Somatic Embryogenesis and Plant Regeneration From Primordial Shoot Explants of Picea abies (L.) H. Karst. Somatic Trees

pls-09-01551 Oc obe 22, 2018 Time: 14:35 # 1 ORIGINAL RESEARCH published: 24 Oc obe 2018 doi: 10.3389/ pls.2018.01551 Edi ed by: Paloma Moncaleán, NEIKER-Tecnalia, Spain Re iewed by: Ta as P. Pas e nak, Albe -Ludwigs-Uni e si ä F eibu g, Ge many Jan Max Bonga, Canadian Fo es Se ice, Canada *Co espondence: Saila Va is [email p o ec ed] Special y sec ion: This a icle was submi ed o Plan E olu ion and De elopmen , a sec ion o he jou nal F on ie s in Plan Science Recei ed: 04 July 2018 Accep ed: 03 Oc obe 2018 Published: 24 Oc obe 2018 Ci a ion: Va is S, Klimaszewska K and A onen T (2018) Soma ic Emb yogenesis and Plan Regene a ion F om P imo dial Shoo Explan s o Picea abies (L.) H. Ka s . Soma ic T ees. F on . Plan Sci. 9:1551. doi: 10.3389/ pls.2018.01551 Soma ic Emb yogenesis and Plan Regene a ion F om P imo dial Shoo Explan s o Picea abies (L.) H. Ka s . Soma ic T ees Saila Va is1*, K ys yna Klimaszewska2and Tuija A onen1 1Na u al Resou ces Ins i u e Finland (Luke), Sa onlinna, Finland, 2Na u al Resou ces Canada, Canadian Fo es Se ice, Lau en ian Fo es y Cen e, Quebec, QC, Canada The ecalci ance o adul coni e issues has p e en ed ege a i e p opaga ion o ees wi h known and desi ed cha ac e is ics. Soma ic emb yogenesis (SE) ini ia ion p o ocol, ecen ly de eloped o whi e sp uce (Picea glauca,Klimaszewska e al., 2011), was applied in o de o examine he easibili y, equency and iming o SE induc ion om p imo dial shoo s (PS) o No way sp uce (P. abies). In o al, 39 geno ypes we e sc eened om 2015 o 2017 using 4–6 yea s old ees o SE o igin as explan dono s. Two geno ypes esponded: 11Pa3794 p oduced six p oli e a ing emb yonal mass (EM) sublines and 11Pa4066 p oduced 23 EM sublines. SE ini ia ions occu ed a he beginning o Ap il, when he empe a u e sum (d.d.) s a ed o accumula e, and a he end o Oc obe o beginning o No embe when he chilling uni (ch.u.) sum was o e 500. EM sublines om bo h geno ypes con ained nume ous ea ly soma ic emb yos as de ec ed by ace oca mine s aining. The sublines o 11Pa4066 p oduced he mean o 78.6 ±12.8 co yledona y soma ic emb yos /g FW, bu 11Pa3794 p oduced only a ew co yledona y soma ic emb yos ha we e able o ge mina e. The o iginal EM lines ( om which he ees we e egene a ed) had p oduced he same numbe o soma ic emb yos in 2011 ma u a ions, which was app oxima ely 120 soma ic emb yos /g FW. Mic osa elli e analyses conduc ed wi h bo h esponsi e geno ypes con i med he gene ic s abili y o he EM sublines compa ed wi h he dono ees g owing in he ield. SE p o ocol de eloped o whi e sp uce PS explan s was also sui able o PS o No way sp uce i he explan s we e in he esponsi e de elopmen al s age. Keywo ds: clonal ees, coni e , No way sp uce, ecalci ance, shoo buds INTRODUCTION Vege a i e, i.e., asexual p opaga ion enables p oduc ion o plan s o uni o m quali y and wi h known, selec ed cha ac e s. Soma ic emb yogenesis (SE) has become he me hod o choice o ege a i e p opaga ion o coni e s (Su on, 2002) due o i s high mul iplica ion a e and he main enance o ju enili y o cell lines ia c yop ese a ion. O he No dic coni e s, SE is cu en ly he mos de eloped in No way (Picea abies (L.) Ka s .) and whi e sp uce (P. glauca (Moench) Voss) (Lelu-Wal e e al., 2013;Adams e al., 2016;Högbe g and Va is, 2016). In he case o No way sp uce, de elopmen o cos -e icien ege a i e p opaga ion echniques is especially impo an , F on ie s in Plan Science | www. on ie sin.o g 1Oc obe 2018 | Volume 9 | A icle 1551 pls-09-01551 Oc obe 22, 2018 Time: 14:35 # 2 Va is e al. Soma ic Emb yogenesis F om Sp uce P imo dial Shoo s since he e is sho age o high-quali y, b ed o es egene a ion ma e ials due o i egula lowe ing o he species, as well as pes and pa hogen p oblems hinde ing seed p oduc ion in he seed o cha ds. The majo d awback o he cu en SE me hodology has been ha emb yogenic cul u es can only be ini ia ed om ju enile plan explan s, in p ac ice om zygo ic emb yos, meaning ha ma u e ees wi h known cha ac e is ics canno be p opaga ed ia SE (Bonga e al., 2010). Ini ia ion o SE om ma u e coni e s would p o ide a sho cu o p oduc ion o plan ing ma e ial om selec ed ees wi h known and desi able ai s. In combina ion wi h cos -e icien mass p opaga ion, his would ha e an eno mous impac on o es y wi h subsequen ly inc eased p oduc i i y and/o p oduc ion o ailo ed aw ma e ials o special end-uses and also o landscaping and Ch is mas ee p oduc ion. To s udy he ecalci ance p oblem, Bonga e al. (2010) sugges ed he use o explan s aken om ma u e ees o soma ic emb yo o igin which po en ially could be mo e esponsi e in issue cul u e. This app oach was ollowed wi h whi e sp uce (P. glauca (Moench) Voss) whe eby p imo dial shoo s (PS) excised om ege a i e buds o 10-yea -old (in 2010) and 17- yea -old (in 2017) soma ic ees p oduced soma ic emb yos ha con e ed o plan s (Klimaszewska e al., 2011;Klimaszewska and Ru ledge, 2016, and Klimaszewska e al., unpublished). Some dono ees in he abo e s udy had gone h ough a phase change om ju enile o ma u e, i.e., lowe ing phase and s ill esponded o SE induc ion ea men . The de elopmen al s age o an explan is c i ical o he ou come o in i o p opaga ion, and o en he ime window o he posi i e esponse has been sho (Selby and Ha ey, 1985;Mon euuis e al., 2010;Bonga, 2017). In whi e sp uce, posi i e esponse o SE induc ions om PS was ob ained bo h in he sp ing and la e summe / ea ly au umn, and he bes esponse was om induc ions made in la e Ap il o ea ly May (Klimaszewska e al., 2011). Bonga (2017) sugges ed ha du ing swi ches in he de elopmen al p og am, like in he ege a i e buds om do mancy o bud b eak, issues may be mo e ac i e mo phogene ically and acqui e he p opensi y o SE. Do mancy has been di ided o di e en physiological phases: a e g ow h cessa ion, he buds en e endodo mancy, which changes o ecodo mancy unde he in luence o chilling empe a u es du ing he au umn (Lang e al., 1987). Du ing ecodo mancy, he shoo buds a e capable o de elopmen , bu low empe a u es du ing he win e hinde hei g ow h onse un il empe a u e ises and days ge longe (Su inen e al., 2012 and e e ences he ein). In No way sp uce, de ining he in e nal de elopmen al s ages o he PS is impossible by obse ing he shoo buds ex e nally, hus he s aging has been made based on compa isons o longi udinally cu buds and he empe a u e (d.d.) and chilling uni (ch.u.) sums (Su inen e al., 2012;Vihe ä- Aa nio e al., 2014). Also he pho ope iod plays a ole in he egula ion o g ow h cessa ion and shoo bud se phenology. The aim o his s udy was o examine he easibili y and equency o SE induc ion om PS o No way sp uce. We used ou o six yea s old ees o SE o igin as dono s, and es ed explan s om clonal ees o 39 geno ypes in 2015–2018. The bes iming o explan collec ion was de e mined by compa ing he posi i e SE ini ia ion esul s wi h empe a u e da a. The gene ic s abili y o emb yonal mass (EM) sublines was examined wi h mic osa elli e ma ke s. MATERIALS AND METHODS Dono T ees No way sp uce SE ees g owing in he expe imen al plan a ion, a Punkaha ju, Finland (61◦4800900N, 029◦1805800E) we e chosen o he ege a i e bud collec ions (Figu es 1A,B). The ees we e p oduced om EMs ini ia ed in 2011 using imma u e o ma u e zygo ic emb yos o igina ing om ull-sib seed amilies be ween supe io ees wi hin Finnish T ee B eeding P og am. The c osses we e made in a g a ed seed o cha d si ua ed in sou he n Finland, and he geno ypes also o igina ed om sou he n Finland. P oduc ion o he SE ees was conduc ed by applying he me hods de eloped by Klimaszewska e al. (2001) and Lelu-Wal e e al. (2008), as desc ibed in Va is e al. (2017). Ma u a ions o soma ic emb yos we e pe o med se e al imes be ween Oc obe 2011 and Feb ua y 2012. Ge mina ions o he co yledona y soma ic emb yos we e ca ied ou in Decembe 2011 un il May 2012. The plan a ion o he soma ic ees was es ablished on a e ile, mounded o es si e in May and June 2014 wi h 280 geno ypes and 10 o 40 clonal ees pe each geno ype. No e iliza ion was applied o soma ic ees unde ield condi ions. Mechanical weeding o he plan a ion was pe o med annually. Shoo Bud Collec ions, Explan P epa a ion and SE Ini ia ions Shoo bud collec ion and SE induc ion p o ocol om PS explan s we e he same as de eloped o whi e sp uce (Klimaszewska e al., 2011). La e al buds (Figu e 1C) we e emo ed mainly om b anches o he uppe pa o he ees whe e buds we e well de eloped, abundan and no co e ed by snow and ice, bu occasionally also om b anches o he lowe pa . A e e y collec ion ime, a minimum o 40 la e al buds we e collec ed pe geno ype om se e al clonal ees, and in o al 39 geno ypes we e es ed (Table 1). Following collec ion, he buds we e s o ed in plas ic ubes a +4◦C o 0 o 4 days be o e being used in he expe imen s. In he sp ing 2015, buds we e collec ed om 25 geno ypes be ween Ma ch 19 and Ap il 21 (Table 1). In he sp ing 2016, collec ion s a ed on Ap il 1 and he las collec ion was done on Ap il 27. Nine new geno ypes we e in oduced and one esponsi e geno ype (11Pa3794) om p e ious yea was collec ed o he second ime. In he all 2016, one geno ype (11Pa4066) ha had been esponsi e in he sp ing was collec ed again on Oc obe 25, and also one new geno ype was collec ed on he same day. Buds o 11Pa4066 we e collec ed om ou g oups o clonal ees: (1) om he same 10 ees as in he sp ing, (2) om 10 ees g owing in a di e en pa o he plan a ion: (a) i e ees om he ex eme igh ow and (b) i e ees om he ex eme le ow, and (3) om one ee g owing a he edge o he plan a ion which was a bo de ee. F on ie s in Plan Science | www. on ie sin.o g 2Oc obe 2018 | Volume 9 | A icle 1551 pls-09-01551 Oc obe 22, 2018 Time: 14:35 # 3 Va is e al. Soma ic Emb yogenesis F om Sp uce P imo dial Shoo s FIGURE 1 | Bud collec ion om soma ic No way sp uce ees and p imo dial shoo (PS) excision. (A) No way sp uce soma ic ees g owing in an expe imen al plan a ion a Punkaha ju, Finland in he sp ing 2018. (B) Fou yea s old soma ic ee a he i s shoo bud collec ion on Ap il 1, 2016. (C) Shoo buds be o e emo al o he ou e mos scales and disin ec ion. (D) Shoo bud cu longi udinally. (E) Qua e s o he PS be o e being placed on he cul u e medium. Ba s: (A) = 1 m, (B) = 50 mm, (C) = 10 mm, (D) =1mm,(E) = 0.5 mm. TABLE 1 | No way sp uce shoo bud collec ion da es, numbe o geno ypes es ed, esponding geno ypes and he numbe o EM sublines. Collec ion da es Numbe o es ed geno ypes, new/ epea ed Responding geno ypes Responding PS EM sublines p oli e a ing EM sublines o ming ma u e SE SE / g FW EM sublines o ming plan s 19 – 29 Ma ch 2015 9/0 0 12 Ap il 2015 4/0 11Pa3794 1 1 1 1.6 (11.2∗) 0 17 – 22 Ap il 2015 12/0 0 1 Ap il 2016 3/0 0 8 Ap il 2016 3/0 11Pa4066 6 17 16 10.7 – 171.3 9 19 – 27 Ap il 2016 3/1 0 25 Oc obe 2016 1/1 11Pa4066 4 6 6 39.9 – 142.5 6 11 Ap il 2017 2/0 0 18 Ap il 2017 2/2 11Pa3794 3 2 1 2.2 0 7 No embe 2017 0/1 11Pa3794 5 2 1 2.1 (15.9∗)∗∗ ∗EM g own wi h nu se issue; ∗∗Plan s a e s ill being acclima ized. SE / g FW ep esen s ange among sublines. In 2017, buds we e collec ed on Ap il 11 and 18 om ou new geno ypes and om 11Pa3794 on Ap il 18 and No embe 8. In he la e collec ion, he buds o 11Pa3794 we e aken om clonal ees g owing in wo di e en ows: 40 buds om i e ees om each he igh and le ow. Be o e he disin ec ion o he shoo buds, he ou e mos scales we e emo ed and hey we e placed in a 50 ml cen i uge ube, up o 25–30 buds pe ube. The disin ec ion s a ed by shaking he buds in 94% e hanol o 1 min ollowed by washing in ap wa e wi h small amoun o Tween-20 o 6 min and hen by shaking in 70% e hanol o 2 min, ollowed by shaking in 10% ( / ) hyd ogen pe oxide o 8 min. A e insing h ee imes in s e ile wa e , he buds we e placed in a Pe i dish on a il e pape mois ened wi h 100 mg l−1poly inylpy olidone (PVP) solu ion. Buds we e cu leng hwise, he wo pa s o PS we e excised and, depending on hei size, hey we e cu leng hwise again in o wo o mo e pa s as desc ibed in Klimaszewska e al., 2011 (Figu es 1D,E). The sec ions o he PS we e placed on he su ace o he semi-solid MLV medium (Li ay e al., 1985, modi ied as in Klimaszewska e al., 2001). The e we e sec ions o ou PS cul u ed in one Pe i dish (90 mm ×15 mm). The medium was supplemen ed wi h 9.5 µM 2,4-dichlo ophenoxyace ic acid (2,4-D) and 4.5 µM 6-benzyladenine (BA). The cul u es we e placed a 24◦C in da kness and we e inspec ed once a week o con amina ions and/o ini ia ions o EM un il he explan s and calli became nec o ic. When he g ow h o EM was clea ly isible i was subcul u ed on o esh medium sepa a ely om each sec ion o a PS and conside ed a subline (Figu es 2A,B,D,E). EM we e subcul u ed a 14-day in e als. Mic oscopic Obse a ion, Nu se Tissue Co-cul u e Expe imen , Ma u a ion o Ea ly Soma ic Emb yos and Con e sion o Plan s To de ec whe he SE was ini ia ed, samples o induced issues we e s ained wi h ace oca mine acco ding o Gup a and Du zan (1987) and obse ed unde he mic oscope (Figu es 2C,F). To enhance soma ic emb yo p oduc ion, he EM subline o 11Pa3794 (induced in 2015) was used in he expe imen wi h he nu se emb yogenic issue (Wes co , 1994). The nu se issue was o 14Pa4623 geno ype induced om a zygo ic emb yo, which consis en ly p oduced la ge numbe s o ma u e soma ic emb yos. Nu se issue clumps we e posi ioned ei he a he ci cum e ence o he Pe i dish (90 mm×15 mm, con aining 20 ml o MLV p oli e a ion medium) su ounding he clumps o 11Pa3794 placed in he cen e o a Pe i dish o ice e sa. The e we e eigh o en clumps a he ci cum e ence and i e o se en clumps in he cen e , espec i ely. Nu se issue expe imen las ed 4 weeks un il he subline was ma u ed in No embe 2015. Simila ly, wo o he EM sublines, induced in F on ie s in Plan Science | www. on ie sin.o g 3Oc obe 2018 | Volume 9 | A icle 1551 pls-09-01551 Oc obe 22, 2018 Time: 14:35 # 4 Va is e al. Soma ic Emb yogenesis F om Sp uce P imo dial Shoo s No embe 2017, we e co-cul u ed wi h nu se issue o 15Pa1029 (Figu e 3A). The ma u a ion o soma ic emb yos and con e sion o plan s we e ca ied ou acco ding o a sligh ly modi ied p o ocol ha was de eloped o zygo ic emb yo SE. EM sublines induced in au umn 2016 and la e we e ma u ed wi h educed concen a ion o abscisic acid (ABA), 30 µM, ins ead o p e iously used 60 µM (Tikkinen e al., 2018a). Also, he in i o ge mina ion was sho ened o 1 o 2 weeks, and he acclima iza ion was done in small con aine s (Tikkinen e al., 2018b). Mic osa elli e Analysis Gene ic s abili y was examined in he pooled EM sublines i induced om a ious sec ions o a single PS o om a single EM subline when i was he only one induced. Mic osa elli e analyses we e conduc ed om he sublines o 11Pa4066 induced in 2016 and 11Pa3794 induced in 2015. The genomic DNA was ex ac ed om he shoo buds o dono ees and om he EM sublines using ei he 500 mg samples and he me hod de eloped by Lodhi e al. (1994), o 150 mg samples and he me hod de eloped by Doyle and Doyle (1990). Mic osa elli e loci, SpAGD1 (P ei e e al., 1997), WS0022.B15, WS00111.K13, WS0016.O09, and WS0092.A19 (Rungis e al., 2004) we e selec ed o PCR ampli ica ion by PTC-100 he mal cycle (MJ Resea chR ,QC, Canada). Ampli ica ion p oduc s we e subjec ed o he elec opho esis in ABI 3500xL (Applied Biosys ems) au oma ed sequence and he geno ypes we e iden i ied using GeneMappe so wa e (Applied Biosys ems). Tempe a u e Da a Fo empe a u e calcula ions, da a om Finnish Me eo ological Ins i u e measu emen si e si ua ed 1 km away om he expe imen al SE plan a ion we e used. The empe a u e sums (d.d.) we e calcula ed by summing he daily mean empe a u es exceeding he h eshold alue 0◦C, which was closely co ela ed wi h he de elopmen o PS in he sp ing (Su inen e al., 2012). Chilling uni sums (ch.u., Sa as, 1974) we e calcula ed based on hou ly empe a u e measu emen s using 3.5◦C as a h eshold alue (Supplemen a y Figu e S1). Chilling uni sums we e used o e alua e PS de elopmen in he au umn when d.d. sums s opped o accumula e. RESULTS In 2015, one PS o 11Pa3794 geno ype ini ia ed EM (Table 1 and Figu es 2A–C). Shoo buds we e collec ed on Ap il 12 and dissec ed 3 days la e (Table 1). The mean empe a u e on he collec ion day was 5.7◦C and he d.d. sum was 91.5 (Supplemen a y Figu e S1). In 2016, di e en clonal ees o 11Pa3794 we e used o he bud collec ion on Ap il 22, when d.d. was 108.9, howe e , he esponse was nega i e. F om he sp ing 2016 collec ions, six PS o 11Pa4066 esponded posi i ely (Figu es 2D–F and Table 1) and in o al 17 EM sublines we e es ablished in cul u e. The esponding buds we e collec ed on Ap il 8, when d.d. sum was 63.1. This geno ype esponded posi i ely also in he au umn, when one bud om he same clonal ee g oup, as in he sp ing, and h ee buds om he new g oup o clonal ees (2b) p oduced six EM sublines. The au umn collec ion was done on Oc obe 25 when he ch.u. sum was 537.6. In he sp ing posi i ely esponding buds we e s o ed o 3 days a +4◦C, and in he au umn ei he o 1 o 2 days. In 2017, h ee buds o 11Pa3794 om he sp ing collec ion (51.6 d.d.) and i e buds om au umn collec ion (632.2 ch.u.) esponded and in o al 17 EM sublines we e ini ia ed (Table 1). Howe e , he p oli e a ion o 13 EM sublines was poo and hey we e disca ded a e 3 mon hs o cul u e. The EM o sublines o 11Pa3794 we e anslucen / whi e a he beginning o he ini ia ion, bu a e 2–4 weeks i changed o opaque, ha d and we . The subline induced om 11Pa3794 in 2015 p oduced low numbe o none o good quali y soma ic emb yos (Figu e 3B), e en hough he ea ly soma ic emb yos we e iden i ied in he s ained issue samples (Figu e 2C). The mean soma ic emb yo numbe was 1.6 pe g FW be o e he co-cul u e expe imen wi h nu se issue, and 11.2 pe g FW a e being p oli e a ed wi h nu se issue (Figu es 3A,C). All he ge mina ed soma ic emb yos om he sublines induced in 2015 died du ing acclima iza ion e en hough hey o med isible oo s and shoo s (Figu e 3D). Sublines induced in 2017 p oduced ma u e emb yos when co-cul u ed o 2 mon hs wi h nu se issue, he iabili y o emb yos will be es ed in an ongoing s udy. The EM sublines o 11Pa4066 we e anslucen and spiky and p oduced he mean o 78.6 ±12.8 iable soma ic emb yos pe g FW. Soma ic seedlings we e es ablished in a g eenhouse (Figu e 3E). Mic osa elli e analyses we e conduc ed wi h bo h esponsi e geno ypes and con i med he gene ic s abili y o EM sublines when compa ed wi h he dono ees g owing in he ield. The mic osa elli e loci de ec ed in 11Pa3794 geno ype we e 188/188 in SpAGD1, 180/234 in WS0022.B15, 226/248 in WS00111.K13, 398/398 in WS0016.O09, and 219/227 in WS0092.A19, and in 11Pa4066 geno ype hey we e 154/166, 178/200, 258/258, 398/404, and 215/223, espec i ely. DISCUSSION The PS explan s and he expe imen al p o ocol de eloped o whi e sp uce was applied o No way sp uce soma ic ees as desc ibed by Klimaszewska e al. (2011). Fi e pe cen o he es ed No way sp uce geno ypes esponded and induced EM. As sugges ed by Klimaszewska e al. (2011) i is necessa y o sc een a su icien numbe o geno ypes o iden i y he ones wi h he abili y o SE in o de o s udy biochemical and molecula bases o he ecalci ance o ma u e ee issues o he SE induc ion. Mo eo e , e en hough we pooled he buds om a g oup o clonal ees, we concluded ha no all o he clonal ees o esponding geno ypes p oduced PS ha we e esponsi e o SE, simila ly o whi e sp uce (Klimaszewska and Ru ledge, 2016;Ru ledge e al., 2017). The causes o he di e en ial beha io in issue cul u e among geno ypes and clonal ees wi hin a geno ype a e no unde s ood bu epigene ically he i able mechanisms could be implica ed. D’U so F on ie s in Plan Science | www. on ie sin.o g 4Oc obe 2018 | Volume 9 | A icle 1551 pls-09-01551 Oc obe 22, 2018 Time: 14:35 # 5 Va is e al. Soma ic Emb yogenesis F om Sp uce P imo dial Shoo s FIGURE 2 | Induc ion o EM sublines om PS explan s. (A-C) 11Pa3794 geno ype, (D–F) 11Pa4066 geno ype. (A) Elonga ing p imo dial needles a e 21 days o cul u e. (B) P oli e a ing EM a e 8 weeks o cul u e. (C) Ea ly soma ic emb yos s ained wi h ace oca mine. (D,E) P oli e a ing EM a e 6 weeks o cul u e. (F) Ea ly soma ic emb yos s ained wi h ace oca mine. Ba s: (A,B,D,E) = 0.5 mm, (C,F)=100µm. FIGURE 3 | (A) Co-cul u e o EM o 11Pa3794 geno ype (A, ou side ci cle) and EM 15Pa1029 nu se issue (B, inside ci cle). (B,C) Co yledona y soma ic emb yos o 11Pa3794 geno ype om di e en ma u a ion e en s be o e and a e co-cul u e wi h nu se issue. (D) Ge mina ed soma ic emb yos om subline o 11Pa3794. (E) Soma ic ees o geno ype 11Pa4066 a e second g owing season. Ba s: (A) = 1 m, (B) = 50 mm, (C) = 10 mm, (D) =1mm,(E) = 50 mm. and B ickne (2014) and e e ences he ein) and He and Li (2018) and e e ences he ein) sugges ed ha p e iously ep essed genes a e equen ly p edisposed o e-ac i a ion, a phenomenon called ansc ip ional o en i onmen al memo y. This mechanism equi es changes in ch oma in s uc u e and a physical in e ac ion wi h nuclea po e p o eins. Such a mechanism allows cells o apidly mobilize a ansc ip ional esponse o an en i onmen al s imuli ha hey ha e p e iously expe ienced. This could, a leas heo e ically, explain he highe p opensi y o soma ic ee-de i ed explan s o unde go SE. The e ha e been, howe e , ma ked di e ences in he esponses o se e al pine species compa ed wi h sp uces, he o me being non- o pa ially esponsi e o induc ion o SE when simila expe imen al app oaches we e applied (Ga cía-Mendigu en e al., 2015;T on in e al., 2016). The posi i e esponse o he g oup o clonal ees o wo No way sp uce geno ypes was epea able in consecu i e yea s un il he end o his s udy when he soma ic ees we e 6 yea s old. We specula e ha based on he esul s wi h whi e sp uce (Klimaszewska and Ru ledge, 2016), his esponsi eness will emain o he nex se e al yea s. The la es expe imen wi h whi e sp uce was ca ied ou in he au umn 2017 and he PS explan o esponding 17 yea s-old soma ic ees ini ia ed EM and he ma u e soma ic emb yos con e ed o plan s (Klimaszewska e al., unpublished). The posi i e esponses o No way sp uce PS explan s we e also epea able be ween he seasons; sp ing and au umn. To ully unde s and he in luence o he PS de elopmen al s age on he ini ia ion o SE, he shoo buds should be collec ed om he beginning o he bud o ma ion in he la e summe h ough he all and win e un il he ull bud b eak in he sp ing. To pe o m an expe imen o his magni ude, he ees should be big enough o collec ion o hund eds o shoo buds, which was no possible in his s udy due o he small size o he dono ees. Howe e , ou s udy and he esul s o Klimaszewska e al. (2011) seem o suppo he sugges ion o Bonga (2017) F on ie s in Plan Science | www. on ie sin.o g 5Oc obe 2018 | Volume 9 | A icle 1551 pls-09-01551 Oc obe 22, 2018 Time: 14:35 # 6 Va is e al. Soma ic Emb yogenesis F om Sp uce P imo dial Shoo s ha du ing swi ches in he de elopmen al s ages issues may be mo e capable o ini ia ing SE. Such a swi ch may occu in la e No embe when he elease o endodo mancy happens. The iming o he elease is dependen on he geno ype and en i onmen al condi ions, bu a leas a pa ial elease s a s when ch.u. sum exceeds 500 and he ull elease occu s when ch.u. sum eaches 800–850 (Vihe ä-Aa nio e al., 2014;Pa anen e al., 2016). The molecula egula ion o bud do mancy includes ho monal signaling and speci ic gene exp ession, bu ecen indings pay a en ion o epigene ic egula ion in ol ing modi ica ions o his ones, DNA me hyla ion and he syn hesis o small non- coding RNAs (D’U so and B ickne , 2014;Rios e al., 2014). The ela ionships be ween genomic DNA cy osine me hyla ion, his one H4 ace yla ion and bud do mancy was desc ibed in Cas anea sa i a (San ama ía e al., 2009), and Valledo e al. (2010) used simila app oach o s udy needle ma u a ion o Pinus adia a. In No way sp uce, empe a u e condi ions du ing zygo ic o SE a ec s he exp ession o speci ic genes and hus iming o deha dening and bud bu s in he sp ing, cessa ion o leade shoo g ow h in he summe , as well as bud se and cold acclima ion in he au umn (Yako le e al., 2014; Ca ne os e al., 2017). T ansc ip ion analysis o he bud-bu s ela ed genes and o he molecula ma ke s like ch oma in s a us may gi e mo e insigh in o iming o he PS explan s esponse o SE. In he sp ing, ini ia ion o he PS g ow h is dependen on he in e se ela ionship be ween accumula ions o ch.u. and d.d. sums: he highe accumula ed ch.u. sum be o e bud b eak, he lowe d.d. sum is equi ed o ini ia e PS g ow h (Vihe ä- Aa nio e al., 2014). Be o e he bud b eak is isible ex e nally, he in e nal mo phological de elopmen begins simul aneously wi h he accumula ion o d.d. sum (Su inen e al., 2012). P imo dial needles elonga e and co e he shoo apex, he whole PS elonga es and he bud scales s a o open. In ou s udy, he i s signs o needles and shoo elonga ion we e isible only a e he PS was cu longi udinally. When he explan was placed on he medium, he p imo dial needles con inued o elonga e (Figu e 3) bu became cu ed. Howe e , ound p o ube ances (nodules) we e no obse ed on he needle p imo dia as was epo ed o whi e sp uce. Such p o ube ances we e o en obse ed o de elop in o EM (Klimaszewska e al., 2011;Klimaszewska and Ru ledge, 2016). In ou s udy wi h No way sp uce, he g ow h o EM appea ed om he con ac a ea be ween explan and medium, and hus he ac ual o igin o he EM was no es ablished. A des uc i e sampling will be necessa y o de e mine he o igin o EM in No way sp uce. Appa en ly, s o ing he buds a +4◦C o 3 days, as was done wi h he h ee ba ches o buds o 11Pa4066 in 2016, did no a ec he esul o he expe imen compa ed wi h esh buds. Despi e simila emb yo p oduc i i y, i.e., a ound 120 emb yos pe g FW in he o iginal EM lines (de i ed om he zygo ic emb yos), he EM sublines o wo esponsi e No way sp uce geno ypes g ew e y di e en ly a e ini ia ion. This may be due o di e en ially exp essed p o eins like in seconda y and e ia y lines o Douglas- i (Gau ie e al., 2018). The EM sublines o 11Pa3794 had much lowe emb yo p oduc i i y han sublines o 11Pa4066, possibly due o he un imely subcul u e, o o he ac o s. The ace oca mine s aining e ealed simila ea ly emb yos in p oli e a ing sublines o bo h geno ypes, bu 11Pa4066 p oduced mo e co yledona y emb yos and plan s. The ini ia ion o SE-like anslucen issue and he p esence o emb yo-like s uc u es do no always esul in he p oduc ion o soma ic emb yos and plan s, which was he case wi h di e en pine species (T on in e al., 2016). The change in he appea ance o EM and dec ease in he soma ic emb yo ma u a ion yield a e usually no iceable a e EMs had been subcul u ed con inuously o se e al mon hs. O e all, i can be a gued ha i ees o SE o igin p o ide explan s ha a e mo e suscep ible o SE ini ia ion han simila explan s om ees o zygo ic o igin, hey a e no comple ely ue- o– ype, and i can ques ion i hey di e om a seedling in o he ai s as well. Högbe g (2003) s udied po en ial connec ions be ween SE success and economically impo an ai s in No way sp uce, and ound no ad e se co ela ions. The on-going ield expe imen s wi h SE ees will p o ide mo e de ailed in o ma ion on his subjec . In his s udy explan s only om SE ees we e used, and i is no possible o know how hei zygo ic coun e pa s would ha e esponded. E en hough he soma ic seedlings o sublines om Pa3794 did no su i e, hey p oduced ma u e soma ic emb yos capable o ge mina ion. The loss o soma ic seedlings du ing acclima iza ion happens also in he SE lines o zygo ic emb yo o igin (Tikkinen e al., 2018b), he e o e, he lack o su i al o Pa4066 soma ic seedlings was no unique. In he sublines o 11Pa3794 ini ia ed in 2015, p oli e a ion in he p esence o nu se issue enhanced he numbe o ma u e soma ic emb yos sligh ly. In ano he s udy wi h P. abies, co- cul u e wi h he emb yogenic nu se issue has been used o s imula e p oli e a ion o emb yogenic issue induced in needle explan s (Wes co , 1994). O he examples o he success ul co-cul u e wi h nu se issue include he enhanced eco e y o c yop ese ed EM o Pinus adia a (Ha g ea es e al., 2002) and acili a ion o SE ini ia ion om imma u e zygo ic hyb id pine emb yos (Ha g ea es e al., 2017). The nu se cul u e is belie ed o modi y cul u e en i onmen in o mo e a o able one, e.g., by me abolizing suc ose and by exc e ing g ow h ho mones in o he cul u e medium. The e ec o co-cul u e wi h nu se issue on ma u a ion and soma ic emb yo p oduc ion has, howe e , no been epo ed hus a . CONCLUSION In conclusion, ini ia ion o SE om PS explan s in No way sp uce was possible wi h ce ain geno ypes. The clonal ees o hese geno ypes esponded epea edly o e he las h ee yea s. Fu he esea ch is needed o lea n how SE induc ion is egula ed since he e is a ia ion among he clonal ees in hei esponse. The di e en esponsi eness o clonal ees wi hin one geno ype p o ides an oppo uni y o use ad anced gene exp ession analysis and compa e di e en ees wi hou a con ounding geno ypic e ec (Ru ledge e al., 2017). In addi ion, o he explan s F on ie s in Plan Science | www. on ie sin.o g 6Oc obe 2018 | Volume 9 | A icle 1551 pls-09-01551 Oc obe 22, 2018 Time: 14:35 # 7 Va is e al. Soma ic Emb yogenesis F om Sp uce P imo dial Shoo s could be es ed such as needle explan s, as sugges ed by Bonga (2017), which also p oduced SE albei a limi ed equency (Ruaud e al., 1992;Ha eng e al., 2001). Fu he mo e, he g ow h o soma ic seedlings p oduced om PS explan s will be ollowed, as well as con inua ion o SE induc ion in he esponding geno ypes. AUTHOR CONTRIBUTIONS SV designed and ca ied ou he expe imen s. SV concei ed he manusc ip , SV, KK, and TA ead, w o e and app o ed he manusc ip . 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The e iewe JMB decla ed a sha ed a ilia ion, hough no o he collabo a ion, wi h one o he au ho s KK o he handling Edi o . Copy igh © 2018 Va is, Klimaszewska and A onen. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (CC BY). The use, dis ibu ion o ep oduc ion in o he o ums is pe mi ed, p o ided he o iginal au ho (s) and he copy igh owne (s) a e c edi ed and ha he o iginal publica ion in his jou nal is ci ed, in acco dance wi h accep ed academic p ac ice. No use, dis ibu ion o ep oduc ion is pe mi ed which does no comply wi h hese e ms. F on ie s in Plan Science | www. on ie sin.o g 8Oc obe 2018 | Volume 9 | A icle 1551