DESICCATION TOLERANCE AND DNA INTEGRITY IN Eugenia pleurantha O. Berg.
(MYRTACEAE) SEEDS
1TATHIANA ELISA MASETTO2, JOSÉ MARCIO ROCHA FARIA3, ANTONIO CLAUDIO DAVIDE4,
EDVALDO APARECIDO AMARAL DA SILVA4
ABSTRACT – The aim of this study was to assess the desiccation tolerance and DNA integrity in Eugenia pleurantha seeds dehydrated to different moisture contents (MCs). Seeds extracted from PDWXUHIUXLWVZHUHVXEPPLWHGWRGU\LQJLQVLOLFDJHODQGHYDOXDWHGDWHYHU\¿YHSHUFHQWXDOSRLQWV of decrease from the initial MC (35.5%, fresh weight basis). The effects of dehydration on seeds ZHUHYHUL¿HGWKURXJKJHUPLQDWLRQWHVWVDQG'1$LQWHJULW\DVVHVVPHQW8QGULHGVHHGVDFKLHYHG JHUPLQDWLRQ YDOXH UHGXFHG WR DIWHU EHLQJ GULHG WR 0&:KHQ GULHG VOLJKWO\ PRUH WR 7.4% MC, seeds were no longer able to germinate, suggesting an intermediate behavior in relation to desiccation tolerance. It was observed DNA degradation in seeds with 7.4% MC, which might have contributed to the loss of seed germination.
Index terms: forestry seeds, DNA degradation.
TOLERÂNCIA À DESSECAÇÃO E INTEGRIDADE DO DNA EM SEMENTES DE Eugenia pleuranthaO. Berg. (MYRTACEAE)
5(6802±(VWHHVWXGRWHYHFRPRREMHWLYRDYDOLDUDWROHUkQFLDjGHVVHFDomRHDLQWHJULGDGHGR'1$ em sementes de Eugenia pleurantha submetidas a secagem até atingir diferentes graus de umidade. 6HPHQWHVH[WUDtGDVGHIUXWRVPDGXURVIRUDPVXEPHWLGDVDVHFDJHPHPVtOLFDJHOVHQGRDYDOLDGDVDSyV FDGDGHFUpVFLPRGHFLQFRSRQWRVSHUFHQWXDLVDSDUWLUGRJUDXGHXPLGDGHLQLFLDOEDVH~PLGD 2VHIHLWRVGDGHVLGUDWDomRIRUDPYHUL¿FDGRVSRUPHLRGHWHVWHVGHJHUPLQDomRHGDLQWHJULGDGHGR '1$6HPHQWHVQmRVXEPHWLGDVjGHVVHFDomRDSUHVHQWDUDPGHJHUPLQDomRYDORUUHGX]LGRSDUD DSyVVHFDJHPDWpGHiJXD&RPDUHGXomRGRJUDXGHXPLGDGHSDUDDVVHPHQWHV QmRPDLVJHUPLQDUDPVXJHULQGRXPFRPSRUWDPHQWRLQWHUPHGLiULRTXDQWRjWROHUkQFLDjGHVVHFDomR 6HPHQWHVFRPGHiJXDDSUHVHQWDPGHJUDGDomRGR'1$RTXHSRGHFRQWULEXLUSDUDDSHUGDGD FDSDFLGDGHGHJHUPLQDomR
7HUPRVSDUDLQGH[DomRVHPHQWHVÀRUHVWDLVGHJUDGDomRGR'1$
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INTRODUCTION
In the last years there has been an increase in the efforts
WRZDUGV FRQVHUYDWLRQ RI WKH JHQHWLF YDULDELOLW\ RI SODQW VSHFLHV OHG E\ WKH JOREDO FRQFHUQ DERXW WKH FRQWLQXRXV
environmental degradation. Ex-situ SODQW FRQVHUYDWLRQ LQ VHHGEDQNVLVUHFRJQL]HGDVRQHRIWKHPRUHHIIHFWLYHZD\V WRSUHVHUYHSODQWGLYHUVLW\6FKRHQDQG%URZQ3ODQW VSHFLHV SURGXFH VHHGV WKDW EHKDYH GLVWLQFWO\ LQ UHVSRQVH
SHUIRUPDQFHV 5REHUWV SURSRVHG WZR FDWHJRULHV RI VHHGVWRJURXSWKRVHWKDWFDQEHGULHGWRYHU\ORZPRLVWXUH FRQWHQWV0&VDQGVWRUHGIRUORQJWHUPSHULRGVRUWKRGR[
and those that can neither be drastically dried or stored for the long term (recalcitrant). Later, another category, called
LQWHUPHGLDWHZDVLQWURGXFHGE\(OOLVHWDOWRJURXS VHHGVZLWKLQWHUPHGLDWHSRVWKDUYHVWEHKDYLRU7KRVHVHHGV
are relatively desiccation tolerant, but do not withstand removal of water to levels as low as orthodox seeds. They survive desiccation to about 10% MC (fresh weight basis)
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seeds are often chilling sensitive, even in the dehydrated
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it is essential to study the behavior of the seeds regarding desiccation tolerance/sensitivity, storability and the intrinsic factors of the desiccation sensitivity.
Eugenia pleurantha 2%HUJDWUHHVSHFLHVEHORQJLQJ WRWKH0\UWDFHDHIDPLO\LVSRSXODUO\NQRZQDV³SLWDQJDGR PDWR´,WJURZVLQWKH%UD]LOLDQVRXWKDQGVRXWKHDVWUHJLRQV 'DYLGHHWDODQGLWVVHHGVZHUHSUHYLRXVO\FODVVL¿HG
DVUHFDOFLWUDQW&DUYDOKR2WKHUEugeniaWUHHVSHFLHV
DOVR SURGXFH UHFDOFLWUDQW VHHGV 0DOXI HW DO 6HHG UHVHDUFK FRQVLGHULQJ WKLV JHQXV LV LPSRUWDQW GXH WR LWV SRWHQWLDO IRU HFRORJLFDO UHVWRUDWLRQ VLQFH WKHLU IUHVK IUXLWV
attract birds and other animals to the recovering area. A better understanding of the basis of desiccation
VHQVLWLYLW\LVDSUHUHTXLVLWHWRSURFHHGWRZDUGDORQJWHUP
storage of non orthodox seeds (Faria et al., 2004). It is known that in dehydrated orthodox seeds the maintenance of DNA
LQWHJULW\ DQGRU WKH DELOLW\ IRU LWV UHSDLU XSRQ UHK\GUDWLRQ LV D IXQGDPHQWDO UHTXLUHPHQW IRU GHVLFFDWLRQ WROHUDQFH 3DPPHQWHU DQG %HUMDN ,W LV WKXV UHDVRQDEOH WR K\SRWKHVL]HWKDWORVVRI'1$LQWHJULW\LVRQHRIWKHFDXVHVRI YLDELOLW\ORVVHVRIVHQVLWLYHVHHGVVXEMHFWHGWRGHK\GUDWLRQ)RU WKLVUHDVRQWKHSUHVHQWVWXG\ZDVFDUULHGRXWWRFKDUDFWHUL]H
the desiccation sensitivity of Eugenia pleurantha seeds and
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MATERIALS AND METHODS
7KHH[SHULPHQWZDVFDUULHGRXWDWWKH/DERUDWRU\RI7UHH 6HHGV'HSDUWPHQWRI)RUHVW6FLHQFHV)HGHUDO8QLYHUVLW\RI /DYUDV/DYUDVFLW\%UD]LO5LSHIUXLWVZHUHFROOHFWHGIURP VHYHUDOWUHHVLQ/DYUDV¶6¶:ORFDWHGLQWKH VRXWK RI 0LQDV *HUDLV 6WDWH 7KH FOLPDWH LQ WKH UHJLRQ LV
FODVVL¿HG DV &ZE DFFRUGLQJ WR WKH FOLPDWLF FODVVL¿FDWLRQ RI .|SSHQ FKDUDFWHUL]HG E\ D GU\ VHDVRQ $SULO 6HSWHPEHUDQGDUDLQ\VHDVRQ2FWREHU±0DUFK7KHDQQXDO DYHUDJHVRIWHPSHUDWXUHUDLQIDOODQGUHODWLYHKXPLGLW\DUH & PP DQG UHVSHFWLYHO\ $IWHU FROOHFWLRQ SDUWRIWKHIUXLWVZDVVWRUHGIRUWZRPRQWKVLQDFROGFKDPEHU &5+XQWLOWKHVWDUWRIWKHH[SHULPHQWVDQGWKH RWKHUSDUWZDVSURFHVVHGLPPHGLDWHO\6HHGSURFHVVLQJZDV
SHUIRUPHGDFFRUGLQJWR'DYLGHHWDO. (1995), consisting of
fruit maceration on a sieve, under running water to seed
VHSDUDWLRQIURPWKHIUXLWUHVLGXHV6HHGVZHUHWKHQVSUHDGRQ WRZHOSDSHURYHUDODEEHQFKDQGNHSWIRUIHZKRXUVLQRUGHU WRUHPRYHWKHVXSHU¿FLDOZDWHU
Assessment of the desiccation tolerance. The
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done according to Hong and Ellis (1996). Dehydration was done by mixing the seeds with silica gel (fast dehydration)
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Seeds were then weighed at intervals in order to detect the
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MC determination.
Germination tests.:HUHSHUIRUPHGLQIRXUUHSOLFDWLRQVRI
25 seeds each on sand in germination boxes, at 30ºC under continuous white light for 90 days (Masetto, 2005)
Moisture content (MC) determination. :DV DVVHVVHG LQ IRXUUHSOLFDWLRQVRIJHDFKE\RYHQGU\LQJDW& IRU KRXUV %UDVLO DQG H[SUHVVHG DV SHUFHQWDJH
moisture content on a fresh weight basis.
DNA integrity assessment. In order to assess the effect of dehydration on DNA integrity, chromosomal DNA
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(24:1) was added and the microtubes were inverted for 5
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70% and, after 10 minutes, the tubes were again centrifuged
DW USP DW & IRU PLQXWHV LQ RUGHU WR UHPRYH UHVLGXHVRI&7$%7XEHVZHUHWKHQLQYHUWHGLQD¿OWHUSDSHU WRGU\WKHSHOOHWZKLFKZDVWKHQGLVVROYHGLQȝ/7(S+ P0 75,6+&O DQG P0 ('7$ )RU FRPSDULVRQ SXUSRVHV GHVLFFDWLRQ WROHUDQW RUWKRGR[ VHHGV RI JXDYD
(Psidium guajava - 0\UWDFHDH ZHUH XVHG 5LSH IUXLWV RI JXDYD ZHUH KDUYHVWHG IURP VHYHUDO WUHHV DW WKH FDPSXV RI WKH)HGHUDO8QLYHUVLW\RI/DYUDV'1$ZDVH[WUDFWHGIURP
1 g seeds freshly harvested (23.2% MC) and from 1 g dried
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DNA were loaded on a 1% agarose gel stained with ethidium
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RESULTS AND DISCUSSION
The freshly harvested seeds of Eugenia pleurantha, with 35.5% MC attained only 20% germination, values close to those found
E\&DUYDOKRIRUWKHVDPHVSHFLHVLQLWLDO0& DQGJHUPLQDWLRQ,QWKHSUHVHQWVWXG\SDUWRIWKHIUXLWV
was stored for two months and, after that, seed germination
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VHHGGHYHORSPHQWZLWKLQWKHIUXLWVVLQFHHPEU\RVRIEugenia
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suggesting the absence of meristematic tissues differentiated
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Dehydration caused accentuated decreases in germination
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thatEugenia pleurantha seeds are desiccation sensitive.
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below 25%, seed germination was gradually reduced until
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The drastic reduction of germination when seeds were dried below 10% MC suggests that this seeds can be
FODVVL¿HGDVLQWHUPHGLDWHVLQFHDFFRUGLQJWR+RQJDQG(OOLV
(1996), intermediate seeds can be safely dried at MC levels about 7-12%.
&DUYDOKRFODVVL¿HGEugenia pleurantha seeds as
UHFDOFLWUDQWUHSRUWLQJWKDWWKHIUHVKO\KDUYHVWHGVHHGV
MC) attained 25% of germination and that after dehydration
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observed that storage of both undried and dried seeds at low
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The difference between the results found by Carvalho (2000)
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seeds were dried right after collection, when the tissues were
VWLOO XQGLIIHUHQWLDWHG %DUURVR ZKLOH LQ WKH SUHVHQW VWXG\IUXLWVZHUHVWRUHGIRUPRQWKVEHIRUHSURFHVVLQJDQG VHHGGHK\GUDWLRQZKLFKFDQKDYHDOORZHGVHHGVWRFRPSOHWH
differentiation and decrease their desiccation sensitivity. It
LVNQRZQWKDWLQDVDPHVSHFLHVLPPDWXUHVHHGVDUHPRUH VHQVLWLYHWRGHVLFFDWLRQWKDQWKHPDWXUHRQHV%HUMDNHWDO
1992, 1993; Faria et al., 2004). Furthermore, in the work done by Carvalho (2000), the dehydration time was much
ORQJHUGD\VWKDQLQWKHSUHVHQWVWXG\GD\V,WLV NQRZQWKDWIRUPRVWVSHFLHVWKDWEHDUGHVLFFDWLRQVHQVLWLYH
seeds, the faster the desiccation rate, the lower the MC that
FDQEHUHDFKHGEHIRUHYLDELOLW\LVWRWDOO\ORVW3DPPHQWHUDQG %HUMDN
The damages caused by dehydration are attributed to the
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0 5 10 15 20 25 30 35 40
0 10 20 30 40
Dehydration time (days)
M o is tu re c ont ent (% ) 0 20 40 60 80 100 5 10 15 20 25 30 35 40
Moisture content (%)
G er m in at io n (% ) B 0 5 10 15 20 25 30 35 40
0 10 20 30 40
Dehydration time (days)
M o is tu re c ont ent (% ) A 0 20 40 60 80 100 5 10 15 20 25 30 35 40
Moisture content (%)
G er m in at io n (% ) B
SURWHFW WKH WROHUDQW VHHGV LQ WKH GHK\GUDWHG VWDWH VXFK DV FHOOXODUSK\VLFDOFKDUDFWHULVWLFVLQWUDFHOOXODUGHGLIIHUHQWLDWLRQ WKH SUHVHQFH RI DQWLR[LGDQWV V\VWHPV DFFXPXODWLRQ RI SURWHFWLQJ PROHFXOHV DV /($ SURWHLQV VXFURVH DQG RWKHU ROLJRVDFFKDULGHVGHSOR\PHQWRIFHUWDLQDPSKLSDWLFPROHFXOHV DQGWKHSUHVHQFHDQGRSHUDWLRQRIUHSDLUPHFKDQLVPVGXULQJ UHK\GUDWLRQ3DPPHQWHUDQG%HUMDN6RWKHGHVLFFDWLRQ VHQVLWLYLW\ LV DVVRFLDWHG WR WKH DEVHQFH RU LQHI¿FLHQF\ RI DW OHDVW RQH RI WKRVH SURFHVVHV WKDW IDFLOLWDWH WKH DFTXLVLWLRQ
and maintenance of desiccation tolerance in orthodox seeds. In desiccation sensitivity seeds, the loss of structural water
GXULQJGHK\GUDWLRQSURFHVVFDXVHVDOWHUDWLRQRIPHWDEROLFDQG
membranes systems, resulting in seed deterioration (Farrant
HWDO7KHRUHWLFDOO\WKHGHVLFFDWLRQWROHUDQFHWHQGVWR LQFUHDVHZLWKWKHQXPEHURISURWHFWLRQPHFKDQLVPVLQDFWLYLW\ %HUMDNDQG3DPPHQWHU
Many Eugenia VSHFLHV EHDU QRQRUWKRGR[ VHHGV VXFK
as ( ÀRULGDand E. rostrifolia FODVVL¿HG DV LQWHUPHGLDWH E\&DUYDOKRDQG6DQWRVHWDOUHVSHFWLYHO\
DQG WKH IROORZLQJ FODVVL¿HG DV UHFDOFLWUDQWE. calycina
(Bülow et al., 1994), E. pyriformis (Andrade and Ferreira, 2000; Silva et al., 2003), E. brasiliensis (Andrade, 2002),
E. involucrata (Maluf et al., 2003), E. divaricata *HQWLO
2003),E. dysenterica, E. unifolia and E. bimarginata (Faiad et al., 2005).
7KHJHUPLQDWLRQSHUFHQWDJHVRIJXDYDVHHGVZHUH
(freshly harvested seeds; 23.2% MC) and 90% (seeds dried
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IURP JXDYD VHHGV NHSW LWV LQWHJULW\ DIWHU GHK\GUDWLRQ DV
shown by the single strong band (lanes 1 and 2 in Figure 2). In orthodox seeds such ability certainly is related to the mechanisms of cell survival under decreased hydration level.
7KHVDPHZDVYHUL¿HGLQ'1$RIE. pleurantha embryos
ZLWKDQG0&)LJXUHDOWKRXJKSUHVHQWLQJ DFRQVWDQWUHGXFWLRQRIWKHJHUPLQDWLRQSHUFHQWDJHV)LJXUH
1B). DNA isolated from E. pleurantha embryos dried to
0&DSSHDUHGKLJKO\GHJUDGHGDVVKRZQE\WKHVPHDUHG
band (lane 7, Figure 2), coinciding with the total loss of
VHHG JHUPLQDELOLW\ )LJXUH % 7KH GHJUDGDWLRQ SDWWHUQ REVHUYHGLVGXHWRWKHSDVVLYHFHOOGHDWKSURFHVV:DQJHW DOZKLFKLVWULJJHUHGZKHQWKHFHOOVDUHVXEMHFWWR
environmental stress. Faria et al. (2005) also related DNA fragmentation in radicles of germinated seeds of Medicago truncatulasubmitted to dehydration.
6HHG0&LVDVVRFLDWHGWRWKHPDLQWHQDQFHRISURWHLQV
integrity, endonucleases activation and DNA conformation
2VERUQH ,Q WKH GHK\GUDWHG VWDWH SURWHLQ GHQDWXUDWLRQPHPEUDQHGDPDJHVORVVRIHQ]\PHVIXQFWLRQ DQG QXFOHLF DFLGV LQWHJULW\ RFFXU DW UDWHV LQÀXHQFHG ERWK E\ WKH HQYLURQPHQWDO FRQGLWLRQV DQG JHQRW\SH 2VERUQH
and Boubriak, 1997). Since seed desiccation tolerance is genetically controlled, it is thought that in intermediate seeds,
DFRPELQDWLRQRIJHQHVSURYLGHVFRQVLGHUDEOHEXWLQFRPSOHWH
desiccation tolerance (Hong and Ellis, 1995).
The maintenance of genetic information is an essential
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M
Psidium guajava Eugenia pleurantha
5
1 2 3 4 6 7
WKH GHK\GUDWLRQ DQG UHK\GUDWLRQ 2VERUQH HW DO &HOO'1$GDPDJHVWDNHSODFHLIGHK\GUDWLRQRFFXUVZKHQ FKURPDWLQRU'1$FRQIRUPDWLRQDUHQRWRUJDQL]HGWRVXSSRUW GHK\GUDWLRQ 2VERUQH DQG %RXEULDN ,Q WKH SUHVHQW
study, the reduction of the MC of E. pleurantha seeds to
FDXVHG'1$GHJUDGDWLRQLQDSRVVLEO\LUUHYHUVLEOHZD\ OHDGLQJWRWKHORVVRILWVIXQFWLRQDOFRQGLWLRQDQGFRQVHTXHQW
seed death.
CONCLUSIONS
:KHQ VXEMHFWHG WR GHK\GUDWLRQ VHHGV RI Eugenia
pleurantha reacted in a way that suggests an intermediate behavior in relation to desiccation tolerance.
The loss of DNA integrity can be counted as one of the causes of loss of viability in E. pleurantha seeds dehydrated to 7% moisture content.
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