plants Article Gentianella lutescens subsp. carpatica J. Holub.: Shoot Propagation In Vitro and Effect of Sucrose and Elicitors on Xanthones Production Dijana Krsti´c-Miloševi´c 1,* , Nevena Banjac 1, Teodora Jankovi´c 2, Dragan Vinterhalter 1 and Branka Vinterhalter 1 1 Department of Plant Physiology, Institute for Biological Research “Siniša Stankovi´c”—NationalInstitute of the Republic of Serbia, University of Belgrade, Bulevar despota Stefana 142, 11000 Belgrade, Serbia; [email protected] (N.B.); [email protected] (D.V.); [email protected] (B.V.) 2 Insitute for Medicinal Plants Research “Dr Josif Panˇci´c”,Tadeuša Koš´cuška1, 11000 Belgrade, Serbia; [email protected] * Correspondence: [email protected] Abstract: In vitro shoot culture of the endangered medicinal plant Gentianella lutescens was estab- lished from epicotyl explants cultured on MS basal medium with 0.2 mg L−1 6-benzylaminopurine (BA) and evaluated for xanthones content for the first time. Five shoot lines were obtained and no significant variations in multiplication rate, shoot elongation, and xanthones profile were found among them. The highest rooting rate (33.3%) was achieved by shoots treated for 2 days with −1 1 5 mg L indole-3-butyric acid (IBA) followed by cultivation in liquid PGR-free ⁄2 MS medium Citation: Krsti´c-Miloševi´c,D.; for 60 days. HPLC analysis revealed the lower content of xanthones—mangiferin, bellidifolin, Banjac, N.; Jankovi´c,T.; demethylbellidifolin, demethylbellidifolin-8-O-glucoside and bellidifolin-8-O-glucoside—in in vitro Vinterhalter, D.; Vinterhalter, B. cultured shoots compared to wild growing plants. The increasing concentration of sucrose, sorbitol Gentianella lutescens subsp. carpatica J. and abiotic elicitors salicylic acid (SA), jasmonic acid (JA) and methyl jasmonate (MeJA) altered Holub.: Shoot Propagation In Vitro shoot growth and xanthone production. Sucrose and sorbitol applied at the highest concentration and Effect of Sucrose and Elicitors on of 233.6 mM increased dry matter percentage, while SA at 100 µM promoted shoot growth 2-fold. Xanthones Production. Plants 2021, The increased sucrose concentration enhanced accumulation of xanthones in shoot cultures 2–3-fold 10, 1651. https://doi.org/10.3390/ compared to the control shoots. Elicitors at 100–300 µM increased the accumulation of mangiferin, plants10081651 demethylbellidifolin-8-O-glucoside, and bellidifolin-8-O-glucoside almost equally, while MeJA at the highest concentration of 500 µM enhanced amount of aglycones demethylbellidifolin and bellidifolin Academic Editor: Iyyakkannu Sivanesan 7-fold compared to the control. The obtained results facilitate conservation of G. lutescens and pave the way for further research on large-scale shoot propagation and production of pharmacologically Received: 28 June 2021 active xanthones. Accepted: 6 August 2021 Published: 11 August 2021 Keywords: shoot culture; secondary metabolites; HPLC; bellidifolin; osmotic stress Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- 1. Introduction iations. The genus Gentianella Moench (Gentianeceae) encompasses about 250 species grow- ing mainly in temperate or mountain habitats in Europe, South America, New Zealand, and Australia [1]. In Europe, the genus Gentianella consists of 22 species with numerous subspecies and taxa that are distributed in the Alps, Carpathians, in the Tatra mountains, Copyright: © 2021 by the authors. as well as in the mountains of the Balkan Peninsula [2]. In Serbia, the genus Gentianella Licensee MDPI, Basel, Switzerland. is represented by six species: G. austriaca, G. bulgarica, G. axilaris, G. ciliata, G. praecox, This article is an open access article and G. crispata [3]. distributed under the terms and Apart their ornamental value, Gentianella species have been well-known as traditional conditions of the Creative Commons medicines since ancient times. In South America they have been used as a traditional rem- Attribution (CC BY) license (https:// edy for the treatment of digestive and liver problems [4,5]. Gentianella amarella is known in creativecommons.org/licenses/by/ the traditional medicine of Mongolia to cure headache, hepatitis, fever, and gallbladder 4.0/). Plants 2021, 10, 1651. https://doi.org/10.3390/plants10081651 https://www.mdpi.com/journal/plants PlantsPlants2021 2021, ,10 10,, 1651 x FOR PEER REVIEW 22of of 2120 disordersgallbladder [6]. disorders Like other [6]. species Like other of the species Gentianaceae of the Gentianaceae family, Gentianella family,plants Gentianella are character- plants izedare characterized by the universal by the occurrence universal of occurrence three main of groups three ofmain secondary groups of metabolites, secondary iridoids,metabo- flavonoids,lites, iridoids, and flavonoids, xanthones and [7]. xanthones [7]. NaturallyNaturally occurringoccurring xanthonesxanthones havehave beenbeen attractingattracting attentionattention forfor aa longlong timetime duedue toto theirtheir specificspecific bioactivitiesbioactivities andand occupyoccupy anan importantimportant positionposition inin thethe pharmacologypharmacology andand chemistrychemistry ofof naturalnatural products.products. XanthoneXanthone compoundscompounds typicaltypical ofofGentianella Gentianellaspecies species belongbelong toto the bellidifolin bellidifolin type type of of xanthones xanthones,, which which mostly mostly occur occur in the in form the form of O- ofglycosides.O-glycosides. They Theyare responsible are responsible for wide for widerange rangeof therapeutic of therapeutic properties properties attributed attributed to Gentianella to Gentianella plants. plants.The xanthones The xanthones bellidifolin bellidifolin and demethylbellidifolin, and demethylbellidifolin, the principal the principal constituents constituents of many ofgentianellas, many gentianellas, have been have reported been to reported show cardioprotective to show cardioprotective effects, antioxidant, effects, antioxidant, antimicro- antimicrobial,bial, and antidiabetic and antidiabetic activity, activity,as well as as displaying well as displaying significant significant potential potential to inhibit to inhibitacetyl- acetylcholinesterasecholinesterase and andmonoamino monoamino oxidase oxidase activity activity [8]. [8 ].Bellidifolin Bellidifolin exhibited exhibited a a variety ofof pharmacologicalpharmacological activities,activities including, including prominent prominent hypoglycemic hypoglycemic [9] [9] and and neuroprotective neuroprotective ac- tivitiesactivities [10 ].[10]. These These findings findings support support the potentialthe potential use of use xanthone of xanthone compounds compounds as new as drugs new indrugs treating in tr aging-relatedeating aging-related neurodegenerative neurodegenerative disorders disorders [11] and [11] also and as also useful as candidatesuseful can- fordidates therapy for oftherapy type 2 of diabetes type 2 diabetes [9]. [9]. AlthoughAlthough findingfinding GentianellaGentianella speciesspecies asas aa reachreach sourcesource ofof important important phytochemicals phytochemicals hashas heightenedheightened researchresearch interest interest in in these these species, species, many many of of themthem havehave notnot yetyet beenbeen properlyproperly investigatedinvestigated duedue toto thethe lowlow availabilityavailability ofof plantplant material.material. AA largelarge numbernumber ofofGentianella Gentianella speciesspecies areare rarerare andand endemicendemic oror growgrow inin inaccessibleinaccessible localities,localities, whilewhile thethe othersothers becamebecame endangeredendangered from excessive harvesting harvesting or or adverse adverse environmental environmental conditions. conditions. Such Such an un- an unfavorablefavorable situation situation is the is thecase case with with the endemic the endemic species species G. lutescensG. lutescens subsp.subsp. Carpaticacarpatica, which, whichwe discov we discoveredered in Serbia in Serbia for the for first the time first during time during field research field research on Povlen on Povlen mountain mountain (Fig- (Figureure 1). 1A). s Aurvey survey of literature of literature indicated indicated that that this this species species has has not not been been phytochemically phytochemically in- investigated so far and very scarce data can be found about it. vestigated so far and very scarce data can be found about it. FigureFigure 1. 1.Gentianella Gentianella lutescens lutescenssubsp. subsp.carpatica carpaticain ain natural a natural habitat habitat on Povlen on Povlen mountain mountain (locality Razbojište),(locality Razbojište), Serbia. Serbia. ItIt isis biennialbiennial 3–40-cm-tall3–40-cm-tall plant,plant, andand isis simplesimple oror branchedbranchedin in thethe upperupper part,part, formingforming aa racemoseracemose inflorescenceinflorescence panicle-like,panicle-like, umbrella-shapeumbrella-shape withwith reddishreddish toto violetviolet flowers.flowers. Gen-Gen- tianellatianella lutescenslutescens subsp.subsp. carpaticacarpatica waswas locatedlocated inin EasternEastern andand CentralCentral EuropeEurope andand northernnorthern partpart ofof thethe BalkanBalkan Peninsula,Peninsula, mainlymainly inin thethe mountains,mountains, withwith ecotypicecotypic variantsvariants inin Austria,Austria, Bulgaria,Bulgaria, Czech, Germany, Germany, Yugoslavia, Yugoslavia, and and Poland Poland [12]. [12]. This This species species was was reported reported as rel- as relativelyatively