Ảnh hưởng của tdz và iaa lên sự phát sinh hình thái từ các lớp mỏng tế bào của lá, cuống lá và thân rễ cây sâm cau (curculigo orchioides gaertn ) nuôi cấy in vitro

ANH Hl/GfNG CUA TDZ VAIAA LEN S i/ PHAT SINH HINH THAI T t/ CAC LOIP MONG TE BAO CUA LA, CUONG LA VA THAN RE CAY SAM CAU (Curculigo orchioides Gaertn.) NUOI CAY I N V IT R O Nguyen Thi Thuy Diem1, Huynh TrUdng Hue1, Nguyen Thi Minh Chau1, Vo Thi Xuan Tuyen1, Nguyen Thi Thuy Tien1, Huynh Thanh Quang1 TOM TAT Mau lat cat mong theo chieu ngang (traverse thin cell layer - tTCL) la, cuong la va than re cua cay sam cau in vitro chide nuoi cay tren moi triidng MS khong co va co TDZ (0,5,1,0 mg/L) ket hdp IAA (1,5,2,0 mg/L) nham khao sat hieu qua cua TDZ va IAA len kha nang tao mo seo va choi trdc tiep Sau 12 tuan nuoi cay, ket qua thu diidc cho thay moi triicrng thich hdp tao mo seo tii tTCL la la MS bo sung 1,0 mg/L TDZ vdi 1,5 mg/L IAA; cuong la la MS co 0.5 mg/L TDZ va 1,5 mg/L IAA; than re la MS co 0,5 mg/L TDZ v6i 2,0 mg/L IAA hoac MS co 1,0 mg/L TDZ vdi 13 mg/L IAA Moi triidng tai sinh choi tii tTCL mau la la MS co 1,0 mg/L TDZ vdi 1,5 mg/L IAA; tTCL cuong la va than re la MS co 0,5 mg/L TDZ va 1,5 mg/L IAA Tii khoa: Sam cau, mo seo, nuoi cay lcfp mong, phat sinh hinh thai, chat dieu hoa sinh triidng ! Tnidng Dai hoc An Giang, Dai hoc Quoc gia TP Ho Chi Minh Tap chiKhoa hoc va Cong nghe Ndng nghiep Viet Nam - So 05(126)/2021 I DAT VAN DE 2.2 Phiidng phap nghien cdu Sam cau (Curculigo orchioides Gaertn.) la loai thao diidc song lau nam co chda nhieu hoat chat dddc lieu quy va dddc dung bien y hqc Nhieu nghien cdu cho rang than re sam cau co chda polysaccharides, glycosides, sapogenin, alkaloid, (Irshad et al., 2006) dddc dung lam thuoc chfla ho, dau lilng, tri, vang da, dau bung, Id loet (Do Tat ldi va ctv., 2004), chong loang xddng, lam thuoc bo tang cddng sinh li (Cao va ctv., 2008), tri dai thao diidng, khang khuan (Nagesh, 2008) Dieu cho thay, day la loai thao dddc co gia tri cao va can dddc phat trien 2.2.1 Bo tri thi nghiem a) Thi nghiem 1: Khao sat sU phat sinh hinh thdi tii cac mau tTCL cua la sam cau Mau la sam cau in vitro dddc cat tdng lat mong theo chieu ngang (traverse thin cell layer tTCL) ki'ch thiidc mm * 5mm Tien hanh cay mau cac mau vao moi trdcmg MS khong co va co bo sung IAA (1,5,2,0 mg/L) ket hdp TDZ (0,5,1,0 mg/L) b) Thi nghiem 2: Khao sat sUphat sinh hinh thdi tii tTCL cua cuong la sam cau Cuong la sam cau in vitro dddc cat ngang tao lat mong tTCL, moi doan cat dai khoang mm, sau cay mau cac mau vao moi trddng MS khong co va co bo sung IAA (1,5,2,0 mg/L) ket hdp TDZ (0,5,1,0 mg/L) c) Thi nghiem 3: Khao sat sU phat sinh hinh thdi tii tTCL cua than ri sam cau Cac mau than re sam cau in vitro la phan choi (da loai bo het la va cuong) diidc cat ngang cac ldp mong co chieu day khoang mm va dddc dat vao moi trddng MS khong co va co bo sung IAA (1,5,2,0 mg/L) ket hdp TDZ (0,5,1,0 mg/L) Cac thi nghiem dddc bo tri theo the thdc hoan toan ngau nhien, moi nghiem thdc lan lap lai, moi lan lap lai la keo, moi keo cay mau Nhan giong cay sam cau theo phddng phap truyen thong nhd gieo hat thildng co ty le mam thap, vdi giam than re thi moi cay giong phai co mot phan cu va phan ngon mdi dam bao cay co the song, nen he so nhan giong rat thap Vi vay, cac ket qua nghien cdu nhan giong in vitro cay sam cau tii cac mau than re, la va choi dinh da diidc thdc hien va cong (Suri et al, 1999; Thomas va Jacob, 2004; Nagesh, 2008; Vo Chau Tuan va ctv., 2011; Triidng Thi Bich Phiidng va ctv., 2018) Tuy nhien, cac ling dung ky thuat nuoi cay lat mong te bao tren doi tiidng sam cau rat ham che niidc ta Trong cac ky thuat vi nhan giong thi nuoi cay lat mong te bao (Thin cell layer - TCL) la ky thuat cho phep kiem soat dieu kien nuoi cay mot each de dang nong hormone noi sinh cua mau thap Svf phan a le cua cac te bao lat mong te bao giam, tao diidc nhieu choi hdn, he so nhan choi cao hdn nhieu so vdi cac phiidng phap nhan giong truyen thong (Le Van Hoa va ctv., 2012) Bai bao nay, trinh bay ket qua nghien ciiu sil tao mo seo va choi td cac vat lieu nuoi cay tTCL cua la, cuong la va than re cay sam cau in vitro dat hieu qua cao nham cai tien quy trinh nhan giong loai sam cau phuc vu cho nhu cau diidc lieu nilcfc n VAT LIEU VA PHU0NG PHAP NGHIEN CtfU 2.1 Vat lieu nghien ciiu - Vat lieu sd dung la la, cuong la va than re cua cac choi sam cau in vitro tai sinh td qua trinh main cua hat sam cau tren moi triidng MS bo sung 2mg/LBA - Moi tnldng MS la moi triidng Murashige vk Skoog (1962) co bo sung agar (8 g/L), dddng (30grt.), than hoat tinh g/L Moi triidng dieu chinh pH = 5A - Dieu kaen nuoi cay in vitro: thdi gian chieu sang M p M o fftt, oldng anh sang khoang 2.000 lux, wMQt&b fiioag nuoi 24 ± 2°C 2.2.2 Chi tieu theo doi Xac dinh ty le mau song, ty le mau tao mo seo, ty le mau tai sinh choi va so choi td cac mau tTCL la, tTCL cuong la va tTCL than re sam cau in vitro 12 tuan nuoi cay 2.2.3 Phan tick d it lieu Cac so lieu thu thap diidc xd ly va phan tich thong ke bang phan mem Excel 2010 va SPSS 20.0 vdi phep thd Duncan de so sanh sil khac biet gida cac nghiem thdc 2.3 Thdi gian va dia diem nghien cdu Thi nghiem dddc thdc hien td thang 01 den thang nam 2020 tai phong thi nghiem Sinh ly ThUc vat, Khoa Nong Nghiep - Tai Nguyen Thien Nhien, Trddng Dai hoc An Giang III KET QUA VA THAO LUAN 3.1 Hieu qua cua TDZ va IAA len sd phat sinh hinh thai td tTCL la 3.1.1 S u p h d tsin h mo seo Sau 12 tuan nuoi cay, nhdng mau lat mong tTCL la sam cau in vitro dddc nuoi cay tren tat ca cac moi triidng deu tao mo seo va dat ty le mau song cao Cac so lieu thu nhan dddc the hien qua bang Tap chiKhoa hoc va Cong nghe Nong nghiep Viet Nam - So 05(126)/2021 Bang Sii hinh mo seo va choi til tTCL la sam cau cl 12 tuan sau cay Chat DHST (mg/L) TDZ IAA Ty le song (%) Al 0 66,7b 89,2 - - A2 0,5 1,5 90,0a 100 51,7b l,2b A3 0,5 2,0 96,7a 100 17,4C l,0b A4 1,0 1,5 96,7a 100 71,7a 3,3a A5 1,0 2,0 92,5 F 90,0a A- ns 11,7d ** l,0b A* CV(%) 10,2 5,7 23,4 36,7 Nghiem thiic Ty le tao mo seo (%) Ty le tai sinh choi (%) So choi (choi/mau) Ghi chu: Cac chit cai theo sau giong thi khdng khac biet thong ke co y nghia thong ke: * = khac biet co y nghta d mite 5%, ** = khac biet co y nghia d mite 1%, ns = khac biet khdng co y nghia thong ke, (-) Khdng ghi nhan choi xudt hien Cac mau tTCL diipc nuoi cay tren moi tnldng MS co bo sung chat dieu hoa sinh tnidng (CDHST) TDZ va IAA dat ty le song, ty le mau tao seo tildng ling lan liigft til 90 - 96,97% va 92,5 - 100% cao hefn so vdi cac mau nuoi cay tren moi tnldng khdng co bo sung CDHST Dieu chiing to la sii ket hop gifla TDZ va IAA cac nong khac deu co hieu qua kich thich sii hinh mo seo Ket qua phu hop vdi nhan dinh cua George va cong tac vien (2008), cytokinin kich thich sii phan chia te bao vdi dieu kien co auxin Tren moi triiclng MS khong TDZ va IAA cung co sii hinh seo tren moi tnlcfng MS khong TDZ va IAA nhilng ty le mau tao mo seo thap hon dat 89,17%, dieu la cac tTCL cua mau la da co san auxin noi sinh nen nuoi cay tren moi triidng MS khong bo sung chat dieu hoa sinh triidng van co sii hinh seo Trong thi nghiem nay, cac mo seo hinh tren moi triidng MS co mg/L TDZ vefi 1,5 mg/L IAA nhieu, phat sinh xung quanh vet cat, lan khap be mat mau cay, mau vang nhat va tap hop nhieu nhom te bao hinh tron nho mau trang co the phat trien thsrnh phoi de hinh choi (Hinh 1) Cac khoi mo seo d nghiem thde lai luc dau cung co bieu hien tang sinh mo nhiing den 12 tuan sau cay chung bat dau thay doi til mau vang sang nau den Rieng cau true mo seo d nghiem thiic A l, A2 va A5 rat ran chac, vi vay kha nang phan biet hoa cua cac mo seo tren cac moi tnidng rat thap 3.1,2 SU tdi sinh choi Kha nang tai sinh choi cua cac mau tTCL la diipc the hien d bang cho thay, nhiing mau song sot d tren moi triidng MS bo sung TDZ va IAA cho hieu qua tai sinh choi cao hon so vdi cac mau nuoi cay tren moi triidng khong co bo sung TDZ va IAA (khong tai sinh choi) Moi triidng MS bo sung 1,0 mg/L TDZ vdi 1,5 mg/L IAA cho ty le mau tao choi cao nhat, choi phat trien tilng cum, than to, chat hldng choi tot dat 71,7% vdi 3,3 choi/mau Ket qua cua thi nghiem cao hdn ket qua nghien cdu cua Prajapati va cong tac vien (2003), nuoi cay cac manh la sam cau (10 mm) tren moi tnidng MS bo sung 0,2 mg/L BAP cho so choi tai sinh chi dat toi da la 1,44 choi/mau Hinh Sii phat sinh hinh thai mo seo va choi til tTCL la sam cau d 12 tuan sau cay Ghi chu: A4x: Khoi mo seo chide quan sat dtidi kinh hien vi soi noi Tap chi Khoa hoc va Cong nghe Nong nghiep Viet Nam - So 05( J26)/2021 Ket qua thi nghiem cho thay, tang nong TDZ td 0,5 mg/L den mg/L ket hop 1,5 mg/L IAA thi sO tai sinh choi tang (tddng dng 1,2 va 3,3 choi/ mau), nhien nong IAA tang len mg/L thi sO tai sinh choi lai giam xuong choi/mau Dieu cho thay, nong TDZ ket hop IAA d nong 1,5 mg/L thi kich thich tai sinh ch6i, nhOng nong IAA tang mg/L thi dc che sd tai sinh choi Ket qua phu hop vdi nhan dinh cua Gaspar va cong tac vien (1996), IAA nong cao lam giam hoat tinh cua TDZ gay dc che sd tai sinh choi cua mau cay 3.2 Hieu qua cua TDZ va IAA len sO phat sinh hinh thai td tTCL cuong la 3.2.1 SUphat sinh mo seo Viec bo sung TDZ va IAA vao moi trUdng nuoi cay cac mau tTCL cuong la sam cau co anh hddng den qua trinh song sot va hinh mo seo cua cac mau sau 12 tuan nuoi cay (Bang 2) Cac mau song sot thu dooc d moi trddng MS khong bo sung TDZ va IAA (23,3%) thap hon so vdi moi trddng co TDZ vdi IAA (26,7 - 60%) Nhin chung, ty le song cua cac mau tTCL cuong la d cac moi trddng nuoi cay khong cao, chi dat toi da la 60% tren moi triidng MS co bo sung 0,5 mg/L TDZ vdi 1,5 mg/L IAA Bang SO hinh mo seo va ch6i td tTCL cuong la sam cau d 12 tuan sau cay CDHST (mg/L) TDZ IAA Ty le song (%) Bl 0 23,3b 100 i B2 0.3 1,5 60,0a 100 ; B3 0,5 2,0 26,7b 88,9 i B4 1,0 1,5 33,3b 100 B5 1,0 2,0 100 F 40,0b * cv(%) 29,9 Nghiem thde ! Ty le tao md seo (%) Ty le tao choi ’(%) S6 choi (chdi/mau) - - ns 11,6 67,0a 2,3' - - 30,3b - l,0b - ** ** 28,2 25,5 Ghi chu: Cac chit cat theo saugiong tthau thi khong khac biet thong ke coy nghia thongke: ** = khac biet coy nghta d mile 1%; * - khde biet co y nghia d mile 5%, ns = khac biet khong coy nghia thd'ng ke, (-): Khongghi nhan choi xuat hien Sd phat sinh mo seo cua mau tTCL cuong la sam cau d bang cho thay, tat ca cac mau song sot ddOc tren cac moi trddng MS co hay khong co bo sung TDZ va IAA deu phat sinh hinh thai chu yeu mo seo, ty le tao mo seo dat 88,9 - 100%, ty le mau hinh mo seo dat cao nhat d nghiem thde Bl, B2, B4 va B5 (dat 100%) Cac mo seo td tTCL cuong la d nghiem thde B2 va B4 mem, mau vang nhat, tap hop nhieu khoi tron nho va co kha nang biet hoa choi d 12 tuan sau cay Ngoai mot so mo seo d nghiem thde Bl, B3, B5 co mau xanh nhat, chac, tang sinh nhanh va khong co kha nang biet hoa choi (Hinh 2) Qua ket qua thi nghiem d io thay, tTCL cuong la ddOc nuoi cay deu co sd phan phan hoa cua cac te bao nhu mo d vet cat, dong vai trd nhd te bao sinh phoi phan chia manh liet tao khoi mo seo Sd phan chia te bao d phan cuong la WOI^ doi hep nen khoi mo seo tao khong ldn M in cay a nghiem thde Bl cung xuat hien mo seo b mo cuong la cat rdi co chda ham ldqng m s m aim sink Theo Nguyen Ddc LdOng va Le Thi i H a ir Tien