The induction and development of somatic embryos from the in vitro cul­ tures of Catharanthus roseus (L.) G. Don

: Catharanthus roseus is containing anticancer alkaloids of vincristine and vinblastine and is an important medicinal plant. Several studies have con­ ducted on in‐vitro culture of this plant. To optimize the somatic embryogenesis, a factorial based on CRD experiment with 10 replications was conducted. Root, hypocotyl and leaf explants grown in­vitro were transferred and cultured on MS media containing diﬀerent combinations of 2,4­D, NAA and 2,4­D×BAP. The results revealed that in callogenesis, the interaction eﬀects of root and hypocotyl explants×2,4­D and NAA as well as hypocotyl×(1 mg l ­1 NAA+1 mg l ­1 BAP) was superior than other treatments (p≤0.01). For calli fresh weight, hypocotyl×NAA and hypocotyl× (1 mg l ­1 NAA+1 mg l ­1 BAP) was the treatment of choice (p≤0.01). The calli produced were sub­cultured to attain the pre­ embryos and somatic embryos. For the number of pre­embryos and somatic embryos; the interaction of hypocotyl×2,4­D was the most eﬃcient treatment. Seemingly, the production of somatic embryos is accessible in this plant by the logical management of growth regulator combinations. Furthermore, the pro­ duction and genetic engineering of the somatic embryos could be a promising trend in the subsequent production of high­valued metabolites from this plant


Introduction
Catharanthus roseus (L.) G. Don, generally known as Madagascar Periwinkle, is a dicotyledonous plant with 2n=16 belongs to the family of Apocynaceae. Catharanthus roseus (L.) G. Don is an herbaceous plant that grows to a height of about 80 Cm and is native of Madagascar (Hogan, 2003). More than 130 types of alkaloids have been extracted from the vegetative parts and roots of this plant, which are used to treat several diseases (Aslam et al., 2009). The most important alkaloids extracted from the shoots of this plant are vincristine and vinblastine, with a well defined anticancer properties (Mujib et al., 2012). The amount of these compounds in this plant is about 0.0005% of dry weight of the plant and their extraction is costly and time consuming (BarralesCureño et al., 2017). These problems have led the scientists to a new approaches of tis sue culture studies in C. roseus L. (Van Der Heijden et al., 2004). The first study done on tissue culture of Catharanthus roseus (L.) G. Don was in 1977. Those, researchers were able to produce callus from the plant (Dhruva et al., 1977). The production of shoots from callus has been successfully done in Catharanthus roseus L. (Ramawat et al., 1987). Somatic embryogenesis is a process by which somatic cells differentiate into embryos, called somatic embryos, which are used as one of the practical in vitro techniques for plant micropropagation (Von Arnold et al., 2002). The first studies on somatic embryogenesis in the Catharanthus roseus (L.) G. Don was done in 1994, which were succeeded in pro ducing somatic embryos by the anther culture (Kim et al., 1994). Furthermore, by hypocotyl explant and 1 mgl 1 of 2,4D (2,4Dichlorophenoxyacetic acid), somatic embryos were obtained (Aslam et al., 2004(Aslam et al., , 2006. Growth regulators (composition and concen tration) and the plant genetic makeup play a role in the success of somatic embryos production. The phe nomenon of genotypedependent plant regeneration also exists in other plant species (Firoozabady and De Boer, 1993).
The physiological conditions, the explant growth stage and embryogenic tissue type affect the produc tion of somatic embryos. The tissue that has the high est metabolism and the least differentiation rate may have the suitable embryogenesis potential (Mikula and Rybczynski, 2001). Auxins, especially 2,4D, are among the most important plant growth regulators initiate the somatic embryogenesis (Choi et al., 1999;Martin, 2003;Gulzar et al., 2019). In medicinal herb Spermatozoa hispida L.; cytokinin, especially BAP (6 Benzylaminopurine), induced frequent somatic embryos (Deepak et al., 2019). With medicinal herb, Coccinia abyssinica; 2,4D and BAP combination helped to produce embryogenic callus, and BAP trig gered the production of somatic embryos (Abate et al., 2019). Lglutamine and Lalanine amino acids have had the most positive effect on somatic embryogenesis process (Ji et al., 2011). Various sources of carbon; such as sucrose, fructose, and glu cose, as energy sources and osmotic regulators; play an important role in the somatic embryogenesis initi ation and frequency (Aslam et al., 2011). MS has been employed as the most common culture medi um for most of the plants (Ji et al., 2011). Somatic embryogenesis, leading to the regeneration of intact plants, is an eminent step in the plant transforma tion. Successful and sustainable transformation requires that a single cell give rise to a whole intact plant. The Ideal transformation takes place through the direct somatic embryogenesisfrom the single cells to reach the intact plants (Aslam et al., 2007).
The aim of the present study was to investigate the different explant types and the diverse plant growth regulators effects to induce somatic embryos and to study of the traits related to the somatic embryogenesis potential in Catharanthus roseus (L.) G. Don.

In vitro seed germination and seedlings production
The present study was conducted in the Research Laboratory, Department of Horticultural Sciences, University of Maragheh, Iran. The seeds were acquired from Pakan Bazr Isfahan Company. The seeds were immersed in water for one day before planting in the dark, and the next day, they were first treated with ethyl alcohol (70%) for one minute and then disinfected with 20% solution of sodium hypochlorite for 10 more minutes. Then, they were washed with sterile distilled water 5 times. The disin fected seeds were cultured in petri dishes containing 20 ml of MS medium (Murashige and Skoog, 1962) without plant growth regulators. In each petri dish; 10 disinfected seeds were cultured at 2528°C. After emergence, the seedlings were transferred to the photoperiod conditions with 16 hours of light at 25°C and 8 hours of darkness at 20°C. After about 10 days, the 24 cm in length seedlings were suitable to con tinue the experiment.

Preparation of culture media
The culture medium was MS basic medium. Sucrose at a concentration of 30 g l 1 was used as a source of carbon. Then, MS culture medium salts were added. pH was set at 5.8. At the last stage, 6 gl 1 of agar was added and placed in an autoclave at 121°C for 20 min. Agarfree culture medium (liquid) was employed for the somatic embryos.

Sampling and culture of explants in different treatments
The explants; root, hypocotyl and leaves were excised from the 24 cm seedling grown in vitro. Hypocotyl explants were cut into a length of about 1 cm and, in order to obtain embryogenic callus; they were cultured in the MS medium supplemented with the same treatments as subcultures. For calli prolifer ation; 3 weeks after the first date of cultivation, the same plant growth regulator treatments were applied to the subcultures. One month after the first subculture; embryogenic calluses were subcultured in MS medium supplemented with concentrations of 0.5, 1, and 1.5 m g l 1 BAP. Then, the resulting calli were transferred to MS medium supplemented with 7 g l 1 of agar, 30 g l 1 of sucrose, 1 mg l 1 glutamine and 3 g l 1 of polyethylene glycol with 6 replications. At the all stages of cultures and subcultures; the sam ples were kept in a growth chamber for 16 h light at 25°C and 8 h dark at 20°C.

Data mining and statistical analysis
The callus production percentage, the percentage of necrotic calli and callus weight were noted. After multiple subcultures and transfer to the embryogen esis culture medium, the number of preembryogenic mass formed were recorded. SAS was used to ana lyze the variance, and mean comparisons were done with Duncan's multiple range test at 5% probability.

Callus production
Hypocotyls of in vitro germinated seeds were used as explant on MS medium supplementary with 2,4D and NAA, which induced white to yellowish cal lus within 10 days of incubation. The embryogenic callus was white, granular, friable, fast growing with in 3 weeks of culture. The number of explants that produced callus and the necrotic calli proportion showed the callogenesis percentage and the percent age of necrotic calli (Fig. 1).
The mean comparison showed that for the callus formation, the interaction between root and 2,4D; hypocotyl and 2,4D; root and NAA; hypocotyl and NAA; and hypocotyl and 1 mgl 1 NAA + 1 mgl 1 BAP were significant at 1% probability level. Figure 2 shows the significant effect of auxin use on the rate of callus production. In this study 2,4D was very effi cient in producing callus and embryos on Catharanthus roseus. Embryonic callus was not observed using leaf explants and hypocotyl explants had the highest embryonic callus.
Furthermore, for the callus fresh weight, the interactions of hypocotyl and NAA; and hypocotyl × 1 mg l 1 NAA + 1 mgl 1 BAP were significantly different from other treatments (p≤0.01) (Fig. 3). Embryogenic callus from the treatment with 1 mg l 1 2,4 D; (B) Embryogenic callus from the treatment with 2 mg l 1 NAA, (C) Embryogenic callus from the treatment with 1 mg l 1 NAA + 1 mg l 1 BAP. Fig. 2 The interaction effects of explant×plant growth regula tors on the callogenesis percentage of Catharanthus roseus. A= 1 mg l 1 2,4D×root; B= 1 mg l 1 2,4D×hypo cotyl; C= 1 mg l 1 2,4D×leaf; D= 2 mg l 1 NAA×root; E= 2 mg l 1 NAA×hypocotyl; F= 2 mg l 1 NAA×leaf; G= 1 mg l 1 NAA+1 mg l 1 BAP×root, H= 1 mg l 1 NAA+1 mg l 1 BAP×hypocotyl; I= 1 mg l 1 NAA+1 mg l 1 BAP×leaf). The calli weight was obtained by weighting 10 samples. The results showed that the growth regula tors and explants type and their interaction were sig nificant on the weight of callus production, Treatment composition H and E with 0.85 and 0.81 g, had the most callus weight respectively. The interac tion of hypocotyl and NAA; and hypocotyl x NAA + BAP were significant (p≤0.01) on callus fresh weight (Fig. 4).

Pre-embryonic and somatic embryogenic tissues
In this study, two different approaches were employed to increase the formation of embryogenic calluses. Both approaches were aimed at choosing the best combination and concentration of plant growth regulators. Different masses were observed on the embryogenic calli tissues and the preembry onic structures were formed evidently. The number of masess of possible pre and somatic embryos formed were counted. Somatic embryos were isolat ed by liquied culture and shown with distinct roots and shoots (Fig. 5).
The results showed that the effect of plant growth regulators and explants, and their interactions were significant (p≤0.01) on the number of preembryos and somatic embryos produced.
Furthermore, the interactions of hypocotyl × 2,4 D produced the highest number of preembryogenic and somatic embryogenic with mean 3.83 and 5.33 respectively in solid culture medium (Fig. 6).
2,4D causes the rapid cell division and the polar ization of cells (Jushee et al., 2007). Our results are almost similar with the findings of Choi et al. (1999) On Eleutherococcus senticosus (75% of somatic embryos). Moreover, the findings of the present study are in line with the results of Martin (2003) on Holostema adakodien, which by using different explants and plant growth regulators; they clearly showed that hypocotyl explants and 2,4D (1 mg l 1 ) attained about 50% of somatic embryos. In another study, hypocotyl explants of Eleutherococcus senticosus in MS medium produced 89% of somatic embryos (Han and Choi, 2003). Also, our results are the same with Aslam et al. (2004) on Catharanthus roseus (L.) G. Don, whom described that hypocotyl and 2,4D (1 m gl 1 ) were the combination of choice. Also, in another study on Catharanthus roseus (L.) G. Don; the most somatic embryos were observed using hypocotyl explants and 2 mgl 1 2,4D. Auxins have synergistic effects with cytokinins and accelerate the cell division. 2,4D may also addup the endogenous levels of IAA in plant tissue, thereby creating dipoles within the cell and forming preembryonic structures (MendezHernandez et al., 2019). 2,4D promotes the accumulation of ROS (oxygen reactive species), and stimulates the ethylene and abscisic acid biosyn thesis in plant tissue and ultimately induces stress behavior in plant tissue. As a result, plant cells change or shift to form somatic embryos (Bharatia et al., 2015;Wojcik et al., 2020). 2,4D application and availability in the culture medium probably results in the expression of cell differentiation genes and the demethylation of DNA. 2,4D plays an important role in the somatic embryos induction and in the stages of maturation and development of somatic embryos. Whereas, 2,4D, has an inhibitory role with the biosynthesis of a number of proteins and mRNAs (Krishnan and Siril, 2017). In our study, calli from leaf explants were unable to produce somatic embryos (Gulzar et al., 2019). Otherwise, Paeonia ostii 'Feng Dan', Asparagus racemosus Willd, and Cnidium officinale Makino, the combinations of auxins and cytokinins produced reasonable somatic embryos (Adil et al., 2018;Chaudhary and Dantu, 2019;Ren et al., 2020).
Auxins had dominant effect on callus production of Catharanthus roseus (L.) G. Don. and the results showed that 2,4D was much efficient than NAA. More somatic embryos were obtained from hypocotyl explants. Due to the medicinal importance of Catharanthus roseus, it is suggested that in the further studies, somatic embryogenesis behavior in this plant should be studied on a larger scale and that the possibility of somatic embryogenesis in a liquid culture medium using suspension culture should be tested in different ways. Therefore, it is possible to study the biosynthesis of secondary metabolites and valuable alkaloids in vitro conditions and to optimize the protocols to extract and purify the above men tioned metabolites.