In the last decade the climate change, especially drought and high temperatures, has become more frequent and severe in Republic of Moldova and inevitably have a significant impact on agriculture. Therefore, to obtain new tomato varieties it is necessary to use not only classical techniques such as interspecific crossings, but also the application of additional factors to extend variability, a useful method in breeding programs of cultivated plants. In this context, biotechnology has many applications. The advantages of in vitro cultures are widely accepted today, because a small amount of explant source is required and multiplication is very efficient. The aim of the research is focused on the development of an in vitro multiplication protocol for tomato necessary both for obtaining regenerated plants and for obtaining a callus line that can be used later for the continuing investigations. The study included 5 tomatoes varieties that differ by the resistance to low temperatures (2) and drought (2): Iulihirsutian, Anatolie, Jacota, CerryDani and as control - Elvira. In order to obtain sterile tomato seedlings, the basal medium MS ½ was used, supplemented with 10 g of sucrose and 7 g of agar. The culture media used for initiation of callusogenesis and morphogenesis started with the modified basal medium Murashige & Skoog 1962, supplemented with growth factors such as auxins (2,4D, IAA, IBA, ANA) and cytokinins (BAP , TDZ) in various concentrations. The prepared culture media was adjusted to pH of 5.7, added 30 g / L sucrose and solidified with 7 g / L agar, then distributed in pots and autoclaved. Most important environmental conditions in a tissue culture growth room are: temperature: 24 ° C ± 1 ° C; white fluorescent light with an intensity of 2000 lux; photoperiod: 16 hours light and 8 hours darkness; with the relative humidity at 70%. As a result, the optimal culture medium to induce callusogenesis was attested to be basal MS medium supplemented with 2.3 BAP and 1.7 AIA, and for morphogenesis - MS supplemented with 2.3 BAP, 1.5AIA and 0.5DTZ.