Cryopreservation, storing biological material in liquid nitrogen (LN, −196 °C), offers a valuable option for the long-term conservation of non-orthodox seeds and vegetatively propagated species in the sector of agrobiodiversity and wild flora. Although large-scale cryobanking of germplasm collections has been increasing worldwide, the wide application of cryopreservation protocol in wild flora is hampered by difficulties in vitro propagation and a lack of universal cryopreservation protocols, among others. This study established a systematic approach to developing an in vitro culture and droplet-vitrification cryopreservation procedure for shoot tips of Scrophularia kakudensis. The standard procedure includes two-step preculture with 10% sucrose for 31 h and with 17.5% sucrose for 16 h, osmoprotection with loading solution C4-35% (17.5% glycerol + 17.5% sucrose, w/v) for 30 min, cryoprotection with A3-80% (33.3% glycerol + 13.3% dimethyl sulfoxide + 13.3% ethylene glycol + 20.1% sucrose, w/v) at 0 °C for 60 min, and cooling and rewarming using aluminum foil strips. After unloading, a three-step regrowth procedure starting with an ammonium-free medium with growth regulators was essential for developing normal plantlets from cryopreserved shoot tips. Liquid overlay on the gelled medium two weeks after inoculation resulted in vigorous growth during subcultures. Moreover, liquid overlay increased LN regeneration by up to 80%, i.e., 23% higher than no liquid overlay.

Grafting is an effective way to increase plant tolerance to biotic and abiotic stressors, it is widely used in watermelon production. However, grafting is labor intensive due to the additional time is required, such as the management of rootstock regrowth. This study used a new grafting tool to destroy (remove) the epidermis of pumpkin and bottle gourd rootstock cotyledon base during grafting, we called this a new grafting method. Compared with the traditional grafting (100%), the new grafting method had significantly lower rate of rootstock regrowth (2-23%), higher watermelon scion dry weight and leaf area. In addition, the time used for the new hole insertion and one cotyledon grafting method to destroy (remove) the epidermis of rootstock cotyledon base (4.2 s/plant, 4.2 s/plant) is significantly shorter than the time required to remove the rootstock regrowth manually in the traditional grafting (9.3 s/plant, 8.8 s/plant). Thus, this study developed a new grafting method for watermelon to inhibit rootstock regrowth and enhance scion growth, and this new method is cost-effective for grafted watermelon seedlings.