ENDOGENOUS GROWTH REGULATOR DETECTION
IN GUINEA GRASS SEEDS'
ROBERTO USBERTI2 and IVANY FERRAZ MARQUES VALIO'
ABSTRACT - Endogenous growth regulator activity/levei was detected lii guinea grass (Panicum
Inaximum Jacq.) seeds, a species widely used as a forage crop in the tropics, aiming at explaining its
high seed dormancy levei. Seeds were previously scarified or not with suiphuric acid and osmoconditioned on PEG-6000. Endogenous growth regulators were detected as follow: gibberellin--like activity (growth of iettuce hypocotyi bioassay); cytokinins (increase of fresh mass of radish cotylcdon bioassay); ABA (spectrophotometer at 230 nm). Exogenous application of GA, showed a germination increasing effect while ABA had a complete effectivenessto prevent it. High-dormancy seed samples had higher gibbereilin-like actívity than low-dormancy ones and intact seeds showed higher gibberellin-like activity than scarified seeds; however, after osmoconditioning, opposite re-sults were recorded. No significant activity of neutral promoters and cytokinins was detected. Average leveis of ABA for untreated, osmoconditioned after zero- and two-month-storage seed saznples were 0.51,0.39 and 0.21 mg. 100 kg 4, respectively. Chemical scarification did not alter either ABA leveis in high-dormancy seed samples (0.66 mg.l00 kg 4 ) or those of low-dormancy (0.23 mg. 100 kg'), lhe fornier being significantly higher than the latter. Finally, the results show that a gibberellin-ABA interaction appears to be the main factor accounting for dormancy, germination and osmoconditioning control in guinea grass seeds.
Index terms: germination, dormancy, osmoconditioning,Panicum rnaxi,num.
DETECÇÃO DE REGULADORES ENDÓGENOS DE CRESCIMENTO EM SEMENTES DE CAPIM-COLONIÃO
RESUMO - Foi detectada a atividade/nível endógeno de reguladores de crescimento em sementes de capim-colonião (Panicum maximun Jacq.), espécie muito usada para formação de pastagens nos trópi-cos, visando explicar o seu alto nível de dormência. As sementes foram previamente escarificadas, ou não, com ácido sulfúrico, e osmocondicionadas em PEG-6000. Os reguladores de crescimento foram detectados como segue: atividade giberelínica(bioteste do hipocótilo de alface), citocininas (bioteste do aumento do peso fresco de cotilédones de rabanete), e ABA (espectrofotometria a 230 nm). A aplicação exógena de (iA, aumentou a germinação, enquanto ABA inibiu-a completamente. Amostras com alta dormência apresentaram maior atividade giberelínica que as de baixa dormência, bem como maior atividade em sementes intactas do que em escarificadas; mas após o osmocondicionamento, essa situaçAo se inverteu. Não foram detectadas diferenças em atividade de promotores neutros e de citocininas. Os valores médios deABA foram 0,51, 0,39 e 0,21 mg.100 kg' nas sementes normais, imediatamente ap6s o seu osmocondicionamento, e dois meses após, respectivamente. O nível endógeno de ABA nas sementes com alta dormência (0,66 mg.l00 kg') foi maior que nas sementes com baixa dormência (0,23 mg. 100 kg'), mas esses valores não foram alterados pela escarificação química. Uma possível interação entre giberelinas e ABA parece ser a causa do controle da dormência, germinação e osmocondicionasnento nessas sementes. - Termos para indexação: germinação, dormência, osmocondicionamento, Panicura inaximum 1 Accepted for publication on March IS, 1997.
Eng. Agr., Dr., Dep. de Sementes, Mudas e Matrizes - CATI. Caixa Postal 1291, CEP 13073-001 Campinas, SI'.
Biólogo, Ph.D. Prof. Titular, Instituto de Biologia, Univer-sidade Estadual de Campinas (UNICAMP). Caixa Postal 6109, CEP13081-970 Campinas, SI'. Bolsista do CNPq. -.
WH4ODUCFION
Growth regulators are considered as prirnary agents of seed germination and dormancy processes (Jan & Amen, 1977). Abscisic acid and gibberellins are lhe growth regulators most frequentiy suggested Pesq. agropec. bras., Brasília, v.32, n.7, p.695-700, jul. 1997
lo confrol seed dormancy and germination. Abscisic acid plays a main role during lhe deveiopment of primar)' dormancy while gibbereiiins are involved in the induction of germination. Changes iii sensitivity lo these growth regulators occur with changes in dormancy (Hilhorst & Karssen, 1992).
Exogenous gibbereliins enhance germination in seeds iii which domiancy (or quiescence) is imposed by severai mechanisms such as immature embryos, bani seed coats and inhibitors. The hypothesis on the mode of action of gibberellins is based on lhe mobilization of endosperm reserves of barley seeds. Thus, it is possibie that for seeds where germination is stimulated by gibbereiiins, lhe first stage ofthis process is via enzymes acting in the breakdown of starch and other substrates (Jones & Stoddart, 1977). Cytokinin activily is high in developing fruits and seeds but lhe leveis drop with the ripening of lhe tissues. Research on the endogenous leveis and ex-ogenously appiied cytokinins suggest that they are involved in the control of bud and seed dormancy Çlliomas, 1977).
Inhibitors may act during seed embryogenesis and ripening mainiy by preventing precocious germina-tion. They can be responsibie for lhe beginning and maintenance ofdormancy, but this effect can be over-come by promoters (Biack, 1980181). ABA (abscisic acid) concentration in acid lime seeds and fruits has been suggested not to determine lhe rale ofgrowth and deveiopment, but iow ABA concen-frations during lhe fmal phase of seed deveiopment may be of significance for seed germination (Murti,
1993
II is weil known that seed osmoconditioning on PEG (polyethylene giycol) solutions has improved seed gerinination rale and uniformity of a wide spec-trum of piant species, including grass seeds (Hardegree, 1994).
Ciuinea grass was chosen for this study in view of its broad application to estabiish forage crops iii lhe tropics. However, lhe freshly harvested seeds of ai-most ali of its cuitivars probably show a low percent-age of germination and a high levei of dormancy, due probably lo uneven maturation (Harty eI ai., 1983).
The main objective of this paper was lhe delec-tion of endogenous growth regulator in dormant and quiescent seeds of Panicum maximum, before and
afier osmoconditioning with PEG-6000, airning aI ex-plaining lIs high seed dormancy levei.
MATERIAL AND MKFHODS
Six samples of seeds (spiketets) of Panicuni maximuin iacq. from two regions of São Paulo State, Brazil, previ-ously grouped according lo lhe degree bf dormancy de-Iected by scarification in 112$04 for five minutes (high dormancy - samples 1 and 6; low dormancy - saniples 2,3; 4 and 5), were used (Table 1).
The optimum conditions for osmoconditioning guinea grass sceds were detected in preliminary tests. Seed samples (1.5 g each) were kept during seven days in gerboxes containing 25 mL of PEG-6000 solution (288 g.kg watert), at 25°C (-0.95 MPa) and 15°C (-1.1 MPa), for intact and scaritied seeds, respectively. Afier imbibition in PEO solution lhe seeds were rinsed in distilled water for one minute, placed in a 5% sodium hypochioride solution for 20 minutes, rinsed again for one minute and surface dried with filter papers. After a week storage at 15% relative humidity (drying cabinet), halfof lhe sampies was immediately extracted for growth regula-tors detection while lhe others were stored at 1(M for two months before analysis.
The foliowing treatments were applied on both intact and scaritied seeds: a)controt seeds (no imbibition in PEG); b) seeds immediately after imbibition in PEG; c) seeds stored for two months aI 10°C, afler imbibition in PEO.
Seed sampies (1.5 g) were then extracted in 80% metha-nol (Valio & Schwabe, 1970) lo detect lhe activity ofgrowth regulators, also lhe possible changes caused by the osmoconditioning. The fractionated exlracts were chrornatographed on glass plates coated with silicagel and developed with ethyl acetate : chloroform : acetic acid (15:5:1). The plates were coated with fluorescent silicagel lo detect ABA lhrough fluorescence and spectroscopy.
TABLE 1. Quality parameters ar guina grau seed
samples used for endogenous growth regu-lators detection.
Seed sample Physical purity Germination (%) (%) Control 112SO4 1 23.9 7 42 2 62.7 IS 25 3 51.6 ló 28 4 57.7 14 22 5 70.2 9 19 6 58.2 4 43
ENDOGENOUS GROWTH REGULATOR DETECTION 114 GUINEA GRASS 697
For bioassays Um chromatograms werc divided in lO horizontal strips corrcsponding to Ri' 0.1 - 1.0. Each strip was also divided in three rcpiicates per Ri' region. Silicagel from plates without application of extracts was used as control. Bioassays were processed only in arcas of the chromatognrns that showed biological activity in previ-ous cxperiments such as Ri' 0.1 - 0.6 for acid and neutral fractions, and Ri' 0.1 - 0.5 for basic fraction.
The growth of lettuce hypocotyi bioassay was used to detect gibbercllin-lilce activity on acid and néutral frac-tions (Frankland & Wareing, 1960). Cytokinins werc de-tected chemicaliy 1»' lhe Wood reagent (Wood, 1955) and biologically through lhe increase of fresh mass using lhe radish cotylcdon bioassay (Letham et ai., 1964). Four let-tuce seedlings or radish cotyiedons were used in these bioassays for cach Ri' strip replicate.
• ABA was detected through fluorescence under an u1 traviolet lamp (230 nm). Extracts of 1.0 g of seeds also standard solution ofABA (1 0 M) were chromatographcd, identifled under ultravio!ct iamp, scraped ofT lhe platcs and analysed by a spectrophotometer at 230 nm, accord-ing lo Kefeli (1978).
Preliminar)' experiments of normal seed germination tests, using different batches of seed samples, were carried out to detect the effects of appiication of growth regula-tors in high- and low-dormancy seeds (0A3 - gibberellic acid 10-3M and 104M; 6-DA- bcnzyl adeninc 10"M and 10 M; Ethrel 60 jig.kg1 and 6 gg.kg'; ABA 10-3 M,
I04M and I0-5M).
The statisticai design was compictely randomized (Sokal & Robif, 1969). ABA standard curve was piotted by linear regression. Comparisons betwcen treatment means in relation lo ABA were carried out through Tukey's test aI the 5% levei. Analysis of variance and F test were also used for lhe sarne Rf of difTerent treatments.
RESIJLTS AND DISCUSSION
Thd appiication of GA3 10 M and GA5 10-4 ti had similar effects on increasinã final germination prcentages while lhe other growth regulators showèd no effect (Table 2). This stimulation ofger-mination by GA3 has been also shown for iettuce (Toyomasu etal., 1994) and for Panicum maximum
seeds (Basra eI ai., 1989). ABA application showed a complete effectiveness to prevent seed germination
o its results are not shdwn at Table 2.
I-ligh-dormancy control seeds (not imbibed in PEG). showed a gibberellin-iike activity aI 0.4 - 0.6 Rf region with higher activity in intact seeds
TABLE 2. ElTects of exogenous growth regulatori lii
guinca grau intact and germlnation.
Treatment Mean germination'
GA3 103M 41.49 a 0A3 104M 39.83 a • Control 33.76 b 6-DA 105M 33.47b Ethrei 6 pg.kg' 33.25 b 6-BAI0M 31.66b Ethrel 60 j.tg.kg' 3 1.08h
'Means followed by differenl 'cnn are statislicaily different. .ccordiag
lo Tukey's test, aI 5%pi-obabiiiiy leve!.
at Rf 0.4 while seeds imbibed in PEG showed it at Rf 0.6, with higher leveis for scarified seeds (Fig. 1). In seeds stored for two months after imbibi-tion in PEG, gibberellin-like activity was also detected but higher differences were found for scarified seeds at Rf 0.2 and at Rf 0.6. Low-dormancy intact and scarified seeds did hot show gibberellin-Iike activity.
!ntact and scarified seeds with high dortnancy presented higher gibbereilin-like activity aI Rf 0.4, though scarified seeds showed one additional higher gibberellin-liké activity aI RI 0.6 (Fig. 2).
lhe presence of higber gibberellin-like activity in control seeds with high dormancy overscarified onés and lhe reversed situation after imbibition in PEG couid be expiained by an increased permeability óf, lhe tissues by H2SO4 treatrnent followed by a faster activation of biochmical processes related to GA poduction. A new approach states that lhe gibber-e!Iins are indispensable for germination and that dor-niancy-releàsetreatments can induce eithertheirsyn-thesis or a change in their compartmentation or a change in sensitivity of lhe tissue (Le Page--Degivi', 1990). 1
Neutral and basic fractiohs obtained froin intact and scarified seeds with high and low dormancy did not show promoting activity, suggesting that neu-trai and cytokinin-like prornoters appear not to be i&ioived in seed kormancy and osmoconditioning ofthis specie's, according to the model proposed by Khan (1975), in which gibberellins, abscisic acid and cytokinins have speciai role iii these processes. On lhe other hand;application of kinetin in Panicum
miiaceum significantly increased seed germination; The mean values detected for. ABA were Iow ABA concentrations however slightiy increased 1.939.1 0' M . (0.51 mg. 100 kg'), 1.48.1 0 M genninationbutinhibitedseedlinggrowth(Dhfr& (0.39 mg.100 kg') . and 1.822.10.6 M sharma, 1991). (0.21 mg. 100 kr') for seeds before imbibition, im-
mediately after imbibition in PEO solution and two-•month-storage later, respectiveiy. These resuits àgree with other data showing a minimum limit of
120 7
- 10 M for germination inhibition, for example in to-
i , ... matoseeds(Finch-Savage&McQuistan,1991),which presented an increase on emergence percentage and 110 - - — a reduction on both time and spread ofemergence
v
/ after exogenous appiication ofABA 10 -5 M. Iii other species, higher values were necessary for seedinhi-100 * ' bition such as for iettuce, 12 mg.100 kg"
1
(Braun & Khan, 1975); Fraxinus americana,3
. - - - / - , 45 mg.i00 kg 1 (Sondheimer et ai., 1968); Acer'5 0,0 0.1 02 0.3 0.4 0.5 . 0,6 saccharum, 82.6 mg.100 kg' (Webb etai., 1973).
!120
(b)'-
- - . * 110 . 7 o o 1:t Jo,r
0.51
t
o Q. 120 1t-.t 110 100901 .z1I.Id'uIr.#lI ril Iv'.a r.r#I t 1 ICI irA iIt1 II'2I1 11-2 1
0.0 0,1 0,2 0,3 0,4 0.5 0,6 0,0 0,1 0,2 -0,3 0,4 0,5 0,6
Rf
FIC. 1. Gibbereltin-like activity lo high-dorrnancy .
intact (hatched) and scarified (open) seeds for FIC. 2. Gibberellin-Iike activity lo high-(hatched) controi (a), irnmediately after imbibition on tow-dorrnancy (open) seed samples for intact PEC (b) and two months after irnbibition in (a) and scarified seeds (b). Signai * shows PEG (c). Signal * shows statistical differences statisticaI differenccs at 5% levei between at 5% levei between values at the sarne R£ values at the sarne Rt-- -
01 cu 2 O) o o o a1 a) —J
ENDOGENOUS GROWTH REGULATOR DETECTION 114 GUINEA GRASS 699
• The amounts of ABA detected in intact and scan-fied seeds, before and after osmoconditioning, were similar. However, high-dormancy seeds showed higher values than low-dormancy ones (2.49.10 M 0.66 mg.100 kg'; 0.87.10 M 0.23 mg. 100 kg-', respectively).
• 'fie localization ofABA in the seeds is irnportant to expIain the domiancy. lii hazelnut, ABA was found mainly iii the penicarp and coat and its removai re-leases the seed from dormancy (Ross, 1984). Never-theiess, in guinea grass the chemicai scariflcation did not reduce the leveis of ABA. It is suggested that the positive effect of H2SO4 in breaking the dormancy ofthis species couid be due to its action
011 dehydration of the tissues thus breaking the
tegu-ments and aliowing more oxygen to the embryo (Durán & Tortosa, 1985).
High-donnancy seed sampies showed higher vai-ues forboth gibbereiiin-iike activity and ABA leveis than iow-dormancy ones. It is suggested that dor. mancy and germination control in guinea grass seeds depends on these growth regulators, with gibberei-Uns piaying a main role in the process. The possibie effect of osmoconditioning 011 high-dormancy seed
sampies couid be expiained by an increase in the leveis of acid promoters afier imbibition in PEG soiu-tion as occurred on scarified seeds.
CONCLUSIONS
1. No statistical differences of neutral promoters and cytokinin-like activity are detected.
- 2. Scaniflcation with concentrated sulphuric acid does not affect endogenous ABA leveis.
3; High-dormancy seed sampies show higher gib-bereiiin-iike activity and ABA leveis than low--dormancy ones.
1 ti) :l(Ø 1
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