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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 ,
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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.
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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
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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
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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)
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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 .
ACKNOWLEDGMENTS
We would like o hank he Eu opean Regional De elopmen
Fund, Sou h Sa o Regional Council, Sa onlinna Business Se ices
L d., and Sa onlinna municipali y o unding his esea ch. We
also hank Eija Ma ikainen and Teijo Nikkanen o pho os.
SUPPLEMENTARY MATERIAL
The Supplemen a y Ma e ial o his a icle can be ound online
a : h ps://www. on ie sin.o g/a icles/10.3389/ pls.2018.01551/
ull#supplemen a y-ma e ial
REFERENCES
Adams, G. W., Kunze, H. A., McCa ney, A., Millican, S., and Pa k, Y. S.
(2016). “An indus ial pe spec i e on he use o ad anced e o es a ion s ock
echnologies,” in Vege a i e P opaga ion o Fo es T ees, eds Y.-S. Pa k, J. M.
Bonga, and H.-K. Moon (Daejeon: Na ional Ins i u e o Fo es Science),
323–334.
Bonga, J. M. (2017). Can explan choice help esol e ecalci ance p oblems in
in i o p opaga ion, a p oblem s ill acu e especially o adul coni e s? T ees
31, 781–789. doi: 10.1007/s00468-016-1509-z
Bonga, J. M., Klimaszewska, K., and on Ade kas, P. (2010). Recalci ance in
clonal p opaga ion, in pa icula o coni e s. Plan Cell Tissue O gan Cul . 100,
241–254. doi: 10.1007/s11240-009-9647-2
Ca ne os, E., Yako le , I., Viejo, M., Olsen, J. E., and Fossdal, C. G. (2017).
The epigene ic memo y o empe a u e du ing emb yogenesis modi ies he
exp ession o bud bu s - ela ed genes in No way sp uce epi ypes. Plan a 246,
553–566. doi: 10.1007/s00425-017-2713-9
Doyle, J. J., and Doyle, J. L. (1990). Isola ion o plan DNA om esh issue. BRL
Focus 12, 13–15.
D’U so, A., and B ickne , J. H. (2014). Mechanisms o epigene ic memo y. T ends
Gene . 30, 230–236. doi: 10.1016/j. ig.2014.04.004
Ga cía-Mendigu en, O., Mon albán, I. A., S ewa , D., Moncaleán, P.,
Klimaszewska, K., and Ru ledge, R. G. (2015). Gene exp ession p o iling
o shoo -de i ed calli om adul adia a pine and zygo ic emb yo-de i ed
emb yonal masses. PLoS One 10:e0128679. doi: 10.1371/jou nal.pone.012
8679
Gau ie , F., Eliášo á, K., Leplé, J.-C., Vond áko á, Z., Lomenech, A.-M., Le Me é,
C., e al. (2018). Repe i i e soma ic emb yogenesis induced cy ological and
p o eomic changes in emb yogenic lines o Pseudo suga menziesii [Mi b.]. BMC
Plan Biol. 18:164. doi: 10.1186/s12870-018-1337-y
Gup a, P. K., and Du zan, D. J. (1987). Bio echnology o soma ic
polyemb yogenesis and plan le egene a ion in loblolly pine. Bio/Technol. 5,
147–151.
Ha g ea es, C., G ace, L., and Holden, D. (2002). Nu se cul u e o
e icien eco e y o c yop ese ed Pinus adia a D. Don emb yogenic
cell lines. Plan Cell Rep. 21, 40–45. doi: 10.1007/s00299-002-
0478-4
Ha g ea es, C., Ree es, C., and Gough, K. (2017). Nu se issue o emb yo escue:
es ing new coni e soma ic emb yogenesis me hods in a F1 hyb id pine. T ees
31, 273–283. doi: 10.1007/s00468-016-1482-6
Ha eng , L., T on in, J. F., Reymond, I., Canle , F., and Pâques, M. (2001).
Molecula e idence o ue- o- ype p opaga ion o a 3yea -old No way
sp uce h ough soma ic emb yogenesis. Plan a 213, 828–832. doi: 10.1007/
s004250100628
He, Y., and Li, Z. (2018). Epigene ic en i onmen al memo ies in plan s:
es ablishmen , main enance, and ep og amming. T ends Gene . doi: 10.1016/j.
ig.2018.07.006 [Epub ahead o p in ].
Högbe g, K.-A. (2003). Possibili ies and Limi a ions o Vege a i e P opaga ion o
No way Sp uce. Ac a Uni e si a is Ag icul u ae Sueciae, Sil es ia 294. Uppsala:
Swedish Uni e si y o Ag icul u al Sciences.
Högbe g, K.-A., and Va is, S. (2016). “Vege a i e p opaga ion o No way sp uce:
expe iences and p esen si ua ion in Sweden and Finland,” in Vege a i e
P opaga ion o Fo es T ees, eds Y.-S. Pa k, J. M. Bonga, and H.-K. Moon
(Daejeon: Na ional Ins i u e o Fo es Science), 538–550.
Klimaszewska, K., Lachance, D., Pelle ie , G., Lelu, A.-M., and Seguin, A. (2001).
Regene a ion o ansgenic Picea glauca,P. ma iana, and P. abies a e
cocul i a ion o emb yogenic issue wi h Ag obac e ium ume aciens.In Vi o
Cell. De . Biol. Plan 37, 748–755. doi: 10.1007/s11627-001-0124-9
Klimaszewska, K., O e on, C., S ewa , D., and Ru ledge, R. G. (2011). Ini ia ion o
soma ic emb yos and egene a ion o plan s om p imo dial shoo s o 10-yea -
old soma ic whi e sp uce and exp ession p o iles o 11 genes ollowed du ing he
issue cul u e p ocess. Plan a 233, 635–647. doi: 10.1007/s00425-010-1325-4
Klimaszewska, K., and Ru ledge, R. G. (2016). “Is he e po en ial o p opaga ion
o adul sp uce ees h ough soma ic emb yogenesis,” in Vege a i e P opaga ion
o Fo es T ees, ed. H. K. Moon (Daejeon: Na ional Ins i u e o Fo es Science),
195–210.
Lang, G. A., Ea ly, J. D., Ma in, G. C., and Da nell, R. L. (1987). Endodo mancy,
pa ado mancy, and ecodo mancy–physiological e minology and classi ica ion
o do mancy esea ch. Ho science 22, 371–377.
Lelu-Wal e , M.-A., Be nie -Ca dou, M., and Klimaszewska, K. (2008). Clonal
plan p oduc ion om sel - and c oss-pollina ed seed amilies o Pinus syl es is
(L.) h ough soma ic emb yogenesis. Plan Cell Tissue O gan Cul . 92, 31–45.
doi: 10.1007/s11240-007-9300-x
Lelu-Wal e , M.-A., Thompson, D., and Ha eng , L. (2013). Soma ic
emb yogenesis in o es y wi h a ocus on Eu ope: s a e-o - he-a ,
bene i s, challenges and u u e di ec ion. T ee Gene . Genomes 9, 883–899.
doi: 10.1007/s11295-013-0620-1
Li ay, J. D., Ve ma, D. C., and Johnson, M. A. (1985). In luence o a loblolly pine
(Pinus aeda L.) cul u e medium and i s componen s on g ow h and soma ic
emb yogenesis o he wild ca o (Daucus ca o a L.). Plan Cell Rep. 4, 325–328.
doi: 10.1007/BF00269890
Lodhi, M. A., Ye, G. N., Weeden, N. F., and Reisch, B. (1994). A simple and e icien
me hod o DNA ex ac ion om g ape ine cul i a s and Vi is species. Plan
Mol. Biol. Rep. 12, 6–13. doi: 10.1007/BF02668658
Mon euuis, O., La de , L., Mon o o, P., Be houly, M., and Ve deil, J.-L. (2010).
“Soma ic emb yogenesis and phase change in ees,” in P oceedings o he IUFRO
Wo king Pa y 2.09.02 “Soma ic emb yogenesis o ees” con e ence on “Ad ances
in soma ic emb yogenesis o ees and i s applica ion o he u u e o es s and
plan a ions”, eds Y. S. Pa k, J. M. Bonga, S. Y. Pa k, and H. K. Moon (Suwon:
Republic o Ko ea), 21–28.
Pa anen, J., Häkkinen, R., and Hänninen, H. (2016). Signi icance o he oo
connec ion on he do mancy elease and ege a i e bud bu s o No way sp uce
(Picea abies) seedlings in ela ion o accumula ed chilling. Sil a Fenn. 50, 1–9.
doi: 10.14214/s .1443
P ei e , A., Oli ie i, A. M., and Mo gan e, M. (1997). Iden i ica ion and
cha ac e iza ion o mic osa elli es in No way sp uce (Picea abies K.). Genome
40, 411–419. doi: 10.1139/g97-055
Rios, G., Leida, C., Coneje o, A., and Badenes, M. L. (2014). Epigene ic egula ion
o bud do mancy e en s in pe ennial plan s. F on . Plan Sci. 5:247. doi: 10.
3389/ pls.2014.00247
F on ie s in Plan Science | www. on ie sin.o g 7Oc obe 2018 | Volume 9 | A icle 1551
pls-09-01551 Oc obe 22, 2018 Time: 14:35 # 8
Va is e al. Soma ic Emb yogenesis F om Sp uce P imo dial Shoo s
Ruaud, J.-N., Be ce che, J., and Pâques, M. (1992). Fi s e idence o soma ic
emb yogenesis om needles o 1-yea -old Picea abies plan s. Plan Cell Rep. 11,
563–566. doi: 10.1007/BF00233093
Rungis, D., Be ube, Y., Zhang, J., Ralph, S., Ri land, C. E., Ellis, B. E., e al. (2004).
Robus simple sequence epea ma ke s o sp uce (Picea spp.) om exp essed
sequence ags. Theo . Appl. Gene . 109, 1283–1294. doi: 10.1007/s00122-00
4-1742-5
Ru ledge, R. G., S ewa , D., O e on, C., and Klimaszewska, K. (2017). Gene
exp ession analysis o p imo dial shoo explan s collec ed om ma u e whi e
sp uce (Picea glauca) ees ha di e in hei esponsi eness o soma ic
emb yogenesis induc ion. PLoS One 12:e0185015. doi: 10.1371/jou nal.pone.
0185015
Sa as, R. (1974). In es iga ions on he annual cycle o de elopmen o o es
ees II. Au umn do mancy and win e do mancy. Communica iones Ins i u i
Fo es alis Fenniae, 84:101.
San ama ía, M. E., Hasbún, R., Vale a, M. J., Meijón, M., Valledo , L., Rod íguez,
J. L., e al. (2009). Ace yla ed H4 his one and genomic DNA me hyla ion
pa e ns du ing bud se and bud bu s in Cas anea sa i a.J. Plan Physiol. 166,
1360–1369. doi: 10.1016/j.jplph.2009.02.014
Selby, C., and Ha ey, B. M. R. (1985). The in luence o na u al and in i o
bud lushing on ad en i ious bud p oduc ion in Si ka sp uce [Picea si chensis
(Bong.) Ca .] bud and needle cul u es. New Phy ol. 100, 549–562. doi: 10.1111/
j.1469-8137.1985. b02801.x
Su inen, S., Pa anen, J., Vihe ä-Aa nio, A., and Häkkinen, R. (2012). De elopmen
and g ow h o p imo dial shoo s in No way sp uce buds be o e isible bud
bu s in ela ion o ime and empe a u e in he ield. T ee Physiol. 32, 987–997.
doi: 10.1093/ eephys/ ps063
Su on, B. (2002). Comme cial deli e y o gene ic imp o emen o coni e
plan a ions using soma ic emb yogenesis. Ann. Fo . Sci. 59, 657–661. doi: 10.
1051/ o es :2002052
Tikkinen, M., Va is, S., and A onen, T. (2018a). De elopmen o soma ic emb yo
ma u a ion and g owing echniques o No way sp uce emblings owa ds la ge
scale ield es ing. Fo es s 9:325. doi: 10.3390/ 9050325
Tikkinen, M., Va is, S., Pel ola, H., and A onen, T. (2018b). Imp o ed ge mina ion
condi ions o No way sp uce soma ic co yledona y emb yos inc eased su i al
and heigh g ow h o emblings. T ees [P ep in ]. doi: 10.1007/s00468-018-1728-
6
T on in, J.-F., A onen, T., Ha g ea es, C., Mon albán, I. A., Moncaleán, P.,
Ree es, C., e al. (2016). “In e na ional e o o induce soma ic emb yogenesis
in adul pine ees,” in Vege a i e P opaga ion o Fo es T ees, eds Y. S. Pa k,
J. M. Bonga, and H. K. Moon (Daejeon: Na ional Ins i u e o Fo es Science),
211–260.
Valledo , L., Meijon, M., Hasbun, R., Jesus Canal, M., and Rod iguez, R. (2010).
Va ia ions in DNA me hyla ion, ace yla ed his one H4, and me hyla ed his one
H3 du ing Pinus adia a needle ma u a ion in ela ion o he loss o in i o
o ganogenic capabili y. J. Plan Physiol. 167, 351–357. doi: 10.1016/j.jplph.2009.
09.018
Va is, S., Ahola, S., Jaakola, L., and A onen, T. (2017). Reliable and p ac ical
me hods o c yop ese a ion o emb yogenic cul u es and cold s o age o
soma ic emb yos o No way sp uce. C yobiology 76, 8–17. doi: 10.1016/j.
c yobiol.2017.05.004
Vihe ä-Aa nio, A., Su inen, S., Pa anen, J., and Häkkinen, R. (2014). In e nal
de elopmen o ege a i e buds o No way sp uce ees in ela ion o
accumula ed chilling and o cing empe a u es. T ee Physiol. 34, 547–556.
doi: 10.1093/ eephys/ pu038
Wes co , R. J. (1994). P oduc ion o emb yogenic callus om nonemb yonic
explan s o No way sp uce (Picea abies (L.) Ka s ). Plan Cell Rep. 14, 47–49.
doi: 10.1007/BF00233297
Yako le , I. A., Lee, Y., Ro e , B., Olsen, J. E., Sk oppa, T., Johnsen, O.,
e al. (2014). Tempe a u e-dependen di e en ial ansc ip omes
du ing o ma ion o an epigene ic memo y in No way sp uce
emb yogenesis. T ee Gen. Genome 10, 355–366. doi: 10.1007/s11295-013-
0691-z
Con lic o In e es S a emen : The au ho s decla e ha he esea ch was
conduc ed in he absence o any comme cial o inancial ela ionships ha could
be cons ued as a po en ial con lic o in e es .
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 .
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F on ie s in Plan Science | www. on ie sin.o g 8Oc obe 2018 | Volume 9 | A icle 1551