In vitro culture of R. rosea has a history of slightly more than 30 years. First studies were done in the former Soviet Union and most of the publications from the initial period are in the Russian language. Most of these report on the effect of different culture media and different plant hormones on the various types of explants, steril- ization of different explant types, callus induction, organogenesis, and regeneration. From the beginning of the in vitro studies, research was conducted on the production of the most valuable secondary metabolites in in vitro cultures as well. The experi- mental work was mostly organized in a number of research units. The following provides a detailed review of the in vitro work on roseroot.
In 1981, Aleksandrova et al. (cited by Furmanowa et al. 1995) patented the method for root regeneration from callus, but did not provide any data on the cal- lus induction and its maintenance. The only information was that Murashige and Skoog (MS) medium was used for root induction from callus supplemented with sucrose (2.5%–3.5%), thiamine, HCl, mesoinositol, NAA (0.8–1.1 mg/L), adenine (0.08–0.013 mg/L), and kinetin (0.01–0.1 mg/L) and 25–28 days were necessary for root formation. First scientific publications about callus induction appeared in the mid-80s (Poletaeva et al. 1984; Baev 1984; Bykov 1986; all cited by Bykov et al. 1999). The explants for callogenesis were sterile-growing 40- to 50-day-old plant- lets. For callus induction MS medium was used both under continuous illumination and in the dark.
7.2.1 explantsFOr in VitRo culture
Leaves of in vitro plantlets derived from seed culture are the explants used the most often and with the most success (Tasheva and Kosturkova 2010, 2012). Furmanowa et al. (1995) germinated immature seeds after being washed in running water for 1 hour, dipped into 70% ethanol for 1 minute, soaked in 5% solution of calcium hypo- chlorite for 10 minutes and finally rinsed three times with sterile water. Ishmuratova (1998) used both seeds and buds successfully for in vitro culture establishment. The sterilization protocol for both included 1 minute in 70% ethanol and either 5 minutes in 3% hydrogen peroxide or 7 minutes in aqueous 0.1% mercuric chloride. The ster- ilization procedure of Martin et al. (2010) included a bath of 3 minutes in 70% etha- nol, followed by 20 minutes in 10% chlorinated lime and finally 20 minutes in 10% sodium hypochlorite. Tasheva and Kosturkova (2010) conducted a broad study with 6 schemes for sterilizing not only seeds but also bud, shoot, or rhizome segments. For seed decontamination, 3 minutes in 70% ethanol followed by 15 minutes of 20% (v/v) bleach is recommended by them. Also apical buds with 1 minute in 70% ethanol, 20 minutes in 15% bleach, and 15 minutes in 0.2% mercuric chloride and rhizome buds with 1 minute in 70% ethanol, 17 minutes in 20% bleach were successfully sterilized and further developed. Ghiorghita et al. (2011) reported about difficulties during decontamination of roseroot explants. This fact was previously concluded by Tasheva and Kosturkova (2010) also. Mature rhizome buds, apices, and shoot segments of young plants were treated with short immersion in 5% chloramine-T solution. Only those explants remained viable after the sterilization process, which
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were previously submitted to low temperature. Based on these reports, a general protocol cannot be determined. The different working groups have contradictory results; what worked for one did not necessarily work for the other. The genotype and the original environment of the plant probably have a major effect on the success of the decontamination.
7.2.2 micrOprOpagatiOn
Micropropagation of R. rosea is in the focus of research from the 1990s. Furmanowa et al. (1995) gave the first report on micropropagation. In their study, the most effec- tive medium for plant development from shoot tips was the one of Nitsch and Nitsch (NN) supplemented with 0.1 or 1 mg/L kinetin or 0.01 mg/L NAA along with 0.1 mg/L IAA. When NAA in higher concentrations (1 mg/L) was added to the medium, the growth was completely stopped. Ishmuratova (1998) conducted micro- propagation of roseroot on MS medium containing 0.2 mg/L BAP and 0.1 mg/L IAA. MS medium containing higher levels of BAP and lower levels of NAA was also found to be the most efficient by Yin et al. (2004).
In 2009, Debnath reported the application of RITA temporary immersion system for the micropropagation of R. rosea (Debnath 2009). Thidiazuron (TDZ) and zeatin were used as growth regulators. TDZ at 2–4 µM stimulated shoot induction, but inhibited shoot elongation. It was also observed that the three studied clones differed significantly with respect to the multiplication rate. In the RITA system, 0.5 µM TDZ sustained rapid shoot proliferation, but at higher concentration induced hyper- hydricity. These hyperhydric shoots produced normal shoots within 4 weeks when transferred to solid MS medium containing 1–2 µM zeatin. Tasheva and Kosturkova (2010) tested a wide range of plant hormones for inducing organogenesis. The best bud formation was achieved on MS medium supplemented with 2 or 0.2 mg/L zeatin along with 0.2 mg/L IAA in case of stem segments with leafy nodes, whereas multi- plication of the developed shoots was the most efficient on MS medium with 1 mg/L zeatin and 0.2 mg/L IAA. Ghiorghita et al. (2011) also studied the possibilities of micropropagation. According to them, the most efficient combination of hormones on MS medium is 0.2 mg/L IAA and 2 mg/L zeatin.
Controversial results have been published about rhizogenesis. According to Ghiorghita et al. (2011), the presence of NAA in the medium leads to the most intense rhizogenesis, but the combination of 0.2 mg/L IAA and 2 mg/L zeatin or 0.5 mg/l NAA and 1 mg/L kinetin is also favorable for root induction. Meanwhile, based on the work of Tasheva and Kosturkova (2010), the presence of NAA induced callogenesis not rhizogenesis. According to them, the most effective root induction was found on medium supplemented with 0.2 mg/L IAA, 2 mg/L IBA, and 1 or 0.4 mg/L giberrelic acid; however, even on simple ½ MS, 58% of the shoots rooted.
Recent studies concerning micropropagation published by Romanian and Bulgarian groups (Ghiorghita et al. 2011; Tasheva and Kosturkova 2010) aim to restore and repopulate the natural habitats of this species, which is endangered in these countries. This strategy might cause a severe genetic drift effect in the natural populations. On the other hand, micropropagated plantlets could be a way to produce uniform material for establishing roseroot plantations.
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7.2.3 aDaptatiOnOF micrOprOpagateD plants
The acclimatization of in vitro propagated plants is always a critical point. Ishmuratova (1998) used a two-step protocol for acclimatization. Plantlets were transplanted into vermiculite for 2 weeks in 85%–90% relative humidity and then to a 1:1:1 mixture of soil, humus, and vermiculite. Tasheva and Kosturkova (2010) first planted the micropropagated plantlets into a mixture of 1:1:2 perlite, peat, and soil in 90% relative humidity. The survival rate was 57%. After 2–3 months, plants were transferred from the adaptation room to greenhouse with a survival rate of 85%. After 6 months, plants were planted in the field and more than 70% survived the winter. Ghiorghita et al. (2011) accommodated the in vitro plantlets in a hydroponic system for a week. This stage was survived by more than 90% of the plantlets. The plantlets were then transferred into soil pots and later planted to their natural habitat to the wild. The first summer was survived by 73.5% of the plants, whereas after the first winter, the percentage of survivors dropped to 57%. Also György and Trócsányi (2012) worked with micropropagated plant material. According to their experience, the key to successful adaptation is the sufficient amount of water. In vitro plantlets were planted in 1:2 mixture of perlite and peat and were directly transferred into the greenhouse. After 3 months, the pots were placed to open-air conditions for the winter. The percentage of surviving plants was 70%.
7.2.4 callus inDuctiOn
The utilization of callus cultures for the production of the bioactive agents is con- sidered as an alternative way, which is faster and independent from environmental conditions. In case of callogenesis, the results of the different working groups are more consistent than in case of micropropagation. Callus culture has been obtained on MS medium supplemented with different plant hormones. Furmanowa et al. (1995, 1998) concluded that callogenesis is the most effective from leaf explants on BAP along with medium containing NAA or IBA or IAA. The best combination for induction and growth of callus was BAP and IBA. Two strains of callus were described: a deep green and a light cream strain. György et al. (2004, 2005) used also leaf explants and the combination of 1.5 mg/L BAP and 0.5 mg/L NAA. Krajewska- Patan et al. (2007a,b) induced callus from hypocotyls of in vitro seedlings on MS medium supplemented with also BAP, NAA, and adenine chloride. Martin et al. (2010) used epicotyls for callus induction and a liquid medium, rather than solid. The most effective combination was found to be 1 mg/L 2,4-D with 1 mg/L IBA or 0.1 or 1 mg/L 2,4-D alone. The main aim of Tasheva and Kosturkova (2010) was the micropropagation of roseroot, but during their thorough experiments, callus- inducing medium composition was also described. Apical buds on MS medium containing 0.2 mg/L BAP and 0.1 mg/L IAA formed a compact, green callus. Leaf explants on medium containing zeatin (2 or 0.2 mg/L zeatin and 0.2 mg/L IAA) produced a poor growing soft callus. Seventy-eight percent of leaf segments cul- tured on medium containing 2-iP (3 mg/L 2-iP and 0.3 mg/L IAA or NAA) formed compact green callus while 22% formed pale and friable callus. The two previously mentioned strains of roseroot callus were also observed by Ghiorghita et al. (2011).
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A compact, green callus was generated on MS medium supplemented with 1 mg/L BAP and 0.5 mg/L 2,4-D from internode fragments, whereas a semi-compact cream color callus developed from leaf fragments inoculated on MS medium with 1 mg/L BAP and 0.5 mg/L 2,4-D.
In 2004, György et al. published an article on the initiation and cultivation of compact callus aggregates (CCA). To establish suspension culture of CCAs, callus from the solid media was gently broken using forceps and was transferred into liquid MS medium containing 0.5 mg/L BAP and 1 mg/L NAA and shaken on a gyratory shaker at 135 rpm. Subcultures were carried out in every 8–10 days by decanting all medium from the flask and adding fresh medium to the cultures. CCA culture was composed of green or light green, spherical, smooth surfaced callus aggregates as described previously by Xu et al. for Rhodiola sachalinensis. CCAs were used later in a number of studies detailed later in Sections 7.3.1 and 7.3.2 (György et al. 2005; György 2006; Krajewska-Patan et al. 2007a,b; György and Hohtola 2009).
7.3 BIOTECHNOLOGICAL METHODS FOR INCREASING