The effects of β-lactam antibiotics and hygromycin B on de novo shoot organogenesis in apple cv. Golden Delicious

Abstract

Since the genetic transformation of the apple is strongly genotype-dependent and generally inefficient, the evaluation of factors affecting shoot regeneration are crucial for the establishment of a successful transformation process. In this report, we evaluated the effects of the β-lactam antibiotics meropenem and timentin on in vitro regeneration via de novo shoot organogenesis from leaf explants of apple cv. Golden Delicious, as well as on the growth of the Agrobacterium tumefaciens strain EHA 105, and compared them with the commonly used β-lactam cefotaxime. Also, we report for the first time the effect of hygromycin B as a selective agent in the domesticated apple, as regards shoot regeneration and shoot multiplication efficiency. We observed that cefotaxime and timentin at concentrations higher than 100 mg L -1 were sufficient to prevent Agrobacterium growth during a two-week period, while meropenem exhibited an inhibitory effect on bacterial growth at all tested concentrations (25-150 mg L -1 ). Cefotaxime at a concentration of 300 mg L -1 increased the number of regenerated shoots per explant (9.39) in comparison with the control (7.67). In contrast to cefotaxime, meropenem and timentin caused a decrease in shoot regeneration efficiency, but larger and more developed shoots were obtained on meropenem (25-125 mg L -1 ) after the same period of cultivation. Hygromycin B at a concentration of 5 mg L -1 or higher completely inhibited shoot regeneration and induced explant tissue necrosis. Therefore, the selection procedure with a final concentration of 4 mg L -1 throughout organogenesis and 10 mg L -1 for further shoot growth and multiplication is recommended for an efficient transformation process in apple cv. Golden Delicious. Abbreviations: BAP -N 6 -benzilaminopurin; BM − basal medium; IBA -indole-3-butyric acid; MIC -minimal inhibitory concentration; MS -Murashige and Skoog mineral solution; NAA -α-naphthaleneacetic acid; HPThygromycin B phosphotransferase; SFC -the index of shoot-forming capacity; SMM -shoot multiplication medium; SRM -shoot regeneration medium; TDZ -thidiazuron How to cite this article: Stanišić M, Ninković S, Savić J, Ćosić T, Mitić N. The effects of β-lactam antibiotics and hygromycin B on de novo shoot organogenesis in apple cv. Golden Delicious. Arch Biol Sci. 2018;70(1):179-90.

In vitro tissue culture of apple and other Malus species: recent advances and applications

“…The effects of antibiotics on shoot growth and development can be important when the objective is to produce transgenic plants. Stanišić et al (2018), using the protocol of Mitić et al (2012) for 'Golden Delicious', noted that when 300 mg/l cefotaxime was used, shoot growth improved, increasing shoot number per explant from 7.67 to 9.69 in the control, whereas other β-lactam antibiotics (meropenem and timentin) stunted shoot growth, a similar response that chrysanthemum and tobacco had displayed when several aminoglycoside antibiotics were tested (Teixeira da Silva and Fukai 2003). Pathak and Dhawan (2012b) found that the growth of rootstock M7 and MM111 shoots could be supported on medium containing between 1 and 5% fructose, glucose, mannitol, sorbitol and sucrose, with the most effective concentration resulting in longest shoots and greatest productivity arising from 4% sucrose and sorbitol for M7 and MM111, respectively.…”

“…Alayón-Luaces et al 2012Shoot tips from 10 cm scionwood cuttings → RTW time NR → 70% EtOH 1 min → 5% NaOCl + 0.1% Tween-20 + 150 mg/l AA 10 min → SDW + 150 mg/l AA 4× Kereša et al (2012) Bud (0.8-1.5 cm) rinsed in water with detergent 10 min → 4% NaOCl 20 min → SDW 5× Mitić et al (2012); Stanišić et al (2018) Seeds → 70% EtOH 1 min → 0.1% HgCl 2 10 min → SDW 3× Dai et al (2013Dai et al ( , 2014 Shoot tips (5-10 mm) → Tween-80 time NR → DW 3-4 × → meristems excised from shoot tips → 0.01% HgCl 2 1 min → SDW 4-5× Mir et al (2013) Single node cuttings → soapy RTW → fungicides (2 g/l each of Benomile + Ridomile) 20 min → 2014Axillary buds (0.2-0.6 cm) → tap water + 0.05% Tween-20 10 min → Zhang et al (2014) Shoot tips → egg yolks + 20% NaOCl + 2 drops of Tween-20 20 min →…”

Cellular, Molecular, and Physiological Aspects of In Vitro Plant Regeneration

“…The medium supplemented with antibiotics such as β-lactam antibiotics and tetracycline has been reported to result in higher regeneration capacity as compared with untreated explants [ 189 , 190 ]. Despite the successful application of antibiotics to suppress bacterial growth in vitro, excessive and regular use of them is detrimental to the regeneration process and results in a reduced regeneration ability of tissue cultures [ 191 ].…”

Tissue culture and genetic transformation of cabbage (Brassica oleracea var. capitata): an overview

“…Their concentration should be optimized before transformation to determine an effective dose leading to plants regeneration as well as a lethal dose for Agrobacterium . This step is extremely important due to the fact that the prolonged presence of Agrobacterium in plant tissues affects the growth and development of explants and can even cause necrosis and consequently their death (Stanišić et al 2018 ). Furthermore, presence of Agrobacterium in tissue of putative transformants could result in false positive results in the molecular analyses.…”

“…In addition, elimination of Agrobacterium from transformed plants prevents the possibility of accidental release of transgenes to the environment when transformants are transferred to the soil (Tambarussi et al 2015 ). Beta-lactam antibiotics (e.g., carbenicillin, cefotaxime and timentin) are most commonly used for combating Agrobacteria after genetic transformation (Stanišić et al 2018 ). Due to the difficulty in the transformation of cabbage, only few papers related to this subject were published.…”