 American Journal of Plant Sciences, 2011, 2, 669-674 doi:10.4236/ajps.2011.25080 Published Online November 2011 (http://www.SciRP.org/journal/ajps) Copyright © 2011 SciRes. AJPS 669 Effect of Vitamins on In Vitro Organogenesis of Plant Peter Abrahamian, Arumugam Kantharajah* Department of Agricultural Sciences, American University of Beirut, Riad El Solh, Beirut, Lebanon. Email: a.kantharajah@hotmail.com Received June 10th, 2011; revised July 25th, 2011; accepted September 1st, 2011. ABSTRACT Vitamins are necessary compounds synthesized and utilized in plants. In tissue culture media, vitamin addition is not always common; since the amount needed by plants is relatively unknown and varies. Vitamins, in combination with other media constituents, have been shown to have direct and indirect effects on callus growth, somatic growth, rooting, and embryonic development. For example, different studies have shown that thiamine is associated with cytokinin and has a role in inducing callus growth and rooting. Moreover, thiamine was essential in facilitating the production of more secondary metabolites such as proteases in pineapple. Both biotin and riboflavin play a role in callus develop- ment as well. Specifically, riboflavin exerts different effects on plant rooting either positively and negatively. Vitamin D known to cause uptake of calcium in animal tissue, exerts a similar effect in plants. In addition, vitamin D causes cell elongation and meristematic cell division. Vitamin C, known for its anti-oxidative properties, has also enhanced shoot growth and rooting. Keywords: Vitamin, Organogenesis, In Vitro, Plant Tissue Culture, Plant Propagation 1. Introduction Plants are a major source of essential vitamins for hu- mans and animals. Their function and synthesis pathways have been extensively studied. Vitamin syntheses in plants are mainly used as essential intermediates in biochemical reactions or as catalysts in various pathways. Vitamins are divided into two main groups, the water-soluble (Ascor- bic acid “C”; thiamine “B1”; riboflavin “B2”; pyridoxine “B6”; nicotinic acid; cobalamin “B12”; folic acid; pan- tothenic acid “B5”; biotin) and fat-soluble (A, D, E, K) vitamins [1]. According to Bonner [2], working on water- soluble vitamins is of higher interest than fat-soluble vi- tamins. In tissue culture, some plants can become defi- cient in vitamin synthesis [3]. Hence, supplementing plant tissue with sub-optimal levels is essential to obtaining vigorous growth. Plant cell requirements for vitamin con- centration vary according to the plant species and type of culture. Thiamine pyrophosphate (TPP) is a derivative of Thia- mine (Vit. B1) [1]. Thiamine’s physiological functions in plants are diverse and serve as cofactors in enzymatic reactions including pentose phosphate pathway, glycoly- sis, tricarboxylic acid cycle (TCA), pyruvate dehyrdro- genase complex, transketolase, and pyruvate decarboxy- lase [4]. Pyruvate decarboxylase has shown to be im- perative in energy production in Arabidopsis [5]. Thia- mine has also been associated with disease resistance, and expression of PR-1 gene with local acquired resis- tance, but not systemic acquired resistance (SAR) [6], however, Ahn et al. [7] showed induced SAR in Arabi- dopsis. Under conditions of abiotic stress in Arabidopsis, endogenous thiamine increases dramatically to cope with oxidative stresses by supplying NADH and NADPH [8]. Vitamin C or ascorbate is oxidized by oxygen, hydrogen peroxides, and superoxides into monodehydroascorbate (MDHA) radicals. Ascorbate oxidase is possibly related to cell wall expansion and growth. MDHA, a product of ascorbate oxidase, radicals obtained depolarize the pla- sma membrane hence causing ion uptake and wall loo- sening [9]. Other vitamins such as riboflavin, a precursor of FAD and FMN coenzymes, and nicotinic acid, pre- cursor of NAD and NADP, participate in cellular redox reactions. In this review paper, we will provide a basic summary of how these pathways are exhibited, at the macro level, upon vitamin addition to plant tissue culture media. 2. Vitamins in Tissue Culture In tissue culture media, thiamine, nicotinic acid, pyri-
 Effect of Vitamins on In Vitro Organogenesis of Plant 670 doxine and myo-inositol found in Murashige and Skoog [10] (MS) medium at 0.1 mg·l–1, 0.5 mg·l–1, 0.5 mg·l–1, and 100 mg·l–1 respectively are the most commonly used, while the addition of other essential vitamins to media is uncertain. Myo-inositol remains a controversial compound being either classified as a water-soluble plant vitamin or as a sugar alcohol [3]. Earlier studies in pea embryos done by Ray [11] have shown that it is possible to achi- eve good in vitro growth by increasing vitamin C content. This finding cannot be broadly applied as some plants are less receptive to increasing concentrations of vitamin C, indicating more autotrophism than other plants (tomato and oat) [2]. It has also been noticed that adding biotin increased the shoot dry weights of peas, similar to the response observed in Ricciocarpus plants treated with pantothenic acid. Unlike other vitamins, thiamine addi- tions to pea embryos in vitro affect rooting and shoot growth simultaneously. In vitro studies have shown that tomato roots are capable of exhibiting prolonged thia- mine dependency. [2]. 2.1. Micropropagation In the presence of 25 mg·l–1 of vitamin D3 micro propa- gated potato plantlets absorbed Ca2+ efficiently [12]. However, vitamin D3 concentrations higher than 25 mg·l–1 i.e. 50 mg·l–1 did not stimulate higher absorption levels. On the other hand vitamin D2 suppressed Ca2+ uptake. It was concluded that combining both vitamins D2 and D3 did not improve calcium absorption hence claiming the superiority of vitamin D3 for calcium ion uptake [12]. 2.2. Callus & Somatic Growth Gamborg et al. [13] cultured soybean root cells unto several media containing different concentrations of thia- mine, and to a complete B5 culture media. An initial amount of 53 mg of soybean cell culture was grown in 0 mg·l–1 and 10 mg·l–1 of thiamine. After 5 days, 138 mg and 203 mg of soybean cells were produced, respectively. Pyridoxine, nicotinic acid and myo-inositol present in the media had no adverse effects on growth individually. Consequently, Gamborg et al. [13] concluded the neces- sity of providing thiamine to the media to sustain growth of soybean root cell. Eriksson [14] have concluded that nicotinic acid and pyridoxine are essential vitamins accompanying thiamine, when studying the optimum growth of Haplopappus gra- cilis Nutt. on a modified medium of MS [10]. Polikarpochkina et al. [15] reported that maize calli decreased in weight from 110 mg/ml to nil after 3 suc- cessive passages when thiamine is eliminated. However, the removal of inositol and pyridoxine from the MS [10] medium did not give any significant difference on gr- owth [15]. The change recorded from the first passage until the third passage, when omitting inositol and pyri- doxine was 9% and 2.5%, respectively. Digby and Skoog [16] found a relation between ki- netin and thiamine in the normal callus culture of tobacco. It has been shown that high levels of kinetin are needed to induce thiamine synthesis in the absence of any ex- ogenous thiamine added. However, sustaining growth on a low level kinetin media was not possible except if thiamine was added. Whereas, Linsmaier and Skoog [17] maintained tobacco cultures with 1000 μg/l of kinetin and nil thiamine over 17 passages. Dravnieks et al. [18] later confirmed that thiamine synthesis was subject to feedback control mechanism thus sensitive to the amount of thiamine in tissue, regardless of kinetin concentration. Both thiamine and biotin significantly affected callus growth of date palm [19]. Increasing thiamine from 0.1 mg·l–1 to 0.5 mg·l–1 caused maximum callus growth; fur- thermore, increasing thiamine to 2 mg·l–1 gave reduced callus weights. Moreover, increasing biotin from 0 to 1 mg·l–1 gave a maximum callus weight similar to thiamine [19]. On the other hand, an earlier report by Drew and Smith [20] showed that presence of riboflavin reduced callus growth of Papaya. A significant decline in mean callus weight was recorded from 89.32 mg to 0.10 mg per explant, in the absence and presence of riboflavin, respectively [20]. Ascorbic acid, functioning primarily as an antioxidant, is used to prevent browning of tissue [1,3]. However, in tobacco cells, ascorbic acid has been shown to function as a stimulant of mitotic cell division [21]. 2.3. Rooting Vitamin D3 stimulates rooting of Phaseolus vulgaris L. in culture [22]. In a control treatment without any vitamins 43.75% of the roots were longer than 14 mm, while vi- tamin D3 addition achieved 78.75%. The effect of vita- min D3 shown by Boland et al. [22] at 10–9 M, on root growth was associated with an uptake of calcium ions, an increase in cell elongation in root zone at 0.5 - 1 mm from the apex, and stimulation of mitotic division of meristematic cells. In vitro rooting of peach rootstock GF677 (Prunus amygdalus × P. persica Batsch.) was studied by adding different concentrations of riboflavin ranging from 0 to 2.0 mg·l–1 [23]. As more riboflavin was added rooting decreased in a linear form until it was completely inhi- bited. The smallest concentration of 0.5 mg·l–1 of ribofla- vin caused the average number, length, fresh weight and dry weight to decrease sharply [23]. Whereas at 1.5 and 2 mg·l–1 of riboflavin chlorotic and necrotic symptoms appeared. Moreover, adventitious root formation in the control was long and thin, while in the treated media, roots were short and thick. Also, callus formation was Copyright © 2011 SciRes. AJPS
 Effect of Vitamins on In Vitro Organogenesis of Plant Copyright © 2011 SciRes. AJPS 671 inhibited in the rooting MS media, due to the suppressing action of auxin by photo-degradation [23]. On the contrary, riboflavin has been shown to stimu- late and help rooting significantly [24-26]. Rooting in apple tissue culture was studied in the presence of ribo- flavin. In the dark riboflavin stimulated rooting signifi- cantly in the presence of auxin (IBA), whereas rooting decreased when the vitamin was omitted and exposed to light [24]. Trindade and Pais [25] showed that Eucalyp- tus globulus Labill. produced 80% rooting ability on a revised De Fossard [27] media containing riboflavin (Ta- ble 1). On the other hand, 60% rooting was achieved on the same media excluding the latter compound [25]. Carica papaya L. rhizogenesis was optimal when 31 μM of riboflavin and 10 μM of IBA were added to the De Fossard [27] media in the dark for 2 days, but losses oc- curred during media preparation [26]. However, Drew et al. [26] found a way to avoid the loss of IBA due to light exposure. The procedure involved injecting riboflavin at 300 μM per ml into 10 ml of media, which is equivalent to the optimum riboflavin level 31 μM, after 1 day of IBA rich medium in the light [26]. Thiamine is another vitamin shown to have significant rooting on pacific yew, an evergreen, Taxus brevifolia Peattie [28]. Upon adding thiamine, Chee et al. [28] ob- tained 61.5% of adventitious rooting in T. brevifolia Pea- ttie compared to 30% without thiamine. In a literature review on Eucalyptus propagation, vitamin E, other than being an antioxidant, affected rooting and speeded up the rooting process upon addition to culture media [29]. Table 1. Effect of vitamins on plant growth and development in in vitro. Vitamin Function1 Culture Medium Concentration Common Name (Species) Effect2 Reference B5 + 2 mg·l–1 2,4-D 10 mg·l–1 Stimulate cell growth Gamborg et al. 1968 MS basal medium + 1.7 gM BAP + 0.2 gM IBA 1.0 μM Soybean (Glycine max L.) Increase embryogenesis Barwale et al. 1986 MS + 2 mg·l–1 2,4-D 0 mg·l–1 Maize (Zea mays L.) Decrease callus weight Polikarpochkina et al. 1979 MS (Hormone free MS) 0.5 mg·l–1; 0.5 mg·l–1 or 2 mg·l–1 Palm (Phoenix dactylifera L.) Increase callus weight, embryo number; embryo length Al-Khayri 2001 MS + 4.2 μM GA 0.3 μM Pineapple (Ananas comosus L.) Reduce shoot fresh mass Pérez et al. 2004 Thiamine (B1) Cofactor in carboxylase reactions and amino acid biosynthesis Linsmaier and Skoog + 5 mg·l–1 2,4-D + 0.1 mg·l–1 BA 0.4 mg·l–1 Turf grass (Zoysia japonica Steud.) Increase embryonic callus Asano et al. 1996 MS + 1 mg·l–1 IBA 0.5 - 2 mg·l–1 Peach (Prunus amygdalus x persica Batsch.) Inhibit rooting and reduces callus Dimassi et al. 2005 De Fossard + 1.11 μM BA + 0.1 μM IBA 7.97 μM Eucalyptus globulus Labill.Stimulate rooting Trindade and Pais 1997 De Fossard + 10 μM IBA 31 μM Papaya (Carica papaya L.) Stimulate rooting Drew et al. 1993 Riboflavin (B2) Oxidation-reduction reactions (Transfer of electrons) MS + 3.2 μM IBA Not Known Apple (Malus domestica Borkh.)Stimulate rooting Van der Krieken 1992 Shenk-Hildebrandt (Hormone free) 10–9 M Common Bean (Phaseolus vulgaris L.) Stimulate rooting, mitotic division, and calcium absorption Boland et al. 1989 Vitamin D3 - MS (Hormone free) 25 mg·l–1 Potato (Solanum tuberosum L.) Enhance Calcium absorption Habib and Donnelly 2003 Biotin Cofactor of en- zymes MS (Hormone free) 2 mg·l–1; 1 mg·l–1 Palm (Phoenix dactylifera L.) Increase callus weight, embryo number; embryo length Al-Khayri 2001 Vitamin C (Ascorbate) Reducing Agent MS + 10 μM IAA + 10 μM Kinetin 4 - 8× 10–4 M Tobacco (Nicotiana tabacumn L.) Increases shoot number Joy et al. 1988 Nicotinic Acid Oxidation-reduction reactions MS basal medium + 1.7 gM BAP + 0.2 gM IBA 32.4 μM Soybean (Glycine max L.) Increase embryogenesis Barwale et al. 1986 1Biochemical pathway in plant cell; 2Effects reported have been due to mixed interaction between vitamin and hormones in media, unless stated otherwise.
 Effect of Vitamins on In Vitro Organogenesis of Plant 672 2.4. Embryo & Organ Development Thiamine and nicotinic acid have been shown to affect embryogenesis [30]. Barwale et al. [30] studied the effect of different concentration of both vitamins on 40 imma- ture soybean embryos cultured to a modified MS [10] medium. Thiamine at 1.0 μM, or more, has induced 68% embryogenesis compared to 0.2 μM, the level of salts in MS medium, at 33% of the immature embryos. Also, a concentration of 32.4 μM nicotinic acid induced 76% embryogenesis [30]. Asano et al. [31] showed that enhancing embryonic callus of Zoysia japonica Steud., a warm season turf grass native to Japan, is obtained by adding thiamine and riboflavin to the media. When thiamine was excluded from the medium 50.3% callus was obtained, on the con- trary, 0.4 and 4 mg·l–1 gave 53 and 60.3% respectively, both insignificantly different. Furthermore, riboflavin was not effective alone, except in the presence of thiamine at 4 mg·l–1 or higher concentration [31]. Thiamine and biotin have shown to be essential com- ponents of tissue culture media for optimizing embryo- genesis of date palm (Pheonix dactylifera L.) [19]. The effect of thiamine has been shown to be dependent on biotin for maximizing the number of somatic embryos, which was also mentioned by Bonner [2]. The highest number of embryos obtained was with a treatment of 0.5 or 2 mg·l–1 thiamine and 2 mg·l–1 biotin. However, the optimum concentration for embryo number was a media containing 0.5 mg·l–1 thiamine and 2 mg·l–1 biotin. Em- bryo elongation also was affected by an interaction be- tween both biotin and thiamin. The maximum embryo length was achieved by 0.5 or 2 mg·l–1 thiamine and 1 mg·l–1 biotin [19]. Pérez et al. [32] studied the effect of thiamine and other compounds on protease excretion in pineapple cul- ture. Exogenous amounts of thiamine in the range of 0.3 - 1.2 μM had a negative effect on pineapple shoot fresh mass, forming a plateau [32]. On the other hand, thia- mine produced a maximum protein content at 0.6 μM, while proteolytic and specific proteolytic activities both at 0.3 μM [32]. Shoot weight of Papaya significantly increased in the presence of both cytokinin and riboflavin, compared to a medium of cytokinin only [20]. While a decrease of shoot weight in the presence of riboflavin and auxin, possibly related to photo-oxidation of auxin, also conveyed in Gorst et al. [33] on Eucalyptus ficifolia F. Muell, was reported in comparison to a medium containing only auxin [20]. Also Drew et al. [34] reported that auxin (Indole-3-butyric acid) concentrations at 10 μM with more or less than, but not, 1 μM of riboflavin caused a small rooting percentage. Moreover, increasing the con- centration of riboflavin gradually from 0.1, 1, to 10 μM degraded IBA, in the presence of light [34]. When 10 μM of IBA is used, a complete destruction of IBA occurs after 16 days versus 2 days when riboflavin is absent and present, in light, respectively [34]. Exogenous application of 8 × 10–4 M and 4 - 8× 10–4 M of ascorbate to a shoot-forming media enhanced shoot formation increased by 45% and 450% when using young callus tissue (4 - 12 subcultures) and old callus (>30 subcultures) of tobacco (Nicotiana tabacum L), re- spectively, after 35 days in culture [35]. In the non-shoot forming media, containing gibberellic acid, shoot-growth of the young callus was significant at 4 × 10–4 M and almost negligible for the old callus [35]. The former phe- nomenon indicates an inhibitory action by ascorbate on gibberellic acid. In addition, ascorbic acid reduced the shoot-forming period [35]. Roest and Bokelmann [36] have shown that a high number of adventitious shoot formation and transferable shoots of Chrysanthemum was obtained when vitamins were kept in the complete MS [10] medium. Whereas, a medium where vitamins were eliminated suppressed shoot formation although all other minerals were retained [36]. On the contrary, omitting vitamins (thiamine, pyri- doxine, nicotinic acid, folic acid, and biotin) from a Bourgin and Nitsch [37] media in vitro did not affect 16 cultivars, except one, of Begonia x hiemalis shoot and root formation [38]. Also, Soczek and Hempel [39] stud- ied the shoot multiplication of three Gerbera cultivars in the presence and absence of thiamine, pyridoxine, nico- tinic acid and other compounds. It was concluded that reducing the concentration, to half or quarter of the Mu- rashige et al. [40] medium, or removing the vitamins, did not have any significance on growth over three passages (each 4 weeks), except in the case of one cultivar requir- ing nicotinic acid [39]. 3. Conclusions Vitamins in culture media should be further studied in order to justify their addition. For instance, little is known about vitamin E (α-tocopherol), a phenol anti-oxidant, presence in culture media. In the last few decades, little interest has been observed in studying certain vitamins, such as biotin and pantothenic acid. Plant species and cultivars require different amount of vitamins, while other do need any at all. For instance, after several passages, thiamine is essential to soybean, rice, and tobacco cul- tures but non-essential to peanut cells, which contain high thiamine concentration [41]. The physiological and morphological output varies between plants when using the same vitamins. According to our desired outcome culture media remain open to modifications, especially Copyright © 2011 SciRes. AJPS
 Effect of Vitamins on In Vitro Organogenesis of Plant673 the common Murashige and Skoog [10]. Although sig- nificant vitamins such as thiamine impose their applica- tion in culture media; others are poorly applied such as ascorbic acid. Scientific knowledge on plant propagation was not the only significant outcome; however, some experiments offered economic solutions. In order to reduce costs, Drew et al. [27,34] suggested adding riboflavin, which degrades auxin, to the tissue culture media rather than transferring the tissue to a hormone (i.e. auxin) free media. In the future, studying the effect on a wider range of vitamins and plant simultaneously is needed for an enhanced fea- sibility outcome. REFERENCES [1] R. H. Horton, “Coenzymes and Vitamins,” Principles of Biochemistry, Pearson Education International, Upper Saddle River, 2006. [2] J. Bonner, “The Role of Vitamins in Plant Development,” Botanical Review, Vol. 3, No. 12, 1937, pp. 616-640. doi:10.1007/BF02872294 [3] E. F. 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