The use of low concentrations of BAP for shoot regeneration
The presence of BAP was required for shoot tip and axillary bud cultures of V. vinifera ‘Napoleon,’ ‘Cabernet Sauvignon,’ ‘Riesling Italian,’ [9-11]; and five Iranian cultivars [12], and for shoot regeneration from foliar tissues of Vitis rootstocks. In the case of explants from rootstock leaves, no calli or shoots were obtained without BAP, but the development of calli and shoots was observed under high BAP concentrations between 2.21 and 8.87 µM [10]. A high BAP of 5 mg/l (22 µM) was also required for shoot regeneration and proliferation of shoot tips and axillary buds of ‘Cabernet Sauvignon’ and ‘Riesling Italian’ [11]. In contrast, a low BAP of 0.5 mg/l (2.2 µM) was used for shoot tip and axillary bud culturing of ‘Napoleon’ [9] and shoot apical meristem (SAM) cultures of five Iranian cultivars [12]. Based on these previous findings, we also used several different concentrations of BAP in this study. The effects of various BAP concentrations on shoot regeneration are shown in Table 1. SAM cultured on BAP-containing medium was induced to produce organogenic calli, which formed shoots 20 days after their transfer to phytohormone-free medium.. After testing with various concentrations, 0. 22µM BAP was selected for later experiments as the optimal shoot induction treatment because of its high viability and rate of shoot regeneration (Table 1). To add, it is notable that the BAP concentration suitable for shoot regeneration of ‘Koshu’ seems to be low as compared with that of other grapes [6], since the regenerated shoots were observed from phytohormone-free medium (Table 1). However, this result does not necessarily mean that BAP concentrations in these SAM tissues were low, and requires further investigation for verification.
Root regeneration of the grapevine ‘Koshu’ by using synthetic auxins
Roots appeared within 15 days after the developed small plantlet was transferred from the leaf explant onto phytohormone-free medium [10]. Phytohormone-free medium was also used for rooting shoots that developed by culturing shoot tips or axillary buds for 6 weeks [9]. On the contrary, the number of roots per shoot for ‘Cabernet Sauvignon,’ ‘Riesling Italian,’ and commercial cultivars of Vitis vinifera L. was increased by pretreating them with 1 to 2 mg/l of Indole-3-butyric acid (IBA) [11, 12]. Since the frequency of root regeneration from regenerated shoots of ‘Koshu’ was low with IBA or NAA, we examined this frequency by using the new synthetic auxins 4-chloroindole-3-acetic acid (4-Cl-IAA), 5,6-dichloroindole-3-acetic acid (5,6-Cl2-IAA), and 4-trifluormethylindole-3-acetic acid (4-CF3-IAA), the shoot regeneration activities of which were, respectively, 15, 4, and 1.5 times as high as that of IBA in black gram [13, 14]. The regenerated shoots obtained were transferred to MS medium containing no auxin, 10-4 M to 10-6 M of 1-naphthaleneacetic acid (NAA), 4-Cl-IAA, 5,6-Cl2-IAA, and 4-CF3-IAA from 20 to 35 days post-cultivation. Calli without and calli with roots were obtained at 7 and 14 days after their transfer onto no auxin and 10-5 M or 10-6 M synthetic auxin (Table 2, Fig. 1 C, D). Notably, the frequency of root formation was highest at the 10-5 M 4-Cl-IAA concentration (Table 2, Fig. 1 C, D). This is the first report of successful root regeneration of grapevines by using synthetic auxins. The rooting plantlets were transferred to hormone-free medium, and their development was observed (Fig. 1 E – H). We have succeeded in obtaining 14 plantlets from the SAM tissues of ‘Koshu.’ The plantlets were morphologically normal (Fig. 1 G, H) and would then be transferred into soil with covering by plastic wrap to maintain humidity. Timing of humidity reduction and/or elevation of light intensity might be important for further plant development.