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Proceeding of the 4 rd International Symposium“NEW RESEARCH IN BIOTECHNOLOGY” USAMV Bucharest, Romania, 2011and necrosis of plant tips. The plants died after the third month. The content of 4%mannitol in the medium blocked the growth and development of plants. The survival ratewas low (20%) and plants died in the first month after inoculation.mm353025Plant heightRoot length20151050МS0 MS 1% MS 2% MS 3%Concentration of mannitolFig. 2 Effect of mannitol on the plant height and root length of A. montana underconditions of slow growthMultiplication of A. montana plants after prolonged in vitro conservation: In vitro culturescould be effectively maintained for 6 months. The plants were transferred to MS free mediumwithout growth regulator and osmoticum in order to return towards normal growth anddevelopment. The plant growth was restored at a temperature of 22 ± 2 °C and light intensity of40 µMm -2 s -1 . The plants subsequently were micropropagated on MS media containing 1.0 mg/lBAP alone or in combination with 0.1 mg /l IAA after three and six months of storage. Shootsthat had been incubated for 6 months multiplied slowly on multiplication medium under normalgrowth conditions. It was necessary one or two passages of plants subcultivation before startedthe process micropropagation. Basal part of plant tissue slowly expanded and induction of newvegatative buds was started. The increase of the swollen tissues and the formation ofadventitious buds were more obviously in cultures grown on MS medium supplemented with 1mg/l BAP and 0.1 mg/l IAA. On this medium also the higher micropropagation rate wasobserved in comparison with plants cultivated on MS medium supplemented with 1 mg/l BAP.The plants produced 2-3 new shoots (MS medium with 1 mg/l BAP) and 3.5- 4 shoots (MSmedium with 1 mg/l BAP and 0.1 mg/l IAA) during sub-cultivation after three months ofstorage. The micropropagation frequency was significantly lower in plants maintained sixmonths in terms of slower growth than in three months (Table 3). Since the height of the plantswas reduced during storage and main axes of the shoots were in a passive state, when the plantswere transferred to fresh medium for micropropagation they grew into viable buds. The plantswere rooted successfully on ½ MS medium containing 0.5 mg/l IBA for rhizogenesis. Rootswere observed within two weeks after the transfer of rooting medium.60
Proceeding of the 4 rd International Symposium“NEW RESEARCH IN BIOTECHNOLOGY” USAMV Bucharest, Romania, 2011Table 3. Micropropagation of A. montana after prolonged in vitro conservation with mannitolNumber of shoots/explantNumber of shoots/explantAfter three monthsAfter six monthsSubcultivationBAP (1 mg/l) BAP (1 mg/l) + BAP (1 mg/l) BAP (1 mg/l) +IAA (0.1mg/l)IAA (0.1 mg/l)1 0.8±0.12 1.2±0.18 0.6±0.09 0.8±0.132 2.7±0.22 3.5±0.27 1.8±0.14 2.2±0.183 3.4±0.25 4.0±0.26 1.9±0.15 2.3±0.20The protocols of in vitro conservation were developed of few plant species. Mannitol at aconcentrations of 20 g/l added to the medium reduced the growth of rose coloured leadwortplants and prolonged subcultivation eight months (Charoensub and Phansiri, 2004). Sarkar andNaik, 1998 reported that 20 or 40 g/l mannitol increased the survival of potato in vitro plants,but concentration of 60 g/l, caused death of plants. The effect of mannitol for in vitro storage ofpotatoes (Mix, 1985), strawberries (Vysotskaya, 1994), bananas (Van den Houwe et al. 1995)and enset (Negash et al., 2001) was reported.4. CONCLUSIONLong-term storage of in vitro plant material of A. montana under slow growth conditionsensures the development of an effective system for protection and preservation of thisendangered medicinal species. In vitro cultures could be effectively maintained on ½ MSmedium supplemented with 0.5 mg/l IBA and 2% mannitol for 6 months at 20 µMm -2 s -1 lightintensity. After prolonged storage the shoots were multiplied on MS medium supplemented with1 mg/l BAP and 0.1 mg/l IAA. This protocol can be used for in vitro conservation of valuable A.montana clones.ACKNOWLEDGEMENTS. The authors are grateful for the financial support provided by theBulgarian National Science Fund, Ministry of Education, Youth and Science (Project DTK-02/38)REFERENCE1. Ashmore, S.E. (1997): Status report on the development and application of in vitrotechniques for the conservation and use of plant genetic resources. IPGRI, Italy.2. Charoensub, R., Phansiri, S. (2004): In vitro Conservation of Rose ColouredLeadwort:Effect of Mannitol on Growth of Plantlets Kasetsart J. Nat. Sci. Vol.38: 97 - 1023. Fay, M.F. (1994): In what situations is in vitro culture appropriate to plant conservation?Biodiversity and Conservation. Vol. 3:176-183.4. Mix, G. (1985): In vitro preservation of potato genotypes. In: Langhe W, Zeven AC &Hogenboom NG (eds) Efficiency in Plant Breeding Pudoc, Wageningen, The Netherlands,pp 194–195.61
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