A cikin wannan binciken, tasirin ƙarfafawa na haɗin gwiwa na maganinmasu kula da haɓakar shukaAn binciki morphogenesis na in vitro da kuma samar da metabolite na biyu a cikin *Hypericum perforatum* L.. An inganta maganin [2,4-D (0.5 mg/L) + kinetin (2 mg/L) + Fe₃O₄-NPs (4 mg/L)] sosai ya inganta sigogin girma na shuka: tsayin shuka ya karu da 59.6%, tsawon tushen sa da 114.0%, adadin tsiro da 180.0%, da nauyin sabon callus da 198.3% idan aka kwatanta da rukunin sarrafawa. Wannan maganin hade ya kuma inganta ingantaccen farfadowa (50.85%) kuma ya kara yawan sinadarin hypericin da 66.6%. Binciken GC-MS ya nuna yawan sinadarin hyperoside, β-patholene, da cetyl alcohol, wanda ya kai kashi 93.36% na jimillar yankin da ya kai kololuwa, yayin da jimillar sinadarin phenolics da flavonoids ya karu da kusan kashi 80.1%. Waɗannan sakamakon sun nuna cewa masu kula da ci gaban tsirrai (PGRs) da kuma Fe₃O₄ nanoparticles (Fe₃O₄-NPs) suna yin tasiri mai kyau ta hanyar ƙarfafa organogenesis da tara sinadarai masu aiki, wanda ke wakiltar wata dabara mai kyau don inganta fasahar halittu ta tsirrai masu magani.
St. John's wort (Hypericum perforatum L.), wanda aka fi sani da St. John's wort, shuka ce mai tsiro mai tsiro na dangin Hypericaceae wanda ke da darajar tattalin arziki.[1] Abubuwan da ke cikinsa sun haɗa da tannins na halitta, xanthones, phloroglucinol, naphthalenedianthrone (hyperin da pseudohyperin), flavonoids, phenolic acid, da mai mai mahimmanci.[2,3,4] Ana iya yaɗa St. John's wort ta hanyoyin gargajiya; duk da haka, yanayin hanyoyin gargajiya, ƙarancin tsiron iri, da kuma saurin kamuwa da cututtuka suna iyakance yuwuwar noman sa mai yawa da kuma ci gaba da samar da metabolites na biyu.[1,5,6]
Saboda haka, ana ɗaukar al'adar nama a cikin vitro a matsayin hanya mai inganci don yaɗuwar shuke-shuke cikin sauri, kiyaye albarkatun germplasm, da kuma ƙara yawan amfanin magungunan da ake samu [7, 8]. Masu kula da girmar shuke-shuke (PGRs) suna taka muhimmiyar rawa wajen daidaita yanayin halittarsu kuma suna da mahimmanci don noman callus da dukkan halittu. Inganta yawansu da haɗuwarsu yana da mahimmanci don kammala waɗannan hanyoyin ci gaba cikin nasara [9]. Saboda haka, fahimtar tsarin da ya dace da yawan masu kula da su yana da mahimmanci don inganta girma da ƙarfin sake farfaɗo da St. John's wort (H. perforatum) [10].
Nanoparticles na iron oxide (Fe₃O₄) wani nau'in nanoparticles ne da aka ƙirƙira ko ake haɓaka su don al'adar nama. Fe₃O₄ yana da mahimman halayen maganadisu, kyakkyawan jituwa tsakanin halittu, da kuma ikon haɓaka haɓakar shuke-shuke da rage damuwa ta muhalli, don haka ya jawo hankali sosai a cikin ƙirar al'adun nama. Amfani da waɗannan nanoparticles na iya haɗawa da inganta al'adun in vitro don haɓaka rarraba ƙwayoyin halitta, inganta ɗaukar abubuwan gina jiki, da kunna enzymes na antioxidant [11].
Duk da cewa ƙwayoyin halittar nano sun nuna kyakkyawan tasirin haɓakawa ga ci gaban shuka, nazarin da aka yi kan haɗakar amfani da ƙwayoyin halittar nano Fe₃O₄ da ingantattun masu kula da ci gaban shuka a cikin *H. perforatum* har yanzu ba su da yawa. Domin cike wannan gibin ilimi, wannan binciken ya kimanta tasirin haɗin gwiwar tasirinsu akan morphogenesis na in vitro da samar da metabolite na biyu don samar da sabbin fahimta don inganta halayen tsire-tsire masu magani. Saboda haka, wannan binciken yana da manufofi biyu: (1) inganta yawan masu kula da ci gaban shuka don inganta samuwar callus yadda ya kamata, sake farfaɗo da harbe-harbe, da kuma tushen shuka a cikin vitro; da kuma (2) kimanta tasirin ƙwayoyin halittar nano Fe₃O₄ akan sigogin girma a cikin vitro. Shirye-shiryen gaba sun haɗa da kimanta ƙimar rayuwa na tsire-tsire da aka sake farfaɗowa yayin daidaitawa (in vitro). Ana sa ran sakamakon wannan binciken zai inganta ingantaccen yaduwar ƙwayoyin halittar *H. perforatum* sosai, ta haka yana ba da gudummawa ga amfani mai dorewa da aikace-aikacen fasahar kere-kere na wannan shuka mai mahimmanci.
A cikin wannan binciken, mun sami ganyen da aka fitar daga shuke-shuken St. John's wort na shekara-shekara da ake nomawa a gona (uwa shuke-shuke). An yi amfani da waɗannan ganyen don inganta yanayin al'adar in vitro. Kafin a noma, an wanke ganyen sosai a ƙarƙashin ruwan da aka tace na tsawon mintuna da yawa. Daga nan aka tsaftace saman da aka fitar ta hanyar nutsar da shi a cikin ethanol 70% na tsawon daƙiƙa 30, sannan aka nutsar da shi a cikin maganin sodium hypochlorite (NaOCl) mai 1.5% wanda ya ƙunshi ɗigon Tween 20 na tsawon mintuna 10. A ƙarshe, an wanke ganyen sau uku da ruwan da aka tace kafin a canza shi zuwa wurin da aka shuka na gaba.
A cikin makonni huɗu masu zuwa, an auna ma'aunin sake farfaɗowar harbe-harbe, gami da ƙimar sake farfaɗowa, adadin harbi a kowace shuka, da tsawon harbi. Lokacin da harbe-harben da aka sake farfaɗowa suka kai tsawon akalla santimita 2, an canza su zuwa wani tushen tushen da ya ƙunshi matsakaicin MS mai ƙarfi, 0.5 mg/L indolebutyric acid (IBA), da 0.3% guar danko. Ci gaban tushen ya ci gaba na tsawon makonni uku, a lokacin an auna ƙimar tushen, lambar tushe, da tsawon tushen. An maimaita kowace magani sau uku, tare da shukar ganye 10 a kowane kwafi, suna samar da kimanin ganye 30 a kowane magani.
An auna tsayin shuka a santimita (cm) ta amfani da ruler, daga tushen shukar zuwa ƙarshen ganyen mafi tsayi. An auna tsawon tushen shukar a milimita (mm) nan da nan bayan an cire shukar a hankali sannan aka cire matsakaicin tsiron. An ƙididdige adadin furannin a kowace shuka kai tsaye a kan kowace shuka. An auna adadin tabo baƙi a kan ganyen, wanda aka sani da nodules, a gani. Ana kyautata zaton waɗannan ƙusoshin baƙi gland ne da ke ɗauke da hypericin, ko kuma tabo na oxidative, kuma ana amfani da su azaman alamar ilimin halittar jiki na martanin shukar ga magani. Bayan an cire duk matsakaicin tsiron, an auna sabon nauyin shukar ta amfani da sikelin lantarki tare da daidaiton milligrams (mg).
Hanyar ƙididdige yawan samuwar callus kamar haka: bayan an noma ƙwayoyin halitta a cikin wani wuri mai ɗauke da nau'ikan masu daidaita girma (kinases, 2,4-D, da Fe3O4) na tsawon makonni huɗu, ana ƙididdige adadin ƙwayoyin halitta da ke iya samar da callus. Tsarin ƙididdige yawan samuwar callus kamar haka:
An maimaita kowace magani sau uku, tare da aƙalla gwaje-gwajen da aka yi wa ƙarin bayani guda 10 a kowane maimaitawa.
Yawan sake farfaɗowa yana nuna adadin ƙwayoyin callus da suka kammala aikin bambance-bambancen budu bayan matakin samuwar callus. Wannan alamar tana nuna ikon ƙwayoyin callus na canzawa zuwa ƙwayoyin halitta daban-daban da kuma girma zuwa sabbin gabobin tsirrai.
Ma'aunin tushen shine rabon adadin rassan da za su iya yin tushe zuwa jimlar adadin rassan. Wannan alamar tana nuna nasarar matakin tushen, wanda yake da mahimmanci a cikin yaduwar ƙwayoyin cuta da yaɗuwar tsirrai, domin kyakkyawan tushen yana taimaka wa tsire-tsire su tsira cikin yanayi mai kyau na girma.
An cire sinadaran Hypericin da kashi 90% na methanol. An ƙara 50 mg na busasshen kayan shuka a cikin 1 ml na methanol kuma an saka shi a cikin sonicated na minti 20 a 30 kHz a cikin na'urar tsabtace ultrasonic (samfurin A5120-3YJ) a zafin ɗaki a cikin duhu. Bayan an yi sonication, an sanya samfurin a cikin centrifuge a 6000 rpm na minti 15. An tattara ruwan da ke sama, kuma an auna shan hypericin a 592 nm ta amfani da na'urar auna haske ta Plus-3000 S bisa ga hanyar da Conceiçao et al. [14] suka bayyana.
Yawancin jiyya tare da masu kula da girmar shuka (PGRs) da ƙananan ƙwayoyin ƙarfe oxide (Fe₃O₄-NPs) ba su haifar da samuwar ƙananan ƙwayoyin baƙaƙe a kan ganyen da aka sake farfaɗowa ba. Ba a ga ƙananan ƙwayoyin ba a cikin kowace maganin da aka yi da kinetin 0.5 ko 1 mg/L 2,4-D, 0.5 ko 1 mg/L, ko ƙananan ƙwayoyin ƙarfe oxide 1, 2, ko 4 mg/L. Wasu haɗuwa sun nuna ɗan ƙaruwa a cikin ci gaban ƙananan ƙwayoyin (amma ba mahimmanci ba ne a kididdiga) a mafi yawan ƙwayoyin kinetin da/ko ƙwayoyin ƙarfe oxide, kamar haɗin 2,4-D (0.5-2 mg/L) tare da kinetin (1-1.5 mg/L) da ƙananan ƙwayoyin ƙarfe oxide (2-4 mg/L). An nuna waɗannan sakamakon a Hoto na 2. Ƙananan ƙwayoyin baƙaƙe suna wakiltar glandar da ke da wadataccen hypericin, waɗanda ke faruwa ta halitta kuma suna da amfani. A cikin wannan binciken, ƙananan ƙwayoyin baƙaƙe galibi suna da alaƙa da launin ruwan kasa na kyallen takarda, yana nuna yanayi mai kyau don tarin hypericin. Jiyya da ƙwayoyin cuta na 2,4-D, kinetin, da Fe₃O₄ sun haɓaka haɓakar ƙwayoyin cuta, rage launin ruwan kasa, da kuma ƙaruwar abubuwan da ke cikin chlorophyll, wanda ke nuna ingantaccen aikin metabolism da yuwuwar rage lalacewar oxidative [37]. Wannan binciken ya kimanta tasirin kinetin tare da ƙwayoyin cuta na 2,4-D da Fe₃O₄ akan girma da haɓaka ƙwayoyin cuta na St. John's wort callus (Hoto na 3a–g). Nazarin da aka yi a baya sun nuna cewa ƙwayoyin cuta na Fe₃O₄ suna da ayyukan fungal da antimicrobial [38, 39] kuma, idan aka yi amfani da su tare da masu kula da haɓakar tsire-tsire, na iya ƙarfafa hanyoyin kare tsirrai da rage alamun damuwa na ƙwayoyin cuta [18]. Kodayake ana tsara tsarin biosynthesis na ƙwayoyin cuta na biyu ta hanyar kwayoyin halitta, ainihin yawan amfanin su ya dogara sosai akan yanayin muhalli. Canje-canje na metabolism da morphological na iya yin tasiri ga matakan metabolite na biyu ta hanyar daidaita bayyanar takamaiman kwayoyin halittar shuka da kuma mayar da martani ga abubuwan muhalli. Bugu da ƙari, masu haifar da ƙwayoyin halitta na iya haifar da kunna sabbin ƙwayoyin halitta, wanda hakan ke ƙarfafa ayyukan enzymatic, wanda a ƙarshe ke kunna hanyoyi da yawa na biosynthesis da kuma haifar da samuwar metabolites na biyu. Bugu da ƙari, wani bincike ya nuna cewa rage inuwa yana ƙara yawan hasken rana, ta haka yana ƙara yanayin zafi a cikin yanayin halitta na *Hypericum perforatum*, wanda kuma yana taimakawa wajen ƙara yawan amfanin hypericin. Dangane da waɗannan bayanai, wannan binciken ya binciki rawar da ƙwayoyin ƙarfe ke takawa a matsayin masu haifar da ƙwayoyin halitta a cikin al'adun nama. Sakamakon ya nuna cewa waɗannan ƙwayoyin halitta na iya kunna kwayoyin halitta da ke da hannu a cikin biosynthesis na hesperidin ta hanyar ƙarfafa enzymatic, wanda ke haifar da ƙaruwar tarin wannan mahaɗin (Hoto na 2). Saboda haka, idan aka kwatanta da tsire-tsire da ke girma a ƙarƙashin yanayi na halitta, za a iya jayayya cewa samar da irin waɗannan mahaɗan a cikin jiki kuma ana iya haɓaka lokacin da aka haɗa matsakaicin damuwa tare da kunna kwayoyin halitta da ke da hannu a cikin biosynthesis na metabolites na biyu. Magungunan haɗin gwiwa gabaɗaya suna da tasiri mai kyau akan ƙimar sake farfaɗowa, amma a wasu lokuta, wannan tasirin yana raguwa. Abin lura shi ne, magani da 1 mg/L 2,4-D, 1.5 mg/L kinase, da kuma yawan da ke tattare da su daban-daban na iya ƙara yawan sake farfadowa da kashi 50.85% daban-daban idan aka kwatanta da ƙungiyar kulawa (Hoto na 4c). Waɗannan sakamakon sun nuna cewa takamaiman haɗuwa na nanohormones na iya yin aiki tare don haɓaka haɓakar shuka da samar da metabolite, wanda ke da matuƙar mahimmanci ga al'adar nama na tsire-tsire masu magani. Palmer da Keller [50] sun nuna cewa maganin 2,4-D zai iya haifar da samuwar callus a cikin St. perforatum, yayin da ƙarin kinase ya inganta samuwar callus da sake farfadowa sosai. Wannan tasirin ya faru ne saboda inganta daidaiton hormonal da ƙarfafa rarrabuwar ƙwayoyin halitta. Bal et al. [51] sun gano cewa maganin Fe₃O₄-NP zai iya haɓaka aikin enzymes na antioxidant daban-daban, ta haka yana haɓaka haɓakar tushe a cikin St. perforatum. Maganin al'adu da ke ɗauke da ƙwayoyin Fe₃O₄ a yawan ƙwayoyin flax da ke cikin 0.5 mg/L, 1 mg/L, da 1.5 mg/L sun inganta yawan farfaɗowar tsirrai [52]. Amfani da ƙwayoyin kinetin, 2,4-dichlorobenzothiazolinone, da Fe₃O₄ sun inganta yawan farfaɗowar ƙwayoyin callus da tushensu sosai, duk da haka, ya kamata a yi la'akari da illolin da ke tattare da amfani da waɗannan hormones don farfaɗowar ƙwayoyin in vitro. Misali, amfani da 2,4-dichlorobenzothiazolinone ko kinetin na dogon lokaci ko mai yawa na iya haifar da bambancin clonal na somatic, damuwa ta oxidative, yanayin callus mara kyau, ko vitrification. Saboda haka, babban farfaɗowar ƙwayoyin ba lallai bane ya annabta kwanciyar hankali na kwayoyin halitta. Ya kamata a tantance duk tsire-tsire da aka farfaɗo ta amfani da alamun kwayoyin halitta (misali RAPD, ISSR, AFLP) ko nazarin cytogenetic don tantance daidaitonsu da kamanceceniya da tsire-tsire a cikin vivo [53,54,55].
Wannan binciken ya nuna a karon farko cewa amfani da na'urorin daidaita girma na shuka (2,4-D da kinetin) tare da ƙwayoyin Fe₃O₄ na iya haɓaka morphogenesis da tara muhimman metabolites na bioactive (gami da hypericin da hyperoside) a cikin *Hypericum perforatum*. Tsarin magani mafi kyau (1 mg/L 2,4-D + 1 mg/L kinetin + 4 mg/L Fe₃O₄-NPs) ba wai kawai ya haɓaka samuwar callus, organogenesis, da yawan metabolite na biyu ba, har ma ya nuna wani ɗan tasiri mai sauƙi, wanda zai iya inganta juriyar damuwa da ƙimar magani na shuka. Haɗin fasahar nanotechnology da al'adun ƙwayoyin shuka yana ba da dandamali mai ɗorewa da inganci don samar da mahadi na magani a cikin vitro. Waɗannan sakamakon suna buɗe hanya don aikace-aikacen masana'antu da bincike na gaba a cikin hanyoyin kwayoyin halitta, inganta allurai da daidaiton kwayoyin halitta, ta haka suna haɗa bincike na asali akan tsire-tsire na magani tare da fasahar biotechnology mai amfani.
Lokacin Saƙo: Disamba-12-2025