In Vitro Clonal Propagation and Germplasm Conservation in the Tropical Timber Tree Spanish White Cedar (C. Montana Moritz Ex Turcz.

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1 In Vitro Clonal Propagation and Germplasm Conservation in the Tropical Timber Tree Spanish White Cedar (C. Montana Moritz Ex Turcz.) (Meliaceae) 1 Gabriela Díaz-Quichimbo, 1 Ruth Poma-Angamarca, 1 Julia Minchala-Patiño, 1 Darlin González-Zaruma, 2 Consuelo Rojas Idrogo and 2,* Guillermo E. Delgado Paredes 1 Laboratorio de Micropropagación Vegetal, Área Agropecuaria y de Recursos Naturales Renovables, Universidad Nacional de Loja, Ecuador. 2 Facultad de Ciencias Biológicas, Universidad Nacional Pedro Ruiz Gallo, Ciudad Universitaria, Juan XXIII 391, Lambayeque, Perú. * guidelg2001@yahoo.es ABSTRACT In Ecuador and Peru Cedrela montana is an important species in both economic and ecological terms and their wood is precious and reported to be highly resistant material. In vitro techniques and clonal propagation can help to develop new plantations and assist in establishing improvement programs for this species. Findings include percentage of germination of seeds and contamination, induction of buds, rooting and germplasm conservation. Seeds germination was 72% in Murashige and Skoog (MS) basal medium supplemented with 2.0 mg/l GA 3 and the fungal and bacterial contamination was 20%. Shoot development was optimized by cultivating in vitro apical bud and node explants in MS basal medium supplemented with 2.0 mg/l BAP. Rooting was also stimulated 2 months after individualization of the propagated plants in MS basal medium supplemented with 1.0 mg/l IBA or 0.5 mg/l IAA, with a mean of 2.4 and 1.4 roots per plant, respectively. The best treatments on germplasm conservation were in 6% mannitol and 0.1 mg/l ABA. Although growth of both mannitol and ABA was consequently inhibited for 6 months of conservation, the re-growth ability was successfully obtained when transferred to MS with ABA 0.5 mg/l and IBA 1.0 mg/l. In vitro plants did not show morphologic differences when compared to ex vitro seeds. Keywords: Cedrela montana, Clonal propagation, Germplasm conservation, In vitro, Tissue culture. 1. INTRODUCTION Tropical hardwood tree species are important economically and ecologically, and play a significant role in the biodiversity of plant and animal species within an ecosystem, similar to temperate hardwood tree species [1, 2]. One of the more well-known tropical tree species grown or harvested as timber for wood included Spanish white cedar. C. montana Moritz ex Turcz. (Cedar, white cedar, Spanish white cedar), is a tropical timber species with a natural distribution from southern Mexico to northern Argentina [3]. C. montana, and others Meliaceae species as Swietenia macrophylla and C. odorata, is a high-value timber species that has been considered vulnerable because of intense exploitation of its natural populations and the lack of intensive propagation systems allowing the establishment of commercial plantations [4, 5]. In the Americas, the development of hardwood plantations of the Meliaceae family is limited because of the long life cycles of these species, their susceptibility to pest attack, and the lack of farmer interest to plant species with profits in the long term [6, 7]. In addition, the attack from Hypsiphyla grandella Seller (Lepidoptera, Pyralidae) have led to serious deterioration of the species. Both, C. odorata and C. montana has been extensively used by the fine-furniture and construction [8], consequently, the demand for this forest resources, which fetches high prices on the world market, is second only to mahogany (Swietenia macrophylla, other tropical Meliaceae) [9]. This demand and uncontrolled overextraction from natural populations during the last 200 years [10] has led to these species, S. macrophylla, C. odorata and C. montana, are included in the CITES list as endangered species and the International Union for the Conservation of Nature (IUCN) as species facing a high risk of extinction in the wild within the medium-term future. Natural cedar is propagated from seed in many parts of Central and South America, but good initial growth is often followed by dieback after 2-3 years; moreover, cedar does not coppice readily nor produce root suckers. Which is why the use of modern in vitro techniques on woody trees has brought new possibilities not only for rapid tree species multiplication, producing homogeneous clonal planting stock useful for commercial plantations, forest restoration [11], and woody biomass production, but also for ex situ conservation and rejuvenation of elite or rare germplasm [12, 13, 14]. However, the in vitro propagation of adult forest trees is experiencing many difficulties in the various steps of the process, including, in particular, extremely high contamination levels in the first explants affecting the efficiency of morphogenesis and plant recovery, and high emission of polyphenols and tannins that 59

2 impede the explants development, interfere with morphogenetic processes, and frequently cause necrosis, vitrification and a deficient rooting capacity [15, 16]. In general, the application of tissue culture on mass clonal propagation of plants is the widest utilization of this technology [17]. As regards seed preservation possibilities, plant species have been divided into two categories [18]: orthodox seeds, which can withstand dehydration to 5% or less (dry weight basis) without damage; when dry, the viability of these seeds can be prolonged by keeping them at the lowest temperature and moisture possible, and recalcitrant seeds, which are high in moisture and are unable to withstand much desiccation; these seeds remain viable only for a short time (weeks or months), even if kept in the required moisture conditions and are predominantly seeds from tropical or subtropical species. In the other hand, in situ conservation has been made almost impossible due to the disappearance of large wild areas, and the conservation ex situ is very difficult to carry out due to several problems [19]. For this, the use of in vitro tissue culture techniques can be of great interest for germplasm collection, storage and multiplication of recalcitrant and vegetatively propagated species; these systems present the following advantages: very high multiplication rates, aseptic system, reduction of space requirements, genetic erosion reduced to zero under optimal conditions, and reduction of the expenses in labour costs; likewise, tissue culture systems greatly facilitate the international exchange of germplasm [19]. It facilitates the availability of planting materials at any time, avoids the transfer of major pests and pathogens, and makes possible virus eradication through meristem culture [20]. In addition, in vitro conservation is less expensive than cryopreservation of field-grown clonal materials [21]. Advances in plant micropropagation of C. odorata are not still in depth [22], and discrete results for C. odorata micropropagation using buds and nodal segments, isolated from juvenile plants generated from seeds, have already been achieved [6, 15, 22, 23, 24, 25, 26, 27]. Recently, some work has been done to develop a protocol to establish in vitro conditions and micropropagation of this species from nodal explants taken from juvenile cuttings field trees [7], rejuvenation of elite mature individuals by ex vitro grafting of mature tree twigs onto 3-month-old juvenile trees [28], and single nodal explants taken from stems of 10-year-old plants, which were maintained in a greenhouse [29]; however, reproducible protocols have not yet been established. To date there are no reports that have been published in vitro cultures of C. montana; therefore, this study was realized to develop a protocol for in vitro establishment and micropropagation of C. montana from seeds, and since there is no genetic improvement program for this species, it seemed necessary to initiate studies with young material. 2. MATERIALS AND METHODS Plant materials: Elite 30-year-old C. montana trees were selected from natural plantations around of the Parque Universitario de Educación Ambiental y Recreación Francisco Vivar Castro (PUEAR), Loja, Ecuador (04 o and 04 o S; 79 o and 79 o W). The selection was based on the trees anatomical features: straight trunks at least 8-12 m long and cm in diameter. Mature fruits were collected from three and five of these trees between August and May in both 2010 and This material was immediately transferred to the laboratory until that spontaneous opening occurred, and mature seeds were released. The resulting seeds were kept at room temperature in a black plastic bag for a maximum period of 2 months. Seeds disinfestation: Seeds were disinfested by immersing them in a 70% (v/v) ethylic alcohol for one min and in 5.25% (v/v) sodium hypochlorite solution (Clorox ) supplemented with 1-2 drops of polyoxyethylene sorbitan monolaurate (Tween 20 ) for 10 min, followed by three rinses of 1 min each with sterile, distilled water. Experimental design: In the first experiment, the disinfested seeds were inoculated and germinated aseptically in the induction medium containing the nutrients of the MS formulation [30] supplemented with three concentrations of GA 3 (0.1 and 2.0 mg/l). In the second experiment, to sprouting of tips and nodes, three types of cytokinins (BAP, KIN and 2iP) were applied in three concentrations (0, 1 and 2.0 mg/l) were tested. In the third experiment rooting of tips and nodes, two concentrations of MS salts (full MS and 50% MS-with 100 and 50% concentration of inorganic salts, respectively and two types of auxins (IBA and IAA), applied at three concentrations (0, 0.5 and 1.0 mg/l) were tested in multifactorial combinations using a completely randomized block design. In the fourth experiment, for germplasm conservation, two types of explants (apical buds with roots and apical buds without roots) and two concentrations of MS salts (full MS and 50% MS-with 100 and 50% concentration of inorganic salts, respectively 60

3 were assessed in factorial combinations with two concentrations of ABA (0.1 and 0.5 mg/l) and two concentrations of mannitol (40 and 60 g/l), respectively. In a separate experiment, after 6 months storage without subculture, stored s under slow growth conditions were transferred to MS medium supplemented with the basal medium supplemented with ABA (0.5 mg/l), IBA (1.0 mg/l) and ABA-IBA ( mg/l) and cultured for 8 weeks. Shoots from all treatments were taken from the culture vessels and planted in potted soil hereafter. In all experiments, the basal MS medium was supplemented with 20 g/l sucrose, 1.0 mg/l thiamine.hcl, 100 mg/l myo-inositol and 7 g/l agar. Culture conditions: The ph of all media was adjusted to 5.8 ± 0.1, with KOH and HCl, prior to autoclaving at 121 o C at 105 kpa for 20 min. Twenty millilitre of medium was poured into 200 ml tissue culture flasks closed with polypropylene screw-on lids. Two tip or nodes explants, with an average length of 25 ± 5 mm (mean SD) were cultured per flask. Each treatment comprised 15 explants and was performed twice. The experiments were evaluated every 30 days, for 120 days. Seed germination and all of the subsequent culture steps were carried out in a culture room at 25 ± 2 o C with a photoperiod of 16 h light (70 µmol m -2 s -1 ) and 8 h dark, and a relative air humidity of approximately 70%. After 6-8 weeks, the emerging seedlings were used as explants for medium optimization experiments. Statistical analysis: Results were processed and analyzed by analyses of variance (ANOVA) and the Tukey HSD multiple range test (p 0.05) in order to compare treatment means. All the statistical analyses were carried out the Statgraphics Plus 5.0 software (StatPoint, Warrenton, Virginia, USA). 3. RESULTS AND DISCUSSION Seeds germination: The results of the C. montana seed treatments are presented in Table 1. The germination of seeds without GA 3 was 50%. The remotion of the wing end portion of the seed and disinfection it in a 5.25% (v/v) sodium hypochlorite solution for 10 min did neither contribute to increase the clean material obtained nor improved the germination percentage. It was necessary to supplement the culture medium with GA and 2.0 mg/l, to increase germination in 62% and 72%, respectively. The not germinated seeds showed the same in vivo behavior of the radicle emergence, without development of the aerial portion i.e. seeds did not germinate completely. On seed germination, radicle emerges in days and complete expansion of cotyledons between 20 to 22 days; in 30 days of culture the length was cm. Phytotoxicity symptoms, as a result of sodium hypochlorite treatment, were also observed as tissue depigmentation with progressive discoloration. In similar study, in in vitro seeds germination of Cedrela odorata was cent per cent; however, the material was contaminated by a bacterium, reducing the number of explants available for trials [15]. Moreover, seeds of some species of Meliaceae have shown antibacterial and antifungal activity. In fact, seeds from C. fissilis put to germinate on synthetic medium made out of the saline formulation of MS diluted to half of its strength and solidified with agar exuded substances into the substrate, which inhibited the growth of fungi normally found as contaminant in tissue culture of explants; was determined that those substances can inhibit the growing of hyphae of Fusarium spp., Phytophthora spp., Penecillium spp., Rhizopus spp., Mucor spp., and yeast; likewise, preliminary analysis indicate bacterial, staphylococcus and streptococcus growth inhibition [31]. It is possible that the biosynthesis of these substances constitute a chemical adaptation of the species to survive in adverse conditions [32]. In the case of C. montana showed the presence of a dark amber halo around the seed in vitro which somehow determine the percentage of contamination was about 20%. It has been speculated that these substances would be associated with sulfur compounds, phenolic acids, nitrogen heterocyclic compounds and / or quaternary amines [31]. Shoot elongation in apical bud and nodal segment explants: After the explants were cultured for a total of 120 days, in apical bud the results showed significantly (P 0.05) higher elongation, number of s, length, number of leaves and nodes per in the treatment with MS medium supplemented with 2.0 mg/l BAP compared to the other treatments. The concentration of 2.0 mg/l BAP was the most effective treatment, generating an average of 3.0 new s per explants and 25.8 mm of elongation; however to increase in elongation in the 2.0 mg/l BAP treatment was not significant (P 0.05) compared to the other treatments. Additionally, treatments with 1.0 and 2.0 mg/l BAP induced the best leaf formation; however to increase in number of leaves formation was not significant (P 0.05) compared to the other 61

4 treatments. Statistical analysis revealed that the cytokinins had a significant effect on growth and development of C. montana plantlets, and the BAP and KIN were superior to the 2iP in number of and length parameters (Table 2). In nodal segment explants the results were very similar (Table 3). Adventitious regeneration (via organogenesis or somatic embryogenesis) through a callus stage (e.g. from zygotic embryos, anthers, meristems, cotyledons, hypocotyls, petioles, leaves, stem internodes or root segments) may not be desirable for clonal propagation of a species because of the possibility of somaclonal variation; however, the development of an efficient organogenesis and somatic embryogenesis protocol in forest species is extremely valuable and needed for genes transfer. Traits such as insect resistance, abiotic stress tolerance, growth speed, and biomass gain are key elements to the genetic improvement of these species that potentially could lead to the establishment of cost-effective commercial plantations [33]. In the multiple adventitious formation in Spanish red cedar (Cedrela odorata) cultured in vitro using juvenile and mature tissues, the concentrations of (3.0 mg/l) 13.5 µm BA was the most effective treatment in the absence of coconut water (CW), generating an average of 2.7 new s per explant. In the presence of 20% CW, none of the cyokinins (BA, 2iP or ZEA) increased the number of s formed; however, at (10 mg/l) 45 µm BA, the same supraoptimal reduction was observed [28]. In our study, 2.0 mg/l BAP, the maximum concentration tested, new s induced per explant was 3.0. These observations are common in tissue culture and can be explained by the presence of a very complex cocktail of cytokinins and other compounds in CW, which makes it irreproducible [34]. In another study on in vitro propagation of C. odorata, the microcuttings from the apical buds of the seedlings showed a somewhat slow in vitro growth, and a good leaf area development. No significant differences were found (p 0.05) among the BAP concentrations ( mg/l); however, an average with of 2.1 axillary buds per explants and a better appearance of the buds was obtained with 0.3 mg/l BAP [15]. These results differ significantly from those reported in our work where 2.9 and 3.0 s were formed at 1.0 and 2.0 mg/l BAP, respectively. Rooting in apical bud and nodal segment explants: When apical bud and nodal segment explants were transferred to regeneration media for root formation (Table 4), media containing MS half-strength concentration and 1.0 mg/l IBA or MS full-strength concentration and 0.5 mg/l IAA could develop roots within 4 months indicating that auxins enhanced root formation. In nodal segment explants the results were very similar (Table 5). Success in micropropagation is dependent on the production of good quality adventitious roots, whose formation has four distinct phases namely, cell dedifferentiation, induction, root primordial developmental and root emergence [35]. In Cedrela odorata the apical buds of the seedling showed a higher percentage of rooted stems in 1.0 mg/l, although the average number of roots per stem was higher with 6.5 roots per stem in 2.0 mg/l IBA; however, the root distribution was better in 1.0 mg/l IBA [15]. In our study, the number of roots formed was significantly lower in MS medium with half-strength or full-strenght IBA with IAA. Germplasm conservation: Growth of s, in both apical buds with roots and apical buds without roots, cultured in medium containing mannitol and ABA was markedly suppresses when compared with those in controls (Table 6 and 7). After 6 months of culture, the best treatment of conservation, in apical buds with roots, was MS full-strength supplemented with mannitol 6% and, in apical buds without roots, was MS half-strength supplemented with mannitol 6%. In these treatments the elongation was 49 and 32 mm, respectively, while the control treatment was 79 mm; in all treatment assays with mannitol the survival rate was 60-70% (Table 6). In the treatments with ABA, in both apical buds with roots and apical buds without roots, the best treatment of conservation was MS half-strength supplemented with ABA 0.1 mg/l; in these treatments the elongation was 60 and 39 mm, respectively, and the survival rate was also 60-70%. In the re-growth process, after 6 months of conservation, the best treatment was MS medium supplemented with ABA 0.5 mg/l and IBA 1.0 mg/l, during 8 weeks of culture; in this treatment, the elongation and the survival rate were 51 mm and 83%, respectively (Table 8). In the in vitro germplasm conservation the methods employed are different, depending on the storage duration requested. For short and medium term storage, the aim is to reduce the growth and to increase the intervals between subcultures (growth limitation method). This is achieved by modifying the culture conditions, mainly by lowering the culture temperature and drastic changes in the composition of the culture medium; however, the risks of the genetic variation increases with in vitro storage duration, and can lead to the loss of trueness to type. 62

5 The addition of osmoticums, such as mannitol or sorbitol, to the medium has proved efficient for reducing growth rates of different plant species. These osmoticums reduce mineral uptake by cell through differences in osmotic pressures thereby retarding plant growth [36]. For instance, the addition of mannitol, a substance with osmotic properties, reduces significantly the growth of Colocasia esculenta and Xanthosoma brasiliense s [37]. Accesions of the potato collection at CIP are conserved in a conservation medium containing 4% sorbitol at a temperature of 6-8 o C, and light intensity of 1000 lux [38]. The use of sorbitol as an osmoticum is applied to many crops without any physiological changes such as callus formation or vitrification, undesirable reactions produced when the media contains mannitol, which can affect potato genetic stability [39]; however, in other studies, the growth of potato is reduced by the addition of 4% mannitol and the same cultures can withstand storage at a lower temperature, thus extending the possible storage duration, if 3% sucrose and 4% mannitol are added to the culture medium [40]. Mannitol at 2-4% has also been used in sweetpotato to extend the interval between subcultures to 1 year [41] but some callus formation and vitrification was reported. In addition, sweetpotato plantlets can remain in 1-4% sorbitol for 6-12 months without subculture [42], sorbitol, however, can be metabolized by the plantlets after some months of storage and exhibit an incremental growth rate, effectively reducing storage time [38]. In cassava (Manihot esculenta) s deteriorate in presence of mannitol, even at 0.1% and with a storage temperature lower than 20 o C [43]. In woody species, the in vitro germplasm conservation of mangosteen (Garcinia mangostana) and longkong (Lansium domesticum), the major fruit crops cultivated in the south of Thailand, was investigated by suppression of growth using growth retardants and increasing the osmotic potential of culture medium. In this study, severe succulence of mangosteen was observed when s were cultured in a medium containing mannitol 1-4% [44]. On the other hand, some growth retardants as abscisic acid (ABA) can be used in order to reduce the growth of s of potato [43, 45]; however, these authors indicate that ABA is detrimental to some varieties. In sweetpotato, although plantlets cultured in growth retardants may have survival rates ranging from 70 to 90%, genotypic effects or toxic effects are seen. For example, sweetpotato plants grown in a medium containing ABA at 5-20 mg/l had a survival rate of 70-85% after 8 months, but showed strong genotypic effects [46]. In G. mangostana and L. domesticum ABA 2.0 mg/l affected to suppress growth of both species, and although grown was consequently inhibited for 12 months of conservation, the re-growth ability was successfully obtained when transferred to proliferation medium [44]. with GA 3 Table 1. Effect of the gibberellic acid (GA 3 ) in in vitro germination of C. montana seeds. Germinated seeds Morphogenic responses (%) Fungal and bacterial contamination (%) + - Emergence of the radicle (days) Emergence of the cotyledons (days) Seedlings length after 30 days Control 50.0 a 50.0 a 18.0 a 13.0 a 22.0 a a b 38.0 b 22.0 a 11.0 a 20.0 a a c 28.0 c 20.0 a 10.0 a 20.0 a b Table 2. Effect of several cytokinins on morphogenic responses induction (buds) in apical bud explants of C. montana. s Shoot length leaves per nodes per Shoot elongation Control 1.3 ± 0.2 d 2.2 ± 0.0 b 3.0 ± 0.3 a 1.3 ± 0.5 a 18.0 ± 2.5 a BAP ± 0.2 b 5.0 ± 0.3 ab 3.0 ± 0.6 a 2.3 ± 1.0 a 19.0 ± 2.6 a BAP ± 0.4 a 6.0 ± 0.5 a 3.0 ± 0.3 a 2.5 ± 1.1 a 25.8 ± 2.6 a KIN ± 0.3 bcd 4.3 ± 0.9 ab 3.0 ± 0.5 a 1.8 ± 0.8 a 26.8 ± 2.0 a KIN ± 0.2 bc 4.5 ± 0.3 ab 3.0 ± 0.4 a 2.1 ± 0.7 a 21.3 ± 2.9 a 2iP ± 0.3 ab 4.2 ± 0.5 ab 4.0 ± 0.5 a 2.4 ± 1.1 a 20.8 ± 2.9 a 2iP ± 0.3 cd 2.2 ± 0.7 b 3.0 ± 0.2 a 1.7 ± 0.6 a 15.3 ± 1.8 a 63

6 Table 3. Effect of several cytokinins on morphogenic responses induction (buds) in nodal segment explants of C. montana. s Shoot length leaves per nodes per Shoot elongation Control 1.3 ± 0.2 b 2.8 ± 0.8 a 2.0 ± 0.7 a 1.3 ± 0.2 a 17.0 ± 2.1 a BAP ± 0.2 a 4.9 ± 2.0 a 3.4 ± 0.5 a 2.3 ± 1.2 a 20.0 ± 2.0 a BAP ± 0.2 a 4.7 ± 0.5 a 2.6 ± 0.3 a 1.8 ± 0.2 a 23.6 ± 0.5 a KIN ± 0.3 a 4.8 ± 1.8 a 3.2 ± 0.5 a 2.3 ± 0.3 a 22.0 ± 1.8 a KIN ± 0.2 ab 5.6 ± 3.0 a 3.0 ± 0.3 a 2.0 ± 0.2 a 23.3 ± 3.0 a 2iP ± 0.2 a 5.0 ± 2.2 a 3.2 ± 0.5 a 2.0 ± 0.3 a 24.0 ± 2.2 a 2iP ± 0.0 ab 3.8 ± 1.2 a 2.2 ± 0.2 a 1.6 ± 0.2 a 15.6 ± 1.2 a Table 4. Effect of several auxins on morphogenic responses induction (roots) in apical bud explants of C. montana. roots per Root length s leaves per nodes per Shoot elongation Testigo 0.0±0.0 b 0.0±0.0 a 1.0±0.0 b 0.3±0.2 c 1.3±0.2 ab 15.1±0.15 b MS IBA 0.7±0.4 ab 7.8±7.3 a 1.3±0.2 b 3.3±0.4 ab 2.0±0.0 ab 28.7±0.0 a MS ½ ±0.8 ab 15.3±9.9 a 1.0±0.0 b 2.3±0.8 bc 1.7±0.2 ab 18.3±0.2 ab IBA MS IBA 0.6±0.3 b 3.5±1.9 a 1.0±0.0 b 1.5±0.3 bc 1.5±0.2 ab 21.3±0.2 ab MS ½ ±0.9 a 30.6±11.4 a 1.1±0.1 b 3.8±0.9 ab 2.9±0.7 a 26.9±0.7 a IBA MS IAA 1.4±1.2 ab 16.6±9.5 a 1.9±1.1 a 5.3±1.2 a 2.6±0.5 ab 27.7±0.5 a MS ½ ±0.4 b 0.0±0.0 a 1.0±0.0 b 1.5±0.4 bc 1.8±1.2 ab 23.4±0.2 ab IAA MS IAA 0.0±0.5 b 0.0±0.0 a 1.0±0.0 b 2.6±0.5 ab 1.4±0.2 ab 26.6±0.2 a MS ½ IAA 0.0±0.2 b 0.0±0.0 a 1.0±0.0 bc 1.4±0.2 b 1.0±0.0 b 14.4±0.0 b Table 5. Effect of several auxins on morphogenic responses induction (roots) in nodal segment explants of C. montana. Number of roots Root length s leaves per nodes per Shoot elongation per Control 0.0±0.0 a 0.0±0.0 a 1.0±0.0 b 0.0±0.0 b 1.3±0.0 ab 16.7±0.0 c MS + IAB ±0.0 a 0.0±00 a 1.0±0.0 b 0.0±0.0 b 2.0±0.0 ab 0.0±0.0 b MS ½ + IBA ±0.0 a 00±0.0 a 1.0±0.0 b 2.7±0.6 ab 1.7±0.0 ab 20.3±0.0 abc MS + IBA ±1.1 a 13.8±8.7 a 1.6±0.3 ab 3.6±0.8 ab 1.5±0.7 ab 27.5±0.3 a MS ½ + IBA ±0.4 a 26.0±13.8 a 1.5±0.2 ab 4.3±1.9 a 2.9±1.0 a 26.7±0.2 ab MS + IAA ±0.1 a 1.1±1.1 a 1.3±0.2 ab 2.2±0.6 ab 2.6±0.2 ab 24.0±0.2 abc MS ½ + IAA ±0.3 a 20.0±13.6 a 1.9±0.1 a 3.0±0.7 ab 1.8±0.3 ab 25.6±0.1 abc MS + IAA ±0.0 a 0.0±0.0 a 1.0±0.0 b 0.0±0.0 b 1.4±0.0 ab 0.0±0.0 a MS ½ + IAA ±0.0 a 0.0±0.0 a 1.0±0.0 b 1.0±0.6 ab 1.0±0.2 b 17.6±0.0 bc 64

7 (%) Table 6. Effect of mannitol on in vitro conservation of C. montana. Shoot Survival elongation roots per leaves per nodes per (%) Explant with roots Control 79.0 ± 3.5 a 2.0 ± 0.6 a 5.0 ± 0.4 a 4.0 ± 0.2 a 60.0 MS + Mannitol 55.0 ± 2.2 b 1.0 ± 0.2 ab 2.0 ± 0.7 b 2.0 ± 0.2 bc MS ½ ± 3.2 b 1.0 ± 0.2 ab 2.0 ± 0.8 b 2.0 ± 0.0 bc 50.0 Mannitol 4.0 MS + Mannitol 49.0 ± 2.8 b 1.0 ± 0.2 ab 2.0 ± 0.0 b 1.0 ± 0.0 b MS ½ ± 3.1 ab 1.0 ± 0.0 b 2.0 ± 0.1 b 2.0 ± 0.1 c 70.0 Mannitol 6.0 Explant without roots Control 51.0 ± 2.7 a 3.0 ± 0.4 a 4.0 ± 0.9 a 3.0 ± 0.3 a 60.0 MS + Mannitol 4.0 MS ½ + Mannitol 4.0 MS + Mannitol 6.0 MS ½ + Mannitol ± 2.3 b 0.0 ± 0.0 b 2.0 ± 0.0 b 2.0 ± 0.0 a ± 1.5 b 0.0 ± 0.0 b 2.0 ± 0.5 b 1.0 ± 0.2 b ± 1.1 b 0.0 ± 0.0 b 1.0 ± 0.4 b 1.0 ± 0.2 b ± 0.9 b 0.0 ± 0.0 b 0.0 ± 0.0 b 1.0 ± 0.0 b 70.0 Table 7. Effect of ABA on in vitro conservation of C. montana. Shoot elongation roots per leaves per nodes per Survival (%) Explant with roots Control 79.0 ± 0.4 a 2.0 ± 0.3 a 5.0 ± 0.4 a 4.0 ± 0.2 a 60.0 MS + ABA ± 0.6 ab 2.0 ± 0.3 a 3.0 ± 0.5 a 2.0 ± 0.2 b 60.0 MS + ABA ± 0.6 b 1.0 ± 0.6 a 3.0 ± 1.0 a 2.0 ± 0.3 b 60.0 MS ½ + ABA 60.0 ± 0.3 b 2.0 ± 0.6 a 3.0 ± 0.5 a 2.0 ± 0.2 b MS ½ + ABA 65.0 ± 0.8 ab 3.0 ± 0.4 a 6.0 ± 0.8 a 4.0 ± 0.3 a Explant without roots Control 51.0 ± 2.7 abc 3.0 ± 0.4 a 4.0 ± 0.6 a 3.0 ± 0.3 a 60.0 MS + ABA ± 8.3 ab 1.0 ± 0.6 ab 3.0 ± 0.5 a 3.0 ± 0.2 a 40.0 MS + ABA ± 2.8 ac 2.0 ± 0.6 b 3.0 ± 0.8 a 2.0 ± 0.5 a 70.0 MS ½ + ABA 0.1 MS ½ + ABA ± 2.1 abc 0.0 ± 0.3 ab 3.0 ± 0.7 a 2.0 ± 0.2 a ± 2.6 abc 2.0 ± 0.8 ab 4.0 ± 0.8 a 3.0 ± 0.4 a

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