Kulolu cwaningo, imiphumela ekhuthazayo yokwelashwa okuhlangene kwe-abalawuli bokukhula kwezitshalo(2,4-D kanye ne-kinetin) kanye nama-nanoparticles e-iron oxide (Fe₃O₄-NPs) ku-in vitro morphogenesis kanye nokukhiqizwa kwe-metabolite yesibili ku-*Hypericum perforatum* L. kuhlolwe. Ukwelashwa okulungiselelwe [2,4-D (0.5 mg/L) + kinetin (2 mg/L) + Fe₃O₄-NPs (4 mg/L)] kuthuthukise kakhulu imingcele yokukhula kwesitshalo: ukuphakama kwesitshalo kukhuphuke ngo-59.6%, ubude bezimpande ngo-114.0%, inombolo yamahlumela ngo-180.0%, kanye nesisindo esisha se-callus ngo-198.3% uma kuqhathaniswa neqembu lokulawula. Lokhu kwelashwa okuhlangene kuphinde kwathuthukisa ukusebenza kahle kokuvuselelwa (50.85%) futhi kwandise okuqukethwe kwe-hypericin ngo-66.6%. Ukuhlaziywa kwe-GC-MS kwembule okuqukethwe okuphezulu kwe-hyperoside, i-β-patolene, kanye ne-cetyl alcohol, okubalwa ku-93.36% wendawo yonke ephakeme, kuyilapho okuqukethwe kwama-phenolics nama-flavonoids kukhuphuke ngamaphesenti angu-80.1. Le miphumela ikhombisa ukuthi abalawuli bokukhula kwezitshalo (ama-PGR) kanye nama-nanoparticles e-Fe₃O₄ (ama-Fe₃O₄-NP) banomthelela wokubambisana ngokuvuselela i-organogenesis kanye nokuqongelela kwamakhemikhali asebenzayo, okumele isu elithembisayo lokuthuthukiswa kwe-biotechnological kwezitshalo zokwelapha.
I-St. John's wort (i-Hypericum perforatum L.), eyaziwa nangokuthi i-St. John's wort, iyisitshalo esihlala isikhathi eside esihlala sikhona somndeni we-Hypericaceae esinokubaluleka kwezomnotho.[1] Izingxenye zayo ezisebenzayo zifaka phakathi ama-tannins emvelo, ama-xanthones, i-phloroglucinol, i-naphthalenedianthrone (i-hyperin kanye ne-pseudohyperin), ama-flavonoid, ama-phenolic acid, kanye namafutha abalulekile.[2,3,4] I-St. John's wort ingasakazwa ngezindlela zendabuko; noma kunjalo, ukuba khona kwezikhathi zesikhathi kwezindlela zendabuko, ukuhluma okuncane kwembewu, kanye nokuthambekela ezifweni kunciphisa amandla ayo okutshala okukhulu kanye nokwakheka okuqhubekayo kwama-metabolites esibili.[1,5,6]
Ngakho-ke, ukukhuliswa kwezicubu ze-in vitro kubhekwa njengendlela ephumelelayo yokusabalala kwezitshalo ngokushesha, ukulondolozwa kwezinsiza ze-germplasm, kanye nokwanda kwesivuno samakhemikhali ezokwelapha [7, 8]. Abalawuli bokukhula kwezitshalo (ama-PGR) badlala indima ebalulekile ekulawuleni i-morphogenesis futhi bayadingeka ekutshalweni kwe-callus kanye nezinto eziphilayo eziphelele ngaphakathi kwe-vitro. Ukwenziwa ngcono kokuhlushwa kwazo kanye nokuhlanganiswa kwazo kubalulekile ekuqedeni ngempumelelo lezi zinqubo zokuthuthukiswa [9]. Ngakho-ke, ukuqonda ukwakheka okufanele kanye nokuhlushwa kwabalawuli kubalulekile ekuthuthukiseni ukukhula kanye namandla okuvuselela i-St. John's wort (H. perforatum) [10].
Ama-nanoparticles e-iron oxide (Fe₃O₄) ayisigaba sama-nanoparticles asethuthukiswa noma asathuthukiswa ukuze kukhuliswe izicubu. I-Fe₃O₄ inezakhiwo eziphawulekayo zamagnetic, ukuhambisana okuhle kwezinto eziphilayo, kanye nekhono lokukhuthaza ukukhula kwezitshalo nokunciphisa ukucindezeleka kwemvelo, ngakho-ke idonsele ukunaka okukhulu ekwakhiweni kwezicubu. Ukusetshenziswa okungenzeka kwala ma-nanoparticles kungafaka phakathi ukuthuthukisa i-in vitro culture ukukhuthaza ukuhlukaniswa kwamaseli, ukuthuthukisa ukumuncwa kwezakhamzimba, kanye nokwenza kusebenze ama-enzyme e-antioxidant [11].
Nakuba ama-nanoparticles ebonise imiphumela emihle yokukhuthaza ekukhuleni kwezitshalo, izifundo zokusetshenziswa okuhlangene kwama-nanoparticles e-Fe₃O₄ kanye nabalawuli bokukhula kwezitshalo abalungiselelwe ku-*H. perforatum* zisalokhu zimbalwa. Ukuze kugcwaliswe lesi sikhala solwazi, lolu cwaningo luhlole imiphumela yemiphumela yalo ehlangene ku-morphogenesis ye-in vitro kanye nokukhiqizwa kwe-metabolite yesibili ukuze kunikezwe ukuqonda okusha kokuthuthukisa izici zezitshalo zokwelapha. Ngakho-ke, lolu cwaningo lunezinhloso ezimbili: (1) ukwenza ngcono ukuhlushwa kwabalawuli bokukhula kwezitshalo ukuze kukhuthazwe ngempumelelo ukwakheka kwe-callus, ukuvuselelwa kwehlumela, kanye nokuhluma kwezimpande ku-vitro; kanye (2) ukuhlola imiphumela yama-nanoparticles e-Fe₃O₄ kumapharamitha okukhula ku-vitro. Izinhlelo zesikhathi esizayo zifaka phakathi ukuhlola izinga lokusinda kwezitshalo ezivuselelwe ngesikhathi sokujwayela (ku-vitro). Kulindeleke ukuthi imiphumela yalolu cwaningo izothuthukisa kakhulu ukusebenza kahle kwe-micropropagation ye-*H. perforatum*, ngaleyo ndlela inikele ekusetshenzisweni okusimeme kanye nokusetshenziswa kwe-biotechnology kwalesi sitshalo esibalulekile sezokwelapha.
Kulolu cwaningo, sithole izitshalo ezikhula emaqabungeni ezivela ezitshalweni zonyaka ze-St. John's wort (izitshalo zikamama). Lezi zitshalo ezikhula emaqabungeni zasetshenziswa ukuthuthukisa izimo zokukhula kwe-in vitro. Ngaphambi kokukhulisa, amaqabunga ahlanzwa kahle ngaphansi kwamanzi agelezayo acwecwe imizuzu embalwa. Izindawo ezikhula emaqabungeni zabe sezihlanzwa ngokucwiliswa ku-70% ethanol imizuzwana engama-30, kulandele ukucwiliswa kusisombululo se-1.5% sodium hypochlorite (NaOCl) esiqukethe amaconsi ambalwa e-Tween 20 imizuzu eyi-10. Ekugcineni, izitshalo ezikhula emaqabungeni zahlanzwa kathathu ngamanzi acwecwe ahlanzekile ngaphambi kokudluliselwa endaweni yokukhulisa elandelayo.
Emasontweni amane alandelayo, kwalinganiswa imingcele yokuvuselelwa kwehlumela, okuhlanganisa izinga lokuvuselelwa, inombolo yokudubula ngesitshalo ngasinye, kanye nobude behlumela. Lapho amahlumela avuselelwe efinyelela ubude obungu-2 cm, adluliselwa endaweni yokukhula kwehlumela equkethe i-MS medium enamandla ayingxenye, i-0.5 mg/L indolebutyric acid (IBA), kanye ne-0.3% guar gum. Ukukhuliswa kwehlumela kwaqhubeka amasonto amathathu, lapho kwalinganiswa khona izinga lokukhula kwehlumela, inombolo yehlumela, kanye nobude behlumela. Ukwelashwa ngakunye kwaphindwa kathathu, kwakhuliswa amahlumela ayi-10 ngokuphindaphindwayo, kwaveza amahlumela angaba ngu-30 ngokwelashwa ngakunye.
Ukuphakama kwesitshalo kwalinganiswa ngamasentimitha (cm) kusetshenziswa irula, kusukela phansi kwesitshalo kuya esiqongweni seqabunga elide kakhulu. Ubude bempande balinganiswa ngamamilimitha (mm) ngokushesha ngemva kokususa ngokucophelela izithombo nokususa indawo ekhulayo. Inani lamahlumela ngesitshalo ngasinye labalwa ngqo esitshalweni ngasinye. Inani lamabala amnyama emaqabunga, aziwa ngokuthi amaqhubu, lalinganiswa ngokubona. Lawa maqhubu amnyama kukholakala ukuthi angama-gland aqukethe i-hypericin, noma amabala e-oxidative, futhi asetshenziswa njengesibonakaliso sempilo yesitshalo ekwelashweni. Ngemva kokususa yonke indawo ekhulayo, isisindo esisha sezithombo salinganiswa kusetshenziswa isikali se-elekthronikhi esinokunemba kwamamiligram (mg).
Indlela yokubala izinga lokwakheka kwe-callus yilena elandelayo: ngemva kokukhulisa ama-explant endaweni equkethe abalawuli bokukhula abahlukahlukene (ama-kinase, ama-2,4-D, kanye ne-Fe3O4) amasonto amane, inani lama-explant akwazi ukwakha i-callus liyabalwa. Ifomula yokubala izinga lokwakheka kwe-callus yilena elandelayo:
Ukwelashwa ngakunye kwaphindwa kathathu, okungenani kwahlolwa ama-explants ayi-10 ekuphindaphindweni ngakunye.
Izinga lokuvuselelwa libonisa isilinganiso sezicubu ze-callus eziqeda ngempumelelo inqubo yokuhlukanisa amahlumela ngemva kwesigaba sokwakheka kwe-callus. Lesi sibonakaliso sibonisa ikhono lezicubu ze-callus lokuguquka zibe izicubu ezihlukene futhi zikhule zibe izitho zezitshalo ezintsha.
I-rooting coefficient iyisilinganiso senani lamagatsha akwaziyo ukuhluma izimpande nenani eliphelele lamagatsha. Lesi sibonakaliso sibonisa impumelelo yesigaba sokuhluma izimpande, okubaluleke kakhulu ekukhuleni kwezitshalo ezincane kanye nokuhluma kwezitshalo, njengoba ukuhluma okuhle kusiza izithombo ukuthi ziphile kangcono ezimweni ezikhulayo.
Ama-compound e-Hypericin akhishwe nge-90% methanol. Ama-milligram angamashumi amahlanu ezinto zezitshalo ezomisiwe afakwe ku-1 ml ye-methanol futhi afakwa ku-sonic imizuzu engama-20 ku-30 kHz kumshini wokuhlanza we-ultrasonic (imodeli A5120-3YJ) ekushiseni kwegumbi ebumnyameni. Ngemva kokufakwa ku-sonication, isampula yafakwa ku-centrifuge ku-6000 rpm imizuzu eyi-15. I-supernatant yaqoqwa, futhi ukumuncwa kwe-hypericin kwalinganiswa ku-592 nm kusetshenziswa i-Plus-3000 S spectrophotometer ngokwendlela echazwe yi-Conceiçao et al. [14].
Ukwelashwa okuningi ngabalawuli bokukhula kwezitshalo (ama-PGR) kanye nama-nanoparticles e-iron oxide (ama-Fe₃O₄-NP) akuzange kubangele ukwakheka kwama-nodule amnyama emaqabunga amahlumela avuselelwe. Awekho ama-nodules abonwe kunoma yikuphi ukwelashwa nge-kinetin engu-0.5 noma 1 mg/L engu-2,4-D, 0.5 noma 1 mg/L, noma ama-nanoparticles e-iron oxide angu-1, 2, noma 4 mg/L. Ukuhlanganiswa okumbalwa kubonise ukwanda okuncane kokuthuthukiswa kwama-nodules (kodwa hhayi okubalulekile ngokwezibalo) ekugxilweni okuphezulu kwama-nanoparticles e-kinetin kanye/noma e-iron oxide, njengokuhlanganiswa kwe-2,4-D (0.5–2 mg/L) ne-kinetin (1–1.5 mg/L) kanye nama-nanoparticles e-iron oxide (2–4 mg/L). Le miphumela iboniswe kuMfanekiso 2. Ama-nodules amnyama amelela izindlala ezicebile nge-hypericin, zombili ezenzeka ngokwemvelo futhi ezizuzisayo. Kulolu cwaningo, ama-nodules amnyama ayehlotshaniswa kakhulu nokunsundu kwezicubu, okubonisa indawo enhle yokuqongelela i-hypericin. Ukwelashwa ngama-nanoparticles angu-2,4-D, kinetin, kanye ne-Fe₃O₄ kukhuthaze ukukhula kwe-callus, ukuncipha kokunsundu, kanye nokwanda kokuqukethwe kwe-chlorophyll, okuphakamisa ukusebenza okuthuthukisiwe kwe-metabolic kanye nokunciphisa okungenzeka komonakalo we-oxidative [37]. Lolu cwaningo luhlole imiphumela ye-kinetin ngokuhlanganiswa nama-nanoparticles angu-2,4-D kanye ne-Fe₃O₄ ekukhuleni nasekuthuthukisweni kwe-St. John's wort callus (Isithombe 3a–g). Izifundo zangaphambilini zibonise ukuthi ama-nanoparticles e-Fe₃O₄ anemisebenzi ye-antifungal kanye ne-antimicrobial [38, 39] futhi, uma esetshenziswa ngokuhlanganiswa nabalawuli bokukhula kwezitshalo, angavuselela izindlela zokuzivikela kwezitshalo futhi anciphise izinkomba zokucindezeleka kwamaseli [18]. Nakuba i-biosynthesis yama-metabolites esibili ilawulwa ngokwezakhi zofuzo, isivuno sawo sangempela sincike kakhulu ezimweni zemvelo. Izinguquko ze-metabolic kanye ne-morphological zingathonya amazinga e-metabolite esibili ngokulawula ukubonakaliswa kwezakhi zofuzo zezitshalo ezithile kanye nokuphendula ezicini zemvelo. Ngaphezu kwalokho, izinto ezibangela ukuvuvukala zingavusa ukusebenza kwezakhi zofuzo ezintsha, okuthi ekugcineni kuvuselele umsebenzi we-enzymatic, ekugcineni kuvuselele izindlela eziningi ze-biosynthetic futhi kuholele ekwakhekeni kwama-metabolites esibili. Ngaphezu kwalokho, olunye ucwaningo lubonise ukuthi ukunciphisa umthunzi kwandisa ukuvezwa kwelanga, ngaleyo ndlela kukhuphule amazinga okushisa emini endaweni yemvelo ye-*Hypericum perforatum*, ephinde inegalelo ekwandeni kwesivuno se-hypericin. Ngokusekelwe kulolu lwazi, lolu cwaningo luhlole indima yama-nanoparticles ensimbi njengezinto ezibangela ukuvuvukala kwezicubu. Imiphumela ikhombisile ukuthi la ma-nanoparticles angavuselela izakhi zofuzo ezihilelekile ku-hesperidin biosynthesis ngokukhuthaza i-enzymatic, okuholela ekuqongeleleni okukhulu kwale nhlanganisela (Isithombe 2). Ngakho-ke, uma kuqhathaniswa nezitshalo ezikhula ngaphansi kwezimo zemvelo, kungaphikiswana ngokuthi ukukhiqizwa kwalezi zinto ezibangela ukuvuvukala emzimbeni kungakhuliswa nalapho ukucindezeleka okulinganiselwe kuhlanganiswa nokusebenza kwezakhi zofuzo ezihilelekile ku-biosynthesis yama-metabolites esibili. Ukwelashwa okuhlanganisiwe ngokuvamile kunomphumela omuhle esilinganisweni sokuvuselelwa kabusha, kodwa kwezinye izimo, lo mphumela uba buthaka. Okuphawulekayo ukuthi ukwelashwa nge-1 mg/L 2,4-D, 1.5 mg/L kinase, kanye nokugxila okuhlukile kungakhuphula ngokuzimela nangokuphawulekayo izinga lokuvuselelwa ngo-50.85% uma kuqhathaniswa neqembu lokulawula (Isithombe 4c). Le miphumela iphakamisa ukuthi inhlanganisela ethile yama-nanohormone ingasebenza ngokubambisana ukukhuthaza ukukhula kwezitshalo kanye nokukhiqizwa kwe-metabolite, okubaluleke kakhulu ekukhuleni kwezicubu zezitshalo zokwelapha. UPalmer noKeller [50] babonise ukuthi ukwelashwa kwe-2,4-D kungadala ngokuzimela ukwakheka kwe-callus ku-St. perforatum, kuyilapho ukwengezwa kwe-kinase kuthuthukisa kakhulu ukwakheka kwe-callus kanye nokuvuselelwa. Lo mphumela ube ngenxa yokuthuthukiswa kwebhalansi yama-hormone kanye nokukhuthaza ukuhlukaniswa kwamaseli. U-Bal et al. [51] bathole ukuthi ukwelashwa kwe-Fe₃O₄-NP kungathuthukisa ngokuzimela umsebenzi wama-enzyme e-antioxidant, ngaleyo ndlela kukhuthaze ukukhula kwezimpande ku-St. perforatum. Imithombo yezolimo equkethe ama-nanoparticles e-Fe₃O₄ ekugxilweni okungu-0.5 mg/L, 1 mg/L, kanye no-1.5 mg/L ithuthukise izinga lokuvuselelwa kwezitshalo ze-flax [52]. Ukusetshenziswa kwama-nanoparticles e-kinetin, i-2,4-dichlorobenzothiazolinone, kanye ne-Fe₃O₄ kuthuthukise kakhulu amazinga okwakheka kwe-callus kanye nezimpande, noma kunjalo, imiphumela emibi engaba khona yokusebenzisa la ma-hormone ukuze kuvuseleleke i-in vitro kudingeka icatshangelwe. Isibonelo, ukusetshenziswa kwesikhathi eside noma okuphezulu kwe-2,4-dichlorobenzothiazolinone noma i-kinetin kungabangela ukuhlukahluka kwe-somatic clonal, ukucindezeleka kwe-oxidative, i-formology ye-callus engavamile, noma i-vitrification. Ngakho-ke, izinga lokuvuselelwa eliphezulu alibikezeli ngempela ukuzinza kwezakhi zofuzo. Zonke izitshalo ezivuselelwe kufanele zihlolwe kusetshenziswa ama-molecule markers (isb. i-RAPD, i-ISSR, i-AFLP) noma ukuhlaziywa kwe-cytogenetic ukuze kunqunywe ukulingana kwazo kanye nokufana kwazo nezitshalo ze-in vivo [53,54,55].
Lolu cwaningo lubonise okokuqala ukuthi ukusetshenziswa okuhlangene kwezilawuli zokukhula kwezitshalo (2,4-D kanye ne-kinetin) ngama-nanoparticles e-Fe₃O₄ kungathuthukisa i-morphogenesis kanye nokuqongelela kwama-metabolites abalulekile asebenzayo (kufaka phakathi i-hypericin kanye ne-hyperoside) ku-*Hypericum perforatum*. Uhlelo lokwelapha olulungiselelwe kahle (1 mg/L 2,4-D + 1 mg/L kinetin + 4 mg/L Fe₃O₄-NPs) alugcinanga nje ngokukhulisa ukwakheka kwe-callus, i-organogenesis, kanye nesivuno sesibili se-metabolite kodwa futhi lubonise umphumela omnene obangela, okungenzeka kuthuthukise ukubekezelelana kokucindezeleka kwesitshalo kanye nenani lezokwelapha. Ukuhlanganiswa kwe-nanotechnology kanye nesiko lezicubu zezitshalo kunikeza ipulatifomu eqhubekayo nephumelelayo yokukhiqizwa okukhulu kwezakhi zokwelapha ngaphakathi kwe-vitro. Le miphumela ivula indlela yezicelo zezimboni kanye nocwaningo lwesikhathi esizayo mayelana nezinqubo zama-molecule, ukulungiswa komthamo kanye nokunemba kwezakhi zofuzo, ngaleyo ndlela kuxhumene ucwaningo oluyisisekelo ezitshalweni zokwelapha ne-biotechnology esebenzayo.
Isikhathi sokuthunyelwe: Disemba 12-2025