Mu kafukufukuyu, zotsatira zolimbikitsa za chithandizo chophatikizana chaowongolera kukula kwa zomera(2,4-D ndi kinetin) ndi tinthu tating'onoting'ono ta iron oxide (Fe₃O₄-NPs) pa morphogenesis ya in vitro ndi kupanga kwachiwiri kwa metabolite mu *Hypericum perforatum* L. adafufuzidwa. Chithandizo chabwino kwambiri [2,4-D (0.5 mg/L) + kinetin (2 mg/L) + Fe₃O₄-NPs (4 mg/L)] chidasintha kwambiri magawo a kukula kwa chomera: kutalika kwa chomera kudakwera ndi 59.6%, kutalika kwa mizu ndi 114.0%, chiwerengero cha mphukira ndi 180.0%, ndi kulemera kwa callus fresh ndi 198.3% poyerekeza ndi gulu lolamulira. Chithandizo chophatikizachi chidathandizanso kukonzanso bwino (50.85%) ndikuwonjezera kuchuluka kwa hypericin ndi 66.6%. Kusanthula kwa GC-MS kunawonetsa kuchuluka kwa hyperoside, β-patholene, ndi cetyl alcohol, zomwe zimapangitsa 93.36% ya malo onse okhala pamwamba, pomwe kuchuluka kwa phenolics ndi flavonoids kunakwera ndi 80.1%. Zotsatirazi zikusonyeza kuti owongolera kukula kwa zomera (PGRs) ndi Fe₃O₄ nanoparticles (Fe₃O₄-NPs) amachita zinthu mogwirizana polimbikitsa organogenesis ndi kusonkhanitsa mankhwala ogwiritsira ntchito bioactive, zomwe zikuyimira njira yabwino yopititsira patsogolo biotechnology ya zomera zamankhwala.
Wort wa St. John (Hypericum perforatum L.), womwe umadziwikanso kuti wort wa St. John, ndi chomera cha herbaceous chosatha cha banja la Hypericaceae chomwe chili ndi phindu pazachuma.[1] Zigawo zake zomwe zimagwira ntchito bwino ndi monga ma tannins achilengedwe, xanthones, phloroglucinol, naphthalenedianthrone (hyperin ndi pseudohyperin), flavonoids, phenolic acids, ndi mafuta ofunikira.[2,3,4] Wort wa St. John ukhoza kufalikira pogwiritsa ntchito njira zachikhalidwe; komabe, nyengo ya njira zachikhalidwe, kumera kochepa kwa mbewu, komanso kufalikira kwa matenda kumalepheretsa kulima kwakukulu komanso kupanga metabolites yachiwiri mosalekeza.[1,5,6]
Motero, kukulitsa minofu ya mu vitro kumaonedwa kuti ndi njira yothandiza yofalitsira zomera mwachangu, kusunga zinthu za germplasm, komanso kuchulukitsa kuchuluka kwa mankhwala [7, 8]. Zowongolera kukula kwa zomera (PGRs) zimagwira ntchito yofunika kwambiri pakulamulira morphogenesis ndipo ndizofunikira pakukula kwa callus ndi zamoyo zonse mu vitro. Kukonza kuchuluka kwawo ndi kuphatikiza kwawo ndikofunikira kwambiri kuti njira izi zitheke bwino [9]. Chifukwa chake, kumvetsetsa kapangidwe koyenera ndi kuchuluka kwa zowongolera ndikofunikira pakukweza kukula ndi mphamvu yobwezeretsa ya St. John's wort (H. perforatum) [10].
Tinthu tating'onoting'ono ta Iron oxide (Fe₃O₄) ndi gulu la tinthu tating'onoting'ono tomwe tapangidwa kapena tikupangidwa kuti tilimidwe minofu. Fe₃O₄ ili ndi mphamvu zazikulu zamaginito, imagwirizana bwino ndi zamoyo, komanso imatha kulimbikitsa kukula kwa zomera ndikuchepetsa kupsinjika kwa chilengedwe, kotero yakopa chidwi chachikulu pakupanga kwa minofu. Kugwiritsa ntchito kwa tinthu tating'onoting'ono timeneti kungaphatikizepo kukonza chikhalidwe cha in vitro kuti chilimbikitse kugawikana kwa maselo, kusintha kudya kwa michere, ndikuyambitsa ma enzymes oletsa antioxidant [11].
Ngakhale kuti tinthu tating'onoting'ono ta nano tawonetsa zotsatira zabwino pakukula kwa zomera, maphunziro okhudza kugwiritsa ntchito pamodzi kwa tinthu tating'onoting'ono ta Fe₃O₄ ndi owongolera kukula kwa zomera mu *H. perforatum* akadali ochepa. Pofuna kudzaza kusiyana kwa chidziwitsochi, kafukufukuyu adawunika zotsatira za zotsatira zake pamodzi pa morphogenesis ya in vitro ndi kupanga kwachiwiri kwa metabolite kuti apereke chidziwitso chatsopano chowongolera makhalidwe a zomera zamankhwala. Chifukwa chake, kafukufukuyu ali ndi zolinga ziwiri: (1) kukonza kuchuluka kwa owongolera kukula kwa zomera kuti alimbikitse bwino kupanga callus, kubwezeretsa mphukira, ndi mizu mu vitro; ndi (2) kuwunika zotsatira za tinthu tating'onoting'ono ta Fe₃O₄ pa magawo okulira mu vitro. Mapulani amtsogolo akuphatikizapo kuwunika kuchuluka kwa kupulumuka kwa zomera zobwezeretsedwa panthawi yozolowera (in vitro). Zikuyembekezeka kuti zotsatira za kafukufukuyu zithandizira kwambiri magwiridwe antchito a *H. perforatum*, motero zikuthandizira kugwiritsidwa ntchito kosatha komanso kugwiritsa ntchito biotechnology kwa chomera chofunikira ichi chamankhwala.
Mu kafukufukuyu, tinapeza zomera zochokera ku masamba a St. John's wort (zomera zazikulu) zomwe zimalimidwa m'munda. Mitengo imeneyi inagwiritsidwa ntchito kukonza bwino momwe zomera zimakulira m'munda. Tisanayambe kubzala, masambawo ankatsukidwa bwino ndi madzi osungunuka kwa mphindi zingapo. Kenako pamwamba pa zomerazo ankatsukidwa ndi mankhwala ophera tizilombo powamiza mu 70% ethanol kwa masekondi 30, kenako n’kuwamiza mu 1.5% sodium hypochlorite (NaOCl) solution yokhala ndi madontho ochepa a Tween 20 kwa mphindi 10. Pomaliza, zomerazo zinatsukidwa katatu ndi madzi osungunuka asanasamutsidwire ku malo ena obzala.
M'masabata anayi otsatira, magawo obwezeretsa mphukira adayesedwa, kuphatikizapo kuchuluka kwa kubwezeretsanso, kuchuluka kwa mphukira pa chomera chilichonse, ndi kutalika kwa mphukira. Pamene mphukira zomwe zabwezeretsedwanso zidafika kutalika kwa osachepera 2 cm, zidasamutsidwira ku chomera chokhala ndi theka la mphamvu ya MS, 0.5 mg/L indolebutyric acid (IBA), ndi 0.3% guar gum. Kukula kwa mizu kunapitilira kwa milungu itatu, pomwe kuchuluka kwa mizu, kuchuluka kwa mizu, ndi kutalika kwa mizu kunayesedwa. Chithandizo chilichonse chinabwerezedwa katatu, ndi zomera 10 zomwe zinalimidwa pa chomera chilichonse, zomwe zinapereka zomera pafupifupi 30 pa chithandizo chilichonse.
Kutalika kwa chomera kunayesedwa mu masentimita (cm) pogwiritsa ntchito rula, kuyambira pansi pa chomera mpaka kumapeto kwa tsamba lalitali kwambiri. Kutalika kwa mizu kunayesedwa mu mamilimita (mm) nthawi yomweyo mutachotsa mbande mosamala ndikuchotsa malo okula. Chiwerengero cha mphukira pa chomera chilichonse chinawerengedwa mwachindunji pa chomera chilichonse. Chiwerengero cha madontho akuda pamasamba, omwe amadziwika kuti timibulu, chinayesedwa m'maso. Timibulu takuda timeneti timakhulupirira kuti ndi tizirombo tokhala ndi hypericin, kapena madontho owopsa, ndipo timagwiritsidwa ntchito ngati chizindikiro cha thupi cha momwe chomeracho chimayankhira chithandizo. Pambuyo pochotsa malo onse okula, kulemera kwatsopano kwa mbande kunayesedwa pogwiritsa ntchito sikelo yamagetsi yolondola ya mamiligalamu (mg).
Njira yowerengera kuchuluka kwa mapangidwe a callus ndi iyi: Pambuyo pokulitsa zomera zomwe zili ndi zinthu zosiyanasiyana zowongolera kukula (kinases, 2,4-D, ndi Fe3O4) kwa milungu inayi, chiwerengero cha zomera zomwe zimatha kupanga callus chimawerengedwa. Njira yowerengera kuchuluka kwa mapangidwe a callus ndi iyi:
Chithandizo chilichonse chinabwerezedwa katatu, ndipo osachepera 10 anawunikidwa pa kubwereza kulikonse.
Kuchuluka kwa kusinthika kwa thupi kumawonetsa kuchuluka kwa minofu ya callus yomwe imamaliza bwino njira yosinthira maluwa pambuyo pa gawo la kupanga callus. Chizindikiro ichi chikuwonetsa kuthekera kwa minofu ya callus kusintha kukhala minofu yosiyana ndikukula kukhala ziwalo zatsopano za zomera.
Kuchuluka kwa mizu ndi chiŵerengero cha nthambi zomwe zimatha kumera mizu ndi chiwerengero chonse cha nthambi. Chizindikiro ichi chikuwonetsa kupambana kwa gawo la mizu, lomwe ndi lofunika kwambiri pakufalikira kwa microbe ndi kufalikira kwa zomera, chifukwa mizu yabwino imathandiza mbande kukhalabe ndi moyo wabwino m'mikhalidwe yokulira.
Mankhwala a Hypericin adachotsedwa ndi 90% methanol. 50 mg ya zinthu zouma za zomera zinawonjezedwa ku 1 ml ya methanol ndipo zinayikidwa kwa mphindi 20 pa 30 kHz mu chotsukira cha ultrasonic (model A5120-3YJ) kutentha kwa chipinda mumdima. Pambuyo pa sonication, chitsanzocho chinayikidwa pa centrifuge pa 6000 rpm kwa mphindi 15. Supernatant inasonkhanitsidwa, ndipo kuyamwa kwa hypericin kunayesedwa pa 592 nm pogwiritsa ntchito Plus-3000 S spectrophotometer malinga ndi njira yomwe Conceiçao et al. [14] adafotokoza.
Mankhwala ambiri okhala ndi owongolera kukula kwa zomera (PGRs) ndi tinthu tating'onoting'ono ta iron oxide (Fe₃O₄-NPs) sanapangitse kuti tinthu tating'onoting'ono takuda tipangidwe pa masamba a mphukira omwe abwezeretsedwanso. Palibe tinthu tating'onoting'ono tomwe tidawonedwa mu mankhwala aliwonse okhala ndi 0.5 kapena 1 mg/L 2,4-D, 0.5 kapena 1 mg/L kinetin, kapena 1, 2, kapena 4 mg/L iron oxide nanoparticles. Kuphatikiza pang'ono kunawonetsa kuwonjezeka pang'ono kwa kukula kwa tinthu tating'onoting'ono (koma osati kofunikira kwambiri) pa kuchuluka kwakukulu kwa kinetin ndi/kapena tinthu tating'onoting'ono ta iron oxide, monga kuphatikiza kwa 2,4-D (0.5–2 mg/L) ndi kinetin (1–1.5 mg/L) ndi tinthu tating'onoting'ono ta iron oxide (2–4 mg/L). Zotsatirazi zikuwonetsedwa mu Chithunzi 2. Tinthu tating'onoting'ono takuda tikuyimira tinthu ta hypericin tochuluka, tomwe timapezeka mwachilengedwe komanso topindulitsa. Mu kafukufukuyu, tinthu tating'onoting'ono takuda tinkagwirizana kwambiri ndi kufiyira kwa minofu, zomwe zikusonyeza malo abwino osungira hypericin. Chithandizo cha 2,4-D, kinetin, ndi Fe₃O₄ nanoparticles chinalimbikitsa kukula kwa callus, kuchepetsa browning, komanso kuchuluka kwa chlorophyll, zomwe zikusonyeza kuti kagayidwe kachakudya kabwinobwino komanso kuchepetsa kuwonongeka kwa okosijeni [37]. Kafukufukuyu adawunika zotsatira za kinetin pamodzi ndi 2,4-D ndi Fe₃O₄ nanoparticles pa kukula ndi chitukuko cha St. John's wort callus (Chithunzi 3a–g). Kafukufuku wakale wasonyeza kuti Fe₃O₄ nanoparticles ili ndi zochita zotsutsana ndi bowa komanso zotsutsana ndi mabakiteriya [38, 39] ndipo, ikagwiritsidwa ntchito limodzi ndi owongolera kukula kwa zomera, imatha kulimbikitsa njira zodzitetezera zomera ndikuchepetsa zizindikiro za kupsinjika kwa maselo [18]. Ngakhale kuti biosynthesis ya metabolites yachiwiri imayendetsedwa ndi majini, phindu lawo lenileni limadalira kwambiri momwe zinthu zilili. Kusintha kwa kagayidwe kachakudya ndi mawonekedwe ake kumatha kukhudza milingo yachiwiri ya metabolite mwa kuwongolera kuwonetsedwa kwa majini enaake a zomera ndikuyankha kuzinthu zachilengedwe. Kuphatikiza apo, zinthu zoyambitsa zimatha kuyambitsa kuyambika kwa majini atsopano, zomwe zimapangitsa kuti ntchito ya enzymatic iyambe, zomwe pamapeto pake zimayambitsa njira zingapo zopangira zinthu zomwe zimapangitsa kuti zinthu ziyambe kupanga. Kuphatikiza apo, kafukufuku wina adawonetsa kuti kuchepetsa mthunzi kumawonjezera kuwala kwa dzuwa, motero kumakweza kutentha kwa masana m'malo achilengedwe a *Hypericum perforatum*, zomwe zimathandizanso kuti phindu la hypericin liwonjezeke. Kutengera ndi deta iyi, kafukufukuyu adafufuza ntchito ya tinthu tating'onoting'ono ta iron ngati zinthu zomwe zingayambitse kukula kwa minofu. Zotsatira zake zidawonetsa kuti tinthu tating'onoting'ono timeneti tingayambitse majini omwe amagwira ntchito mu hesperidin biosynthesis kudzera mu kukondoweza kwa enzymatic, zomwe zimapangitsa kuti pakhale kuwonjezeka kwa chigawochi (Chithunzi 2). Chifukwa chake, poyerekeza ndi zomera zomwe zimamera m'mikhalidwe yachilengedwe, zitha kutsutsidwa kuti kupanga zinthu zotere mu vivo kumathanso kuwonjezeka pamene kupsinjika pang'ono kumaphatikizidwa ndi kuyambika kwa majini omwe amagwira ntchito mu biosynthesis ya zinthu zachiwiri. Mankhwala ophatikizana nthawi zambiri amakhala ndi zotsatira zabwino pa kuchuluka kwa kubwezeretsedwa, koma nthawi zina, izi zimafooka. Chodziwika bwino n'chakuti, chithandizo cha 1 mg/L 2,4-D, 1.5 mg/L kinase, ndi kuchuluka kosiyanasiyana kungawonjezere kuchuluka kwa kubwezeretsedwa kwa thupi ndi 50.85% poyerekeza ndi gulu lolamulira (Chithunzi 4c). Zotsatirazi zikusonyeza kuti kuphatikiza kwapadera kwa ma nanohormones kumatha kugwira ntchito mogwirizana kuti kulimbikitse kukula kwa zomera ndi kupanga metabolite, zomwe ndizofunikira kwambiri pakukula kwa minofu ya zomera zamankhwala. Palmer ndi Keller [50] adawonetsa kuti chithandizo cha 2,4-D chingayambitse kupangika kwa callus mu St. perforatum, pomwe kuwonjezera kwa kinase kunathandizira kwambiri kupangika kwa callus ndi kubwezeretsedwa kwa thupi. Zotsatirazi zidachitika chifukwa cha kusintha kwa mahomoni ndi kukulitsa kugawikana kwa maselo. Bal et al. [51] adapeza kuti chithandizo cha Fe₃O₄-NP chingathe kuwonjezera ntchito ya ma enzymes oletsa antioxidant, motero chimalimbikitsa kukula kwa mizu mu St. perforatum. Zomera zokhala ndi Fe₃O₄ nanoparticles pamlingo wa 0.5 mg/L, 1 mg/L, ndi 1.5 mg/L zinasintha kuchuluka kwa kusinthika kwa zomera za fulakesi [52]. Kugwiritsa ntchito kinetin, 2,4-dichlorobenzothiazolinone, ndi Fe₃O₄ nanoparticles kunasintha kwambiri kuchuluka kwa callus ndi mizu, komabe, zotsatirapo zoyipa zomwe zingachitike chifukwa chogwiritsa ntchito mahomoni awa pakukonzanso mu vitro ziyenera kuganiziridwa. Mwachitsanzo, kugwiritsa ntchito 2,4-dichlorobenzothiazolinone kapena kinetin kwa nthawi yayitali kapena mopitirira muyeso kungayambitse kusintha kwa somatic clonal, kupsinjika kwa okosijeni, mawonekedwe osazolowereka a callus, kapena vitrification. Chifukwa chake, kuchuluka kwa kusinthika kwakukulu sikuneneratu kukhazikika kwa majini. Zomera zonse zobwezeretsedwa ziyenera kuyesedwa pogwiritsa ntchito zizindikiro zama molecular (monga RAPD, ISSR, AFLP) kapena kusanthula kwa cytogenetic kuti mudziwe kufanana kwawo ndi kufanana kwawo ndi zomera za mu vivo [53,54,55].
Kafukufukuyu adawonetsa koyamba kuti kugwiritsa ntchito pamodzi kwa owongolera kukula kwa zomera (2,4-D ndi kinetin) ndi Fe₃O₄ nanoparticles kungathandize kukulitsa kapangidwe kake ndi kusonkhanitsa ma metabolites ofunikira a bioactive (kuphatikiza hypericin ndi hyperoside) mu *Hypericum perforatum*. Njira yabwino kwambiri yothandizira (1 mg/L 2,4-D + 1 mg/L kinetin + 4 mg/L Fe₃O₄-NPs) sikuti idangowonjezera kupanga kwa callus, organogenesis, ndi phindu lachiwiri la metabolite komanso idawonetsa zotsatira zochepa zomwe zimapangitsa kuti chomera chikhale chopirira kupsinjika komanso kufunika kwa mankhwala. Kuphatikiza kwa nanotechnology ndi chikhalidwe cha minofu ya zomera kumapereka nsanja yokhazikika komanso yothandiza yopanga mankhwala ambiri mu vitro. Zotsatirazi zimatsegula njira yogwiritsira ntchito mafakitale ndi kafukufuku wamtsogolo pankhani ya njira zama molekyulu, kukonza mlingo ndi kulondola kwa majini, potero kulumikiza kafukufuku woyambira pa zomera zamankhwala ndi biotechnology yothandiza.
Nthawi yotumizira: Disembala-12-2025