Effect of Abscisic Acid on Pollen Germination and Tube Growth of Maize Genotypes1

Plant Breeding 110, 250—254 (1993)© 1993 Paul Parey Scientific Publishers, Berlin and HamburgISSN 0179-9541

Effect of Abscisic Acid on Pollen Germination and Tube Growthof Maize Genotypes^

E. FRASCAROLI and R. TUBEROSA

Institute of Agronomy, University of Bologna, Via Filippo Re 6, 1-40126 Bologna, Italy.

With one figure and one table

Received April 20, 1992 I Accepted August 18, 1992Communicated by G. Wricke

AbstractAs a preliminary step to using gametophytic selec-tion to obtain genotypes of maize, Zea mays L.,differing for abscisic aeid (ABA) content and/or sen-sitivity, the influence of ABA on the in vitro germi-nation and tube growth of maize pollen and thepresence of genetic variability for these traits wasinvestigated. In a first experiment, pollen samples ofthree genotypes were germinated on substrates con-taining increasing amounts of ABA. Low ABA con-eentration (0.5 juM) significantly increased pollengermination and tube growth, while 500 juM ABAsignificantly decreased both traits, thus indicatingthat ABA can influence the growth of maize pollenin vitro. In a second experiment, eleven inbred lineswere screened for the in vitro reactivity of theirmierogametophytes to ABA. The lines differed sig-nificantly for the response (% of control) of tubelength, but not of pollen germination to ABA.

Key words: Zea mays — abseisie acid — pollengermination — pollen tube growth — gametophyticselection.

An accumulation of abscisic acid (ABA) iscommonly observed in plants under conditionsof water shortage (LARQUE-SAAVEDRA and WAIN

1976). To understand better the adaptive re-sponse of the plant to an insufficient watersupply, ABA metabolism has been thoroughlyinvestigated at different morphophysiologicallevels (DAVIES and JONES 1991). An increasing

Research supported by National Research Councilof Italy, Special Project RAISA, sub-projeet N. 2Paper N. 526.

interest has been devoted to the influence ofABA at flowering and in the early stages ofgrain filling, since water shortage at these timesis particularly harmful to final yield (ZENG andKING 1986, WESTGATE and BASSETTI 1990). Al-teration of the content of ABA and/or cellsensitivity to it has been proposed as a possiblemeans to select genotypes with an increaseddrought tolerance.

In cereals, as well as in other species, geneticvariability for the capacity to accumulate ABAunder water shortage has been found (QUARRIE

1991) and selection has altered the expressionof this trait in bread wheat (QUARRIE 1987),maize (QUARRIE 1991) and rice (HENSON et al.1985). Typically, the selection is carried out ona small segregating population (usually a fewhundred plants), thus limiting the possibilityof selecting the most desirable genotypes. Asan alternative to sporophytic selectionmethods, selection at the gametophytic levelallows the screening of a much larger popula-tion, provided that the corresponding genesare expressed during both the gametophyticand the sporophytic phase of the plant's lifecycle. Recent studies (reviewed by OTTAVIANO

and MuLCAHY 1989) have indeed demonstratedthat in higher plants a large portion of thegenome is expressed during both thegametophytic and sporophytic generations. Asa result, gametophytic selection has proved tobe successful in changing the allele frequenciesof loci controlling tolerance of low and hightemperatures (ZAMIR and GADISCH 1987.

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Effect ol Abscisic Acid on Pollen Germination and Tube Growth ol Maize Genotypes 251

Ct al. 1990), salinity (SAI,III;R ct al.1983) and herbicides (SARI GORI.A ct al. 1989).Gametophytic selection could also be utilizedto select genotypes differing in ABA contentand/or sensitivity.

To the best of our knowledge, no inlorma-tion IS available on the effects ot ABA onpollen grain germination and tube growth inniaize {7.ea mays L.). The present sttidy dealswith two experiments with niaize to investigateI) tlie effects of ABA on pollen germinationand tube growth and 2) the variability, amongvarious genotypes, oi the response of theirmale gametophytes to ABA.

Materials and Methods

Experiment 1: In July 1991 pollen \va.s collected onsix consecutive d.iys from tassels of field-grownplants of tlie inbred lines A188 and BMS and of thesingle cross A188 x B73; each day represented areplicate. Each pollen sample was split into five lotswhicli were germinated on an agarized medium(CHI^NC; and pREl'LlNG 1976) supplemented eitherwith 0 (control). 0.5, 5.0, 50 or 500/iM (±) cis-transABA. ABA was liker-sterilizcd and added to thecooled, auloelaved medium. E.ieh pollen lot wasused to inoculate one Petri dish which representedthe experimental unit. Petri dishes were incubated in[he d.irk at 27 °C for 3 h and then the pollen tubeswere lixed with Farmer's solution (acetic acid/ethanol; I : 3 v/v). For each Petri dish, pollen germi-nation was assessed with 100 grains; tube length wasnieasured from 50 germinated grains using a MINI-MOP image analysis system.

Experiment 2: The pollen ol the eleven inbred lineslisted in Table I was collected on two cimseciitivedays in July 1991; each day represented a replicate.I'ollen samples were split into three lots which wereused to inoculate three Petri dishes containing anagarized medium supplemented, respectively, with 0(control), 50 and 500/(M ABA. Pollen traits werenieasured using the procedures previously described.

Results

por experiment I, the analysis of variance (datanot reported) for pollen germination and tubelength showed highly significant effects due toABA doses. Interestingly, the lower doses ofABA had a significant stimulatory effect onpollen germination and tube growth (I'ig. 1).A significant decrease in both traits was evi-dent only on substrates containing 500 /(MABA. Wlien the mean responses ol the single

genotypes to ABA concentrations were ex-amineci, significant differences were detectedtor both traits (data not reported). Mean values(expressed as 'Xi of control) ranged from 73(A 188) to 116% (A 188 X B73') for pollengermination and from 92 (BMS) to 106 %(A 188 X B73) for pollen tube length. The"genotype X dose" interaction was not signifi-cant, which implies that differences amotiggenotypes were similar over the range ol ABAconcentrations tested.

Significant effects due to the ABA doseswere detected for pollen germination and tubelength in experiment 2 (Fig. 1). As with thefirst experiment, ABA caused contrasting ef-fects according to its concentration. As com-pared to the control, 50 /*M ABA significantlyincreased pollen germination and tube length,while 500//M ABA caused a significant reduc-tion. It should be noted that in the previousexperiment, 50 /(M ABA did not affect thetraits investigated to any appreciable extent.Signilicant genotypic differenees were also de-teeted among the responses (% of control) oftube length to ABA, but not of pollen germi-nation. Also in this experiment the "genotypeX dose" interaction was not significant. Theresponses of the inbreds were averaged overthe two ABA concentrations tested and were

Table I. Kflects of ABA on pollen tube length ofeleven maize inbreds. Each value is the mean of theresponses (as % of control) to two ABA concentra-tions (50, 500 //M)

Inbred line Tube length (% ol control)

B88Lol045Va26Lo876Os420Mes44Wl'9Polil7

M o 17I-'2B79

Mean

126 a

110 ab

104 a—c

99 be

98 be

94 be

94 be

88 be

86 c

80 c

78 c

96

Values not followed by the same letter are signifi-cantly difterent at P £ 0.05 (Duncan's test).

252 FRASCAROLI and TUBEROSA

A) B)

30 1 L S D LS D

30 -

I—1.-^

500

400

300

0.5 50 500 50 500

ABA CONCENTRATION (pM)

Fig. 1. Effect of ABA concentration on germination and tube length of maize pollen:A) mean value of three genotypes; B) mean value of eleven inbred lines

found to range, as compared to the control,from 78 % in B79 to 126 % in B88 (Table 1).

DiscussionPollen tube growth depends on the rates ofbasic metabolic activities, such as those in-volved in energy production and the biosyn-thesis of cell wall constituents, the variabilityof which is likely to be controlled by genesexpressed in both sporophytic and gametophy-tic tissues (OTTAVIANO and MULCAHY 1989).The stimulatory effects noticed for the lowerconcentrations of ABA could tentatively beascribed to greater sucrose availability for thegerminating pollen grain and growing tube,promoted by an increase in the permeability ofthe microgametophyte's membrane to sucrose.ABA has been shown to increase sucrose trans-port in sugar beet tissue (SAFTNER and WYSE

1984) as well as in the seed coat apoplast ofsoybeans (ScHUSSLERet al. 1984) and peas (Rosset al. 1987). Pollen tubes growing in vitroutilize the carbohydrates present in the sub-strate, which, under in vivo conditions, are

normally supplied by the style. Alternatively,ABA could directly affect pollen germinationand tube growth by altering membrane per-meability to ions or other compounds normal-ly required, and/or indirectly by influencingthe expression of genes regulating the develop-ment of the gametophyte. Tomato pollen ex-posed to increasing ABA concentrationsshowed, as compared to the control, a signifi-cant decline in germination coupled with asignificant increase in pollen tube length(MCLEOD 1975). The author speculated that thegrowth of tomato pollen tubes is subject tonatural restraints which can be overcome by avariety of substances (ABA itself) likely to bepresent in the style tissue. In tobacco, a short-lived increase in the endogenous ABA level inthe pollen grains within anthers cultured invitro, considerably affected the developmentalpattern of the microspores (IAMAMURA andHARADA 1980).

The different dose-response relationships (inabsolute terms only) between the first andsecond experiment are probably due to the factthat different groups of genotypes were evalu-

Effect of Abscisic Acid on Pollen Germination and Tube Growth of Maize Genotypes 253

ated under differerit environmental conditions.Seemingly, the ABA content of pollen grainsmight be influenced by genotypic and environ-mental factors as has been observed with otherplant organs (KOSMKIN and TARARINA 1990). Insuch cases, a specific ABA concentration maynot have the same effects on the germinationand the early phase of tube growth of pollengrains differing in ABA eontent. An artificialwater stress applied to tobacco anthers cultur-ed in vitro, induced a two-fold increase in theABA content of the pollen grains (IAMAMURA

and HARADA 1980). However, it was not possi-ble to ascertain whether the de riovo synthesisof ABA occurred in the anther tissue or in thepollen grains. Considerable amounts of ABAhave been found to accumulate in the pollen ofmaize as well as of other anemogamous speciessurveyed by Lii'i' (1991). The same authorspeculated that ABA might have a protectivefunction by improving the level of droughtresistance of the pollen. Further investigationis currently in progress to determine the extentof ABA biosynthesis during pollen germina-tion and tube growth in maize.

The results of the present research indicatethat ABA affects the germination and tubegrowth of maize pollen in vitro and that signif-icarit variation exists among the genotypestested for the in vitro response of their pollento ABA. If such results are due to genes expre-ssed at both the gametophytic and sporophyticlevels, it may be possible to use gametophyticselection effectively to obtain genotypes differ-ing in ABA content and/or sensitivity.

ZusammenfassungDie Wirkung von Abscisinsaure auf die Pol-lenkcimung und auf das Pollcnschlauch-wachstum von Maisgenotypen

Als ein erster vorlaufiger Schritt zur Anwen-dung gametophytischer Selektion auf Maisge-notypen {Zea mays L.), die sich in ihrem Ge-halt an Abscisinsaure (ABA) und/oder in ihrerEmpfindlichkeit gegeniiber Abscisinsaure un-terscheiden, wurde untersucht, ob Abscisin-saure einen EinfluB auf die in vitro-Kdmun^und auf das in w>ro-Schlauehwachstum vonMaispolkn hat und ob cs eine genetische Va-riabilitiit dieser beiden Eigenschaften gibt. In

einem ersten Versuch wurden Pollen von dreiGenotypen auf Substraten mit ansteigendenAbscisinsaurekonzentrationen zum Keimengebraeht. Durch eine niedrige ABA-Konzen-tration (0,5 fiM) wurden Keimung und Pollen-schlauchwachstum deutlich gefordert, wiih-rend bei einer Konzentration von 500 /JM beibeiden Merkmalen eine signifikante Abnahmeerkennbar war. Somit konnte gezeigt werden,dafi Abscisinsaure das Waehstum von Maispol-len in vitro zu beeinflussen vermag. In einemzweiten Versuch wurden elf Inzuehtlinien aufdie in vitro-Kcaktion ihrer Mikrogametophy-ten gepruft. Die Linien untersehieden sich si-gnifikant in ihrer Reaktion auf Abscisinsaurenur in bezug auf die PoUenschlauchlange (inProzent gegeniiber der Kontrolle); die Pollen-kornkeimung war nicht beeinflufit).

We thank Prof. P. LANDI and Prof. S. CONTI forhelpful discussions and suggestions.

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