Influence of Plant Growth Regulators on Somatic Embryogenesis Induction in Seriphidium herba-album

Seriphidium herba-album (syn. Artemisia herba-alba) is a medicinal, aromatic, greenish-silver herb. It is used widely in folk medicine for treatment of diarrhea, abdominal cramps and in the healing of external wounds. It's also used for the treatment of diabetes mellitus, neurological disorders as epilepsy, Alzheimer's disease, depression and jaundice. In this study we assessed the protocol for callus induction, maturation of somatic embryogenesis, frequency of germination and conversion into plantlets for leaf explants of Seriphidium herba-album using different concentrations of PGRs. Highest induction frequencies of embryogenic calli occurred after 35 days on MS medium supplemented with 1.5 mg L-1 2,4-D and 0.5 mg L-1 BAP. Optimum MS medium for higher frequency of matured somatic embryos was recorded using 5.0 mg L-1 BAP and 0.5 mg L-1 NAA and somatic embryos also induced young in vitro grown plantlets when cultured in the medium containing GA3 and kinetin. Hence, attempts to induce direct somatic embryogenesis have been achieved up to embryo regeneration and maturation.


I. INTRODUCTION
Seriphidium herba-album (previously named Artemisia herba-alba Asso.) known also as desert wormwood (known in Arabic as shih, white wormwood, armoise herbe blanche). Seriphidium herba-album is a small perennial shrub native to northern Africa, western Asia, Southwestern Europe, and the Arabian Peninsula, where it grows on dry steppes. It grows from 8 to 16 inches tall, is strongly aromatic, and is covered with fine glandular hairs which give it a grayish aspect (Segal et al., 1987;Nahla et al., 2011). Seriphidium herba-album is one of the most important medicinal plants that have been used in folk medicine for the treatment of gastric disturbances and healing external wounds, remission of diabetic symptoms, activating the function of the liver, and healing rashes, joint pains, inflammations and rheumatoid arthritis, with no side-effects (Feuerstein et al., 1986;Essawi and Srour, 2000). Moreover, experimental evidence showed that S. herba-album decoctions have beneficial effects as antioxidants as they contain compounds like tannins (Ben Abid et al., 2007). S. herba-album was found to have antifungal activity against Penicillium citrinum and Mucor rouxii due to the presence of carvone and piperitone isolated from the fresh leaves of the plant (Mahmoud et al., 2007;Boutkhil et al., 2011). The tissue culture of various Artemisia species of various Artemisia species is largely focused on improving artemisinin production. In vitro cultures of this species have been the object of great amount of research (Gulati et al., 1996;Nin et al., 1996;Honda et al., 2001;Liu et al., 2004;Sujatha and Rajnitha Kumari, 2007a;Mannan et al., 2008;Rezvan et al., 2010;Gopinath et al., 2014). It is widely known that plants from a natural resource or plants propagated by conventional means generally produce small quantities of secondary metabolites which makes their price very high; hence the studies on plant tissues and plant micropropagation offer various advantages for obtaining massive amounts plant material. The plant micropropagation is an efficient method for propagating disease-free and genetically uniform plants. It also appears that both liquid and solid shoot cultures are a good choice for micropropagation in laboratories because they reduce costs related to the automation of the procedure (Honda et al., 2001).The artemisinin production in shoot cultures of such s pecies as A. pontica, A. judaica, A. vulgaris, A. scoparia, A. absinthium and A. annua was investigated by a number of researchers (Gulati et al., 1996;Pan et al., 2003;Sujatha and Rajnitha Kumari, 2007b;Singh and Sarin, 2010;Kour et al., 2014;Dangash et al., 2015). The present study is aimed to develop an appropriate and efficient regeneration protocol for the medicinal plant Seriphidium herba-album from leaf explants using different concentrations of PGRs for the first time, which has a high potential to be used in conservation and genetic transformation of this important medicinal plant.

II. MATERIALS AND METHODS Plant material and sterilization.
Plant materials of Seriphidium herba-album were collected from North West coast of Egypt. The leaf explants were washed under running tap water for two hours. Surface sterilization was carried out under complete aseptic conditions in the Laminar Air Flow Hood by using aqueous mercuric chloride solution (HgCl 2 ) (0.1% w/v) for 5 minutes, then rinsed once with sterile distilled water and transferred to 2.5% aqueous solution of sodium hypochlorite for 20 minutes at the end of sterilization treatment; the explants were rinsed 3-4 times with antioxidant solution. Sterilized explants were cut into leaf segments (0.5-1.0 cm) and implanted into sterilized media containing various combinations of plant growth regulators.

Callus induction.
Leaf explants were cultured on solidified MS medium containing 2, 4-D (1.0-2.0 mg L -1 ) alone or in combination with BAP (0.5-1.5 mg L -1 ). Media were solidified with 2.5 g/L g L -1 , subjected to pH 5.8 before autoclaving (121°C). A total of 12 different hormonal combinations were tested. The effect of the hormonal composition was evaluated by counting the calli obtained after 35 days of culture in the dark at 25 ± 2°C. Percentage of induced callus in each culture was evaluated as follows: Number of callus/Total number of explants x100.

Somatic embryos induction.
Induced primary somatic embryonic calli were sub cultured for further production of embryonic callus and their subsequent germination. Medium for induction of somatic embryonic callus, their proliferation and germination was conducted by transferring the somatic embryos into concentration of BAP alone or in combination of NAA and different complex additives. In defining the media composition, 15 different media containing BAP (1.0, 2.0, 3.0, 4.0 and 5.0 mgL -1 ) alone or in combination with NAA (0.5 and 1.5 mgL -1 ) were tested separately for induction of somatic embryogenesis. Calli were incubated for eight weeks period in this medium. For germination of somatic embryos, MS medium supplemented with GA3 (1.0, 2.0 and 3.0 mgL -1 ) in combination with Kn (0.5 and 1.0 mgL -1 ) were tested. The culture was kept in growth room temperature at 25 ± 2°C with light intensity 3000 lux for 16 h photoperiod using cool white fluorescent lamps in the growth room for 8 weeks. Data were recorded as number and percentage of somatic embryogenesis formation. Statistical analysis.
The experiment was carried out based on complete randomized design. Each of the experiments, excluding field performance study, was executed in five replicates with 20 samples per replication. For in vitro culture experiments, every single explant was treated as an experimental unit. Analysis of variance (ANOVA) was used to statistical analysis of experimental data using MSTAT Software (2009). Differences between individual means were estimated according to Snedecor and Cochran (1982). All values are reported as means ± standard deviation.

III. RESULTS AND DISCUSSION Embryogenic callus induction.
Source of plant growth regulator is an important factor for impacting callus induction, somatic embryogenesis and plant regeneration. In most cases, successful plant regeneration needs a mixture of the different auxin and cytokinin. Callus were produced in all media containing 2,4-D alone or in combination with BAP. A considerable variation in callus induction percentage and biomass of Seriphidium herba-album was detected in parallel with a mixture of the different auxin and cytokinin. After 35 days, the highest Callus induction percentage (95.44%) was recorded with 1.5 mg L -1 2, 4-D and 0.5 mg L -1 BAP ( Table 1 Nin et al. (1996) reported that no callogenic response from leaf explant on PGR-free medium and explants died after few days. 2, 4-D as callus inducing hormone produced light green, soft, friable and compact callus from leaf and stem explants. But at all concentration of 2, 4-D organogenic response was not observed within observation time. Etienne et al. (1991) stated that water status in the callus is apparently important for initiating somatic embryogenesis, and RWC appear to be good physiological marker of it embryogenic state. Ganesan and Paulsamy (2011) observed 98.66% callogenic response from leaf discs with NAA at 0.9 mg L -1 in A. annua L. whereas Benjamin et al. (1990) observed callus induction from shoot buds using BAP plus IAA for Artemisia pallens. 2, 4-D at varying concentration (0.05-0.25 mg L -1 ) in combination with BAP (0.5 mg/l) also produced light green and soft callus when supplemented in MS medium. When IBA and NAA were combined with Kin, the callogenic response was also low and callus was not good in texture. Xu and Jia (1996) observed best callus result in the presence of 2, 4-D with Kin for Artemisia sphaerocephala. Values are presented by mean ± SE Same letters represent no significant differences between means at P ≤ 0.05 level.  (Fig. 3A and B) compared with other treatments. Results also show that the mean number of shoots on the explant (5.87) treated with MS containing 3.0 mg L -1 GA3 and 0.5 mg L -1 kinetin was significantly higher than the other treatments (Fig 3A and B). While, the best results of mean length of shoots formed per explant (14.28 mm) were produced on concentration of 3 mg L -1 GA3 and 1.0 mg L -1 kinetin compared to the other treatments ( Fig. 4A and B).   Values are presented by mean ± SE same letters represent no significant differences between means at P ≤ 0.05 level.

IV. CONCLUSION
The different concentrations and combinations of PGRs, used in our study, were effective to induce calli, maturation of somatic embryogenesis, frequency of germination and conversion into plantlets. The combinations of 2,4-D and BAP supplemented medium induced friable calli in the leaf explants of Seriphidium herba-album. The creamish green calli with good growth were observed under dark regimes on MS medium supplemented with 2,4-D (2 mg L -1 ) and BAP (1.5 mg L -1 ). Somatic embryogenesis was induced from callus explant in presence of BAP and Kn as cytokinin. The regeneration system developed in this study will be useful for plant improvement through indirect somatic embryogenesis and genetic engineering of Seriphidium herba-album Moreover, this system can be available for the clonal propagation in order to obtain the strain containing a constant concentration of artemisinin in Seriphidium herba-album.