DEVELOPMENT A PROTOCOL FORIN ...

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S.A. Bekheet, A.M.M. Gabr, M.K. El-Bahr. ... Plant Biotechnology Department, National Research Center, El-Tahrir Str., Dokki, Cairo (EGYPT) .... Recently, Ahmed et al. ..... El-Gizawy, A.M.; El-Bahr, M.K.; El-Oksh, I.I. and Bekheet, S.A. (1993).

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S.A. Bekheet, A.M.M. Gabr, M.K. El-Bahr. Development a protocol for in vitro morphogenesis and multiplication of globe artichoke (Cynarascolymus L). International Journal of Academic Research Part A; 2014; 6(6), 297-303. DOI: 10.7813/2075-4124.2014/6-6/A.41 Library of Congress Classification: QK640-(707)

DEVELOPMENT A PROTOCOL FORIN VITROMORPHOGENESIS AND MULTIPLICATION OF GLOBEARTICHOKE (Cynarascolymus L) S.A. Bekheet*, A.M.M. Gabr, M.K. El-Bahr Plant Biotechnology Department, National Research Center, El-Tahrir Str., Dokki, Cairo (EGYPT) *Corresponding author: [email protected] DOI: 10.7813/2075-4124.2014/6-6/A.41 Received: 01 Sept, 2014 Accepted: 02 Nov, 2014

ABSTRACT Areliable protocol for in vitro morphogenesis, regeneration and multiplication of globe artichoke was developed. In vitro sprouted shootlets (cotyledons) were taken from in vitro grown seedlings and re-cultured on MS medium contained 2 mg/l BA for establishment tissue cultures. Calli cultures were induced from leaf, petiole and stem explants of in vitro grown shootlets. The highest frequency of callus induction was observed on leaf explants cultured on MS medium supplemented with 5 mg/l kin + 0.5 mg/l IAA. Among different concentrations of picloram added to callus culture medium, 3 mg/l picloramgave the highest fresh weight and growth value.Concerning in vitro regeneration, the highest percentage of direct organogenesis (from leaf explants) was observed with medium contained 1 mg/l BA + 2 mg/l NAA. However, transferring callogenicpieces to 2 mg/l kin + 2 mg/l NAA containing medium resulted in the highest percentage of indirect organogenesis presented as shoot bud formation.In concern ofin vitro multiplication of shootlets, it was found that addition of BA to multiplication medium more effective to produce multiple shoots, while kin shows the longest shootlets. It is apparent from the results that both BA and kin have negative effect on the number of proliferated leaves. Key words: Globe artichoke, In vitroculture, Callus induction, Regeneration, Multiplication 1. INTRODUCTION Globe artichoke (Cynarascolymus L.) is an herbaceous perennial plant widely grown in the Mediterranean region. Its immature flower buds are eaten by man and its residues are used as animal feed. In addition, globe artichoke provides a rich dietary source of bioactive compounds derived from phenylpropanoid metabolism, notably caffeoylquinic acids and flavonoids [1,2].Therefore, globe artichoke is used in liquor and pharmaceutical industries. The globe artichoke is cross-pollinated plant species. This result in a high level of heterogeneity among the seed population. For this reason, globe artichoke is traditionally propagated by vegetative means [3]. These handicaps could be overcome only if homogenous seed-propagated cultures were available. However, the low rate of vegetative multiplication and the potential for disseminating diseases are two major factors that hinder the expansion and development of globe artichoke [4].The spread of this species through in vitroculture of shoot apices leads to plants free from systemic pathogens, with agreater multiplication rate than that obtained by traditional agamic multiplication. In recent years, micropropagationhas been successful in raising plants in vitro to a commercial level in many plant species. Multiple shoots can be produced in vitro and these can be developed into plantlets by regenerating their roots. Thus a single explant source, shoot tip or nodal segment could conceivably provide thousands of new true to type plantlets per year. In this concern, Growth and morphogenesis in vitro are regulated by the interaction and balance between the growth regulators supplied in the medium, and the growth substances produced endogenously. In this concern, picloram (4-amino-3,5,6-trichloropicolonic acid), known for its auxin-like activity, could be more effective than other auxins such as 2,4-D [5]. Low concentrations of picloram can stimulate RNA, DNA, and protein synthesis leading to uncontrolled cell division and growth, and, ultimately, vascular tissue destruction [6]. In vitro propagation of globe artichoke has been attempted. Several explants have been used to initiate in vitro cultures but shoot tips are the most widely used to produce virus-free plants. However, difficulties in explant disinfection have prompted some researchers to use seeds for culture initiation. In this respect [7] described a method of micropropagation and sanitation of globe artichoke using meristem culture. Taking advantage of new culture media [8], growth regulators [9] and mycorhizalinocula [10], micropropagation was improved for globe artichoke, facilitating the large-scale production of numerous cultivars and their clones. Otherwise, in vitrocallus induction is considered to be an important aspect in genetic manipulation as well as in production of

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secondarymetabolites of globe artichoke. In spite of micropropagation techniques have been established for this species, but in vitro cultures have not yet been extended to generate an efficient system for the induction of callus tissue. In this respect, callus formation and in vitro morphogenic responses in globe artichoke had been studied by several researchers [11,12,13,14,15]. In the present work, a reliable protocol for in vitro morphogenesis andmultiplication of one of the important globe artichoke cultivar (Green Globe) cultivated in Egypt was reported. 2. MATERIALS AND METHODS Establishment of in vitro cultures Seeds of globe artichoke (Green Globe cv.) were washed with distilled water and then immersed in 70 % ethanol for 1 min followed by 50 % commercial Clorox (containing 5.25 % sodium hypochlorite) for 20 min and finally washed three times with distilled sterilized water. The steps of disinfestation were took place under aseptic conditions in a laminar air-flow cabinet.The disinfected seeds were placed in 250 ml Erlenmeyer flasks contain 50 ml liquid free MS-basal medium [16] on a rotary shaker at 110 rpm as a sprout culture technique [17]. Shoot tips were isolated from the aseptic grown seedlings and re-cultured on fresh MS medium contained 2 mg/l BA to get of stock in vitroshootlet cultures (Fig.1-A). Callus induction For callus induction, three types of explant i.e., leaf, petiole and stem were excided (1 cmlength) from the in vitro grown shootlets and cultured on MS medium containing the following combinations of growth regulators: - 1 mg/l BA + 2 mg/l NAA.- 2 mg/l BA + 5 mg/l NAA. - 2 mg/l kin + 0.5 mg/l IAA.- 5 mg/l kin + 0.5 mg/l IAA. Three segments per jar of eachtype of explants were used. Percentages of callus formation (callus frequency) were registered after five weeks of culturing from20 replicates. Effect of picloram on callus growth In order to study the callus growth, about 250 mg of callus derived from leaf explants were re-culturedon MS medium supplemented with 5 mg/l kin + 0.5 mg/l IAA (the best medium for callus induction) supplemented with 1, 2, 3 and 4 mg/l of picloram. Fresh weight and growthvalue of callus were determined after five weeks of sub-culturing. Final fresh weight - Initial fresh weight *Growth value = _________________________ Initial fresh weight Morphogenesis andregeneration To investigate the ability of organogenesis and subsequent in vitroshootletproliferation, different explants i.e., leaf, petiole and stem as well as calli tissue were cultured on MS-medium supplemented with different combinations of cytokinins and auxins as follow: - 1 mg/l BA + 2 mg/l NAA.- 1 mg/l kin + 2 mg/l NAA. - 2 mg/l BA + 2 mg/l NAA.- 2 mg/l kin + 2 mg/l NAA. Direct organogenesis observed as mean of responses of the three expalnts (leaf, petiole and stem) and indirect organogenesis (from callus) were recorded after five weeks of culturing. In vitro multiplication of propagules To assess the effect of cytokininsonshootlet multiplication through formation of adventitious shoot buds of globe artichoke, the proliferated shootlets (1cm length) were cultured on MS medium supplemented with different concentrations of BA and kin as follow: - MS hormone-free.- MS + 5 mg/l kin. - MS + 2 mg/l BA.- MS + 1 mg/l BA + 5 mg/l kin. The number of proliferated shootlets, shootlet height (cm) and number of proliferated leaves were recorded after six weeks of culturing Culture media and incubation conditions Tissue culture media were solidified with 0.7 % agar and contained 30 g/l sucrose.pHwas adjusted to 5.8 beforeautoclaving at 121°C and 1.5 Ib/M² for 25 min. In all treatments, the growth regulators were added to the culturemedium prior to autoclaving. Cultures were maintained at 25 ± 2°C and 16 hr photoperiod provided by whitefluorescent tubes (3000 lux light intensity). Experimental design and statistical analysis Each experiment was set up as a separate completely randomized design. Data were statistically analyzedusing standard error (SE) according to the method described by [18].

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3. RESULTS AND DISCUSSION Callus induction Results demonstrated that callusinduction affected by type of explant (leaf, petiole and stem) and combinations of growth regulators is presented in Table (1). Data observed indicated that callus was induced on all explants within five weeks of culturing. A range of varying callus frequencies was obtained depending on growth regulator combinations and type of explant. Generally, IAA was more effective when combined with kin compared with thosecombinations of BA and NAA. From the four combinations of growth regulators, it was found that supplementation of culture medium with 5 mg/l kin + 0.5 mg/l IAA gave rise to the maximum frequency of callus formation. However, among the three explants examined, leaf segments were the most suitable for callus induction since it gave the best results of callus frequency with all combinations of growth regulators used. The highest value of callus frequency (80%) was registered when leaf explant cultured on medium contained 5 mg/l kin + 0.5 mg/l IAA (Table 1 and Fig. 1-B). Table 1. Callus frequency (%) induced on globe artichoke explants i.e., leaf, petiole and stem after five weeks of culturing on MS medium containing different combinations of auxins and cytokinins Growth regulators 1 mg/l BA + 2 mg/l NAA 2 mg/l BA + 5 mg/l NAA 2 mg/l kin + 0.5 mg/l IAA 5 mg/l kin + 0.5 mg/l IAA

Leaf 30 40 60 80

Explant Petiole 20 30 40 50

Stem 10 20 30 30

Mean n= 20

Responses of explants culturedin vitrodepend on the explant source and the combination of exogenously applied plant growth regulators. Callus cultures usuallyare produced fromany differentiated plantstructure i.e. leaf, stem and root by placing explants on media containing relatively high level of auxin and low level of cytokinin[19]. It is well known that callus formation is due to cell division and cell elongation of existing cells. Auxins increase cell growth while cytokinins are responsible for cell division. In the present work, it was observed that kin in combination with IAA was more effective on callus induction of globe artichokecompared with BA in combination with NAA. Furthermore, leaf explant generally registered the highest percentages of callus induction. The influence of growth regulators used in our study is in harmony with those used byOniseiet al. [12] and El-Bahr et al. [14]. They mentioned that, leaf was best explants used for in vitro callus initiation of artichoke. However, Ordaset al [13] found that most prolific callusing response of immature flower buds of globe artichoke was obtained by using MS medium supplemented with 2 mg/l BA + 5 mg/l NAA. Recently, more than 100 combinations of media supplements (e.g., phytohormones, absorbers of polyphenols, and inhibitors of polyphenol oxidase), along with various light regimes, and three different genotypes of globe artichoke were investigated to define the optimal conditions for callus induction from leaf explants [20]. This led to the elaboration of an in vitro culture protocol which resulted in a high frequency of callus induction after just 1 week in culture. Effect of picloramon callus growth The effects of picloramadded to culture medium on development and growth of callus proliferated from leaf segments ofglobe artichoke are illustrated in Table (2).The data showed that the callusfresh weight was obviously affected by addition of picloram into culture medium. Growth value also was alsoenhanced by supplementation of culture medium with picloram. It was notable that addition of 3 mg/l picloram to culture medium registered the highest values of callus growth presented as fresh weight (1.95 g) and growth value (6.8) (Table 2 and Fig. 1-C). However, the lowest growth parameters were recorded with picloram-free medium. Picloram supports growth of explants in tissue culture, inhibits root growth, induces cell wall loosening, produces stem curvature and other formative effects. In the present study, it was found that supplementation of culture medium with picloram generally enhanced the growth of globe artichoke callus cultures.It gave rise to the best responses towards callus fresh weight, and growth value especially at the concentration of 3 mg/l.In this connection,it was proved thatpicloram could be used to induce embryogenic callus from leaf explants of Gentianapneumonanthecultured on medium containing 8 µM, whereby embryogenic callus of various appearances such as yellow, white or crystalline was formed [21]. Recently, Ahmed et al. [22] mentioned that the best embryo callus induction of Phyla nodiflora (L.) Greene was obtained using medium contained 0.1 mg/l picloram. Furthermore, Castellar et al. [23] In their study on establishment of callus and cell suspension cultures of Petiveriaalliacea Lreported that cells grown in the presence of picloram entered the exponential phase after a lag phase of four weeks, with a 4-fold increase in fresh weight associated to approximately 2-fold increase in dry weight by the end of the 5th week.

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Table 2. Fresh weights and growth values of callus of globe artichoke grown for five weeks on MS medium contained various concentrations (1, 2, 3 and 4 mg/l) of picloram Picloram(mg/l) 0.0 0.1 0.2 0.3 0.4

Fresh weight (g) 1.15 ± 0.22 1.60 ± 0.30 1.80 ± 0.14 1.95 ± 0.50 1.70 ± 0.24

Growth value 3.6 5.4 6.2 6.8 5.8

± Standard error (SE)

Morphogenesis and regeneration The growth, differentiation and organogenesis of tissues become feasible only on the addition of one or more plant regulators to a medium. This part of study aimed to investigate the ability of in vitro direct and indirect organogenesis of globe artichoke using NAA as auxin combinedwith BA or kin as cytokinin. For this purpose leaf segments and callus pieces were cultured on MS medium amended with the different combinations of the three growth regulators. Dataillustrated in Table (5) revealed that BA when combined with NAA obviously resulted the highest values of direct organogenesis from leaf segments. The medium contained 1 mg/l BA + 2 mg/l NAA gave the highest percentage (70 %) of direct organogenesis (Fig. 1-D). However, combinations of kin and NAA showed remarkable increasing of indirect organogenesis from callus. The medium containing 2 mg/l kin + 2 mg/l NAA gave the highest percentage (60 %) of indirect organogenesisobserved as shootlet proliferation (Fig. 1-E). The availability of efficient direct (from explants) or indirect (from callus) regeneration systems is of primeimportance in the application of plant improvement using in vitro cultures. In vitro plant regeneration is a complex phenomenon involving different biochemical mechanisms for its progression. It is the process of the activation and regulation of certain enzymes at specific times for organogenesis [24]. Direct organogenesis asproliferation of shoot buds is usually used to produce clonal plants that are true-to-type, and therefore unorganized callusphase was avoided.Regeneration presented here as direct or indirect organogenesis of globe artichoke was varied depending on cytokinins added to culture medium. BA was more effective on direct organogenesis. However, Kin was more suitable for indirect organogenesis.The results are in line with those reported Petri and Ricci [25]. They used BA or Kin in combination with NAA for in vitro shoot regeneration of globe artichoke. The use of NAA x BA combination for in vitro morphogenesis of globe artichoke has been also described byEl-Bahr et al. [14]. They mentioned that indirect morphogenic response as shoot organogenesis from calli of globe artichoke was achieved by using MS medium containing 5 mg/l BA+ 2 mg/l NAA. They added that the ability of organogenesis varied depending on genotype and type of explants. In this respect, a successful in vitro regeneration of shoots from bracts of globe artichoke was recognized [13]. In this work, the most rapid and prolific callusing response was obtained using a medium containing 5 mg/l NAA + 2 mg/l BA. Shoot differentiation was obtained following transfer to MS medium containing 0.5 mg/l IBA+ 5 mg/l BA. Table 3. In vitro direct (from leaf explants) and indirect (from callus) organogenesis frequencies of globe artichoke after five weeks of culturing on MS medium containing NAA combined with BA or kin Growth regulators 1 mg/l BA + 2 mg/l NAA 2 mg/l BA + 2 mg/l NAA 1 mg/l kin + 2 mg/l NAA 2 mg/l kin + 2 mg/l NAA.

Direct organogenesis (%) 70 50 40 30

Indirect organogenesis (%) 30 20 50 60

Mean n= 20

In vitro multiplication of propagules The effects of kin and BA on shootlet multiplication are presented in Table (4). Addition of BA or kin individually or in combination was sufficient to enhance the capability of explants to produce more shoots than control treatment. Moreover, presence of BA generally resulted in a higher number of shoot per explants than when not present.Although the number of shoot per explants increased with BA, this also resulted in a decrease of length of the shoot produced. The highest number of shootlets(6.50) was observed on medium supplemented with 2 mg/l BA (Fig. 1-F). The data procured on the length of primary shoot indicated thatKin treatments produced the longest shoots than the BA application. The medium containing 5 mg/l kin gave the maximum value (3.50 cm) whiles the minimum one (1.90 cm) was attained by the medium containing 2 mg/l BA. Contrary, the highest average of leaves number (4.00) was noticed with MS hormone free medium. Table 4. In vitro multiplication from shootlet of globe artichoke after six weeks of culturing on MS medium containing kin or BA individually and in combination Medium

Number of shootlets

Shootlet height (cm)

Number of proliferated leaves

MS hormone-free. MS + 5 mg/l kin

1.50 ± 0.14 4.50 ± 0.11

2.50 3.50

4.00±0.10 3.50± 0.15

MS + 2 mg/l BA MS + 1 mg/l BA + 5 mg/l kin

6. 50 ± 0.20 5.00 ± 0.20

1.90 2.30

3.00± 0.19 2.70± 0.10

± Standard Error (SE)

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In vitro shoot buds multiplication is mainly phytohormone dependent. Cytokinins are generally known to reduce the apical meristem dominance and induce both axillary and adventitious shoots formation from meristematic explants. In this work, multiplication experiment were performed with shoot buds derived from in vitro grown seedlings. We found that addition of BA to multiplication medium was very effective to produce multiple shoots. Otherwise, kin treatment showed the longest shootlets which it can be used for elongation of propagules before rooting stage. Regardingleaf number, it is apparent from the results obtained that both BA and kin have negative effect on the number of proliferated leaves. Several articles have been published describing the micropropagation of globe artichoke. An improvement in shoot proliferation and overall development on medium containing 5 mg/l kin was claimed [3]. However, medium containing 1 mg/l IAA+ 1 mg/l 2ip was used to promote shootlet multiplication [7]. Otherwise, Suelzuet al. [26] obtained three shoot per explants by using medium supplemented with 10 mg/l kin + 0.5 mg/ l IAA. In this respect, a micropropagation method of globe artichoke was developed starting from seeds [27]. Shoot multiplication occurred through a proliferation of axillary buds and the best multiplication rates were obtained when different BA concentrations and 0.5 mg/l NAA were combined. Otherwise, micropropagation protocol for plants of globe artichoke cv. Early French was reported employing cyclodextrins during the rhizogenesis [28]. It was found that, with BAP a multiplication rate was obtained greater to that with Kin and 2iP. Highest multiplication rate of globe artichoke grown in vitrowas also achieved with MS medium supplemented with 5 mg/l kin+0.5 mg/l IAA [29]. Recently, El-Zeinyet al. [30] mentioned thatincreasing the levels of both cytokinins (BA and Kin) from 2.5 to 10 mg/l in tissue culture medium was sufficient to enhance the capability of explants of globe artichoke to produce more shoots especially at high level. There were relationship between number of subculture and rates of shoots productions. Increasing the number of subcultures till fifth times increased gradually the number of shoot production and decreased shoot length.

Fig. 1. (A)Shootlet cultures of globe artichoke proliferated on MS medium supplemented with 2 mg/l BA, (B) Callus induced from leaf expalnt on medium contained 5mg/l kin + 0.5 mg/l IAA, (C) callus grown on medium contained 5 mg/l kin + 0.5 mg/l IAA + 3 mg/l picloram, (D) Direct shootlet proliferation from leaf explants on 1 mg/l BA + 2 mg/l NAA containing medium, (E) indirect organogenesis using medium contained 2 mg/l kin + 2 mg/l NAA and (F) shoot bud multiplication using medium supplemented with 2 mg/l BA

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ABBREVIATIONS BA, benzyladenine; Kin, kinetin; NAA, naphthaleneacetic acid; IAA. REFERENCES 1. Adzet, T. (1987). Hepatoprotective activity of polyphenolic compounds from Cynara Scolymus against CC14 toxixity in isolated rat hepatocytes. J. Nat. Prods. 50:612-617. 2. Bekheet, S.A. (2011). In vitro biomass production of liver-protective compounds from globe artichoke (Cynarascolymus L.) and milk thistle (Silybummarianum) plants. Emir. J. Food Agric. 23 (5): 473-481. 3. Ancora, G.; Belli-Donini, M.L.; and Cuozzo, L. (1981). Globe artichoke plants obtained from shoot apices through rapid in vitro micropropagation. Scientia Hortic., 14: 207-213. 4. Elia A.; Conversa G.; Montervino C. and Lotti C. (2007). Micropropagation of the early artichoke cultivar 'violet du provence'. VI International Symposium on Artichoke, Cardoon and Their Wild Relatives.ActaHorticulturae, 1: 127-134. 5. Steinmacher, D.A.; Clement, C.R. and Guerra, M.P. (2007). Somatic embryogenesis from immature peach palm inflorescence explants: Towards development of an efficient protocol, Plant Cell Tissue Organ Culture 89 (1) 15-22. 6. Tu, M., Hurd, C. and Randall, J. M. (2001). Weed Control Methods Handbook: Tools and Techniques for Use in Natural Areas. The Nature Conservancy, http://tncweeds.ucdavis.edu, Version: April 2001. 7. Harbaoui, Y.; Smaiyn, G.; Welvaert, W. and Debergh, P.C. (1982). Assainissement viral de l’artichaut (CynarascolymusL.) par la culture in vitro d’apexméristématiques. Phytopathologie Méditerrannée 21: 15-19. 8. Tavazza R.; Papacchioli V. and Ancora G. (2004). An improved medium for in vitro propagation of globe artichoke (CynarascolymusL.) cv. Acta Hortic., 660: 91-97. 9. Schneider F. (2005). Effect of different cultural conditions on micropropagation of rose (Rosa sp. L.) and globe artichoke (CynarascolymusL.).Thèse de doctorat, Université Technique de Munich. 145p. 10. Ruta C.; Tagarelli A. and Fortunato I. (2005). Mycorrhization on micropropagated artichoke. Acta Horicolturae 681: 407-412. 11. Fortunato, I.M.; Vanadia, S. and Latanizo, V. (1979). In vitro culture of globe artichoke. In: Biotechnology in Aagriculture and Forestry. Vol. 2: Crop (ed Bajaj, Y.P.) pp. 474-480, Springer verlag. Berlin 12. Onisei, T.; Ecaterina, T. and Doina, A. (1988). Callus induction and plant regeneration of Cynarascolymus L. Rev. Roum Biol. Veget., Teme, 33 (2): 115-119. 13. Ordas, R.; Tavazza, R. and Ancora, G. (1990). In vitro morphogenesis in the globe artichoke (Cynarascolymus L). Plant Science, 71: 233-237. 14. El-Bahr. M. K.; Okasha, Kh. A. and Bekheet, S. A. (2001).In vitro morphogenesis of globe artichoke (Cynarascolymus L.). Arab J. Biotech. 4 (1): 119 - 128. 15. Bekheet S.A.; El-Bahr M.K.; Ali S. A. and Hamed M.A. (2014). Callus production of Globe artichoke and Milk thistle: In vitro hypolipidemic and antioxidant activities. World Journal of Pharmaceutical Research, 3 (4):1-17. 16. Murashige, T. and Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473 - 497. 17. Shevchenko, Y., Smetanska, I. and Wendt, A. (2010). Stevia rebaudianaBertoni- Überblicküber die Forschung an einerverbotenenPflanze und derermöglichenEinsätze. Journal für Verbraucherschutz und Lebensmittelsicherheit, 5(2): 193-198. th 18. Snedecor, G.W. and Corchran, W.G. (1967). Statistical Methods.6 Edition. Iowa State University Press, Iowa. 19. Robert, J.G. (1984). An introsuction in somatic cell genetic. Hort. Sci., 19 (3): 214-217. 20. Menin, B.; Moglia, A.; Comino, C.; Hakkert, J.C.; Lanteri, S. and Beekwilder, J. (2013). In vitro callusinduction in globe artichoke (Cynaracardunculus L. var. scolymus) as a system for the production of caffeoylquinic acids.The Journal of Horticultural Science & Biotechnology.88 (5): 537-542. 21. Bach, A. and Pawlowska, B. (2003). Somatic embryogenesis in GentianapneumonantheL. Acta Biol. Crac. Ser. Bot. 45(2): 79-86. 22. Ahmed, A.B.A.; Rao, A.S.; Rao, M.V. and Taha, R.M. (2011). Effect of picloram, additives and plant growth regulators on somatic embryogenesis of Phyla nodiflora (L.) Greene. Braz. Arch. Biol. Technol. 54: 7-13. 23. Castellar, A.; Gagliardi, R.F. and Mansur, E. (2011). In vitro propagation and establishment of callus and cell suspension cultures of Petiveriaalliacea L., a valuable medicinal plant. J Medicinal Plants Research. 5: 1113-1120. 24. Abbasi, B.H.; Saxena, P.K.; Murch, S.J. and Liu, C.Z. (2007). Echinacea biotechnology: challenges and opportunities. In Vitro Cell Dev. Biol-Plant. 43: 481-492. 25. Petri, P.S. and Ricci, A. (1981). Micropropagation of globe artichoke from tissues in vitro. Industria Grafica Laterza, 231-238. 26. Suelzu, R.; Tognoni, F. and Lereari, B. (1989). Further studies on micropropagation of globe artichoke. InformatoreAgrario. 45: 71-72.

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27. Lauzer, D. and Vieth, J. (1990). Micropropagation of seed-derived plant of Cynarascolymus L., cv Green Globe. Plant Cell, Tissue and Organ Culture. 12: 237-244. 28. Brutti, C.; ApoÂstolo, N.M.; Ferrarotti, S.A.; Llorente, B.E. and Krymkiewicz, N. (2000). Micropropagation of Cynarascolymus L. employing cyclodextrins to promote rhizogenesis. Scientia Hortic., 83: 1-10. 29. El-Gizawy, A.M.; El-Bahr, M.K.; El-Oksh, I.I. and Bekheet, S.A. (1993). In vitro multiplication of globe th artichoke. 4 Agric. Dev. Res., Ain Shams Univ., Cairo, Annals Agric. Sci. Sp. Issue. 2: 765-777. 30. El-Zeiny, O.A.H.; El-Behairy, U.A., Zocchi, G. and. Rashaan, M.M. (2013). Commercial production of globe artichoke (Cynarascolymus L.) in-vitro. Egypt. J. Agric. Res., 91(3): 993- 1007.

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