Impaginato 179 Adv. Hort. Sci., 2019 33(2): 179-185 DOI: 10.13128/ahs-24090 Embryogenesis in Valerian (Valeriana officinalis L.) using leaf segments Gh. Abdi 1 (*), M. Khush-Khui 2, A. Shekafandeh 2 1 Department of Biotechnology, Persian Gulf Research Institute, Persian Gulf University, Bushehr, 7516913817, Iran. 2 Department of Horticulture, College of Agriculture, Shiraz University, Shiraz, Iran. Key words: glutamic acid, in vitro culture, MS medium, somatic embryogen- esis, Valeriana officinalis L. Abstract: A protocol for direct embryogenesis induction without an intervening callus production was developed in valerian (Valeriana officinalis L.) using the leaf segments. Direct somatic embryogenesis was induced using by half- strength MS medium supplemented with 2,4-D (0.5 mg·l-1), glutamic acid (100 mg·l-1), 4% sucrose and 8 g·l-1 agar. The embryo germination and plantlet forma- tion were enhanced on MS medium supplemented with NAA (0.1 mg·l-1) and Kin (2 mg·l-1). Regenerated plants with well-developed root and shoot systems were successfully (72%) transferred to the greenhouse. 1. Introduction Valerian (Valeriana officinalis L.) is an herb belongs to Valerianaceae family which grows in several geographic areas of the world including Iran. The genus Valeriana encompasses nearly 250 species, found mainly in temperate zone regions. Some species of this genus have high econom- ic demand in Iran and also, around the world. Plants of this genus have sedative, antispasmodic and anxiolytic properties mainly due to the pro- duction of iriod esters, known as valepotriates, in the plant rhizomes (Hiller and Zetler, 1996). The conventional methods of propagation of valerian are from seed and rhizome. Considering the importance of the genus Valeriana in the medicinal world (O’Hara et al., 1998) concentrated efforts are being made to improve the propagation using biotechnological approaches. Plant regeneration has been described from shoot tip, axil- lary bud explants, via callus and embryo-like structures derived through suspension cultures of V. wallichii (Mathur et al., 1988; Mathur and Ahuia, 1991; Mathur, 1992). Also, plant regeneration from adventitious shoots, seedlings, suspension cultures of V. edulis (Enciso-Rodriguez, 1997; Castillo-España et al., 2000), shoot regeneration from leaf segments of V. officinalis (Abdi and Khosh-khui, 2007), shoot organogenesis and somatic embryogenesis from leaf explants of V. jatamansi (Rong et al., 2014) and from shoot buds of V. jatamansi (Kaur et al., 1999) and shoot (*) Corresponding author: abdi@pgu.ac.ir Citation: ABDI GH., KHUSH-KHUI M., SHEKAFANDEH A., 2019 - Embryogenesis in Valerian (Valeriana offi- cinalis L.) using leaf segments. - Adv. Hort. Sci., 33(2): 179-185 Copyright: © 2019 Abdi Gh., Khush-Khui M., Shekafandeh A. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 9 November 2018 Accepted for publication 28 January 2019 AHS Advances in Horticultural Science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2019 33(2): 179-185 180 organogenesis and somatic embryogenesis from leaf explants of Valeriana jatamansi Jones have been described (Chen et al., 2014). Somatic embryogenesis is an efficient and high volume propagation system for the large number of plants within a short period. Successful genetic transformation attempts have mostly employed embryogenic callus or cell cultures as the target tissue in several medicinal plants (Leena and Jaindra, 2003). However, a major limitation of this callus system is the repeated subculture to select embryogenic callus portions among highly proliferat- ing non-embryogenic tissue. This process is not highly producible and furthermore increases the chance of somaclonal variation. As these limitations have become unavoidable, strategies to improve plant regeneration must necessarily include manipulation of the medium to embark upon new morphogenetic pathways (Pedroso and Pais, 1995). Direct somatic embryogenesis offers several advantages in medici- nal plant improvement, as cost effective and large- scale clonal propagation is possible using bioreactors, ultimately leading to automation of somatic seed production and development of artificial seeds. Besides, such a system could provide a new source for use in genetic transformations. The plant derived from direct somatic embryogenesis usually is unicel- lular in origin and hence genetically uniform. The leaf segments of valerian are an excellent source for the induction of indirect embryogenesis and the factors affecting this process had been studied (Castillo- España et al., 2000). Direct somatic embryogenesis and factors controlling it have been studied in many plant species (Pedroso and Pais, 1995; Chen et al., 1999; Desai et al., 2004; Kuo et al., 2005; Quiroz- Figueroa et al., 2006; Thengane et al., 2006; Jayanthi et al., 2011). Based on our knowledge, there is no report about direct somatic embryogenesis on Valeriana officinalis. The aim of this study was to establish a method for asexual multiplication of Valeriana officinalis through direct somatic embryo- genesis. 2. Materials and Methods Plant material and culture methods Fresh leaves of valerian were collected from 4- month old greenhouse-grown plants (Fig. 1A). They were washed with tap water and a few drops of Rica (a commercial detergent). They were surface steril- ized by 70% Ethyle Alcohol for 1 min. and rinsed twice with sterile distilled water. The leaves were immersed in a solution of 1.5% sodium hypochlorite for 10 min and rinsed four times with sterile distilled water. The leaves were cut into 7-8 mm2 segments and transferred to 150 ml glass jars with 25 ml of half-strength MS medium (Murashige and Skoog, 1962). The pH of media was adjusted to 5.8 by 0.1 N HC1 before autoclaving for 15 min at 121°C and 1.5 kg·cm-2 pressure. Cultures were placed initially under the dark condition for 2 weeks and thereafter they were maintained at 25±3°C under 16 h photoperiod provided by cool white fluorescent lamps (45 µmol·m-2·S-1) with relative humidity of 75-85%. Embryo induction and germination For embryo induction, after preliminary experi- ments, MS media were supplemented with 0.5 mg·l-1 2,4-D, 0.5 mg·l-1 Naphthalene acetic acid (NAA), 100 mg·l-1 glutamic acid (Glu) or different concentrations of sucrose (3, 4, and 5%) (Table 1). For embryo germi- nation, two separate experiments were conducted. In the first experiment, the explants that formed embryo were divided to 4 segments (each fragment was about 0.2±0.03 g) and transferred to MS medium containing NAA (0.5, 1, 2 and 5 mg·l-1), gibberellic acid (GA3) (0.5, 1 and 2 mg·l-1), or MS added to differ- ent combinations of NAA (0.1 and 0.2 mg·l-1) and Fig. 1 - Effects of different treatments on in vitro culture of vale- rian, (A) 4-month old greenhouse-grown plant of vale- rian, (B) Initiation of small embryo-like structures in the 10 to 13 day after culture in induction media, (C) and (D) Embryo formation in top and cut end of valerian leaf explant after 4 weeks. (E) Callus initiated in M2 medium with great potential for regeneration. (F) Germination of embryo mass in medium supplemented with 2 mg l-1 Kin and 0.1 mg l-1 NAA. Abdi et al. - Valeriana officinalis L. embryogenesis 181 kinetin (1.5 and 2 mg·l-1). In the second experiment, embryos were transferred individually to MS medi- um supplemented with NAA (0.1 and 0.2 mg·l-1) and kinetin (1.5 and 2 mg·l-1) to stimulate the germina- tion and roots and shoots development. Acclimatization The 4 cm plantlets were transferred to small pots containing 1/3 vermiculite, 1/3 perlite and 1/3 sand (v/v). The pots were placed in transparency box and maintained under 25±5°C temperatures and 70% rel- ative humidity for 4 weeks and then transferred to greenhouse. Data analysis The experiment was conducted as a completely randomized design in a factorial arrangement with 4 replications and each replicate with 12 explants. The means were compared with Duncan’s new multiple range test (DNMRT) at 5% probability level. To deter- mine the efficiency of embryo induction medium, responsive leaves that formed embryo was recorded after 4 weeks. For embryo germination experiments the percent of germinated embryo was recorded after 3 weeks and the number of plantlets was recorded 6 weeks after culture. Plantlets were recog- nized when they developed roots and shoots. 3. Results Embryo induction The leaf segments showed swelling and initiation of small embryo-like structures in the 10-13 days after culture (Fig. 1B). In the following weeks, embryogenic clumps were visible at the cut end and surface of the explants (Fig. 1C and D). Embryoid for- mation in cut edges of leaf explants was higher than other parts of leaves. Well-developed embryos were observed all over the cultured explants within four weeks of culture. Different medium showed different embryogenesis response. When Glu and sucrose (4%) were added to medium, embryogenesis response increased. Maximum embryogenic response of leaf explants (57.21±2.7%) was observed on M8 medium supplemented with 2, 4-D, 4% sucrose, and 100 mg·l-1 glutamic acid. NAA addition instead of 2, 4-D decreased percentage of explants response in M9 (43.31±2.33%) medium. Increasing sucrose concentration more than 4% reduced embryogenic response on M 12 (25.28±1.24%), and M13 (13.39±2.28%) (Table 1). The explants on M2 and M3 media did not exhibit any embryogenic response. However, explants on these media initially showed slight swelling and sub- sequently resulted to callus production. M2 showed higher percentage of callus proliferation compared to M3 (data not shown). Callus initiated in M2 medium had a great potential for regeneration (Fig. 1E). The explants on M1 medium did not exhibit any response. Embryo germination For embryo germination experiment, two kinds of explants were used. In the first experiment, the explants that formed embryo were divided to 4 seg- ments (each fragment was about 0.2±0.03 g) and used as mass embryo. In the second experiment the embryos were transferred individually to MS medium supplemented with various supplements. Somatic embryo germination response varied greatly in vari- ous hormone supplements (Table 2). MS without growth regulators showed low response to embryo germination. In this treatment, germination percent- age per number of embryoid pieces and also number of plantlet per pieces was not considerable (Table 2). MS+NAA (0.5, 1, and 2 mg·l-1) led to rooting of somat- ic embryos (Fig. 2A). The inclusion of GA33 (0.5, 1, and 2 mg·l-1) in the germination medium increased the germination percentage and consequently plantlets with both well-developed shoots and roots. The com- bination of Kin-NAA enhanced somatic embryo ger- mination percentage more than other treatments. Among Kin-NAA treatments, 2 mg·l-1 Kin and 0.1 mg· l-1 NAA showed high frequency of embryos and also plantlet formation (Table 3). The somatic embryos were easily isolated with a pair of forceps, each developing into a single plant. The highest recovery Table 1 - Different media used for induction of direct somatic embryogenesis in Valeriana officinalis L. PGR Additives (mg·l-1) M 1 M 2 M 3 M 4 M 5 M 6 M 7 M 8 M 9 M 10 M 11 M 12 M 13 NAA - - 0.5 - 0.5 - 0.5 - 0.5 - 0.5 - 0.5 2,4-D - 0.5 - 0.5 - 0.5 - 0.5 - 0.5 - 0.5 - Glutamic acid - - - 100 100 - - 100 100 - - 100 100 Sucrose (%) 3 3 3 3 3 4 4 4 4 5 5 5 5 Response (%) - - - 12.4±0.3 11.21±1.1 5.28±0.1 3.4±0.03 57.21±2.7443.31±2.33 9.8±0.15 8.8±0.1 25.28±1.2413.39±2.28 Adv. Hort. Sci., 2019 33(2): 179-185 182 of somatic embryos (78+3.21%) was possible on MS medium supplemented with NAA (0.1 mg·l-1) and Kin (2 mg·l-1), this condition showed considerable germi- nation percentage and plantlet formation while the fewest somatic embryos germination and developing into a single plant (41+1.73%) was observed in MS medium supplemented with a low concentration of NAA (0.1 mg·l-1) and Kin (1.5 mg·l-1) (Table 4). 4. Discussion and Conclusions Using different plant tissue culture technique and plantlet regeneration have been reported in different species of Valeriana genus, such as V. officinalis (Abdi and Khosh-Khui, 2007; Abdi et al., 2008; Reza et al., 2009), Valeriana wallichii (Mathur et al., 1988), Valeriana edulis ssp. procera (Enciso-Rodriguez, 1997), V. jatamansi (Kaur et al., 1999; Das et al., 2013), Valeriana glechomifolia (Salles et al., 2002; Bello de Carvalho et al., 2004), Valeriana glechomifo- lia (Bello de Carvalho et al., 2004). However, direct somatic embryogenesis have never been reported from any explant of the species. In this study, the effects of different plant growth regulators (PGRs) and different supplement on the induction of direct somatic embryogenesis were investigated. We estab- lished an efficient embryo induction, somatic embryogenesis and plant regeneration system from leaf explants using various supplement and different types and concentrations of PGRs. The induction and development of in vitro somatic embryos comprise complex processes including cell division, differentia- tion, growth, and pattern formation (Capron et al., 2009)., the composition of the basal culture medium, the type and levels of plant growth regulators (PGR), the level of carbon sources and the balance of organ- ic and inorganic nitrogen sources are key factors for in vitro embryogenesis system. Primary nitrogen sources, including Glu, is very important in plant tis- sue culture in order to stimulate the cellular growth Fig. 2 - Effects of different treatments on in vitro culture of vale- rian (A) Rhizogenesis response of embryo in medium containing NAA. (B) Individual embryos in medium con- taining 2 mg l-1 Kin and 0.1 mg l-1 NAA. (C) Well germina- ted embryos in medium containing 2 mg l-1 Kin and 0.1 mg l-1 NAA. (D) Plantlets with developed shoots and roots obtained from embryo germination. Table 2 - Effects of various supplements on germination of Valeriana officinalis L. embryos Media Response MS + without growth regulators Germination and plantlet production was low MS + NAA Rhizogenesis MS + Kin and NAA Plantlets with both well-developed shoots and roots MS +GA 3 Plantlets with both well-developed shoots and roots with vigorous growth of plantlets Table 3 - Effects of various supplements on frequency and plan- tlet/embryo mass in Valeriana officinalis L. Plant growth regulator Explants response (%) Plantlet per each embryo mass PGR free 13.71 g 1.27+0.1 e GA3 (mg·l-1) 0.5 23.24 f 2.71+0.01 d 1.0 38.00 d 3.00+0.10 c 2.0 31.70 e 2.71+0.11 d Kin+NAA (mg·l-1) 1.5+0.1 43.37 d 3.21+0.12 c 1.5+0.2 37.75 d 4.00+0.02 b 2.0+0.1 61.32 a 5.21+0.12 a 2.0+0.2 51.37 b 4.27+0.13 b PGR (mg l-1) Explant response (%) Plantlet per each embryo mass Kin + NAA 1.5 + 0.1 49+1.2 c 23 bc 1.5 + 0.2 41+1.73 d 19 c 2.0 + 0.1 78+3.21 a 37 a 2.0 + 0.2 58+2.41 b 25.6 b Table 4 - Effects of growth regulators on percentage of embryo germination and number of plantlets Abdi et al. - Valeriana officinalis L. embryogenesis 183 embryos induction (Luo et al., 1996; Nakagawa et al., 2001). The positive effect of sucrose may related to the vital function of the sucrose such as controlling the several developmental processes in the cells (Gibson, 2000; Smeekens, 2000), nutritional function (serve as a carbon source during somatic embryogen- esis) and osmotic regulator or cell osmolarity (Biahoua and Bonneau, 1999 ). The positive role of sucrose in the present study may be interpreted as both nutritional and osmotic regulatory functions of this carbohydrate. The conversion of somatic embryos to plantlets is a multi-step process. In many embryogenetic systems the transfer of somatic embryos into PGR free culture medium enhances the development of the somatic embryos and their con- version to plantlets. One of the determinative factors for the low rates of somatic embryo conversion to plantlets is associated with the residual effects of 2, 4-D. Prolonged expositions to this PGR normally reduces the conversion and increases the number of abnormal somatic embryos (Cruz et al., 1990). When somatic embryos of V. officinalis were culture in medium supplemented with Kin-NAA and GA3, the number of plantlets increased. The positive role of cytokinins may be related to reversion of negative effects caused by 2, 4-D to the cultures (Parrot et al., 1988). The role of the GA3 in promoting the germina- tion of somatic embryos is well documented in other embryogenic systems (Deng and Cornu, 1992). Our results are in agreement with the findings of Castillo- España et al. (2000) who used combination of Kin- NAA for embryo germination in Valeriana edulis ssp. Procera. In conclusion, the induction of direct somat- ic embryogenesis in valerian using leaf segments as described in this study could be useful in rapid propa- gation of the elite plant, which has best characters for medicinal purposes. Furthermore, direct embryo- genesis can be beneficial for gene transformation via particle bombardment or Agrobacterium in short time, avoiding somaclonal variation. In addition, the identified protocol does not seem induce a prolifera- tion of callus before the differentiation of somatic embryos. Regenerated plants with well-developed root and shoot systems were successfully (72%) transferred to greenhouse. Acknowledgements The authors are thankful to the Prof. Tafazoli for useful comments. and the connection between cells and tissues (Young et al., 1999). The productions of direct somatic embryos on leaf explants of V. officinalis are possible by the addition of the Glu to the culture medium and increasing the sucrose level to 4%. Absence of any callus formation indicated that the process of embryo development was direct. Glutamate occupied a cen- tral role in amino acid metabolism in plant. It can form by action of the glutamate synthase utilizing glutamine (Bohinski, 1991). Glutamine as a nitrogen source for purins and pirimidins biosynthesis, was sig- nificantly stimulated the direct embryogenesis in Valeriana officinalis in this study. This finding is in parallel with the results of Chowdhry et al. (1993), Gex et al. (2006) and Shahsavari (2011). This could explain the enhanced rate of induction and develop- ment of somatic embryos in the present investiga- tion. Additionally, during the metabolism and protein synthesis the nitrogen originated from amino acids is quickly assimilated into carbonic skeletons (Lea, 1993). This stimulation suggested that organic nitro- gen was a growth-limiting factor in Valerian cultures and the inclusion of glutamine decreased the culture lag phase, which indicated that glutamine was much more readily assailable than inorganic nitrogen. The somatic embryos in this study were formed more in the cut edges of leaf explants. Increasing in embryo- genic competence of wounded tissue probably was related to the endogenous growth regulators changes in leaf tissue (Ivanova et al., 1994). This study also demonstrated that 2, 4-D is more efficient than NAA to induce somatic embryos formation in valerian. Shoot organogenesis and somatic embryo- genesis were also reported in cucumber in the pres- ence of 2,4-D and NAA (Kuijpers et al., 1996) and the same results were reported in Cassava leaf explants by Sofiari et al. (1997). Plant tissue studies on V. edulis revealed that 2,4-D induced somatic embryos while NAA induced shoots (Castillo-España et al., 2000). Increasing the level of the sucrose from 3% to 4% showed a positive effect in embryo induction, while the induction response decreased at the pres- ence of 5% sucrose in the culture medium. 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