Ultrasonic treatment stimulates multiple shoot regeneration and explant enlargement in recalcitrant squash cotyledon explants in vitro

Abstract

Ultrasonic treatment (0.5-2 min) stimulated multiple shoot regeneration to high levels in vitro from recalcitrant cotyledon explants of commercial squash (Cucurbita pepo L.) cultivars Ma'yan and Bareqet, on Murashige and Skoog [Physiol Plant 15:473-497, 1962] (regeneration) medium augmented with 4.4 microM benzyladenine. At this stage, unsonicated control explants regenerated only a few very small shoots or bud-like structures. Ultrasound also stimulated massive explant growth. Ultrasound treatment resulted in further multiple shoot production (five times greater than control) after explant transfer to elongation medium (Murashige and Skoog [Physiol Plant 15:473-497, 1962] medium with 0.44 microM benzyladenine and 2.9 microM gibberellic acid). Longer ultrasonic treatments (5 or 10 min) promoted multiple shoot regeneration and explant growth accompanied by hyperhydration. Scanning electron microscope observations showed that 2 min ultrasound changed the joint area between epidermal cells and removed some of the surface from the cotyledon epidermal cells, without gross surface injury to the explants. Longer periods of ultrasound (5-10 min) caused further surface erosion. Rubbing the explant contact surface with chloroform or sandpaper emulated the effect of sonication on shoot regeneration and explant growth, demonstrating that ultrasound exerts its morphogenic influence by surface removal. Sonication of explants from other batches of squash seeds (of cultivars Ma'yan and True French), that regenerated without such treatment, reduced regeneration and caused hyperhydration. This is the first report of stimulation of in vitro regeneration by ultrasound treatment.

Sonication assisted Agrobacterium-mediated transformation enhances the transformation efficiency in flax (Linum usitatissimum L.)

“…of soybean (Trick and Finer 2000), black locust (Zaragozá et al 2004) and Chenopodium rubrum (Flores Solís et al 2007). Sonication could also stimulate massive explant growth and the regeneration response of some species recalcitrant in tissue cultures, such as squash (Ananthakrishnan et al 2007). Our introductory experiments with cv.…”

“…This means that the intensity and period of sonication should be very carefully evaluated and optimized for each species, type of tissue and purpose of the experiment. It is speculated that the microwounds produced by sonication could either disturb or eliminate the surface barrier preventing the regeneration of cells located deeper in the plant tissues (Ananthakrishnan et al 2007) and/or allow Agrobacterium to efficiently infect deep within the tissue and stably transform. In our preliminary experiments performed with a powerful sonicator (source ca.…”

In vitro organogenesis in tomato cultivars is enhanced by gas exchange and application of ultrasound

“…Other interesting physical treatment is ultrasound, a widely used tool for the manipulation of plant and animal cells and organs. Following exposure to non-lethal levels of ultrasound, many plants exhibit altered characteristics at different organizational levels (Ananthakrishnan et al, 2007;Dong et al, 2002). This effect may be related to the cavitation waves, and further violent collapse of bubbles, generating high pressure and temperature.…”

“…In in vitro cultivation, ultrasound is used to obtain the largest number of branches in the genetic transformation procedure known as Sonication-assisted Agrobacterium-mediated genetic transformation (SAAT) (Koetle et al, 2017). Ananthakrishnan et al (2007) stimulated multiple shoot regeneration from recalcitrant cotyledon explants from seeds before germination of commercial squash (Cucurbita pepo L.) that was the first demonstration that ultrasound exerts morphogenic influence by surface removal, however the technique has been caused hyper hydratation of the explants. The association of in vitro cultures with gas exchange and ultrasound to make regeneration optimization in vitro cultures can be helpful.…”

Direct Organogenesis from Cotyledonary Node Explants of Cucurbita pepo (L.)—An Important Zucchini Type Vegetable Crop

“…A wide range of other C. pepo cultivars regenerate to the same extent (with one exception) using the same technique [12]. Brief sonication treatment stimulate shoot regeneration reported [13]. In vitro shoot regeneration of a seedling-derived organ caused by prolonged seed storage period reported [14].…”