Tiiu ban: Khoa hgc cong nghe Biin • ISBN: 978-604-913-015-1
BUOfC DAU LUtJ GILT MOT SO LOAI RONG BIEN
\6l MUC DICH BAO TON
PRELIMINARY STUDY ON STORAGE OF SEAWEEDS TO ESTABLISH EX-SITU CONSERVATION
Nguyen Thi Huong, Le Nhu Hau, Vii Thi Mo"
Vien Nghien ciiu va iTng dung Cdng nghe Nha Trang 02 - Hiing Vuong, Nha Trang, Khanh Hda
Email: [email protected] Tom tat:
Cd nhiiu bdng chirng cho thdy cdc lodi rong dang cd nguy ca tuyet chung vd thue su da cd mot sd lodi tuyet chimg trong vdng ndm gdn ddy. Hiin nay, chua cd nhirng huang ddn cu thi nao cho viec duy tri cdc lodi cd nguy ca tuyet chimg ndy vd cd le cdc nhd khoa hoc nen xdy dung phirang phdp bdo tdn rong bien thdng qua nudi trdng nhirng ddi tuang ndy. Xudt phdt tir thue ti dd, chiing tdi da nghien ciru Idm the ndo de luu giir rong biin trong phdng thi nghiim vai thai gian ddi nhdm bdo tdn cdc lodi rong quy hiim.
Ngudn Kappaphycus alvarezii (Doty), Gracilariopsis bailiniae (Zhang et Xia) va Gracilaria salicornia ((C. Ag.) Dawson) dugc xir ly bdng Malachit Green (I- 2ppm) trong 5 phiit. Mdi trudmg nudi la nuac biin dugc bd sung MS (Murashige and Skoog Basal Salt Mixture) (0,05 mg/l). Rong biin da dugc luu trir trong tu dn dinh nhiet, vai nhiet do nuac: 24 - 28 °C, do man: 30-32 %o, pH; 7-8, chiiu sdng 12:12 (L: D) vai cuang do 20-40^mol photon m' s', sue khi nhe. Trong sudt qud trinh nudi, nuac biin vd chdt dinh duang dugc bd sung hai tudn I ldn. Kit qud chi ra rdng rong bien cd the dugc luu tru Idu ddi (it nhdt, cho I ndm) trong phdng thi nghiem vai tdc do tdng trudmg: 0,2-0,9%/ngdy. Trong bdo cdo ndy, chung tdi trinh bdy kit qud ban ddu vi qud trinh luu giir mdt sd lodi rong biin trong diiu kien thi nghiim vai muc dich thdnh lap ngdn hdng gidng rong biin a Viet Nam trong tuong lai.
Abstract
There is much evidence that algae are endangered and indeed that some of them have become extinct within recent years. Currently, there are no guidelines for maintaining endangered species of algae. Perhaps the scientific should
establish guidelines for ex-situ conservation of algae in culture collections. Thus, we have been studying how to maintain macroalgae in the laboratory for long time to preserving rare species of algae.
The source of Kappaphycus alvarezii (Doty), Gracilariopsis bailiniae (Zhang et Xia) and Gracilaria salicornia ((C Ag.) Dawson) were treated with Malachite Green (I-2ppm) for 5 minutes. The medium used for culture is seawater which was enriched MS (Murashige and Skoog Basal Sah Mixture) (0.05 mg/l). The seaweeds were stored in thermal stability cabinet, water temperature fluctuated between 24 and 28oC, salinity ranged from 30-32 %o, pH from 7-8, the photoperiod was maintained at 12:12 (L; D) and illuminated with 20-40pmol photon m-2s-I, light aeration conditions. During this experiment, the culture
medium was replenished every two weeks. The results indicate that these species can be stored for a long term (at least, for 1 year) in the laboratory with growth rate; 0.2-0.9%/day. In this report, we presented initial results on the culture collection in laboratory to establish gene seaweeds bank in Vietnam in the future.
1. Mddau
Mdt sd loai rong cd gia trj kinh te da dugc nhieu nude tren the gidi nghien ciiu va dua vao nudi trdng de chii ddng ngudn nguyen lieu, nhien lieu cho cdng nghiep va thirc pham cho con ngudi. Ddng thdi, giai quyet cdng an viec lam, nang cao ddi sdng cho khdng it dan cu ven bien. Tren thue te, ngudn gidng rong bien dua vao nudi trdng chii yeu la chgn loc nhiing cay khoe, sinh trudng tdt d vu trudc lam gidng cho vu sau ma chua cd ngan hang gidng thuan chiing de luu giir va chii ddng tao ra ngudn gidng khi can thiet. Mot sd cdng trinh nghien cuu nhan gidng rong bang phuong phap nudi cay md thanh cdng tren the gidi va trong nude da md ra nhieu trien vong cho muc dich luu giir va ling dung nudi trdng rong bien.
Mdt sd cdng trinh nghien ciiu thanh cdng ciia cac nha khoa hoc tren the gidi ve nudi cay md, nhan gidng nhan tao, nudi trdng trong phdng thi nghiem tren ddi tugng Kappaphycus alvarezii bang mdi trudng ESS-1, PES da md ra nhieu trien vong cho muc dich luu giir va ling dung nudi trdng rong bien [2, 3, 4, 5].
Trong khi dd d Viet Nam, mdi chi cd nghien ciiu thir nghiem cac phuong phap nhan gidng rong Kappaphycus alvarezii bang nhanh vdi viec sir dung dung djch khang sinh de khir triing va bd sung mdi trudng MS cho ket qua tuang ddi kha quan. Tuy nhien, ket qua nay vdi muc dich nudi tang nhanh sinh khdi va chi nghien ciiu trong thdi gian ngan [1].
Xuat phat tir thirc te va nhiing thanh tun da dat dugc, chiing tdi di sau nghien ciiu lun giir mdt sd loai rong bien cd gia trj kinh te d dieu kien thi nghiem trong thdi gian dai, theo nhiem vu ciia de tai "Nghien ciru hiin trgng phdn bd vd gidi phdp bdo vi ngudn lgi rong Dd"
cap Vien KH&CNVN (2009-2010), nham bao tdn dugc nhiing loai rong cd gia trj va chii ddng dugc ngudn gidng cho cac hoat ddng nghien cuu, nudi trdng khac.
2. Doi tuong va phuong phap nghien ciru 2.1. Doi tirang
Ddi tugng dugc nghien cuu: Kappaphycus alvarezii (Doty) , Gracilariopsis bailinae{Zhang et Xia), Gracilaria salicornia {{C. Ag.) Dawson) dugc thu tir viing bien Ninh Thuan va Nha Trang (thang 4 - 6 , nam 2009). Cac loai rong nay dugc dua ve phdng thi nghiem, thuan dudng trong be kinh 5 ngay trudc khi tien hanh luu giii.
Day la nhirng loai rong cd gia trj kinh te va da dugc nghien ciiu trong thdi gian gan day nen chiing tdi di sau nghien cim nham thiet lap quy trinh luu giii, budc dau hinh thanh ngan hang gidng rong bien Viet Nam.
2.2. Phirang phdp nghien ciru
Chon nhiing nhanh rong khde, mau sac tuoi sang, khdng bj tray xudc. Id loet. Cat rong thanh nhiing doan 7 - 10 cm ddi vdi rong K. alvarezii va giir nguyen nhanh rong ddi vdi Gracilariopsis bailinae, Gracilaria salicornia (10-12 g/nhanh).
Rong dugc khu triing bang hon hgp khang sinh: Erymycine: 20 mg/l, Gentamycin:
12.8 mg/l. Rifampin: 36 mg/l, Nergamycin: 4 mg/l hoac Malachite Green (2ppm). Thdi gian khir triing: 2-5 phiit. Sau khi khu triing, rira rong bing nude biin da loc qua ludi loc Phytoplanton de loai bd khang sinh hay hda chit cdn bam lai tren rong [1].
Tieu ban: Khoa hgc cong nghe Bien ISBN: 978-604-913-015-1
Mdi trudng dugc sir dung de luu giir: MS. Nude biin dugc loc qua tiii loc Phytoplanton de loai bd thue vat ndi. Hap nude bien trong ndi hip khir triing. Thilt bj luu giii:
binh nhua 600mL dugc dat trong tu In djnh nhiet. Dilu kien lun giii: T°: 24-28°C, S%o: 30- 32%o, pH: 7-8, sue khi nhe, cudng do chieu sang: 30-50|imol photon, 12 h/ngay.
Sau khi khir triing, rong dugc cho vao mot binh da cd sin 600mL nude bien va mdi trudng nudi. Hang ngay, theo ddi su bien ddi ve mau sac, tinh trang rong va mdi trudng nude luu giii. Thay nude, ve sinh binh va bd sung dinh dudng lian/2tuan. Djnh ky kiem tra tdc do tang trudng cua rong (llan/2 tuan), khir triing rong 1 lan/thang.
Tien hanh luu giii rong d cac mdi trudng nude bien loc va khir triing vdi ndng do 1/40MS (0.1 g/1) va 1/80MS (0.05g/l) dl chon loc mdi trudng tdi uu.
cac yeu td mdi trudng: Nhiet do: dieu chinh bang he thdng dieu nhiet trong tii dn djnh nhiet. Do man: xac djnh bang khue xa ke Salinometer (Shibuya - Japan). Cudng do anh sang:
xac djnh bang may Li-2502. pH: do bang may do pH (Eco testr pHl).
Khdi lugng rong dugc can bang can dien tir (KP, Maxel 500 - Taiwan). Rong dugc tham khd trudc khi can. Xac djnh tdc do tang trudng theo cdng thiic Penniman et al (1986).
SGR IV
100
Trong dd:
- SGR: tdc do tang trudng tuong ddi ngay (%/ngay).
- WQ: trong lugng ban dau.
-1: thdi gian giira hai lan can (ngay).
- Wt: trong lugng sau thdi gian t ngay.
3. Ket qua va thao luan
3.1. Lu-u giii- lodi rong Sun (Kappaphycus alvarezii (Doty))
Nghien ciiu mdi trudng nude ya ndng do dinh dudng thich hgp ddi vdi cac loai rong luu giir, chiing tdi nghien cim trudc hit ddi vdi loai K. alvarezii. Do ylu td khach quan, mot sd mau rong chit ddng loat sau 130 ngay luu giir nen chiing tdi danh gia tdc do tang trudng ciia loai d cac mdi trudng sau 126 ngay.
N g i y
-Loc KhCr trving
- L p c + 1/40MS -KhLP tmng •>• 1/40MS
Lpc + 1/80MS Khi> tmng + 1/80MS
Hinh 2: Biiu do thi hiin tdc do tdng truang cua rong K. alvarezii a cdc mdi truang Qua bieu do (Hinh 2) thay dugc rong luu giir trong timg mdi trudng nude loc hay nude khir triing cd sir khac nhau ve tie do sinh trudng ddi vdi timg ndng do dinh dudng bl sung. Trong mdi trudng nude loc: rong dugc bl sung 1/80MS cd khdi lugng tang din theo
thdi gian lun giir nhung toe do tang trudng tuong ddi lai thay ddi theo timg giai doan. Tdc do tang trudng tuong ddi ciia rong cd bd sung 1/80MS tang dan den thang thir 2 rdi giam d cac thang tiep theo. Tuy nhien, tic do tang trudng d thang thii 4 cd cao hon thang thir 3. Trong khi dd, rong khdng b l sung dinh dudng hay bd sung 1/40MS, tdc do tang trudng tuong ddi chi tang nhe d thang diu tien, thang thii 2 giam dan va tang trudng am vao thang thii 3 va thang thir 4. Dac biet la vao thang thir 3, rong cd bd sung 1/40MS tdc do tang trudng ciia rong la -5%/ngay. Rong bj thdi nhiin va diit thanh nhieu doan, mat mau iron bdng tu nhien. Than rong bj teo quat lai ddi vdi nhirng mau luu giir trong mdi trudng khdng bd sung dinli dudng.
Ddi vdi mdi trudng nude khu triing: khdi lugng cac mau rong da sd tang len theo thdi gian luu giii ngoai trir mot sd mau rong dugc bd sung 1/40MS thi khdi lugng giam dan tir thang thii 3. Tdc do tang trudng tuong ddi ciia rong bd sung 1/40MS tang trong 42 ngay dau sau dd giam dan, dac biet trong giai doan tir 84 tdi 98 ngay tdc do tang trudng la -
1.46%/ngay. Rong bj thdi nhiin d mot sd dinh nhanh va diit doan. Trong khi dd rong khdng bd sung dinh dudng lai cd tdc do tang trudng tang d 42 ngay dau va nhiing ngay trong thang thii 4. Cac thdi gian cdn lai hau nhu khdng tang trudng, mau sac iron bdng tu nhien.
Bdng I; Tdc do tdng truang tuang ddi ve khdi lugng cua rong Kappaphycus alvarezii
Moi truong Loc
Loc+ 1/40MS LOC+1/80MS Khir triing
Khir triing + 1/40MS Khir triing + 1/80MS
Toe do tang trudng tuong doi ve khoi luffng (±SD) (%/ngay) -0.6511'"'± 1.4959
-0.9079' ± 1.9839 0.30517'" ±0.2896
0.1904"* ±0.2103 -0.2200'''±0.6011 0.0003'" ± 0 .2066
Ghi chii: sd trung binh trong cdt ling vdi cac chii sd (a, b, c, d) khac nhau thi khac nhau vdi (P<0,05)
Bang 1 cho thay rong luu giir trong mdi trudng dugc bd sung 1/80MS d ca nude loc va nude khir triing cd tdc do tang trudng cao hon va khdng khac vdi mdi trudng nude khir triing ddi chimg (P<0,05). Tdc do tang trudng 0,00-0,35%/ngay trong sudt qua trinh luu giii.
cac mdi trudng cdn lai cho tdc do tang trudng kem, dac biet la mdi trudng Loc + 1/40MS, Loc va Khir triing + 1/40MS (-0,91 din -0,22%/ngay).
Nhu vay cd the thay mdi trudng bd sung 1/80MS ed anh hudng tdt tdi qua trinh luu giii rong Sun va cd the xem day la mdi trudng va ndng do tuong img phii hgp hon de luu giir loai rong nay trong phdng thi nghiem.
3.2. So sdnh ket qud liru giir ba lodi Kappaphycus alvarezii (Doty), Gracilariopsis bailinae (Zhang etXia), Gracilaria salicornia ((C. Ag.) Dawson).
Qua nghien cim hon bdn thang ddi vdi loai Kappaphycus alvarezii thi thay mdi trudng nude bien loc cd bd sung 1/80MS cho hieu qua luu giii tuong ddi tdt va it chi phi nhat nen chiing tdi tien hanh luu giii ba loai Kappaphycus alvarezii, Gracilariopsis bailinae, Gracilaria salicornia d mdi trudng nay.
Tiiu ban: Khoa hgc cong nghe Biin ISBN: 978-604-913-015-1
-6 00
-K.alvarezii —•—Gracilariopsis bailinae Gracilaria salicornia
Hinh 3: Biiu dd thi hiin tdc do tdng truang cua cdc lodi rong sau qud trinh luu giir Sau 14 thang luu giii, tie do tang trudng tuong ddi ve khdi lugng ciia ca ba loai rong khac nhau va giao ddng khdng theo quy luat. Qua qua trinh luu giir, khdi lugng ciia rong bj giam d mot sd thang do rong bj thii nhiin than da dan den tdc do tang trudng am tai thdi diem dd. Bilu dd (Hinh 3) da thi hien tic do tang trudng ciia loai Kappaphycus alvarezii dn djnh hon so vdi hai loai cdn lai, dao ddng 0.2-0.9%/ngay. Tdc do tang trudng ciia loai Gracilariopsis bailinae cd khoang dao ddng ldn nhat va cd nhieu thang tdc do nay tang trudng dudi 0%/ngay. Day la loai cd than nhd va de dirt doan nen sire chju dung kem hon so vdi hai loai K alvarezii, G. salicornia. Rieng ddi vdi loai G salicornia, tdc do tang trudng d thang thir tam giam manh (-5.73%). Cd dieu nay la do d thang thii 6, 7 cac nhanh rong hinh thanh thdi sinh san. Sau thdi gian nay cac nhanh chinh bj bac mau dan va men nhun, chiing tdi phai loai bd sau mdi lan ve sinh rong dan den khdi lugng rong giam.
Nhin chung, ca ba loai van tdn tai sau 14 thang lun giir. Loai Kappaphycus alvarezii thich nghi nhat trong dieu kien thi nghiem. Thdi gian dau (tir thang thir nhat den thang thii 7) chiing tdi tien hanh khir triing rong bang hdn hgp khang sinh nhung ket qua chua dugc tot:
mdi trudng nudi bj nhiem ddng vat phii du, nhieu nhanh rong bj thdi nhiin d dinh nhanh. Khi tien hanh khir triing rong bang dung djch Malachite green (2ppm) d giai doan gan day (tir thang thii 8 den thang 14) thi thay ket qua tuong ddi kha quan, mdi trudng nudi it nhiem ddng vat phii du. Tuy nhien ket qua nay van dang trong qua trinh thir nghiem, can theo ddi d giai doan tiep theo.
4. Ket luan
- Rong dugc luu giiir trong mdi trudng nude bien khir triing, nude biin loc + 1/80MS (0.05g/l) va nude bien khir triing + 1/80MS cd tic do tang trudng tdt hon so vdi cac mdi trudng cdn lai.
- ca ba Kappaphycus alvarezii (Doty), Gracilariopsis bailinae (Zhang ET Xia), Gracilaria salicornia ((C. Ag.) Dawson). DIU cd kha nang tdn tai hon 14 thang trong dilu kien thi nghiem.
- Loai Kappaphycus alvarezii thich nghi vdi dieu kien thi nghiem tot hon so vdi loai Gracilariopsis bailinae, Gracilaria salicornia.
TAI LIEU THAM KHAO
1. Huynh Hoang Nhu Khanh, (2006). "Nghien cii-u thir nghiim cdc phuang phdp nhdn gidng rong sun Kappaphycus alvarezii bdng ky thuat cdng nghi sinh hoc". Vien nghien cim va iTng dung Cdng nghe Nha Trang. Bao cao tdng kit dl tai nhanh, 14 trang.
2. C. J. Dawes & E. W. Kock, (1991). "Branch, micropropagule and tissue culture of the red algae Echeuma denticulatum and Kappaphycus alvarezii farmed in the philipines". Journal of Applied Phycology 3: 247-257.
3. C. A. Penniman et all, (1986). "Reproductive phenology and groMth of Gracilaria tikvahiae McLachlan (Gigartinales, Rhodophyta) in the Great Bay Estuary, New Hampshire". Bot. Mar. 29: 147-154.
4. C. R. K. Reddy et all, (2003)."/A? vitro somatic embryogenesis and regeneration of somatic embryos from pigmented callus of Kappaphycus alvareii (Doty) (Rhodophyta, Gigartinales)"- J. Phyeol. 39: 610-616.
5. C. R. K. Reddy et all, (2005). "Process for cultivation of algae". United States patent (US6858430B1). 16 pp.
6. R. C. Salvador & A. E. Serrano, (2005). "Isolation of protoplasts from tissue fragments of Philippine cultivars of Kappaphycus alvarezii (Solieriaceae,
Rhodophyta)". Journal of Applied Phycology 17: 15-22.