Young leaves and petioles from previous in vitro-generated plants grown using standard Murishage and Skoog (MS) medium ¹² were used as explants. The intact leaf were sliced into 1 cm ² of lamina and 1 cm length of petiole. The explants were then inoculated to the MS containing plant growth regulator of 2,4-dichlorophenoxyacetic acid (2,4-D) (Merck, Cat. No.31518, Germany) combined with 6-benzylaminopurine (BAP, Merck, Cat. No. B3408, Germany) to induce calli. The explants were grown and placed at a sterilized 600 ml bottle containing around 20 ml solidified medium for a month at the culture room at a temperature of 25±2°C. The composition of the treatments were Z ₁ (MS + 0 mg L ^-1 2,4-D + 0 mg L ^-1 BAP), Z ₂ (MS + 1 mg L ^-1 2,4-D ¹ + 0.5 mg L ^-1 BAP), Z ₃ (MS + 2 mg L ^-1 2,4-D ¹ + 0.5 mg L ^-1 BAP), Z ₄ (MS + 2 mg L ^-1 2,4-D ¹ + 1 mg L ^-1 BAP), Z ₅ (MS + 3 mg L ^-1 2,4-D ¹ + 0.5 mg L ^-1 BAP), Z ₆ (MS + 3 mg L ^-1 2,4-D ¹ + 1 mg L ^-1 BAP), Z ₇ (MS + 4 mg L ^-1 2,4-D ¹ + 0.5 mg L ^-1 BAP), and Z ₈ (MS + 4 mg L ^-1 2,4-D ¹ + 1 mg L ^-1 BAP). All treatments were replicated five times. In total there were two types of explants x eight treatments x five replication x five explants per replication/bottle = 400 experiment units.

The green, fresh and compact calli produced at the first experiment were selected and transferred to MS medium with half the usual level of nutrients containing 0, 5, 10, 15 and 20% PEG as selection agent medium of drought tolerance. All treatments were replicated three times. In total there were five treatments x three replications x four explants per replication/bottle = 60 experiment units. The surviving calli in the PEG selection-agent medium were regenerated in MS medium containing 0, 0.5, 1 or 1.5 mg L ^-1 BAP to induce shoots and roots. The growth variables during in vitro culture, such as the number of calli induced per treatment, percentage of responsive explants to induce calli, percentage of surviving calli under drought stress simulated by polyethylene glycol, and number of regenerated plants, were observed.

All media applied in the experiment could successfully induce callus formation except the basal medium containing no growth regulator (control). The existence of 2,4-D and BAP in various concentration combination was able to induce callus formation but the number of callus per explant and the percentage of responsive explant were varied ( Table 1, Figure 1A, B). The highest number of induced calli per treatment was observed using the Z ₃ treatment medium. These results were observed using both leaf and petiole ( Figure 1A, B). Callus formation was successfully induced in sweet potato using both explants ^{13, 14} . The use of 2,4-D to induce callus formation is common ^{13, 15} . Callus formation can be induced by other plant growth regulators such as zeatin, or 1-naphthaleneacetic acid combined with gibberellin (GA ₃) ^{14, 16} . These calli emerged and was developed from the mesophyll cells in the leaf and protoplast from the stem or petiole ^{13, 14} .

The fresh, green and compact calli produced in the first experiment was transferred to the drought stressed medium simulated by MS medium containing different concentration of PEG to discover the putative drought tolerant calli of purple sweet potato. At least one callus survived in all PEG-containing medium, but the survival rate was varied ( Table 2, Figure 1C). The higher the concentration of the PEG in the medium the lower survival rate of the calli in the selection medium. This indicates that drought stress caused by PEG simulation affects callus survivability and can be used as drought-tolerant selection medium, as also reported in other crop plants such as soybean ^{9, 17– 20} , tobacco ²¹ , grass ²² , rice ²³ and chili ²⁴ . The putative drought-tolerant plants are screened from regenerated plants derived from the survived calli in the highest concentration of PEG. There was no significant different response between calli derived from the leaf and petiole ( Table 2).

The surviving calli grown in medium containing 40% PEG were incubated in the regeneration medium to initiate shoot and/or root. The only medium MS containing 1.5 mg L ^-1 BA can successfully induce shoot and root growth ( Table 3, Figure 1D). This indicates that the calli exposed with high concentration of PEG are still viable to regenerate into whole plant. The results increase the possibility to get a new purple sweet potato variant tolerant to drought stress as also reported in soybean ^{19, 20} .

The raw data for calli and explant growth are available on OSF ²⁵ .