Computer-Assisted Recovery of Threatened Plants: Keys for Breaking Seed Dormancy of Eryngium viviparum

ORIGINAL RESEARCH published: 12 Decembe 2017 doi: 10.3389/ pls.2017.02092 F on ie s in Plan Science | www. on ie sin.o g 1Decembe 2017 | Volume 8 | A icle 2092 Edi ed by: Juan Caballe o, Uni e sidad Au ónoma de Que é a o, Mexico Re iewed by: Emiliano Villo do Pineda, Poly echnic Uni e si y o Guanajua o, Mexico Rena o Vicen ini, Uni e sidade Es adual de Campinas, B azil *Co espondence: Ped o P. Gallego [email p o ec ed] Special y sec ion: This a icle was submi ed o Bioin o ma ics and Compu a ional Biology, a sec ion o he jou nal F on ie s in Plan Science Recei ed: 08 Sep embe 2017 Accep ed: 24 No embe 2017 Published: 12 Decembe 2017 Ci a ion: Ayuso M, Ramil-Rego P, Landin M, Gallego PP and Ba eal ME (2017) Compu e -Assis ed Reco e y o Th ea ened Plan s: Keys o B eaking Seed Do mancy o E yngium i ipa um. F on . Plan Sci. 8:2092. doi: 10.3389/ pls.2017.02092 Compu e -Assis ed Reco e y o Th ea ened Plan s: Keys o B eaking Seed Do mancy o E yngium i ipa um Manuel Ayuso1, Pablo Ramil-Rego2, Ma iana Landin3, Ped o P. Gallego1*and M. Es he Ba eal1 1Applied Plan and Soil Biology, Facul y o Biology, Uni e si y o Vigo, Vigo, Spain, 2GI-1934 TB-Biodi e si y, IBADER, Uni e sidad de San iago, Lugo, Spain, 3Depa men o Pha macology, Pha macy and Pha maceu ical Technology, Facul y o Pha macy, Uni e si y o San iago de Compos ela, San iago de Compos ela, Spain Many endange ed plan s such as E yngium i ipa um (Apiaceae) p esen a poo ge mina ion a e. This ac could be due o in insic and ex insic seed a iabili y in luencing ge mina ion and do mancy o seeds. The objec i e o his s udy is o be e unde s and he physiological mechanism o seed la ency and, h ough a i icial in elligence models, o de e mine he ac o s ha s imula e ge mina ion a es o E. i ipa um seeds. This desc ip ion could be essen ial o p e en he disappea ance o endange ed plan s. Ge mina ion in i o was ca ied ou unde di e en do mancy b eaking and incuba ion p ocedu es. Pe cen ages o ge mina ion, iabili y and E:S a io we e calcula ed and seeds we e dissec ed a he end o each assay o desc ibe emb yo de elopmen . The da abase ob ained was modeled using neu o uzzy logic echnology. We ha e ound ha he mos o E yngium seeds (62.6%) we e non- iable seeds ( ully emp y o wi hou emb yos). Excluding hose, we ha e es ablished he ge mina ion condi ions o b eak seed do mancy ha allow ob aining a eal ge mina ion a e o 100%. Ad an ageously, he bes condi ions poin ed ou by neu o uzzy logic model o emb yo g ow h we e he combina ion o 1 mg L−1GA3(Gibbe ellic Acid) and high incuba ion empe a u e and o ge mina ion he combina ion o long incuba ion and sho wa m s a i ica ion pe iods. Ou esul s sugges ha E. i ipa um seeds p esen mo phophysiological do mancy, which educe he a e o ge mina ion. The knowledge p o ided by he neu o uzzy logic model makes possible no jus b eak he physiological componen o do mancy, bu s imula e he emb yo de elopmen inc easing he a e o ge mina ion. Undoub edly, he s a egy de eloped in his wo k can be use ul o eco e o he endange ed plan s by imp o ing hei ge mina ion a e and uni o mi y a o ing hei ex i o conse a ion. Keywo ds: Apiaceae, a i icial in elligence, conse a ion, ge mina ion, do mancy, endemic plan , emb yo seed a io, unde de eloped emb yo Ayuso e al. Compu e -Assis ed Reco e y o Th ea ened Plan s INTRODUCTION E yngium i ipa um is a small biennial aqua ic plan ha belongs o Apiaceae amily, endemic o he Eu opean A lan ic egion and wi h dis ibu ion in NW F ance, NW Po ugal and NW Spain (Rome o e al., 2004). Thei na u al habi a is la a eas subjec ed o seasonal looding, li ing subme ged o 7–9 mon hs o he yea . Aqua ic plan s a e one o he mos h ea ened g oups in he Eu opean lo a, mainly due o an h opic habi a al e a ion and des uc ion (Rome o e al., 2004; Ramil-Rego and Dominguez- Conde, 2006; Magnanon e al., 2012). In 1997, E. i ipa um was classi ied as ulne able by In e na ional Union o Conse a ion o Na u e (IUCN) and included in ed lis o h ea ened plan s (Wal e and Gille , 1998). Mo eo e , E. i ipa um has been lis ed in Annex I o he Be ne Con en ion and conside ed by Di ec i e 92/43/EEC as a p io i y species (Annex II). I s classi ica ion has ecen ly been changed o endange ed due o he loss o many subpopula ions, he d as ic educ ion o he a ea o occupancy (<80 km2in he wo ld) and he dec ease in he quali y o i s habi a and o he numbe o indi iduals (Lansdown, 2011). The main h ea s o endange ed plan s a e human p essu e o e he e i o y, compe i ion wi h o he species, o e g azing and collec ing ha cause deg ada ion, agmen a ion, and massi e educ ion o hei na u al habi a s (Ramil-Rego and Dominguez-Conde, 2006; González-Beni o and Ma ín, 2011; Lansdown, 2011; Magnanon e al., 2012). Al hough high p o ec ion and access es ic ions o hei na u al habi a s ha e been implemen ed, o in si u conse a ion, he si ua ion is c i ical o some species as E. i ipa um. New ex si u conse a ion s a egies, such as in i o p opaga ion a e needed and should be u gen ly designed and implemen ed in o de o p o ec hem. Di e en mic op opaga ion p ocedu es ha e been de eloped o endange ed and endemic plan s in he pas 30 yea s and, also o his pa icula species (Ba-a es e al., 2004; González-Beni o and Ma ín, 2011). The c i ical popula ion size o E. i ipa um does no allow su icien ma e ial om i s na u al habi a o mic op opaga ion. The e o e, Fay (1992) has p oposed i s seeds as he ma e ial o choice o ex si u conse a ion, which allows p ese ing hei biodi e si y. Many species o Apiaceae amily a e well known by a non-uni o m and asynch onized seed ge mina ion (Mozumde and Hossain, 2013), which p omo es ge mina ion a es lowe han 10% and limi s hei in si u conse a ion. Mo eo e , low ge mina ion in Apiaceae is due o se e al kind o do mancy s a e in seeds and by he p esence o high amoun o non- iable seeds (Robinson, 1954; Ojala, 1985). Di e en s a egies ha e been de eloped o o e come do mancy p oblems in se e al Apiaceae, including cold and/o wa m s a i ica ion (Necaje a and Ie insh, 2013), he use o plan g ow h egula o s such as gibbe ellic acid (GA3) and kine in (KIN) (Mozumde and Hossain, 2013) o he incuba ion empe a u e (Finch-Sa age and Leubne -Me zge , 2006). As a as we know no s udies ha e add essed he low ge mina ion a e p oblem in E. i ipa um, which emains a challenge. In ecen yea s, se e al a i icial in elligence ools ha e been applied o model and p edic he e ec o di e en a iables in plan issue cul u e (Zielinska and Kepczynska, 2013), moni o ing seed g ow h and igo (Chaugule, 2012) o es ing ge mina ion (Dell’Aquila, 2004). In his wo k, neu o uzzy logic has been applied o in es iga e he cause-e ec ela ionships be ween se e al ge mina ion ac o s (do mancy b eaking s a i ica ion and ge mina ion condi ions) and seed ge mina ion esponses (pe cen age o ge mina ion, emb yo seed a e, e c.). Neu o uzzy logic is a hyb id app oach ha combines he adap i e lea ning capabili ies om a i icial neu al ne wo ks wi h he gene ali y o ep esen a ion om uzzy logic h ough simple “IF-THEN” ules. This me hodology has been p e iously and success ully used as ad anced decision suppo ool (Gallego e al., 2011; Gago e al., 2014). On his pu pose, his s udy a emp s o ge insigh on he ge mina ion o E. i ipa um seeds and he causes o i s low ge mina ion a e. Neu o uzzy logic is applied o model ge mina ion esul s as a unc ion o se e al ge mina ion condi ions in o de o ind he key ac o s ha con ol o s imula e he emb yo de elopmen , acili a ing he comple ion o ge mina ion. Unde s anding he physiological mechanism esponsible o seed ge mina ion should help o design new p ocedu es o any o he endange ed plan wi h low ge mina ion a es. MATERIALS AND METHODS Plan Ma e ial F ui s (schizoca ps) o E. i ipa um Gay we e collec ed on he ma gin o he Cospei o Lake, Lugo, Spain (43◦14′30.16′′N, 7◦ 32′55.539′′W) in Sep embe 2013 and 2014. Ha es ed ma u e b own ui s we e kep in d y pape bags unde oom labo a o y condi ions un il used. Indi idual me ica ps (seeds) we e ob ained by mechanical ic ion and s o ed d y in plas ic Pe i dishes a 4◦C o 12 weeks in da kness. Ge mina ion Condi ions E yngium i ipa um Gay is an endange ed plan and i s use is legally limi ed, being ex emely di icul o ob ain bo h ui s and/o seeds, he e o e wo ba ches o jus 750 seeds (2013) and 1,440 seeds (2014) we e used in his s udy. The ini ial expe imen s we e adjus ed o maximize he numbe o seeds pe ea men (55–100), whe eas in he ollowing expe imen s he ea men s we e pe o med wi h 80 seeds each (see Table 1). Se e al ea men s ha e been ca ied ou o imp o e E yngium ge mina ion including seed su ace s e iliza ion and s a i ica ion (cold and wa m), o s imula e seed do mancy b eaking and o induce emb yo g ow h and de elopmen . Seed S e iliza ion Seeds we e su ace s e ilized be o e (we s a i ica ion) o a e (d y s a i ica ion) o he do mancy-b eaking ea men s. In bo h cases, seeds we e soaked in 2% sodium hypochlo i e o 5 min. A e , in lamina low cabine , seeds we e washed wi h s e ile dis illed wa e o h ee imes, and s i ed in 50% sul u ic acid o 40 min. Seeds we e emo ed om sul u ic solu ion, washed again o h ee imes du ing 5 min, and soaked o e nigh in s e ile dis illed wa e , p e ious do mancy b eaking and incuba ion expe imen s. F on ie s in Plan Science | www. on ie sin.o g 2Decembe 2017 | Volume 8 | A icle 2092 Ayuso e al. Compu e -Assis ed Reco e y o Th ea ened Plan s TABLE 1 | Design o E yngium seed do mancy-b eaking and incuba ion p ocedu es. Do mancy-b eaking Incuba ion T ea men Seeds (N◦) S a . 4◦C (Weeks) S a . 25◦C (Weeks) Mois u e condi ion GA3 (mg L−1) Subs a e Temp (◦C) GA3 (mg L−1) KIN (mg L−1) Time (weeks) 1 95 8 4 D y 0 MS 24 0 0 20 2 95 8 4 D y 0 MS 24 1 0 20 3 95 8 4 D y 0 MS 24 0 1 20 4 55 8 4 D y 0 MS 18-13 0 0 20 5 55 8 4 D y 0 MS 18-13 1 0 20 6 55 8 4 D y 0 MS 18-13 0 1 20 7 100 8 24 D y 0 MS 18-13 0 0 20 8 100 8 24 D y 0 MS 18-13 0 1 20 9 100 8 24 D y 0 MS 18-13 0 5 20 10 80 0 4 D y 0 Wa e 18-13 0 0 10 11 80 0 4 D y 0 MS 18-13 0 0 10 12 80 0 4 D y 0 MS 18-13 0 1 10 13 80 0 4 D y 0 MS 18-13 0 5 10 14 80 0 4 We 0 Wa e 18-13 0 0 10 15 80 0 4 We 0 MS 18-13 0 0 10 16 80 0 4 We 0 MS 18-13 0 1 10 17 80 0 4 We 0 MS 18-13 0 5 10 18 80 0 4 We 2 Wa e 18-13 0 0 10 19 80 0 4 We 2 MS 18-13 0 0 10 20 80 0 4 We 2 MS 18-13 0 1 10 21 80 0 4 We 2 MS 18-13 0 5 10 22 80 8 0 We 0 Wa e 18-13 0 0 10 23 80 8 0 We 0 MS 18-13 0 0 10 24 80 8 0 We 0 MS 18-13 0 1 10 25 80 8 0 We 2 Wa e 18-13 0 0 10 26 80 8 0 We 2 MS 18-13 0 0 10 27 80 8 0 We 2 MS 18-13 0 1 10 Seed we e p e iously s o ed in da kness a 4◦C du ing 12 weeks (MS, Mu ashige and Skoog medium; GA3, gibbe ellic acid, and KIN, kine in). Do mancy-B eaking P ocedu e On he i s ba ch o seeds, long pe iods o cold s a i ica ion (12 +8 weeks a 4◦C) ollowed by a a iable pe iod o wa m s a i ica ion (4–24 weeks a 25◦C) we e es ed. All seeds (750) we e s a i ied in d y pape il e in Pe i dishes ( ea men s 1–9; Table 1). Fu he , wi h he second ba ch o seeds, also sho e pe iods o cold s a i ica ion (12 +0) and wa m s a i ica ion (0–4 weeks a 25◦C) han in 2013 we e es ed ( ea men s 10–13). We ea men s (wa e o 2 mg L−1GA3solu ions) we e also included ( ea men s 14–27). In hese ea men s, seeds we e su ace s e ilized p e iously. Incuba ion P ocedu e Once comple ed do mancy b eaking p ocedu es, su ace s e ilized seeds we e sowed in s e ilized glass cul u e essels on 25 mL MS medium (Mu ashige and Skoog, 1962) supplemen ed wi h GA3(0 and 1 mg L−1) o KIN (0, 1, and 5 mg L−1) o in plas ic Pe i dishes on double il e pape laye mois ened wi h 20 mL s e ile dis illed wa e (Table 1). S e iliza ion was ca ied in au ocla e a 121◦C o 20 min a 105 kPa. Incuba ion was ca ied ou in g ow h chambe s a 24 o 18– 13◦C he mope iod (simila o he habi a empe a u e) and 12/12 h pho ope iod (Flux densi y o 55 µmol m−2s−1) du ing 10 and 20 weeks. All combina ions a e shown in Table 1. Ge mina ion Tes Ge mina ed and no-ge mina ed seed pe cen age was calcula ed a e 20 o 10 weeks (Table 1) ea men s 1–9 and 10–27, espec i ely. Seeds wi h longe han 1 mm isible adicle we e conside ed as ge mina ed. No-ge mina ed seeds we e dissec ed by i s longi udinal axis and e alua ed using a s e eomic oscope (Nikon SMZ-U). In o de o measu e and calcula e he emb yo and seed leng h a io (E:S a io; Vandelook e al., 2007a), he came a so wa e package (0.7X DXM Lens Nikon) was used. Ano he ba ch o seeds (50 in 2013 and 2014) we e s o ed du ing 12 weeks in da kness a 4◦C, dissec ed and hei a io E:S calcula ed in o de o know he emb yo de elopmen in he momen o s a he ge mina ion ea men s. This E:S a io was used as con ol. Once ge mina ed o dissec ed, seeds we e spli ou in o wo g oups: iable and non- iable seeds. All ge mina ed seeds and F on ie s in Plan Science | www. on ie sin.o g 3Decembe 2017 | Volume 8 | A icle 2092 Ayuso e al. Compu e -Assis ed Reco e y o Th ea ened Plan s hose no ge mina ed bu wi h emb yo isible we e included as “ iable seed” and employed o calcula e he ollowing pa ame e s: -Seed ge mina ion pe cen age: %G =(N◦ge mina ed seeds/N◦ seeds) ∗100 -Real seed ge mina ion pe cen age: %RG =(N◦ge mina ed seeds/N◦ iable seeds) ∗100 -No ge mina ed seeds pe cen age: %NG =(N◦no ge mina ed iable seeds/N◦seeds) ∗100 -E:S a io =Emb yo leng h/Seed leng h. Ge mina ed seeds we e conside ed as E:S =1 and included in he calcula ion a e age E:S a io. Non-ge mina ed seeds we e conside ed as “non- iable,” including emb yoless seeds and hose comple ely emp y. Two new pa ame e s we e calcula ed o he non- iable seeds: -Emb yoless seeds pe cen age: %EL =( o al N◦emb yoless seeds/ o al N◦seeds) ∗100 -Emp y seeds pe cen age: %EM =( o al N◦emp y seeds/ o al N◦seeds) ∗100 S a is ical Analysis S uden - es was pe o med o es he signi ican di e ence (P <0.05) o E:S a io espec o con ol. Neu o uzzy Logic A da abase wi h esul s om 27 ea men s was modeled using he comme cial neu o uzzy logic so wa e, Fo mRules 4.03 (In elligensys L d., UK). Neu o uzzy logic combines he lea ning capabili ies o neu al ne wo ks wi h he linguis ic capabili ies o uzzy logic. Neu o uzzy model allows o model ge mina ion esul s as a unc ion o he se e al ac o s s udied and p edic esul s o a no s udy combina ion o ac o s. Addi ionally, i allows exp essing he model h ough simple IF...THEN ules p o iding unde s anding and knowledge. E e y IF-THEN ule is associa ed o a membe ship deg ee which ep esen s he deg ee o u h om 0 o 1 (Gallego e al., 2011; Gago e al., 2014; Nezami-Alanagh e al., 2014, 2017). Fo he modeling, he inpu s we e he nine ge mina ion a iables s udied du ing do mancy b eaking ( ime o s a i ica ion a 4◦C, ime o s a i ica ion a 25◦C, mois u e condi ion, and GA3concen a ion) and incuba ion (subs a e, empe a u e, GA3concen a ion, KIN concen a ion and ime) and he ou pu s we e he six ge mina ion pa ame e s de ined abo e (%G, %NG, %EL, %EM, %RG, and E:S a io). A sepa a e model was de eloped o each ou pu . The pa ame e s used o modeling by Fo mRules R a e shown in Table 2. RESULTS Seed S e iliza ion Field sampled E. i ipa um seeds p esen ed se e e con amina ion, which was no elimina ed by he p ocedu es desc ibed o o he Apiaceae. Despi e o se e al disin ec ion p ocedu es as e hanol 70% and sodium hypochlo i e 1–2%, E yngium seeds emained highly con amina ed (60–100%). Addi ional s ong ea men s including seed soak in sodium TABLE 2 | The aining pa ame e s se ing wi h Fo mRules 3.31. MINIMIZATION PARAMETERS Ridge eg ession ac o : 1 e−6 MODEL SELECTION CRITERIA S uc u al Risk Minimiza ion (SRM) C1 =0.8–0.854 C2 =4.8 Numbe o se densi ies: 2 Se densi ies: 2. 3 Adap nodes: TRUE Max. inpu s pe SubModel: 4 Max. nodes pe inpu : 15 hypochlo i e 2% o 5 min, long ise in dis illed wa e and s i in 50% sul u ic acid o 40 min we e necessa y o comple ely elimina e con amina ion. Seed Ge mina ion Table 3 p esen s E. i ipa um ge mina ion pe cen ages (%G) o all he condi ions s udied. As i can be seen, he ge mina ion alues a e eally poo o all he ea men s; being he maximum seed ge mina ion pe cen age 27.3% o ea men 2. The highes ge mina ion alues we e eached on ea men s 2, 3, and 8 (>20%G), which sha ed he nex condi ions: long cold d y s a i ica ion pe iod (12 +8 weeks) wi hou GA3and long incuba ion o 20 weeks (Table 1). T ea men s 18, 19, 20, 21, and 25, gi e no ge mina ion a all (Table 3). Seeds on hese ea men s we e s a i ied in we condi ions (wi h 2 mg L−1GA3) and he mope iod o 10 weeks (Table 1). Looking o he easons o hose low ge mina ion a es, no ge mina ed seeds we e dissec ed. As i can be seen in Figu e 1, he images show ha seeds could be classi ied as emp y (A), emb yoless (B), seeds wi h unde de eloped emb yo (C), and wi h ully de eloped emb yo (D). Pe cen ages o no ge mina ed seeds (%NG), emb yoless (%EL), and emp y seeds (%EM) help o explain p e ious ge mina ion pe cen ages (Table 3). The o e all low ge mina ion a e was due o a high numbe o NG seeds (mean 30.4%) and non- iable seeds (mean 62.6%), om which 25.5% whe e emb yoless and 37.0% emp y seeds, espec i ely (Table 3). These esul s sugges ha , only a maximum o 37.4% could be ge mina ed in op imal condi ions (7.1 %G +30.4 %NG). I he ge mina ion pe cen age is ecalcula ed conside ing jus he iable seeds, named as eal seed ge mina ion (%RG; Ma e ials and Me hods) he esul s a e p omising, as some o he ea men s each 89–100% ( ea men s 2 and 4; Table 3). Table 3 also shows he E:S a io ob ained a he end o each ea men . All ea men s ha e alues highe han 0.31 (con ol E:S a io om seeds s o ed du ing 12 weeks a 4◦C) al hough no signi ican di e ences we e ound o some o he ea men s agains con ol (Table 3). This esul s poin ou he impo an ole o some o he condi ions used du ing do mancy–b eaking and incuba ion p ocedu es on emb yo de elopmen and jus i ies he modeling in o de o unde s and which and how hey a ec ge mina ion. F on ie s in Plan Science | www. on ie sin.o g 4Decembe 2017 | Volume 8 | A icle 2092 Ayuso e al. Compu e -Assis ed Reco e y o Th ea ened Plan s TABLE 3 | Pe cen age o ge mina ion (%G), no ge mina ion (%NG), emb yoless seeds (%EL), emp y seeds (%EM), iable (%G+%NG), non- iable (%EL+%EM) seeds, eal ge mina ion (%RG), and mean and s anda d e o o he E:S a io. T ea men %G %NG %EL %EM %Viable %Non- iable %RG E:S a io (±SE) 1 7.1 14.3 28.6 50.0 21.4 78.6 33.3 0.51 (±0.25) 227.3 0.0 45.5 27.3 27.3 72.7 100.0 1.00 (±0.00)* 322.2 33.3 0.0 44.4 55.6 44.4 40.0 0.50 (±0.21) 4 17.8 2.2 11.1 68.9 20.0 80.0 88.9 0.98 (±0.02)* 5 12.8 10.3 23.1 53.8 23.1 76.9 55.6 0.71 (±0.13)* 6 16.0 12.0 8.0 64.0 28.0 72.0 57.1 0.74 (±0.14)* 7 13.6 27.3 8.0 51.1 40.9 59.1 33.3 0.54 (±0.06)* 822.2 20.8 15.3 41.7 43.1 56.9 51.6 0.68 (±0.06)* 9 15.1 19.2 19.2 46.6 34.2 65.8 44.0 0.62 (±0.07)* 10 5.4 39.5 34.0 21.1 44.9 55.1 12.1 0.48 (±0.05)* 11 3.3 36.7 34.0 26.0 40.0 60.0 8.3 0.47 (±0.05)* 12 1.3 34.2 36.2 28.2 35.6 64.4 3.8 0.43 (±0.04)* 13 4.0 28.7 20.7 46.7 32.7 67.3 12.2 0.57 (±0.08)* 14 2.1 37.6 27.0 33.3 39.7 60.3 5.4 0.43 (±0.04)* 15 5.0 43.3 22.7 29.1 48.2 51.8 10.3 0.45 (±0.04)* 16 4.8 32.9 29.5 32.9 37.7 62.3 12.7 0.49 (±0.06)* 17 2.8 40.0 27.6 29.7 42.8 57.2 6.5 0.42 (±0.04)* 18 0.0 35.7 32.2 32.2 35.7 64.3 0.0 0.36 (±0.02) 19 0.0 38.5 29.4 32.2 38.5 61.5 0.0 0.33 (±0.01) 20 0.0 42.8 26.9 30.3 42.8 57.2 0.0 0.35 (±0.01) 21 0.0 42.4 32.6 25.0 42.4 57.6 0.0 0.36 (±0.01)* 22 2.3 32.6 28.0 37.1 34.9 65.1 6.6 0.45 (±0.06)* 23 1.7 28.2 31.6 38.5 29.9 70.1 5.8 0.49 (±0.08)* 24 1.1 39.5 29.4 29.9 40.7 59.3 2.8 0.37 (±0.03) 25 0.0 41.7 30.3 28.0 41.7 58.3 0.0 0.37 (±0.01)* 26 1.7 41.0 28.3 28.9 42.8 57.2 4.1 0.40 (±0.04)* 27 1.1 45.4 30.5 23.0 46.6 53.4 2.5 0.37 (±0.03) Mean 7.1 30.4 25.5 37.0 37.4 62.6 22.1 0.51 (±0.03) T ea men wi h E:S di e en signi ican ly wi h con ol (0.31) a p <0.05 is indica ed (*). FIGURE 1 | Dissec ed E yngium seeds emp y (A), emb yoless (B), wi h unde de eloped emb yo (C) and ully de eloped emb yo (D). Neu o uzzy logic succeeded in simul aneously modeling he six ge mina ion pa ame e s. Table 4 shows he inpu s ha explain he a iabili y o each ou pu oge he wi h he quali y pa ame e s o he models ob ained: co ela ion coe icien s (R2) and ANOVA pa ame e s (calcula ed a io, deg ees o eedom and c i ical o α=0.01). As i can be seen R2- alues anged om 74.85 o 95.92% indica ing high model p edic abili ies. Mo eo e , ANOVA - a io alues we e always highe han he co esponding c i ical alues (α<0.01) indica ing hei accu acy. I is in e es ing o no e se e al gene al ea u es om hese esul s: (a) Only 6 ou o 9 inpu s help o explain he a iabili y o he ou pu s s udied, (b) The ac o ime o F on ie s in Plan Science | www. on ie sin.o g 5Decembe 2017 | Volume 8 | A icle 2092 Ayuso e al. Compu e -Assis ed Reco e y o Th ea ened Plan s TABLE 4 | C i ical ac o s om he neu o uzzy logic T ain R2and ANOVA pa ame e s o aining [ a io, deg ee o eedom (d 1: model and d 2: o al) and c i ical alue o α<0.01] o each ou pu . Ou pu s C i ical ac o s T ain se R2 a io d 1, d 2 c i ical %G Time 79.69 47.0883 2, 26 5.53 %NG Time GA3(I) 74.85 22.8146 3, 26 4.64 %EL Time Temp ×KIN GA3(I) 85.97 20.4282 6, 26 3.59 %EM Time Temp S a . 25◦C GA3(I) 81.32 18.278 5, 26 5.80 %RG S a . 25◦C×Time Temp ×GA3(I) GA3(D-B) 95.92 52.8306 8, 26 3.29 E:S Temp ×GA3(I) Time S a . 25◦C GA3(D-B) 90.39 25.5405 7, 26 3.42 Concen a ion o GA3du ing do mancy b eaking (D-B) o incuba ion (I) pe iod. The inpu s wi h s onge e ec on each ou pu ha e been highligh ed. incuba ion has been highligh ed by he model as impo an o all he pa ame e s, (c) %RG and E:S a e explained by he same se o inpu s including wo om do mancy b eaking phase (S a . 25◦C and GA3) and h ee om incuba ion (Time, GA3and Temp), and (d) The a ia ion o he pa ame e s %NG, %EL, and %EM, which a e associa ed wi h low pe cen ages o ge mina ion, a e explained by he ime o incuba ion and he concen a ion o GA3 du ing he incuba ion pe iod, bu also in ol es o he a iables such as he ime o s a i ica ion a 25◦C o he empe a u e and he concen a ion o KIN du ing incuba ion. IF-THEN ules gene a ed by he neu o uzzy logic so wa e wi h an associa ed membe ship deg ee a e shown in Table 5. All ac o s included in he models we e uzzy ica ed as low o high indica ing a linea e ec on ge mina ion pa ame e s in he ange o he s udy, independen ly i hey ha e a signi ican e ec alone o in in e ac ion wi h o he ac o . Those ules g ea ly simpli y he p ocess o unde s anding he cause-e ec ela ionship among ac o s and ge mina ion pa ame e s. As an example, he pe cen age o ge mina ion a iabili y is explained by jus one ac o : ime o incuba ion (Table 4). IF ime o incuba ion is low, THEN he %G will be low (Rule 1; Table 5). The membe ship 0.93 indica es ha in his condi ion he pe cen age o ge mina ed E yngium seeds p edic ed by he model will be close o he lowes % G alues in he da abase. The e o e, 20 weeks o incuba ion p omo es highe ge mina ion pe cen ages han 10 weeks, as i is o ea men s 2, 3, and 8 wi h he highes %G (highligh ed in Table 3). On he con a y, he %NG a iabili y is explained by wo ac o s: ime and concen a ion o GA3du ing he incuba ion pe iod (Table 4). Neu o uzzy logic highligh s he key ole o incuba ion ime bu logically, in he opposi e way han o %G (Table 5): i low incuba ion ime is used, he pe cen age o no ge mina ion o E yngium seeds is high ( ule 3; membe ship 0.90) and i he ime o incuba ion is high hen %NG is low ( ule 4; 0.95). In he same sense i he plan g ow h egula o GA3 is p esen in he incuba ion solu ion (1 mgL−1) hen he %NG will be low. These ules explain o example, he high alue o %G oge he wi h low alue o %NG ob ained o ea men 2 (Table 3). Two kinds o non- iable seeds we e de ec ed: emb yoless bu wi h endospe m (EL; Figu e 1B) o wi hou endospe m (EM; Figu e 1A). The pe cen age o bo h in E yngium seeds is de e mined by incuba ion ime, bu in a di e en way (Table 5). While a long pe iod o incuba ion p omo es always low emb yoless seeds pe cen ages ( ule 8; membe ship 1.0), also p omo es high pe cen ages o emp y seeds ( ule 16; 1.0). The in e ac ion o high empe a u e and high kine in concen a ion gi es low emb yoless seeds pe cen ages ( ule 14; 1.0), as can be seen when compa ed ea men s 3 (24◦C) s. 12 (18–13◦C) in Table 3. The p esence (high) o GA3du ing incuba ion ( ules 10 and 22) also educes he %EL and %EM, in ag eemen wi h he esul s ob ained o %NG ( ule 6). The pe cen age o eal seed ge mina ion (RG) a iabili y can be explained by a selec ion o i e inpu s ( he in e ac ion o ime o s a i ica ion a 25◦C and ime o incuba ion, he in e ac ion o empe a u e and concen a ion o GA3du ing incuba ion and he concen a ion o GA3du ing do mancy b eaking p ocess). F om he ules co esponding o RG pa ame e (Table 5) i is easy o deduce ha ; (a) a sho pe iod o wa m s a i ica ion (low) combined o a long pe iod o incuba ion (high weeks) ende ed, always, high %RG ( ule 25; membe ship 1.0); (b) high empe a u e in combina ion wi h 1 mg L−1GA3du ing incuba ion also p omo es high % eal ge mina ion ( ule 30; 1.0); and (c) he addi ion o GA3du ing do mancy b eaking pe iod, ully inhibi s E yngium seed ge mina ion ( ules 31–32). All his condi ions can be obse ed in ea men 2 (Table 1) wi h high %RG (100%; Table 3). High E:S a io a o seed ge mina ion. Neu o uzzy poin ed ou , ha he in e ac ion high incuba ion empe a u e and 1 mg L−1GA3p omo es emb yo g ow h ( ule 38; membe ship 1.0) as in ea men 2 (Table 3). Long pe iods o incuba ion ( ule 34; 1.0) oge he wi h sho wa m s a i ica ion pe iods ( ule 39; 0.96) imp o e E:S (Table 5). On he con a y, he p esence o GA3in he we solu ion du ing he b eak do mancy p ocedu e educe E:S a io ( ule 42; 1.0). These esul s a e in ully ag eemen wi h hose desc ibed o %RG. DISCUSSION Acco ding o li e a u e e iew, no disin ec ion o gene al an isep ic p ocedu es such as e hanol 70% o 30s ollowed by sodium hypochlo i e a low concen a ion 1% o 5–10 min we e enough o su ace seed s e iliza ion in Apiaceae amily (Walmsley and Da y, 1997; Vandelook e al., 2008; Thiem e al., 2013). Those ypical p ocedu es did no wo k a all o E. i ipa um seeds. All seeds e ealed se e e con amina ion by F on ie s in Plan Science | www. on ie sin.o g 6Decembe 2017 | Volume 8 | A icle 2092 Ayuso e al. Compu e -Assis ed Reco e y o Th ea ened Plan s TABLE 5 | Rules gene a ed by neu o uzzy logic. Rules S a . 25◦C GA3(D-B) Temp GA3(I) KIN Time G NG EL EM RG E:S Membe ship deg ee 1 IF Low THEN Low 0.93 2 High High 0.63 3IF Low THEN High 0.90 4High Low 0.95 5 Low High 0.77 6 High Low 0.82 7 IF Low THEN High 0.54 8 High Low 1.00 9 Low Low 1.00 10 High Low 0.61 11 Low Low High 1.00 12 Low High High 1.00 13 High Low High 1.00 14 High High Low 1.00 15 IF Low THEN Low 1.00 16 High High 1.00 17 Low High 0.89 18 High Low 1.00 19 Low High 0.98 20 High Low 1.00 21 Low High 0.67 22 High Low 1.00 23 IF Low Low THEN Low 1.00 24 High Low Low 1.00 25 Low High High 1.00 26 High High Low 0.59 27 Low Low Low 0.61 28 High Low Low 1.00 29 Low High Low 1.00 30 HIgh High High 1.00 31 Low Low 0.51 32 High Low 0.71 33 IF Low THEN Low 1.00 34 High High 1.00 35 Low Low High 0.60 36 High Low Low 1.00 37 Low High Low 1.00 38 High High High 1.00 39 Low High 0.96 40 High Low 1.00 41 Low Low 0.54 42 High Low 1.00 The inpu s wi h he highes membe ship deg ee (s onge e ec ) on each ou pu indica es by he model a e highligh ed. ungi du ing ge mina ion and none we e able o ge mina e. Only a e applica ion o s ong an isep ic p ocedu es including sul u ic acid, a me hod commonly used o su ace-s e ilize seeds hea ily con amina ed wi h ungi (La chs and Ch is ensen, 1985; Siegel e al., 1987), we succeeded in achie ing he o al elimina ion o ungal con amina ion. Poo ge mina ion a e is a common p oblem in he Apiaceae amily (Robinson, 1954; Ojala, 1985; Baskin and Baskin, 2014). F on ie s in Plan Science | www. on ie sin.o g 7Decembe 2017 | Volume 8 | A icle 2092 Ayuso e al. Compu e -Assis ed Reco e y o Th ea ened Plan s E yngium genus is no an excep ion. E yngium ma i imum (Walmsley and Da y, 1997; Necaje a and Ie insh, 2013), E yngium planum (Thiem e al., 2013) and E yngium oe idum (Mozumde e al., 2011; Mozumde and Hossain, 2013), ha e shown lowe han 20, 12, and 10% ge mina ion espec i ely, wi hou do mancy b eaking ea men s. Li le in o ma ion on E yngium i ipa um is a ailable. Ou esul s anging om 0 o 27% o ge mina ion a e in ag eemen wi h Magnanon and cowo ke s ha epo ed low ge mina ion pe cen ages (10–40%) on popula ions om F ance and Spain (Magnanon e al., 2012). Low ge mina ion a es in he Apiaceae amily ha e been co ela ed wi h he nex h ee causes (Robinson, 1954; Ojala, 1985; Baskin and Baskin, 2014): (1) p esence o high pe cen age o non- iable seeds wi hou emb yo; (2) p esence o high pe cen age o seed wi h unde de eloped emb yos; and (3) p esence o do man seeds. In ag eemen wi h he i s cause ou esul s showed a high non- iable seeds pe cen age (62.5%) close o o he Apiaceae as Ane hum g a eolens ( anged 39–62%; Robinson, 1954) o An h iscus caucalis (49%; Rawnsley e al., 2002). Among he po en ial causes o non- iable seeds (zygo e degene a ion, dea h o emb yo, mu a ions, e c.; Baskin and Baskin, 2014), wo ha e been desc ibed o Apiaceae amily: (1) insec in es a ion; hese seeds ha e an endospe m bu no emb yo (Flemion and Hen ickson, 1949) and (2) sel -pollina ed umbels; seeds p oduced in hese umbels a e usually emp y (wi hou endospe m and emb yo; Ojala, 1985). In his wo k, he high pe cen age o non- iable seeds ha e been desc ibed as emb yoless (25.5% EL) and emp y (37% EM), suppo ing he hypo hesis ha he poo ge mina ion pe cen age o E. i ipa um seeds can be explained as a consequence o he high pe cen age o non- iable seeds due o insec in es a ion and sel -pollina ed umbels. The second cause o low ge mina ion in Apiaceae is he p esence o unde de eloped emb yos a he momen o dispe sal (Ma in, 1946). The emb yo needs o g ow up o a c i ical leng h be o e ge mina ion. E:S a io was in es iga ed o check he p esence o unde de eloped emb yos in E. i ipa um (Table 3). The E:S a io seeds s o ed du ing 12 weeks a 4◦C in da kness, used as con ol, a e aged 0.31, simila o hose alues ound o o he Apiaceae species as To ilis japonica wi h a a io 0.25, Angelica syl es is wi h 0.29, o Selinum ca i olia wi h 0.31 a ha es . Howe e , a e 20 weeks a 5◦C, hose emb yos g ow h un il 0.29, 0.39, and 0.34, espec i ely, a li le highe han he E:S a io a ha es (Vandelook e al., 2007a, 2008). This delay in seed ge mina ion due o he unde de eloped and di e en ia ed emb yo is called mo phology do mancy (MD, Nikolae a, 1977), and hose Apiaceae has been cha ac e ized as MD seed (Vandelook e al., 2007a,b, 2008). Ou esul s, clea ly demons a ed a high pe cen age 30.4% o unde de eloped emb yo seeds sugges ing ha E. i ipa um also showed MD do mancy, in ag eemen wi h o he E yngium sp. (Necaje a and Ie insh, 2013). Seeds wi h MD, as Apiaceae, need speci ic condi ions as a mois subs a e, sui able empe a u e and pho ope iod as g ow h emb yo equi emen s (Baskin and Baskin, 2014). I seeds emain unde hese condi ions, emb yos should g ow h and ge mina e in 4 weeks o less. No p ecise p o ocol o o e come his p oblem ( he need o igh condi ions enabling emb yo g ow h and de elopmen un il each a c i ical size o ge mina e) has been p e iously desc ibed in he li e a u e o E. i ipa um. The hi d cause o low ge mina ion a es in Apiaceae is he exis ence o an addi ional physiological mechanism o do mancy, which also inhibi s ge mina ion (Baskin and Baskin, 2004). Some Apiaceae p esen bo h do mancies mo phological (MD) and physiological (PD) a he same ime, known as mo phophysiological do mancy (MPD), and need a conside ably longe pe iod o ge mina e han MD seeds (Baskin and Baskin, 2004). The combina ion o cold and wa m s a i ica ion can be e ec i e o b eak MPD do mancy and allow he emb yo o g ow h. Wa m s a i ica ion was e ec i e in some species o Osmo hiza and E y h onium genus (Baskin e al., 1995). In addi ion, sho wa m s a i ica ion inc eases he seed sensi i i y o GA3imp o ing ge mina ion a e in E. ma i imum and sugges ing a key ole o GA3 o o e come he do mancy (Vandelook e al., 2008; Necaje a and Ie insh, 2013). E. i ipa um p esen s low pe cen age o seeds (mean 7.1%) ha ge mina e a e b eaking he MPD. In e es ing, some ea men , such as 2, eached 27.3%G and 100%RG, which means ha all iable seeds we e able o b eak MPD do mancy and ge mina e. To unde s and he physiological mechanism o do mancy b eaking, a ep esen a i e s udy o seed ge mina ion equi e es se e al ac o s, which implies many di e en ea men s and eplica es, and inally la ge ba ches o seeds. Those equi emen s a e essen ial o an accu a e in e p e a ion o he cause-e ec o hose ac o s and hei in e ac ions on seed ge mina ion (In e na ional Seed Tes ing Associa ion, 1985). As s a ed abo e, E. i ipa um is an endange ed species and he e o e, no la ge seed ba ches a e a ailable. Based on hese limi a ions, wo expe imen s including jus 27 ea men s we e p oposed. We es he mos impo an ac o s well known as do mancy b eaking in Apiaceae such as cold and wa m s a i ica ion and wo PGR (GA3and KIN) well known as induce s o emb yo de elopmen (Vandelook e al., 2008; Mozumde and Hossain, 2013; Necaje a and Ie insh, 2013). The au ho s a e awa e ha he design space was no well-sampled, which hinde s a con en ional s a is ical ea men o analyse he esul s and d aw conclusions. Neu al ne wo ks has demons a ed o be a p ac ical app oach o deciphe ing he key ac o s in se e al biological p ocess and an excellen al e na i e o con en ional s a is ical me hods (Gago e al., 2010a,b; Gallego e al., 2011; Nezami-Alanagh e al., 2014; A ab e al., 2016). Ad an ageously, neu o uzzy logic echnology allows wo king wi h no well- de ined design spaces and di e en kind da a a he same ime (Nezami-Alanagh e al., 2017). In his pape , ha echnology was used o educe he numbe o ea men s and he e o e, he needed seeds, wi hou losing key in o ma ion o ex ac scien i ic conclusions. Neu o uzzy logic succeeded in modeling all ou pu s simul aneously wi h high p edic abili y and accu acy (Table 3). Addi ionally, i allowed ob aining a se o ules ha explain he cause-e ec among he ac o s (inpu s) and he ge mina ion pa ame e s ob ained (Table 4). The model pinpoin ed ha he F on ie s in Plan Science | www. on ie sin.o g 8Decembe 2017 | Volume 8 | A icle 2092 Ayuso e al. Compu e -Assis ed Reco e y o Th ea ened Plan s ac o ime o incuba ion was he mos signi ican ac o o E yngium seed ge mina ion; long pe iods (a ound 20 weeks) a e s ongly ecommended o ob ain high %G, low %NG, low %EL, high E:S, and high %RG, bu in his case in combina ion wi h low wa m s a i ica ion. In ac only a e 20 weeks o incuba ion he highes eal ge mina ion a es can be eached (see ea men 2 and 4; Table 3). In e es ing, also wi h high incuba ion pe iods a high pe cen age o emp y seeds was ob ained, sugges ing a high co ela ion be ween endospe m deg ada ion and du a ion o he ge mina ion pe iod. The model also iden i ied he impo an ole o he in e ac ion be ween incuba ion empe a u e and GA3concen a ion. I empe a u e is a ound 24◦C and 1 mg L−1GA3is p esen , he highes eal ge mina ion and E:S was ob ained. The hi d mos impo an ac o was he ac o s a i ica ion a 25◦C. Low pe iod o wa m s a i ica ion (4 weeks) is p e e ed o ob ain he highes % o eal ge mina ion and E:S a io (1.00 membe ship). Some au ho s (Necaje a and Ie insh, 2013) demons a ed ha seed o E. ma i imum seed ge mina ed o a high inal ge mina ion pe cen age only a e 12–16 weeks cold s a i ica ion. Ou model sugges s ha E. i ipa um p esen s seeds wi h MDP, and needs o b eak do mancy. Long incuba ion ime (20 weeks) combined wi h low wa m s a i ica ion (4 weeks) a con inuous high empe a u e 24◦C and 1 mg L−1GA3a e s ongly ecommended. AUTHOR CONTRIBUTIONS MA: Pe o med he expe imen s; PR-R: Con ibu ed wi h eagen s/ma e ials; ML and PG: Con ibu ed modeling/analysis ools; PG and MB: Concei ed and designed he expe imen s. All au ho s con ibu ed o w i ing o he manusc ip . FUNDING This esea ch was suppo ed/pa ially suppo ed by TREMEDAL—Inland we lands o No he n Ibe ian Peninsula: managemen and es o a ion o mi es and we en i onmen s Eu opean Union (LIFE11 NAT/ES/000707, 2012–2015) and “Red de Uso Sos enible de Recu sos y Residuos” unded by XUNTA DE GALICIA (R2014/019). ACKNOWLEDGMENTS We hank o Ms Vale ia Ribe a who p o ided insigh and expe ise o his pape . REFERENCES A ab, M. M., Yadollahi, A., Shojaeiyan, A., and Ahmadi, H. (2016). A i icial neu al ne wo k gene ic algo i hm as powe ul ool o p edic and op imize in i o p oli e a ion mine al medium o G ×N15 oo s ock. F on .Plan Sci. 7:1526. doi: 10.3389/ pls.2016.01526 Ba-a es, A., Blanca, G., Güemes, J., Mo eno, J., and O iz, S. (2004). 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