6- benzylaminopurine (BAP) and α-naphthalene acetic acid (NAA) for shoot regeneration. The optimal medium for inducing shoots contained 3 mgl-1 BAP and 1 mgl-1 NAA, which produced a shoot induction percentage of 53.33% and a mean number of 0.43 shoot per explant. The shoots were subsequently rooted in MS medium that contained 0.2 mgl-1 of indol-3-butyric acid (IBA). Different potting media were assessed during plantlet acclimatization. The highest percentage of plant survival (83.33%) was on the medium that contained sand and soil (1:1), while maximum root length (4.37 cm) and plant height (7.87 cm) were attained in potting medium that consisted peat moss, perlite and vermiculite (3:1:1).Key words: Brassica oleracea, broccoli, 6-benzylaminopurine, α-naphthalene acetic acid, indole-3- butyric acid

Plant regeneration of Brassica oleracea subsp. italica (Broccoli) CV Green Marvel as affected by plant growth regulators

Hypocotyls and shoot tips were used as explants in in vitro plant regeneration of broccoli (Brassica oleracea subsp.italica) cv. Green Marvel. The explants were excised from sterile germinated seedlingsand placed on shoot induction medium containing basal salts of Murashige and Skoog (MS) and various concentrations of 6-benzylaminopurine (BAP) and -naphthaleneacetic acid (NAA). The highest percentage of hypocotyl explant producing shoot (96.67%) and the highest mean number of shoots produced per hypocotyl explant (6.03) were obtained on 3 mgL¹ BAP. Meanwhile, the highestpercentage of shoot tip explant producing shoot (100%) and highest number of shoot produced per shoot tip explant (3.76) were recorded on 5 mgL¹ BAP. For rooting of shoots, NAA, indoleacetic acid(IAA) and indolebutyric acid (IBA) at 0, 0.2 and 1 mgL¹ were applied. Highest percentage of shoots with roots (100%) and highest mean number of roots produced per shoot (6.5) occurred on medium with 0.2mgL¹ IBA, while the maximum root length (2.46 cm) was attained on MS medium without plant growth regulator (MSO). Plantlets were successfully acclimatized in potting medium containing peatmoss,perlite, and vermiculite (3:1:1)

Shoot tip regeneration and optimization of Agrobacterium tumefaciens-mediated transformation of Broccoli (Brassica oleracea var. italica) cv. Green Marvel

A protocol of plant regeneration from shoot tips and optimization of Agrobacterium tumefaciens-mediated transformation of broccoli (Brassica oleracea var. italica) cv. Green Marvel have been developed. Shoot tip response was assessed on Murashige and Skoog (MS) medium supplemented with different concentrations of zeatin. The highest regeneration with a maximum of 13 shoots per explant was obtained on MS medium containing 1.5 mg l-1 zeatin. Primary selection of putative transformed explants was performed on the optimized regeneration medium (MS medium containing 1.5 mg l-1 zeatin and 80 mg l-1 kanamycin) for 60 days. The effects of preculture, acetosyringone and growth of bacterial culture were studied. Explants precultured on callus induction medium for 4 days prior to inoculation with A. tumefaciens with 200 lM acetosyringone resulted in improved transformation frequency. The Agrobacterium culture dilution of 1:5 and inoculation time of 30 min increased the efficiency of transformation of shoot tip explants. The results also indicated that 150 mg l-1 ampicillin alone was adequate to eradicate Agrobacterium growth in the SRM incorporated with the respective minimum inhibitory concentration of 80 mg l-1 kanamycin. The polymerase chain reaction (PCR) and Southern blot assays confirmed the transgenic status of the broccoli cv. Green Marvel regenerants. A transformation efficiency of 5 % was achieved based on the positive PCR results using the optimized procedure. The expression of luciferase reporter gene in the transformed cells and the transcription of AtHSP101 using RT-PCR further confirmed the transgenic status of the regenerated plants