Africn Journl of Biotechnology Vol. 12(5), pp. 446-452, 30 Jnury, 2013 Aville online t http://www.cdemicjournls.org/ajb DOI: 10.5897/AJB12.2529 ISSN 1684 5315 2013 Acdemic Journls Full Length Reserch Pper Fruit mturtion nd in vitro germintion of mcw plm emryos Priscil Oliveir Silv 1, Leonrdo Monteiro Rieiro 1*, Mri Olívi Mercdnte Simões 1, Pulo Sérgio Nscimento Lopes 2, Teddy Mrques Fris 2 nd Queil Souz Grci 3 1 Biologicl Sciences, Universidde Estdul de Montes Clros, Cmpus Montes Clros, Mins Geris, Brzil. 2 Agriculturl Sciences, Universidde Federl de Mins Geris, Cmpus Montes Clros, Mins Geris, Brzil. 3 Biologicl Sciences, Universidde Federl de Mins Geris, Cmpus Belo Horizonte, Mins Geris, Brzil. Accepted 3 Jnury, 2013 Acrocomi culet (mcw plm) is oil producing plm tree with significnt gro-industril potentil. Seed dormncy in plm species my e due to emryo immturity, which could result from delyed emryogenesis. We evluted the correspondence etween the visul chrcteristics of mturing fruits nd their physiologicl spects nd the in vitro germintion cpcity of the emryos. 11 fruit unches in different stges of mturity were collected nd clssified in terms of the degree of mturtion of the endosperm, the color of the exocrp, nd the occurrence of scission. The wter nd oil contents of the mesocrp nd seed were determined, nd lipids nd proteins were identified through histochemicl nlyses of the mesocrp, endosperm, nd emryo. The emryos from ech fruit unch were cultivted in vitro in 75% Murshige nd Skoog (1962) medi with dded orgnic compounds. The wter contents of the seeds vried from 71.2 to 21.1% mong the different stges of fruit ripening nd were relted to the visul mrkers of fruit mturtion (exocrp color rnging from drk green to rown). Lipid ccumultion in the mesocrp occurred lter thn in the endosperm, nd only occurred in fruits from unches showing signs of scission. Emryos from unches in different stges of mturtion showed similr germintive cpcities, s well s similr ptterns of lipid nd protein storge. Emryogenesis in A. culet is precocious, nd the emryos of immture fruits cn e utilized for in vitro cultivtion. Key words: Acrocomi culet, emryo culture, emryogenesis, wter content, oil content. INTRODUCTION The mcw plm tree [Acrocomi culet (Jcq.) Lodd. ex. Mrt.] is very common in the Cerrdo (Brzilin svnn) iome (Lorenzi et l., 2010). Due to the lrge quntities of high-qulity oil in the mesocrps nd seeds of these plnts nd their dpttion to dry tropicl climte, this species hs significnt economic potentil, especilly in terms of iofuel production (Hine et l., 2005; Mour et l., 2010). The estlishment of commercil cultivtions of mcw plm plms hs een limited, y their seed dormncy, which results in slow germintion speeds nd low germi- *Corresponding uthor. E-mil: leomri@hotmil.com. Tel: 66 38 32298154. ntion rtes (tking up to four yers) (Rieiro et l., 2011). Dormncy in plms is often relted to the immturity of their emryos (Bskin nd Bskin, 1998; 2004; Orozco-Segovi et l., 2003), which reflects delyed emryogenesis in reltion to fruit development (Werker, 1997; Hrtmnn et l., 2002). In vitro cultivtion of plm tree emryos offers the possiility of overcoming dormncy in mny species nd fcilitting studies of emryogenesis nd germintion (Bhojwni nd Rzdn, 1996; Rghvn, 2003; Pechy Aké et l., 2004; 2007). Rieiro et l. (2011) reported high germintion percentges of mcw plm emryos derived from mture fruits, lthough there re no pulished reports tht relte emryo development nd germintive cpcity to fruit mturtion. The development of Acrocomi culet fruits is slow,
Silv et l. 447 requiring out 13 months (Figures 1A to C) (Scriot nd Llers, 1991). A etter understnding of the ptterns of mesocrp, endosperm, nd emryo mturtion, s well s the iochemicl profile of the seeds, could help optimize strtegies for industril hrvesting nd for otining the est emryos for in vitro propgtion (DeMson, 1988; Pnz et l., 2004; Mour et l., 2010). Considering the economic importnce of mcw plm nd the scrcity of studies focusing on plm dispores (Orozco-Segovi et l., 2003), the present study evluted the iochemicl nd histochemicl chrcteristics of their fruits in different stges of mturtion, s well s the in vitro germinility of their emryos to ddress the following questions: i) re there esily identifile visul chrcteristics tht cn e ssocite with the wter contents nd metolic reserves of fruits nd seeds? ii) Are there differences in the in vitro germintive cpcities of emryos from fruits t different stges of mturtion? iii) Wht re the reltionships of emryogenesis with fruit development? MATERIALS AND METHODS Six individul plm trees were selected mong popultion of A. culet (Figure 1A) in the municiplity of Montes Clros, Mins Geris, Brzil. 11 fruit unches (Figure 1B) in different stges of mturity were selected nd clssified sed on their visul chrcteristics: 1) fruits with drk green exocrp, endosperm not completely developed; 2) fruits with green exocrp nd developed endosperm; 3) fruits with rownish exocrp; nd 4) fruits with rown exocrp, extrcted from unches in which the scission strted (Figures 1D to G). Four smples of ten fruits were chosen t rndom from ech unch for evlution of their wter nd oil contents. After rupturing the exocrp (using wooden mllet), the mesocrps were removed using sclpels nd the seeds were then extrcted from the endocrp (fter reking it in vise). The fresh weights of the mesocrps nd seeds were determined for ech smple. The dry weights of the mesocrps nd seeds were determined seprtely fter drying t 105ºC for 24 h; their wter contents were clculted considering the differences etween their fresh nd dry weights. The oil contents of the mesocrps nd seeds were determined y extrcting the dehydrted smples in Soxhlet extrctor using petroleum ether s the solvent (AOAC, 2005). Regression nlysis ws used to djust the curve indictive of the vrition of the seed wter contents s function of the mturtion sttge of the fruit unches (SAS Institute, 1990). The presence of reserve compounds in the mesocrp, endosperm, nd emryo of five fruits from nine different unches were determined y histochemicl nlyses. The fruit nd seed smples were fixed in FAA 50 (Johnsen, 1940) or Krnovsky solution (Krnovsky, 1965) for 24 h, conserved in 70% lcohol, nd dehydrted in n ethnol series for emedding in hydroxyethyl-methcrylte (Leic ) following the protocol descried y Piv et l. (2011). These smples were sectioned trnsversely (10 μm) nd stined with Sudn IV to indicte the presence of lipids (Foster, 1949) nd with romophenol lue to indicte the presence of proteins (Mzi et l., 1953). The slides were exmined using Nikon Eclipse E200 opticl microscope. The effect of the degree of fruit mturtion on the in vitro germinility of plm emryos ws evluted using seeds from the fruits of the 11 unches. The seeds were removed using ench vise nd disinfected in 6% sodium hypochlorite for 10 min; the emry- os were susequently extrcted using sclpels while working in lminr flow cinet nd then plced in 100 mg L -1 solution of scoric cid. After disinfecting the emryos in 0.5% chlorine for 10 min, followed y three rinses in utoclved distilled wter, the emryos were inoculted into 7.5 x 1 cm test tues contining 2 ml of 75% strength MS medium (Murshige nd Skoog, 1962) with 0.4 mg L -1 thimine; 1 mg L -1 pyridoxine; 0.5 mg L -1 nicotinic cid; 100 mg L -1 myo-inositol; 0.5 g L -1 hydrolyzed csein; 3 g L -1 ctivted chrcol; 30 g L -1 sucrose; 6 g L -1 gr; the ph ws djusted to 5.7 (Rieiro et l., 2012). The experiment ws estlished in rndomized design with 11 different tretments (fruits t different stges of mturtion) nd five repetitions of 10 tues with one emryo ech. After inocultion with the emryos, the tues were wrpped in luminum foil nd mintined in the drk t 30 C. After 30 dys, the emissions of the primry roots nd the lef sheths were determined, s well s the dry weights of the hustorium, cotyledon petiole, roots, nd lef sheths (Figure 1H) fter drying t 105 C for 24 h. The dt concerning the emissions of roots nd lef sheths ws registered y counting the numer of emryos or plntlets showing those events nd then trnsforming those numers into percentges, which were susequently rc sine trnsformed [(x/100) 0.5 ] for comprison. Anlysis of vrince ws used for ech vrile, nd the Tukey test used for comprisons etween the verges, fter significnt differences were determined mong the different tretments using the F test; regression nlysis ws used to define the tendency curve (SAS Institute, 1990). RESULTS The fruits of A. culet re gloose drupes with firous exocrp, firous mesocrp hving mucilginous nd oily spect, nd hrd endocrp. The seed ws covered y thin tegument lyer nd hd n oily endosperm nd smll emryo (Figure 1C). The wter content of the mesocrp ws greter thn 70% in ll of the studied stges of fruit mturtion, so tht this prmeter cnnot e used s n indictor of fruit mturity (Figure 2). The wter content of the seeds, however, ws more closely relted to the visul chrcteristics of fruit mturtion. Oil ccumultion occurred in the seeds efore eing oserved in the mesocrp. Fruits with rownish exocrps were found to hve considerle quntities of oil in the endosperm, while significnt increses in the lipid contents of the mesocrps were only seen in fruits of the unches where the scission strted. These seeds contined developed emryos tht could e extrcted with reltive ese, independent of the mturtion sttges of the fruits (Figure 1C). Histochemicl evlutions did not indicte the presence of protein in ny significnt quntities in the mesocrps during ny of the fruit mturtion stges evluted (Tle 1). In reltion to lipids, higher concentrtions of lipidic reserves in mesocrps from fruit unch showing signs of scission ws noted (Tle 1; Figure 3A). Vritions were oserved in terms of the presence of lipids in the endosperm, nd fruit unches with the lowest seed wter contents hd the gretest lipid reserves (Tle 1; Figure 3B). High concentrtions of proteins were lso identified in the endosperms of fruit unches tht were in the most dvnced sttes of mturtion (Tle 1; Figure 3C).
448 Afr. J. Biotechnol. Figure 1. Individuls, fruits, nd plntlets of A. culet: A) A nturl popultion in northern Mins Geris Stte, Brzil. B) Fruit unch nering scission. C) Mture fruit indicting its contents nd the emryo (rrow). D) Fruits with drk green exocrps in which the endosperms re not completely developed. E) Fruits with greenish exocrps. F) Fruits with rownish exocrps. G) Fruits with rown exocrps tht hve scised. H) Plnt with fter 30 dys of in vitro cultivtion. cp, Cotyledon petiole; ed, endosperm; en, endocrp; ex, exocrp; h, hustorium; li, ligule; me, mesocrp; ro, root; sh, lef sheth. Aundnt reserves of proteins nd lipids were oserved in ll of the emryos, ut no lrge vritions were noted mong fruits t different stges of mturtion (Tle 1; Figure 3B, D). The results of the in vitro cultivtion of emryos indicte tht ll fruits contined emryos cple of germinting independent of their stte of mturtion (Figure 1H). Nonetheless, the level of fruit mturity did ffect the cpcity of the plntlets to emit roots nd lef sheths. Plntlets otined from emryos from extremely mture or immture fruits showed the highest cpcity to emit lef sheths, while fruits of intermedite ge showed greter cpcity to emit roots (Figure 4). Emryos from fruits in their initil or finl stges of mturtion yielded plntlets; higher cpcity to produce oth lef sheths nd roots. Emryos from the most immture fruits (hving seeds with wter contents of 71.2%) hd the sme cpcity to produce plntlets with lef sheths nd roots s did emryos from seeds from unches with scising fruits. It ws not possile to identify ny definitive tendency in reltion to influence of the stges of mturity of the fruit
(%) ) Silv et l. 449 seed wter content seed oil content mesocrp wter content mesocrp oil content 90 80 y = 66,803x -0,487 R² = 0,9833 70 60 50 40 30 20 10 0 Endosperm immture Exocrp greenish Exocrp rownish Ascission Figure 2. Wter nd oil contents of the mesocrp nd seeds of A. culet tht were removed from fruits with different visul chrcteristics indicting mturity. unches in plntlet development (Figure 5), s dry weights of their structures did not vry gretly in plntlets otined from fruits with greter or lesser degrees of mturity. The unches with seeds hving the lowest wter contents gve rise to seedlings with more developed hustori (except for one unch with fruits hving rown exocrps). No differences were oserved in the weights of the lef sheths mong fruits derived from unches of differing mturities. DISCUSSION The wter content of mcw plm seeds cn e used s n indictor of fruit mturity. The wter content of the mesocrp, however, ws not found to e relted to fruit mturity, s hd een oserved in Buti cpitt (Mrt.) Becc. (Areccee) (Neves et l., 2010). Iossi et l. (2007), on the other hnd, reported tht oth seeds nd the mesocrps of Phoenix roeelenii O Brien (Areccee) fruits undergo significnt reductions in their wter contents during mturtion. Mour et l. (2010) noted the undnce of protein nd lipidic reserves in the endosperms nd emryos of mture A. culet seeds. The high lipidic concentrtions in the mesocrps of fruits in unches tht showed signs of scission, s well s the precocious ccumultion of this sme reserve sustnce in their endosperms is in greement with the results found for Eleis guineensis Jcq. (Cheng et l., 1985; Trnrger et l., 2011). In this ltter species, the oil content of the mesocrp tripled in the three weeks efore scission; the oils found in their seeds lso incresed precociously (nd to greter extent) ner the hlfwy-point of fruit development. Seed development usully involves phses of morphogenesis, mturtion (time during which reserves re ccumulted), nd then desicction (Bewley nd Blck, 1994). The synchrony of emryo development in reltion to the other seed components cn vry, however, resulting in dry seeds with emryos in diverse sttes of development, depending on the species considered (Werker, 1997). In the present study, we determined tht reserve ccumultion occurs reltively erly with mcw plm emryos, indicting tht emryogenesis in this plnt is precocious in reltion to fruit development. Emryos derived from immture fruits, without completely developed endosperms, re cple of producing seedlings with fully formed roots nd lef sheths confirming tht emryogenesis is precocious in A. culet nd tht immture fruits cn e used s sources of emryos for in vitro cultivtion, s ws seen in the plm trees Astrocryum ulei Mrt. (Pereir et l., 2006) nd B. cpitt (Neves et l., 2010). The reduced cpcity of fruits in intermedite stges of development to produce norml seedlings (with lef sheths nd roots), s oserved in the present work, is comptile with reports from other species. A numer of studies hve demonstrted vritions in the levels of scisic cid (ABA) tht restrict germintion during emryo development. ABA is often ssocited with preventing vivipry
450 Afr. J. Biotechnol. Tle 1. Presence nd ccumultion of lipids nd proteins in the mesocrps, endosperms, nd emryos of A. culet s evluted y histochemicl tests from fruits with different seed wter contents. Seed wter content (%) Mesocrp Endosperm Emryo Lipid Protein Lipid Protein Lipid Protein 71.2 + - ++ ++ ++ +++ 44.3 + - ++ ++ ++ +++ 39.1 + - + ++ ++ +++ 29.9 + - ++ ++ +++ +++ 28.7 + - + ++ ++ +++ 28.2 + - ++ ++ +++ +++ 23.2 + - + ++ ++ +++ 21.4 ++ - ++ +++ ++ +++ 21.1 +++ - +++ +++ ++ +++ Figure 3. Histologicl sections of the mesocrp, endosperm, nd emryo of A. culet sumitted to histochemicl nlyses: A) Mesocrp of n scising fruit stined with Sudn IV, indicting n undnce of lipids (red color). B) Endosperm nd emryo of n scising fruit, indicting the undnce of lipids (red color). C) Endosperm of n scising fruit, stined with romophenol lue, indicting the presence of proteins (lue color). D) Emryo of fruit with greenish exocrp, indicting the presence of proteins (lue color). e, emryo; en, endosperm. the emryo is well-developed nd the dehydrtion level of the seed is not sufficient to inhiit germintion (Bewley nd Blck, 1994; Hrtmnn et l., 2002; Nmr et l., 2010). Another possiility is tht vritions of the equilirium etween uxins nd cytokinins tht control orgnogenesis during fruit mturtion cn fvor either root or eril portion development (Tiz nd Zeiger, 2002). Future studies quntifying hormone contents during m-
dry weight (mg) Plntlets with roots nd/or sheths (%) Silv et l. 451 90 80 70 60 50 40 30 20 10 0 c c c root sheth root nd sheth c c cd d cd c y = 0,0525x 4-1,3963x 3 + 13,139x 2-47,311x + 93,607 R² = 0,8928 cd c cd cd cd c c endosperm immture exocrp greenish exocrp rownish scission Figure 4. Percentges of plntlets of A. culet tht emitted roots nd folir lef sheths tht were otined y the in vitro germintion of emryos derived from fruits with different visul chrcteristics indictive of mturtion. The dotted line indictes the tendency to produce plntlets producing oth roots nd lef sheths during the process of fruit mturtion. 10 8 6 cotyledon petiole hustorium root 4 2 0 endosperm immture exocrp greenish exocrp rownish scission Figure 5. Averge dry weights of the petiole cotyledons, hustori, nd roots of plntlets of A. culet otined y the in vitro germintion of emryos derived from fruits hving different visul chrcteristics indictive of mturity. cw plm fruit development could contriute to etter understnding of the fctors controlling the germintive cpcity of emryos during their mturtion. There ws no well-defined effect of fruit unch mturtion on plntlet development s mesured y the verge dry weight of the cotyledon petiole, roots, nd lef sheths; indicting tht the growth cpcity of the plntlets must e hevily influenced y their genotype. Nonetheless, the more mture fruit unches yielded more developed hustori thn most of the others. The hustori function in the sorption of endosperm reserves in the post-germintive period (Orozco-Segovi et l., 2003), nd it is possile tht the developmentl cpcity of this structure increses in fruits pproching scission. Mny plm tree species hve een descried s sho-
452 Afr. J. Biotechnol. wing morphologicl dormncy (Bskin nd Bskin, 1998) relted to the immturity of the emryo (Orozco-Segovi et l., 2003). Evidence presented y Rieiro et l. (2011), however, such s the rpid germintion of oth isolted emryos cultivted in vitro nd of seeds with their operculr tegument removed nd treted with gierellic cid, indictes type of physiologicl dor-mncy in mcw plm seeds. Precocious emryogenesis in reltion to fruit development nd the ility of mcw plm emryos derived from immture fruits to germinte (s demonstrted in the present study) reinforce the supposition tht dormncy in this species is not relted to emryo immturity due to morpho-ntomicl fctors. Form the results of this study, it cn e concluded tht: 1) the wter content of the seeds is ssocited with visul indictors of fruit mturity; 2) the emryos of immture fruits hve ccumulted reserves nd high germintive cpcity, nd cn e used for in vitro propgtion; nd 3) emryogenesis is precocious in reltion to fruit development. ACKNOWLEDGMENTS The uthors would like to thnk the Fundção de Ampro Pesquis do Estdo de Mins Geris for the Bols de Incentivo à Produtividde em Pesquis grnt to L.M. Rieiro nd for the resources from the project PPM (process n 00500-11) received y Q.S. Grci, nd the Conselho Ncionl de Desenvolvimento Científico e Tecnológico (CNPq) for resources from the project MCT/CNPq (process n 560812/2010-8) nd for the Produtividde em Pesquis grnt wrded to Q.S. Grci Pulo. S. N. Lopes thnks CNPq for the technologicl development nd innovtive extension grnt (process numer 313116/2009-1). REFERENCES Assocition of Officil Anlyticl Chemists - Interntionl [AOAC] (2005). Officil Methods of Anlysis. 18ed. AOAC, Githersurg, MD, USA. Bskin CC, Bskin JM (1998). Seeds: ecology, iogeogrphy nd evolution of dormncy nd germintion. Acdemic Press, Sn Diego, CA, USA. Bskin CC, Bskin JM (2004). A clssifiction system for seed dormncy. Seed. Sci. Res. 14: 1-16. Bewley JD, Blck M (1994). 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