Kolu phononongo, iziphumo ezivuselelayo zonyango oludibeneyo lweabalawuli bokukhula kwezityalo(2,4-D kunye ne-kinetin) kunye ne-iron oxide nanoparticles (Fe₃O₄-NPs) kwi-in vitro morphogenesis kunye nemveliso yesibini ye-metabolite kwi-*Hypericum perforatum* L. ziphandwe. Unyango oluphuculweyo [2,4-D (0.5 mg/L) + kinetin (2 mg/L) + Fe₃O₄-NPs (4 mg/L)] luphucule kakhulu iiparameter zokukhula kwesityalo: ubude besityalo bunyuke nge-59.6%, ubude beengcambu nge-114.0%, inani leentyatyambo nge-180.0%, kunye nobunzima obutsha be-callus nge-198.3% xa kuthelekiswa neqela lolawulo. Olu nyango ludibeneyo lukwaphucule ukusebenza kakuhle kokuvuselelwa (50.85%) kwaye lwanyusa umxholo we-hypericin nge-66.6%. Uhlalutyo lwe-GC-MS lubonise ukuba kukho i-hyperoside eninzi, i-β-patolene, kunye ne-cetyl alcohol, ebalelwa kwi-93.36% yendawo iyonke, ngelixa kukho i-phenolics kunye ne-flavonoids eyonyukile nge-80.1%. Ezi ziphumo zibonisa ukuba abalawuli bokukhula kwezityalo (ii-PGR) kunye ne-Fe₃O₄ nanoparticles (ii-Fe₃O₄-NPs) zisebenza ngokubambisana ngokuvuselela i-organogenesis kunye nokuqokelelana kwee-bioactive compounds, nto leyo emele icebo elithembisayo lokuphucula i-biotechnology yezityalo zonyango.
I-St. John's wort (Hypericum perforatum L.), eyaziwa ngokuba yi-St. John's wort, sisityalo esihlala sikhula se-herbaceous sosapho lwe-Hypericaceae esinexabiso lezoqoqosho.[1] Izinto ezinokubakho eziphilayo ziquka ii-tannins zendalo, ii-xanthones, i-phloroglucinol, i-naphthalenedianthrone (hyperin kunye ne-pseudohyperin), ii-flavonoids, ii-phenolic acids, kunye nee-oyile ezibalulekileyo.[2,3,4] I-St. John's wort ingasasazwa ngeendlela zemveli; nangona kunjalo, ixesha lexesha lemveli, ukuhluma kwembewu okuncinci, kunye nokuchaphazeleka zizifo kunciphisa amandla ayo okulima kakhulu kunye nokwakheka okuqhubekayo kwee-metabolites zesibini.[1,5,6]
Ngoko ke, inkcubeko yezicubu ze-in vitro ithathwa njengendlela esebenzayo yokwandisa izityalo ngokukhawuleza, ukugcinwa kwezixhobo ze-germplasm, kunye nokwanda kwemveliso yeekhompawundi zamayeza [7, 8]. Abalawuli bokukhula kwezityalo (ii-PGR) badlala indima ebalulekileyo ekulawuleni i-morphogenesis kwaye bayimfuneko ekukhuliseni i-callus kunye nezinto eziphilayo ezipheleleyo kwi-in vitro. Ukuphucula uxinano lwazo kunye nokudibanisa kwazo kubalulekile ekugqityweni ngempumelelo kwezi nkqubo zophuhliso [9]. Ke ngoko, ukuqonda ukwakheka okufanelekileyo kunye noxinano lwabalawuli kubalulekile ekuphuculeni ukukhula kunye namandla okuvuselela i-St. John's wort (H. perforatum) [10].
Ii-nanoparticles ze-iron oxide (Fe₃O₄) luhlobo lwee-nanoparticles eziye zaphuhliswa okanye ezisaphuhliswayo ukuze kukhuliswe izicubu. I-Fe₃O₄ ineempawu ezibalulekileyo zemagnethi, ukuhambelana okuhle kwezinto eziphilayo, kunye nokukwazi ukukhuthaza ukukhula kwezityalo kunye nokunciphisa uxinzelelo lokusingqongileyo, ngoko ke itsale ingqalelo enkulu kuyilo lwenkcubeko yezicubu. Ukusetyenziswa okunokwenzeka kwezi nanoparticles kunokuquka ukuphucula inkcubeko ye-in vitro ukukhuthaza ukwahlukana kweeseli, ukuphucula ukufunxwa kwezondlo, kunye nokusebenza kwee-enzymes ze-antioxidant [11].
Nangona ii-nanoparticles zibonakalise iziphumo ezilungileyo zokukhuthaza ukukhula kwezityalo, izifundo malunga nokusetyenziswa okudibeneyo kwee-nanoparticles ze-Fe₃O₄ kunye nabalawuli bokukhula kwezityalo abaphuculweyo kwi-*H. perforatum* zisanqabile. Ukugcwalisa lo msantsa wolwazi, olu phononongo luvavanye iziphumo zeziphumo zazo ezidibeneyo kwi-morphogenesis ye-in vitro kunye nemveliso yesibini ye-metabolite ukuze kubonelelwe ngolwazi olutsha lokuphucula iimpawu zezityalo zonyango. Ke ngoko, olu phononongo luneenjongo ezimbini: (1) ukuphucula ukuxinana kwabalawuli bokukhula kwezityalo ukuze kukhuthazwe ngempumelelo ukwakheka kwe-callus, ukuvuselelwa kwehlumela, kunye nokuhluma iingcambu kwi-vitro; kunye (2) nokuvavanya iziphumo zee-nanoparticles ze-Fe₃O₄ kwiiparameter zokukhula kwi-vitro. Izicwangciso zexesha elizayo ziquka ukuvavanya izinga lokusinda kwezityalo ezivuselelweyo ngexesha lokuqhelana (in vitro). Kulindeleke ukuba iziphumo zolu phononongo ziya kuphucula kakhulu ukusebenza kakuhle kwe-micropropagation ye-*H. perforatum*, ngaloo ndlela zinegalelo ekusetyenzisweni okuzinzileyo kunye nokusetyenziswa kwe-biotechnology kwesi sityalo sibalulekileyo samayeza.
Kolu phononongo, sifumene izityalo ezisandul’ ukuvela emagqabini ezivela kwizityalo zonyaka zeSt. John’s wort (izityalo ezingumama). Ezi zityalo zisetyenziselwe ukuphucula imeko yokulima kwi-vitro. Ngaphambi kokulima, amagqabi ahlanjwa kakuhle ngamanzi acociweyo imizuzu embalwa. Emva koko iindawo ezisandul’ ukuvela zahlanjwa ngokuntywiliselwa kwi-70% ye-ethanol imizuzwana engama-30, kwalandela ukuntywiliselwa kwisisombululo se-1.5% ye-sodium hypochlorite (NaOCl) esiqulethe amaconsi ambalwa e-Tween 20 imizuzu eli-10. Ekugqibeleni, izityalo ezisandul’ ukuvela zahlanjwa kathathu ngamanzi acocekileyo ngaphambi kokuba zidluliselwe kwindawo elandelayo yokulima.
Kwiiveki ezine ezalandelayo, iiparameter zokuvuselelwa kwehlumela zalinganiswa, kuquka izinga lokuvuselelwa, inani lokudubula ngesityalo ngasinye, kunye nobude bokudubula. Xa amahlumela avuselelwe afikelela kubude obuyi-2 cm ubuncinane, adluliselwa kwindawo yokuhluma enesiqingatha se-MS medium, i-0.5 mg/L indolebutyric acid (IBA), kunye ne-0.3% guar gum. Ukukhula kwehlumela kwaqhubeka iiveki ezintathu, apho izinga lokukhula kwehlumela, inani leengcambu, kunye nobude beengcambu zalinganiswa. Unyango ngalunye lwaphindwa kathathu, kunye namahlumela ali-10 akhuliswe ngokuphindaphindiweyo, okuvelisa malunga namahlumela angama-30 ngonyango ngalunye.
Ukuphakama kwesityalo kwalinganiswa ngeesentimitha (cm) kusetyenziswa irula, ukusuka kwisiseko sesityalo ukuya kwincam yegqabi elide. Ubude beengcambu balinganiswa ngeemilimitha (mm) emva nje kokususa ngononophelo izithole nokususa indawo ekhulayo. Inani lamahlumela kwisityalo ngasinye labalwa ngokuthe ngqo kwisityalo ngasinye. Inani lamabala amnyama kumagqabi, aziwa ngokuba ngamaqhuqhuva, lalinganiswa ngokubonakalayo. La maqhuqhuva amnyama kukholelwa ukuba ngamagland aqulethe i-hypericin, okanye amabala e-oxidative, kwaye asetyenziswa njengesalathisi sempilo yesityalo kwinyango. Emva kokususa yonke indawo ekhulayo, ubunzima obutsha bezithole balinganiswa kusetyenziswa isikali se-elektroniki esichanekileyo seemiligram (mg).
Indlela yokubala izinga lokwakheka kwe-callus yile ilandelayo: emva kokukhulisa ii-explants kwindawo equlethe abalawuli bokukhula abahlukeneyo (ii-kinases, i-2,4-D, kunye ne-Fe3O4) kangangeeveki ezine, inani le-explants ezikwaziyo ukwenza i-callus liyabalwa. Ifomyula yokubala izinga lokwakheka kwe-callus yile ilandelayo:
Unyango ngalunye luphindwe kathathu, ubuncinane ii-explant ezili-10 zihlolwe kuphindaphindo ngalunye.
Izinga lokuvuselelwa libonisa umlinganiselo wezicubu ze-callus ezigqibezela ngempumelelo inkqubo yokwahluka kwamahlumela emva kwesigaba sokwakheka kwe-callus. Esi sibonakaliso sibonisa amandla ezicubu ze-callus okuguquka zibe zizicubu ezahluliweyo kwaye zikhule zibe ngamalungu amatsha ezityalo.
I-rooting coefficient yi-ratio yenani lamasebe anakho ukuhluma iingcambu kwinani lilonke lamasebe. Esi sibonakaliso sibonisa impumelelo yesigaba sokuhluma iingcambu, esibaluleke kakhulu ekukhuleni kwezityalo ezincinci kunye nokukhula kwazo, njengoko ukuhluma iingcambu kakuhle kunceda izithole ukuba ziphile ngcono kwiimeko zokukhula.
Iikhompawundi zeHypericin zikhutshwe nge-90% ye-methanol. I-50 mg yezinto zezityalo ezomileyo zongezwa kwi-1 ml ye-methanol kwaye zafakwa kwi-sonic imizuzu engama-20 kwi-30 kHz kwisicoci se-ultrasonic (imodeli A5120-3YJ) kubushushu begumbi ebumnyameni. Emva kokufakwa kwe-sonication, isampuli yafakwa kwi-centrifuge kwi-6000 rpm imizuzu eli-15. I-supernatant yaqokelelwa, kwaye ukufunxwa kwe-hypericin kwalinganiswa kwi-592 nm kusetyenziswa i-Plus-3000 S spectrophotometer ngokwendlela echazwe nguConceiçao et al. [14].
Uninzi lonyango olusebenzisa abalawuli bokukhula kwezityalo (ii-PGR) kunye nee-nanoparticles ze-iron oxide (ii-Fe₃O₄-NPs) aluzange lubangele ukwakheka kwamaqhuqhuva amnyama kumagqabi amahlumela avuselelweyo. Akukho maqhuqhuva abonweyo nakweyiphi na indlela yonyango nge-0.5 okanye 1 mg/L 2,4-D, 0.5 okanye 1 mg/L kinetin, okanye i-1, 2, okanye 4 mg/L iron oxide nanoparticles. Iindidi ezimbalwa zibonise ukunyuka okuncinci kophuhliso lwamaqhuqhuva (kodwa akunabalulekanga ngokwezibalo) kumanqanaba aphezulu e-kinetin kunye/okanye i-iron oxide nanoparticles, njengokudibanisa i-2,4-D (0.5–2 mg/L) kunye ne-kinetin (1–1.5 mg/L) kunye nee-nanoparticles ze-iron oxide (2–4 mg/L). Ezi ziphumo ziboniswe kuMfanekiso 2. Amaqhuqhuva amnyama amele amaqhuqhuva atyebileyo e-hypericin, avela ngokwendalo kwaye aluncedo. Kolu phononongo, amaqhuqhuva amnyama ayenxulunyaniswa kakhulu nokumnyama kwezicubu, okubonisa indawo efanelekileyo yokuqokelelana kwe-hypericin. Unyango olusebenzisa ii-nanoparticles ze-2,4-D, kinetin, kunye ne-Fe₃O₄ lukhuthaze ukukhula kwe-callus, ukunciphisa i-browning, kunye nokwanda komxholo we-chlorophyll, nto leyo ebonisa ukuba ukusebenza kwe-metabolic kuphuculwe kunye nokunciphisa umonakalo we-oxidative [37]. Olu phononongo luvavanye iziphumo ze-kinetin xa idibene nee-nanoparticles ze-2,4-D kunye ne-Fe₃O₄ ekukhuleni nasekuphuhlisweni kwe-St. John's wort callus (Umzobo 3a–g). Izifundo zangaphambili zibonise ukuba ii-nanoparticles ze-Fe₃O₄ zinemisebenzi yokubola kunye neyokubulala iintsholongwane [38, 39] kwaye, xa zisetyenziswa kunye nabalawuli bokukhula kwezityalo, zinokuvuselela iindlela zokuzikhusela kwezityalo kwaye zinciphise izalathisi zoxinzelelo lweseli [18]. Nangona i-biosynthesis yee-metabolites zesibini ilawulwa ngokwezakhi zofuzo, isivuno sazo sokwenyani sixhomekeke kakhulu kwiimeko zendalo. Utshintsho lwe-metabolic kunye ne-morphological lunokuchaphazela amanqanaba e-metabolite yesibini ngokulawula ukubonakaliswa kwezakhi zofuzo ezithile zezityalo kunye nokuphendula kwizinto zendalo. Ngaphezu koko, izinto ezibangela ukuvuvukala zinokubangela ukusebenza kweejini ezintsha, nto leyo evuselela umsebenzi we-enzymatic, ekugqibeleni isebenze iindlela ezininzi ze-biosynthetic kwaye ikhokelele ekwakhiweni kwee-metabolites zesibini. Ngaphezu koko, olunye uphando lubonise ukuba ukunciphisa umthunzi kwandisa ukukhanya kwelanga, ngaloo ndlela kuphakamisa amaqondo obushushu emini kwindawo yendalo ye-*Hypericum perforatum*, ekwanegalelo ekwandeni kwemveliso ye-hypericin. Ngokusekelwe kolu lwazi, olu phononongo luphande indima yee-nanoparticles zesinyithi njengezinto ezinokubangela ukuvuvukala kwinkcubeko yezicubu. Iziphumo zibonise ukuba ezi nanoparticles zinokuvuselela iijini ezibandakanyeka kwi-hesperidin biosynthesis ngokuvuselela i-enzymatic, okukhokelela ekwandeni kokuqokelelwa kwale compound (Umzobo 2). Ke ngoko, xa kuthelekiswa nezityalo ezikhula phantsi kweemeko zendalo, kunokuthiwa imveliso yee-compounds ezinjalo kwi-vivo inokwandiswa xa uxinzelelo oluphakathi ludibaniswa nokusebenza kweejini ezibandakanyeka kwi-biosynthesis yee-metabolites zesibini. Unyango oludibeneyo ngokubanzi lunempembelelo entle kwizinga lokuvuselelwa, kodwa kwezinye iimeko, esi siphumo sibuthathaka. Okuphawulekayo kukuba, unyango olusebenzisa i-1 mg/L 2,4-D, 1.5 mg/L kinase, kunye noxinzelelo olwahlukeneyo lunokunyusa ngokuzimeleyo nangokubonakalayo izinga lokuvuselelwa nge-50.85% xa kuthelekiswa neqela lolawulo (Umzobo 4c). Ezi ziphumo zibonisa ukuba indibaniselwano ethile yee-nanohormones inokusebenza ngokubambisana ukukhuthaza ukukhula kwezityalo kunye nemveliso ye-metabolite, nto leyo ebaluleke kakhulu kwinkcubeko yezicubu zezityalo zamayeza. UPalmer noKeller [50] babonise ukuba unyango lwe-2,4-D lunokubangela ngokuzimeleyo ukwakheka kwe-callus kwi-St. perforatum, ngelixa ukongezwa kwe-kinase kwandisa kakhulu ukwakheka kwe-callus kunye nokuvuselelwa. Esi siphumo sasibangelwa kukuphuculwa kokulinganisela kwehomoni kunye nokukhuthaza ukwahlukana kweeseli. UBal et al. [51] bafumanise ukuba unyango lwe-Fe₃O₄-NP lunokuphucula ngokuzimeleyo umsebenzi wee-enzymes ze-antioxidant, ngaloo ndlela lukhuthaza ukukhula kweengcambu kwi-St. perforatum. Imithombo yenkcubeko equlethe ii-nanoparticles ze-Fe₃O₄ kumanqanaba e-0.5 mg/L, 1 mg/L, kunye ne-1.5 mg/L iphucule izinga lokuvuselelwa kwezityalo zeflakisi [52]. Ukusetyenziswa kwe-kinetin, i-2,4-dichlorobenzothiazolinone, kunye nee-nanoparticles ze-Fe₃O₄ kuphucule kakhulu amazinga okwakheka kwe-callus kunye neengcambu, nangona kunjalo, iziphumo ebezingalindelekanga zokusebenzisa ezi hormones ekuvuseleleni i-in vitro kufuneka ziqwalaselwe. Umzekelo, ukusetyenziswa kwexesha elide okanye okuphezulu kwe-2,4-dichlorobenzothiazolinone okanye i-kinetin kunokubangela utshintsho lwe-somatic clonal, uxinzelelo lwe-oxidative, i-morphology engaqhelekanga ye-callus, okanye i-vitrification. Ke ngoko, izinga eliphezulu lokuvuselelwa alithethi ukuba kufuneka liqikelele uzinzo lwe-genetic. Zonke izityalo ezivuselelwe kufuneka zihlolwe kusetyenziswa iimpawu ze-molecular (umz. i-RAPD, i-ISSR, i-AFLP) okanye uhlalutyo lwe-cytogenetic ukumisela ukulingana kwazo kunye nokufana kwazo nezityalo ze-in vivo [53,54,55].
Olu phononongo lubonise okokuqala ukuba ukusetyenziswa okudibeneyo kwabalawuli bokukhula kwezityalo (2,4-D kunye ne-kinetin) kunye ne-Fe₃O₄ nanoparticles kunokuphucula i-morphogenesis kunye nokuqokelelwa kwee-metabolites ezibalulekileyo ze-bioactive (kubandakanya i-hypericin kunye ne-hyperoside) kwi-*Hypericum perforatum*. Inkqubo yonyango ephuculweyo (1 mg/L 2,4-D + 1 mg/L kinetin + 4 mg/L Fe₃O₄-NPs) ayigcinanga nje ngokwandisa ukwakheka kwe-callus, i-organogenesis, kunye nemveliso yesibini ye-metabolite kodwa ikwabonise nesiphumo esincinci esibangela ukuba izityalo zingakwazi ukunyamezela uxinzelelo kunye nexabiso lezonyango. Ukudibanisa i-nanotechnology kunye nenkcubeko yezicubu zezityalo kubonelela ngeqonga elizinzileyo nelisebenzayo lokuveliswa okukhulu kweekhompawundi zamayeza kwi-in vitro. Ezi ziphumo zivula indlela yezicelo zoshishino kunye nophando lwexesha elizayo kwiindlela ze-molecular, ukulungiswa kwedosi kunye nokuchaneka kwezakhi zofuzo, ngaloo ndlela zidibanisa uphando olusisiseko kwizityalo zamayeza kunye ne-biotechnology esebenzayo.
Ixesha leposi: Disemba-12-2025