Efficient Adventitious Morphogenesis from In vitro Cultures of the Medicinal Plant Cymbopogon schoenanthus

Cymbopogon schoenanthus subsp. proximus is a medicinal grass, the source of sesquiterpene proximadiol and is well known in folk medicine as renal antispasmodic. The effect of growth regulators, different sugar types, methyl jasmonate and vitamins on de novo organogenesis from seed cultures was studied. Among different concentrations of NAA and BA, 4 mg/l NAA in combination with 0.5 mg/l BA produced 100% callus induction. Both 1.0 and 4.0 mg/l NAA in combination with 0.5 mg/l BA yielded the highest number of shoots. High concentration of sucrose 6% was efficient in root induction from organogenic shoots. Sugar types at a constant concentration of 3% had a significant effect on raising shoot numbers. Different concentrations of methyl jasmonate, biotin and Ca-pantothenate showed a decline in de novo shoot induction, but root formation frequency and numbers were significantly improved with all concentrations used.


Introduction
Cymbopogon schoenanthus subsp. proximus is a medicinal herb commonly known as Halfa bar, the plant is well known in folk medicine as diuretic, anti-diabetic and renal anti-spasmodic (Boulos 1983, Batanouny et al. 1999. Many researches proved that the plant extract of this subspecies possesses many biological activities (Selim 2011, El-Nezhawy 2014. The plant extract is rich with terpenoids mainly sesquiterpenes.The antispasmodic bioactive compound was identified as proximadiol (El-Askary et al. 2003).
Plant in vitro culture is an ideal method to generate a large number of genetically uniform plants and also to conserve the rare and endangered plants (Sidhu 2010). This technique is also used widely to propagate a wide range of the medicinal plants like Allium sativum L., Aloe vera L. and Artimisia annua (Chaturvedi et al. 2007). In vitro regeneration of the genus Cymbopogon was reported for the economically important species, Cymbopogon winterianus was efficiently regenerated through callus culture using shoot tips and leaves as explants (Bobade et al. 2010), through organogenesis (Gahukar et al. 2014). Somatic embryogenesis of C. flexuosus was reported by (Nayak et al. 1996).
Sugar plays an essential role in in vitro plant growth and development, its accumulation inside the plant helps in new organ induction and differentiation (Gabryszewska 2010). Carbon source significantly affected somatic embryogenesis of Theobroma cacao (Da Silva et al. 2008) and indirect regenerated shoots from Pinuspinea (Sul 2004).
Plants produce vitamins to use them as intermediates in the biochemical reactions or as catalysts in different metabolic pathways. Biotin has an essential role as a cofactor and possesses a role in plant metabolism (Nikolau et al. 2003, Alban et al. 2000. Requirements of the vitamins concentrations in the plant tissue culture media differ from plant to plant and according to the type of culture (Abrahamian and Kantharajah 2011). Exogenous addition of other vitamins was found to improve regeneration capacity of some plants (Joy et al. 1988, Mehta et al. 2000. Role of vitamins in root induction and development has been reported in many studies (Drew et al. 1993, Der Krieken et al. 1992. Methyl jasmonate (MeJa) is widely distributed in plant kingdom and its accumulation varied depending on the tissue type and function, developmental stage and in response to biotic and a biotic stress (Creelman and Mullet 1997). Effect of MeJa on Pharbitis nil growth, elongation of shoot and root was reported in Maciejewska and Kopcewicz (2002). Jasmonate enhanced the development of protocorm-like bodies from Cymbidium (Jaime et al. 2012) and enhanced shoot tip culture and rooting from Pistaciavera (Dolcet-Sanjuan and Claveria 1995).
Although C. schoenanthus subsp. proximus is of a high economic importance both in folk medicine and as the source of the bioactive sesquiterpene proximadiol, very few reports has been published on the species. The species is endangered due to extensive collection from the wild. The application of different biotechnology tools including in vitro culture, will aid in providing an alternative source for the bioactive compounds, propagules for ex situ conservation and a source of genetic variation for the species gene pool. Our present study reports an efficient organogenesis system of regeneration from in vitro cultures, that is a continuous with our previous efforts to establish different regeneration systems for the species. We studied the effect of different concentration of growth regulators, vitamin types and concentrations, sugar types and methyl jasmonate on organogenesis. We report an efficient, short-term regeneration system that can be used for the production of bioactive compounds and as a source for genetic conservation.

Materials and Methods
Seeds were collected from Aswan University, Egypt in March 2014 and stored in paper bags in the dark at 25ºC.
Tissue culture media were purchased from Phyto-Technology (USA). Plant growth regulators, methyl jasmonate, sugars and vitamins were purchased from Sigma Aldrich (St. Louis, MO, USA).
Surface sterilized seeds (El-Bakry and Abdelsalam 2012) were cultured on Murashige and Skoog MS salts (1962) with Gamborg's B5 vitamins (Gamborg et al. 1968) (MSB5) supplemented with different concentrations of NAA (0.5, 1.0, 2.0, 4.0, and 8.0 mg/l) in combination with two different concentrations of BA (0.2, 0.5 mg/l). Subculture was carried out after 4 weeks on the same media. Regenerated shoots cultured on MSB5 medium with 4.0 mg/l NAA and 0.5 mg/l BA were transferred after four weeks to rooting media supplemented with different concentration from NAA (0.0, 0.1, 0.2 mg/l) in combination with 2 sucrose concentrations 3 and 6%. Number of shoots and roots were screened after 4 and 8 weeks. Plantlets developed on solid media were screened and acclimatized after 12 weeks from seed culture.
Effect of different sugar types: MSB5 medium supplemented with 4 mg/l NAA, 0.5 mg/l BA and 3% sugars: fructose, glucose, lactose, maltose and sucrose were used separately with surface sterilized seeds culture. Subcultures were carried out after 4 weeks onto the same media composition and sugar type. Eight weeks old cultures were transferred to hormone free media with 6% sugar concentration. Twelve weeks old plantlets were screened and acclimatized.
Effect of different methyl jasmonate concentrations: Surface sterilized seeds were cultured on media supplemented with 4 mg/l NAA, 0.5 mg/l BA and different concentrations from methyl jasmonate (0.02, 2.24, 22.4, 44.8 mg/l). Subcultures were carried out after 4 weeks on the same media. All cultures were screened for number of shoots and roots after 4 weeks from first subculture.
Effect of Ca-panthothenate and biotin: Seeds were cultured on media supplemented with 4 mg/l NAA, 0.5 mg/l BA and different concentrations from each of Ca-panthothenate and biotin (0.5, 1.0, 1.5, 2.0 mg/l). Subculture was carried out after 4 weeks on the same media. Explants were screened for root number after 4 weeks from first subculture.
Healthy plantlets from the experiments (effect of NAA concentrations and sugar types) were carefully removed from the Magenta box and excess medium removed by washing with tap water. Plantlets were transferred to pots containing garden soil, covered with polyethylene bags to maintain high humidity. Plantlets were watered daily with tap water and incubated at 25°C under white fluorescent light for one week. Humidity was gradually decreased. After eight weeks plants were transferred to the greenhouse under ambient condition with programmed watering.
For each experiment, data were collected from each seed explant on number of shoots, fresh weight and dry weight. For each treatment, data were collected from 16 -32 seed explants cultured in 4 -8 Magenta boxes with 4 seeds in each. Data were analyzed using Minitab 17 software by one way ANOVA. Treatment means were compared by Fisher least significant difference (LSD) method with 95% confidence level.

Results and Discussion
Different physiological treatments were used to initiate and control adventitious organogenesis from C. schoenanthus subsp. proximus cultures. Using NAA and BA in different concentrations, followed by culturing on maltose selected as a carbon source, we were able to produce shoots efficiently within 6 weeks. This was followed by successful rooting on high sucrose concentration and greenhouse acclimatization (Fig. 5). Adventitious roots were produced on the addition of each of Ca-pantothenate, biotin and methyl jasmonate to the culture media in the presence of growth regulators. Such roots are a potential rich source of bioactive compounds.
Callus developed on MSB5 medium supplemented with different concentrations of NAA and BA. After three weeks of seed culturing, the highest callus formation response frequency (100%) was obtained from MSB5 medium supplemented with 4 mg/l NAA and 0.5 mg/l BA (Fig. 1).
Number of indirect regenerated shoots and roots were recorded after one and eight weeks of seed culture are shown in Table 1. After four weeks of culture, the best mean number of shoots (5.0 ± 2.8 shoot/explant) was observed on a medium supplemented with 0.5 mg /l NAA in combination with 0.5 mg/l BA, after 8 weeks both 1.0 and 4.0 mg/l NAA in combination with 0.5 mg /l BA gave the highest mean number of shoots 5.00 ± 0.2 and 4.50 ± 0.4, respectively. Auxin is required in polar growth of all organ primordia (Benjamins andScheres 2008, Delker et al. 2008). Auxins play an important role in axillary meristem initiation in vegetative branching of grasses (McSteen 2010). Biosynthesis and transport of auxins have an important role in stem elongation, leaf initiation and development. Reduction in auxin transportation decreased internodes length in mutant maize (Phillips et al. 2009), narrow leaf and dwarf stem in mutant rice (Qi et al. 2008) and decreased number of leaves in Arabidopsis (Bennett et al. 1995). Auxin has a role in the control of tiller angle in rice, which enhanced rice growth and density (Wang et al. 2007). Indirect organogenesis from Vitex trifolia plant required higher concentration from BA comparing with NAA in the culture medium support indirect organogenic shoot formation (Arulnndam and Ghanthikumar 2011). In rooting stage, higher concentrations of NAA 4.0 mg/l with 0.5 mg/l BA and 8.0 mg/l NAA with 0.25 mg/l BA produced highest mean number of roots 1.71 ± 0.5 and 0.75 ± 0.5, respectively. Healthy shoots (2 -3 cm length) produced on MSB5 medium with 4.0 mg/l BA and 0.5 mg/l BA were transferred after 8 weeks to a rooting medium composed of MSB5 medium fortified with different concentrations of sucrose and NAA. The results indicated that different concentrations of NAA did not show a significant difference (p < 0.05). However, result from higher concentration of sucrose (6%) was significantly better than that of 3% with p value = 0.08. Higher concentrations from sucrose in combination with 0.1 mg/l NAA produced higher mean number of roots 7.0 ± 2.0 roots/explant (Table 2). Similar result of enhanced root formation from regenerated sugarcane shoots cultured in medium containing high concentration of sucrose (5%) was reported (Gopitha et al. 2010), glucose with 9% concentrations induced root proliferation in Peonies plants (Gabryszewska 2010    Sugar types at 3% concentration were highly significant on indirect regeneration of shoots and subsequent rooting (Fig. 2) at p = 0.0. Maltose gave rise to the best mean number of shoots (13.5 ± 0.7 shoots/explant) after eight weeks. On the other hand, the highest mean number of roots (12.4 ± 1.0 roots/explant) was obtained from MSB5 medium supplemented with sucrose (Table 3). Our previous results reported that maltose was the best sugar in somatic embryogenesis (Abdelsalam et al. 2015) and micropropagation of C. schoenanthus subsp. proximus (Abdelsalam et al. 2017). Maltose produced the best results in shoot induction from soybean callus culture (Sairam et al. 2003), and is considered as a favorable sugar for in vitro plant regeneration of many grasses including, rice and wheat (Last andBrettell 1990, Navarro-Alvarez et al. 1994). In maize plant regeneration, both maltose and sucrose showed better results in shoot and root induction experiments (Gauchan 2012). Maltose enhanced embryo induction four times higher than sucrose in wheat anther culture (Last and Brettell 1990). Maltose enhanced shoot proliferation from protoplast derived tissue of indica and japonica rice varieties (Jain et al. 1997).  Effect of methyl jasmonate on indirect regeneration is shown in Table 4, Fig.  4B. Methyl jasmonate had an inhibitory effect on shoot production; shoot induction was retarded or completely inhibited using 0.02 mg/l MeJa in the culture media. Although, for rhizogenesis 2.24 mg/l MeJa gave the highest mean number of regenerated roots (13.33 ± 3.40), the data were not statistically significant different when compared to control and other concentrations. Exogenous addition of methyl jasmonate inhibited the in vitro shoot regeneration of tobacco and leaf blade formation of lily bulbs plants (Biondi et al. 2001, Jasik andKlerk 2006). Certain concentration from methyl jasmonate reduced shoot formation in thin cell layer tobacco culture through over inducing of mitotic division and cell expansion around shoot primordia leading to thickening and suberization (Capitani et al. 2005). MeJa also showed an inhibitory effect in different stages from somatic embryogenesis of Medicago sativa (Rudus et al. 2006). MeJa was reported to produce an antagonistic effect on cytokinin activity in many studies such as, inhibition of anti-senescence effect of Kn in oat, and effect of cytokinin in soyabean callus growth (Ueda and Kato 1982). Effect of different concentrations of Ca-pantothenate and biotin on indirect organogenesisis is shown in Figs 3 and 4C-D. Although none of the vitamin treatments showed any organogenic shoot regeneration, root number was enhanced in media supplemented with vitamins. Lower concentration of Capantothenate (0.5, 1.0 mg/l) yielded a higher mean number of roots compared to the same concentration of biotin. Moreover, higher concentrations of biotin (1.5, 2.0 mg/l) were better than their respective concentrations from Ca-pantothenate and control. 1.5 mg/l biotin produced the best mean number of roots (17.00 ± 4.04 roots/ explant). Roots of Cymbopogon species are important source of terpenoids. Roots extract from Cymbopogon citratus contained 10 sesquiterpenoid that differed in their chemical composition than those present in shoots (Li et al. 2005). Cymbopogon nardus root extract contained the greatest inhibitory activity in comparing with leaf and stalk (Suwitchayanon et al. 2014).  The effect of vitamins in organ induction and developments were widely described in previous studies. Biotin enhanced root growth of Cymbidium and improved the colonization of alfalfa roots (Streit et al. 1996). Ca-pantothenate and biotin enhanced plant regeneration through organogenesis of Cymbopogon martinii (Baruah and Bordoloi 1989). Addition of coconut water and biotin to the culture medium also significantly enhanced shoot bud induction and multiplication from Cymbopogon pendulus (Bhattacharya et al. 2010). Thiamine improved root formation in Taxus species (Chee 1995). Addition of vitamin E in Eucalyptus tissue culture media enhanced adventitious root formation (Roux et al. 1991). The use of in vitro cultures is important in the genetic manipulation, conservation of species, for the production of bioactive compounds and as a source of variation for different characters. In the present work, we established an efficient, short term de novo regeneration system that can be used for propagation, ex situ conservation, as a source of bioactive compounds and a potential source of variation for the species. Shoots in high numbers (13.5 ± 0.7 shoots/explant) were produced when maltose was used as carbon a source and acclimatized plants were successfully established in the greenhouse within 3 months.
Sensitivity of the species tissues to auxins was demonstrated on two occasions: (a) The use of NAA at high concentrations than BA resulted in shoot regeneration both directly from zygotic embryo germination, mixed with adventitious shoots produced from seed callus tissue. Tillering in grasses both in vivo and in vitro has been shown be positively affected by auxins. (b) The interaction between NAA and each of Ca-pantothenate and biotin, where the vitamins used seemed to nullify the ability of auxins to produce de novo shoots, and exhibited high ability for adventitious root production. Producing adventitious roots using different vitamins treatments can be used to enrich the production of the bioactive terpenoids in vitro and can lead to future studies with hairy root culture transformation of the species for the production of such bioactive compounds.
Due to the known relatively narrow geographical distribution of Cymbopogon schoenanthus subsp. proximus in sub-tropical Africa, and due to the harsh climatic conditions in such natural habitats, the present work may aid in producing genetic variation, within a relatively short time span, that can be useful for the production of different bioactive compounds. It can also provide propagules for ex situ conservation.