Acta Hortic. 1113. ISHS 2016. DOI 10.17660/ActaHortic.2016.1113.40 XXIX IHC – Proc. Int. Symp. on Micropropagation and In Vitro Techniques Eds.: M. Lambardi and S. Hamill 271 In vitro collection methods for Malus shoot cultures used for developing a cryogenic bank in Kazakhstan N.V. Romadanova 1 , S.A. Mishustina 1 , G.N. Matakova 1 , S.V. Kushnarenko 1 , I.R. Rakhimbaev 1 and B.M. Reed 2,a 1 Institute of Plant Biology and Biotechnology, Ministry of Education and Science, Almaty, Republic of Kazakhstan; 2 United States Department of Agriculture, Agricultural Research Service, National Clonal Germplasm Repository, Corvallis, OR, USA. Abstract Initiation of shoot cultures from field-grown plants is often difficult due to the high level of bacterial and fungal organisms that populate trees. This study compared shoot initiation techniques for apples. Shoots were forced from dormant bud wood in the laboratory or directly taken from the field as new growth in the spring. Fourteen promising apple cultivars, five clonal rootstocks (Malus × domestica Borkh.) and 10 wild Malus sieversii (Ledeb. M. Roem.) were introduced into culture. All shoots were treated with 0.1% HgCl 2 for 10 min. Cultures were initiated on liquid Murashige and Skoog medium containing 30 g L -1 of sucrose, 0.5 mg L -1 of 6-benzylaminopurine, 0.01 mg L -1 of indole-3-butyric acid, 1 mg L -1 of gibberellic acid and 1 mg L -1 of L- ascorbic acid, рН 5.7, for 2-4 weeks with daily transfers to fresh medium, and then moved to solid MS medium for propagation. Cultures were indexed for bacteria and fungi using the 523 detection medium and any infected shoots were discarded. Shoot initiation from forced dormant vegetative buds varied from 22.3 to 82.3% with a mean of 55.0% for the 35 genotypes. Shoots, collected from new growth under field conditions, initiated clean shoots with a range from 5.1 to 44.8% and a mean of 18.4% for all genotypes. Shoot cultures were established from all of the tested genotypes. Many of these genotypes had never been cultured before, so this study is strategic for the security of important Malus germplasm. These shoot cultures will be used for the creation of Kazakhstan’s apple cryobank. Keywords: Malus × domestica, micropropagation, aseptic culture, cultivar, rootstock, wild form INTRODUCTION Biotechnological methods for micropropagation of plant tissues and organs on artificial nutrient media is now common practice (Brischia et al., 2002). Micropropagation of valuable cultivars, rootstocks, and unique forms from a small amount of starting material has several advantages compared with conventional vegetative propagation. These include the ability to obtain new shoots throughout the year, not depending on the season, a reduction in time for the breeding process due to the selection of relevant features directly from the in vitro culture and a high rate of reproduction. Using aseptic plants in vitro in international germplasm exchange facilitates the passage of quarantine control, as modern standards for planting material require planting stock free of viral and mycoplasma infection (Brischia et al., 2002; Matushkina, 2008; Pence et al., 2011; Engelmann et al., 2011). Micropropagation involves several steps. In the first place it is the selection of the primary explant, sterilization, and the selection of the optimum cultivation conditions for shoot growth and development on the nutrient medium (Brischia et al., 2002). On the other hand, problems are related to the difficulty of introducing tree crops, especially apples, in aseptic conditions. These problems are associated with high rates of fungal and bacterial infection of the plant material when it is selected in the field, as well as the substantial a E-mail: Reedba@onid.oregonstate.edu