Intrageneric fusions of isolated protoplasts from Ulva and Porphyra

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Title
Intrageneric fusions of isolated protoplasts from Ulva and Porphyra by
electrofusion method
Author(s)
Reddy, C.R.K.; Saito, Munehisa; Migita, Seiji; Fujita, Yuji
Citation
長崎大学水産学部研究報告, v.68, pp.21-27; 1990
Issue Date
1990-10
URL
http://hdl.handle.net/10069/29895
Right
This document is downloaded at: 2015-02-01T01:37:41Z
http://naosite.lb.nagasaki-u.ac.jp
Bull. Fac. Fish. Nagasaki
21
Univ. , No.68 (1990)
Intrageneric
fusions
of isolated
Ulva and Porphyra
C. R. K. Reddy*,
by electrofusion
Munehisa
Isolated algal protoplasts
protoplasts
from
method
SAITO*, Seiji MIGITA and Yuji FUJITA
of 1. Ulva pertusa (sterile mutant)
and U conglobata, 2.
Porphyra yezoensis normal and P. yezoensis green type, were electrically
fused.
The
protoplasts from each alga were mixed together with its algal partner in a 1 : 1 ratio in
low conductivity electrofusion solution at a density of 1 × 105-6 cells/ml.
Protoplasts
were aligned into short chains in high frequency (1 MHz) alternate current (AC) field and
sudsequently
fused by the application of a single short duration direct current (DC) pulse.
Protoplasts
aligned at 200 V for 10 s and 40 V for 20 s yielded maximum pairs about 25
and 40% in Ulva and Porphyra respectively.
pulse of 200 V resulted optimum
The application
of 20-25 it s duration DC
binary fusion percentages
about 12% in Ulva and
whereas 250 V of 40 µ s duration yielded maximum fusions about 16% in Porphyra.
application
V of 40 µ s duration for Porphyra) to the aligned protoplasts
Key words : electrofusion,
protoplasts,
Somatic
hybridization
develop
new
diversity¹).
recently
plants
fusions
in higher
methods,
plants²–4).
fusion5) has been widely
ing protoplast
are several
on protoplast.
However
fusion
reports
in higher
effects
Therefore
an electrofusion
been
successfully
higher
*
plants10-¹²)
Graduate
school
to
used
the
plants6).
of U. pertusa (sterile mutant)¹³),
There
been published
are non specific
treated
or caused
which has
protoplasts
was employed
of Marine
and
thalli : Young
clean vegetative
thalli
U. conglobata, P.
yezoensis normal and green type were used for the
isolation
of protoplasts.
All the
above
mentioned
plants
are
being
cultures
in our laboratory.
Isolation
of protoplasts
grown
as unialgal
protoplasts.
technique
to fuse
Meterials and Methods
Vegetative
fusion frequencies
cytotoxic
the
mediated
fusion of algae by PEG method7-9).
have either variable
between
Ueda normal with P. yezoensis green type.
for accomplish-
have recently
all fusion methods
heterokaryons
protoplasts of Ulva pertusa Kjellm. with U. conglobata Kjellm. and between Porphyra yezoensis
all fusion
(PEG)
applied
have
ducing binucleate
protoplast
Among
glycol
to
genetic
methods
for inducing
polyethylene
has
fusion
greater
of fusion
been described
plants
protoplast
with
A number
lysis.
protoplasts. This study essentially investigates
the suitable electrical conditions required for in-
in higher
through
induced protoplast
Ulva, Porphyra
Introduction
been accomplished
The
of a high intensity DC pulse ( > 300 V of 30 µ s duration for Ulva and > 350
science
of
to fuse algal
and Engineering.
pertusa
produced
and
: Protoplasts
U. conglobata
by incubating
were
the thallus
from
U.
separately
(about
25 mg
22
Reddy et al．： Electrofusion of algal protoplasts
fresh wt．） in 50／o cellulase R−10 and 20／o abalone
were counted for every fusion event to caliculate
crude enzyme powderi‘）． The isolated proto−
the rate of protoplast alignment and fusions．
plasts from both the species were further incubat−
The rate of protoplast alignment and fusion
ed in 10／o protease P6 （Amano pharmacy Co．，
were caliculated as follows and expressed as a
Japan） enzyme prepared after Fujita and Saitoi5）
percentage． Total alignment rate ＝（total num−
for about 30 min in dark prior to start of the
ber of protoplasts involved in alignment into
electrofusion． Similarly protoplasts from P．
chains）／（total number of protoplasts）× 100．
ye20ensis normal and green type were prepared
Total fusion frequency ＝＝（total number of proto−
following the methods of Fujita and Saito’5）．
plasts involved in binary and multi （＞3 cells）
fusion products）／（total number of protoplasts
Electrofusion： Protoplasts of U． Perlblsa with U．
involved in fusion event十number of unfused
conglobata and P． yezoensis normal with green
cells）×100． Binary fusion products＝（total
type were subjected to electrofusion at 200C using
number of protoplasts involved in binary
a Shimadzu somatic hybridizer SSH−2 （Shimadzu
fusions）／（total number of protoplasts involved in
Co．，Japan）． Protoplasts from each fusion part−
binary fusions十number of unfused cells） × 100．
ner were mixed together with its algal partner in
Results
1 ： 1 ratio in the electrofusion solution （O．2 mM
tris（hydroxymethyl）aminomethane， 1．0 mM
CaC12 ． 2H20 and 1．0 mM MgC12 ・ 6H20 and O．9
Electrofusion of Ulva and Po？Phyra proto−
Mmannitol（0．7 M for Po rp宮町in distilled water，
plasts was performed in two steps． ln the first
adjusted to pH：7．5）at a density of 1．0×10ト6 cells／
step protoplast adhesion with adjacent protoplast
ml． Aliquots of 200 ptl protoplast suspension of
（Figs． IA， C） was generated by dielectrophoresis in
the two fusion partners were placed between the
an AC field at 1 MHz． The results of alignment
two electrodes （1 mm spacing） in a fusion chamber
rate of protoplasts as a function of the interaction
（FTC−02 of Shimadzu Co．） and allowed to settle
of alignment voltage and time are shown in Table
for few minutes prior to the start of the
1 for Ulva and Poz？bhyra respectively． The num−
electrofusion． Protoplasts were initially aligned
ber of protoplasts in chain increased with align−
into short chains preferably Pairs in alternate
ment voltage and time． The application of low
current （AC） field and subs equently fused by the
AC fields （20V） for shorter duration （10s） yielded
application of a single short duration direct cur−
high percentage of （about 25％） of paired proto−
rent （DC） pulse． To investigate the necessary AC
plasts （Fig． I B） in Ulva． Maximum percentage
and DC fields required for induction of protoplast
（about 400／o） of paired protoplasts （Fig． ID） in
alignment and fusions， protoplast suspension of
Porphyra were obtained at higher AC field （40V）
each combination were initially aligned in an AC
and longer duration （20s）． The prolonged expo−
field （1 MHz） at different voltages ranging from
sure （＞25s） to above mentioned respective AC
10 to 40 V for different durations ranging from 10
fields induced long protoplast chains （Figs． IA， C）
to 25 s to find out the appropriate voltage and
in both Ulva and Po7Phyra ． Though the percent−
length of electric fields necessary for establishing
age of total aligned protoplasts and multi−
protoplast pairs． Similarly pulse voltage （100
protoplast chains increased with AC field strength
−350V） and pulse width （10−60pt s） have also been
and time， but the latter one however reduced the
calibrated to obtain optimum fusion frequencies
pairing protoplasts number （Table l） than the
of binucleate fusion products， in order to facilitate
former one．
easy regeneration and subsequent genetic analysis
The second step was induction of protoplast
of regenerated plants． Generally， five random
fusion by the application of a single high intensity
microscopic fields （each with about 100−150 cells）
DC rectangular pulse of microsecond duration．
Bull． Fac． Fish． Nagasaki Univ．， No．68 （1990）
蘇
斌
撰嚢．、，璽鳶綴温石癬一
難毅瓢箪隔垣戸
欝轡
購
〆醐・
難
、触も
蔓
売タ﹃ ‘
夷執瞬
5
㌧郵轄
縛1
羅特
Fig． 1．
Intrageneric electrofusion of Ulva and Po2Phyra protoplasts． A： Protoplast alignment into
long chains of U． Pertusa （stained with neutral red） and U． conglobata， exposed to long durations
to high frequency AC fields at 1 MHz， 20 V for 25 s． B ： lnduction of protoplast pairs of U．
pertusa （stained with neutral red） and U． conglobata， exposed to short durations to AC field at
1 MHz， 20 V for 10 s． C ： Protoplast alignment into long chains of P． yezoensis normal and green
（with arrows） type， exposed to long durations to high frequency AC fields at 1 MHz， 40 V for
25 s． D： lnduction of protoplasts pairs of P． ye20ensis normal and green （with arrows） type，
exposed to short durations to AC fields at 1 MHz， 40 V for 20 s． E： Protoplast fusion of U．
Pertusa with U． conglobata soon after application of a single DC pulse of 200 V for 20 ” s
duration． F：Round heterokaryons of Ulva， 3 minutes after application of DC pulse． G：
Protoplast fusion of P． ye20ensis normal with green soon after application of a single DC pulse
of 250 V for 40 ＃ s duration． H ： Round heterokaryons of PoiPhyra ， 5 minutes after applica−
tion of DC pulse．
Bar in all figures is 20 ＃m．
23
24
Reddy et al．： Electrofusion of algal protoplasts
The yield of fusion products as a result of the
plasts has been rep’ortedi8）， however there are no
interaction of pulse voltage and pulse width are
detailed studies on optimizing the electrical condi−
shown in Fig．2 A＆B for Ulva and Porphyra
tions for obtaining high fusion frequencies of
respectively． The fusion process in Ulva proto−
viable fusion products in algal protoplasts． Pro−
plasts was initiated by the application of a DC
toplasts from normal P． ye20丁目sis were previously
pulse of ＞150 V of 15 pt s duration （Fig． IE）． The
fused with green type following the PEG
delivery of a short duration （20−25pt s） DC pulse of
200 V to aligned protoplasts in Ulva resulted
Table 1． Effect of AC voltage and AC voltage
optimum binary fusions about 120／o （Fig． IF）， and
applied time on protoplast induction
into pairs in Ulva and Po7Phyra
whereas 250 V of 40 pt s duration yielded optimum
binary fusions about 160／o in Po7Phyra （Figs． IG，
Parameters
O／o protoplasts involved in pairs
Protoplast suspension of
H）． Although percentage of heterokaryons were
Ulva PorPhyra
not determined， but 40−500／o of total fusion prod−
Voltage （V）i）
ucts were found to be heterokaryons． However
the application of a high intensity DC pulse （＞
300V of 30pt s duration for Ulva ＞350 V of 40pt s
10
19 （33）＊
20
25 （44）
32 （68）
o （o）＊
30
15 （50）
36 （80）
and duration for Porphyra） to aligned protoplasts
40
10 （47）
40 （98）
induced protoplast lysis．
Time （s）2）
Discussion
Electrofusiop has， been developed to an effi−
cient and routine technique to ・fuse both animal
cells and plant protoplastsi6・i7）． Though in one
instance electrofusion of Enteromorpha proto一
10
25 （44）
18 （44）
15
16 （48）
35 （90）
20
10 （52）
40 （95）
25
9 （60）
28 （97）
Protplqsts of Ulva and PoiP）hyra were constantly
ekposed 1）to AC fields for 10 and 20s and 2）to AC
voltages 20 and 40 V respectively．
＊：O／o total protoplasts involed in long and short
（pairs） chains．
［コ Total fusion products
Eli8 Binary fusion products of
Ul va
rw Binary Eusion products of Porρ妙ra
20
30
A
，
，
■
●
o・●● ●
怐怐怐C●・
●oo﹁■9●o・
oo●●■．o●9
150
．・．●●
・・．・9
●
o
■
9
・
200 250 300
Pulse voltage （V）
Fig． 2．
350
o
。㌦
。．・．。．
■
●
o
10
o
，
o
o
㌧
・
㌧
㌦
㌔
●
㌔
・
●
．
・
．
％．●
・．
●●
。．
％
o
B
9
㌦
・
。
・
。
。
・
．●
●・
㍉θ
10
20
．
︵巴。αぢう層。臼ΩgO葛づh
30
●。
j
15 20 25 3035 40 50 60
Pulse widt二h （ps）
Effect of pulse voltage （A） and pulse width （B） on fusion frequencies of prealigned protoplasts of
Ulva and Po7pdyra． Prior to fusion pulse， protoplasts of both Ulva and Po7PhNra were aligned
to pairs by applying 20 and 40 V AC fields for 10 and 20 s respectively． Pulse width in （A） for
Ulva is 20 pt s and for Pomphyra is 40pt s， similarly pulse votage in （B） is 200 V for Ulva and 250
VforPoゆ勿η．
25
Bull． Fac． Fish． Nagasaki Univ．， No．68 （1990）
method8）， Later protoplasts from several species
with CaC12 and MgC12 concentration in the fusion
of Porp勿ra were electrically fused and high
solution23）． Electrofusion in Ulva was complete−
fusion frequencies were reported． The regenera−
ly blocked by increasing the CaC12 and MgC12
tion rate of post fusion products was also higher
concentration to 3 mM in the fusion medium．
than with the PEG methodi5）． Although PEG
Similarly fusion medium without CaC12 also lim−
induced fusions to occur in，U． Pertusa with U．
ited the electrofusion （〈1％） in Ulva． Therefore
conglobata， it did not yield satisfactory fusion
it is essential to investigate the right concentra−
frequencies and viable fusion products’9＞． Conse−
tions of CaC12 needed for inducing high fusion
quently electrofusion has been performed as an
rates． The frequency of protoplast fusion by
alternative to the PEG method． Protoplasts of
PEG method in higher plants has been reported to
U．Pertzasa with U． conglo bata and P． ye20ensis
vary with the nature （i． e． ultra structure） of the
normal with green type were electrically fused by
protoplastS2‘）’and fusion conditions． Unlike the
a combined approach of 1． cell adhesion by AC
Po77）hyra protoplasts Ulva protoplasts with their
fields and 2． subsequent cell fusion by DC pulses．
big vacuoles might have less tendency to involve
The fusion medium prepared in seawater with
in fusion events． Thus the fusion petcentage of
mannitol did not induce protoplast fusion due to
Ulva protoplasts is’ comparatively less than the
high conductivity． The subsequent preparation
Po7Phyra． The regeneration and development of
of protoplast suspension in low conductivity
heterokaryons， following the electrofusion
medium prepared in distilled water however in−
method were earlier reported for Po7Phyra i5） and
duced electrofusions in both cases． The proto−
Ulva’9）． Thus this study demonstrates the suit−
plast alignment and fusions occurred at lower
ability of electrofusion methods for fusing algal
field strength in Ulva than in P（）ゆ勿ra． How−
protoplasts as in higher plants．
ever the alignment rate and fusion percentages in
Acknowledgements
Porphyra were greater than Ulva． The rate of
cell alignment is usually attributed to both the
magnitude of the electric field and ionic strength
This work was partially supported by a grant
of the fusion medium． The former is however
in Aid for Scientific Research from the Ministry
dependent on the radius of the cell （the smaller the
of Education， Science and Culture， Japan． We
cell， the larger the electric field that must be
are indebted to Prof A． Gibor， University of
applied to achieve alignment）． The induction of
California for critically reading and revising the
protoplast alignment and fusion at lower voltages
manuscript． We are also thankful to Mrs．
in Ulva despite the same cell size and fusion
Jhansi Lakshmi for skilfully typing this manu−
medium （except mannitol concentration） might be
script on word processor．
due to the differences between protoplast mem−
References
branes （composition and structure） of Ulva and
P（）ゆ勿ra． Secondly the protease treatment of
Ulva protoplasts prior to the fusion might have
1） Gleba， Y． Y． and Sytnik， K． M．（1984）： Proto−
resulted in the induction of alignment and fusion
plast fusion． Genetic engineering in higher
at lower voltages． lt is assumed that the
plants． pp．220， ln Frankel R．（ed．）， Mono−
protease treatment prior to fusion， enhances the
graphs on Theoritical and Applied Genetics．
fusion ability of protoplasts by removing the
Vol． 8． Berlin， Springer Verlag．
surface glycoproteins20，2i） or generation of
2） Kao， K：N． and Michayluk， M． R．（1974）：A
fusogenic polypeptides22） on membrane compo−
method for high frequency of intergeneric
nent． However the electrofusion frequencies in
fusion of plant protoplasts． Planta， 115， 355
P． ye20ensis normal．with P． Psezado linearis varied
−367．
26
Reddy et al．： Electrofusion of algal protoplasts
3） Kameya，． T．（1975）：Induction of hybrids
（1989）：． Protoplast isolation and regeneration
through somatic cell fusion with dextran
of three species of Ulva in axenic culture．
sulphate and gelatin． Jpn． J． Genet．，50， 235
−246．
Bot． Mar．， 32， 483−490．
15）
4） Nagata， T．（1978）：A novel cell fusion
isolation and fusion in Porph二yra．
method of protoplasts by polyvinyl alcohol．
Naturwissenschaften， 65， 263−264．
Fujita， Y． and Saito， M．（1990）：Protoplast
Hydrobiologia （in press）．
16）
Vienken， J．，Ganser， R．，Hampp， R． and Zim−
5） Kao， K． N．（1977）：Chromosomal behaviour
mermann， U．（1981）：Electricfield induced
in somatic hybrids of Soybean−Nicotiana
fusions of isolated vacuoles and protoplasts
glazaca． Molec． Gen． Genet．，150， 225−230．
of different developmental and metabolic
6） Evans， D． A．（1983）：Protoplast fusion． pp．
291−321， ln Evans D． A．，Sharp， W． R．，Am−
provenience． Physiol． Plant．，53，64−70．
17）
mirato， P． V． and Yamada， Y．（eds．）， Hand
ed fusion and related electrical phenomenon．
book of Plant Cell Culture Vol．1． Tech−
niques for propagation and breeding． New
Zimmermann， U．（1982）： Electricfield mediat−
Biochem． Biophys． Acta．，694， 227−277．
18）
Saga， N．，Polne−Fuller， M． and Gibor， A．
York Macmilan Publishing Co．
（1986）： Protoplasts from seaweeds： Produc−
7） Ohiwa， T．（1975）：Behaviour of cultured
tion and Fusion． Beih． Nova． Hedwigia．，
fusion products from Zygnema and SPirogyra
83， 37−43．
protoplasts． Protoplasma， 97， 185−200．
19）
8） Fujita， Y． and Migita， S．（1987）：Fusion of
Reddy， C． R． K． and Fujita， Y．（1989）： proto−
plast isolation and fusion of Ulva Pertusa and
protoplasts from thalli of two different color
U． conglobata． pp．235−238 ln Miyachi， S．，
types in Porphyra ye20ensis Ueda and develop−
Karube， 1． and lsida， Y．（eds．）， Marine Biote−
ment of fusion products． Jap． J． Phycol．，35，
chnology， Tokyo， The Society for Marine
Biotechnology，
201−208．
9） Lee， Y． K and Tan， H． M （1988）： Genetic
20）
Ohno−Shosaku， T． and Okada， Y．（1984）：
Facilitation of electrofusion of mouse
transformation through protoplast fusion in
algae． pp．101−109， ln Stadler， T．，Mollin，
lymphoma cells by the proteolytic action of
J．，Verdus， M． C．，Karamanos， Y．，Morvan，
protease． Biochem． Biophys． Res． Com−
mun．，120， 138−143．
H and Christiaen， D （eds．）， Algal biotech−
nology， London， Elsevier Applied Science．
21）
Kameya， T．（1979）：Studies’on plant cell
10） Bates， G． W． and Hasenkampf， C． A．（1985）：
fusion： effect of dextran and pronase E on
Culture of plant somatic hybrids following
fusion． Cytologia， 44， 449−456．
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22）
Lucy， J． A．（1984）：Fusogenic mechanisms．
227−233．
pp． 28−39， ln Everd， J． and Whelan， J．（eds．），
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（1985）： Somatic hybrids in tobacco mediated
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23）
Saito， M． and Fujita， Y．（1990）： Optimization
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of fusion conditions in both polyethylene
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24）
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Bull. Fac. Fish. Nagasaki
Univ. , No.68 (1990)
27
電気刺激法によるアオサとアマノリのプロトプラストの属内融合
C.R.K.レ
1.ア
ディー*,齋
ナアオサUlva pertusa(不
藤
宗久*,右
稔型)と
田
清治,藤
田
雄二
ボタンアオサU.conglobata,2.ス
サビノリ
の野生型と緑色変異型との間で,葉体から単離したプロトプラストを電気
Porphyra yezoensis
刺激法によって融合させた。それぞれの葉体から単離したプロトプラストは、低導電性の融合
緩衝液で洗浄後1×105-6ce11s/mlに
調製し,1:1の
したプロトプラスト混合懸濁液に高周波電圧(AC)を
チェーンが形成され,パルス電圧(DC)の
V,10sの
割合で混合した。融合チャンバーに滴下
印加することによってプロトプラスト
印加により融合が開始された。アオサではAC200
印加,スサビノリでは40V,20sの
印加により,それぞれ最大25%お
ロトプラスト対が形成された。そしてアオサではDC200V,20∼25μsの
サビノリでは250V,40μsの
印加で約16%の
μs,スサビノリでは350V,40μs以
よび40%の
プ
印加で最大約12%,ス
融合率が示された。またアオサでは300V,30
上の印加によって細胞の破壊が生じた。
(*:長
崎大学海洋生産科学研究科)

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