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Repeated subculturing of the micro-shoots has been suggested as a method to rejuvenate adult tissues (Kanwar et al., 2010). Gantait et al. (2010), noticed no difference in the number of shoots between the first and fifth subcultures, whereas Kanwar et al. (2015) obtained 22.7 shoots/explants after the sixth subculturing, with an average length of 9.0 cm, compared to the first with only 5.5 shoots/explant with an average length of 3.5 cm. Hashemabadi and Kaviani (2008) obtained 12 shoots/explants in the fourth subculturing. Kanwar et al. (2015) observed that the in vitro rooting response increased as a function of the in vitro culture cycle applied. At the sixth subculturing, the length root was 3.7 cm. The same authors, in another experiment, transferred explant as juvenile leaf, after every 3 days, into fresh shoot induction full strength MS medium supplemented with 0.04% AC and incubated it at 25 2C under 16 h photoperiod. They obtained a phenolic-free microshoot; AC provided the dark conditions required for the root initiation and growth and also adsorbed the substances presumed to be detrimental.
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