全 文 ：第 25卷　第 4期　　　　　　　　　　　　　植　　　物　　　研　　　究 2005年　10月
Vo .l 25　No. 4　　　　　　　　　　　　BULLETIN OF BOTAN ICAL RESEARCH Oc.t , 　2005
Foundat ion item:Supported by th e S tateKey Basic ResearchP roject(G19990160) and theKey Project of Ch ineseM inistry ofEducat ion (104191)
Au thor in troduction:Zu Yuangang (1954— ), M ale, Ph. D. , P rofessor, M ajor in p lan t science. E-m ail:zygorl@ pub lic. h r. h .l cn
Received date:2005 - 07 - 07
超声破碎拟南芥原生质体后的再生培养初报
祖元刚　孟庆焕　郭晓瑞　孙佳音　刘红梅
(东北林业大学森林植物生态学教育部重点实验室 , 哈尔滨　150040)
摘　要　拟南芥原生质体在超声破碎过滤后 ,对其叶绿体 、细胞核等细胞器进行再生培养 。观察
到超声破碎对叶绿体有一定破坏作用导致叶绿体在蓝色荧光下不发荧光 ,在培养 115 h后叶绿体
有聚集现象;细胞核明显增多;出现内质网的明亮绿色荧光点 。细胞核和内质网都是形成新原生
质体的必要细胞器 ,通过对其再生培养可为细胞器重组乃至细胞重建提供启示。
关键词　拟南芥;原生质体;细胞器重组;荧光探针
The study on secondary culturation after the protoplasts of
Arabidopsis tha liana broken by ultrason ic
ZU Yuan-G ang　MENG Q ing-Huan　GUO X iao-Ru i　SUN Jia-Y in　LIU Hong-Mei
(Key Laboratory of Fo rest P lant E co lory, M inistry of Educa tion, Northea st Fo restry Unive rsity, H arb in　150040)
Abstract　A fter the pro top lasts o fA rabidopsis thalianaw as b roken by the ultrasonic and filtra ted by the
filtermembrane, we subcu ltured the organs such as chlorop last and nucleus. It’ s obse rved that som e
ch lo roplastsw ere no fluorescence under the b lue exc ita ted ligh.t The result indicated the ultrasonic had
ce rta in functions of destroying the chlorop lasts. A fte r being cultu red fo r 115 h, the ch lo roplasts began to
gather;and the nuclei obv iously increased;also the b righ t green do ts of endop lasm ic re ticulum ap-
peared. As w e a ll known the nuc le i and endop lasm ic reticu lum are essential ce ll o rgans which fo rm the
new p ro toplast, so cultu ring cell o rgans m ay offe r us some enlightenments abou t organs recombina tion
and even for the cells reconstruction.
Key words　Arabidopsis thaliana;pro top last;organ recombination;fluorescen t probe
The preparation o f p ro toplasts of Arabidopsis
thaliana has a lready become an ex tensive technology
now
[ 1]
. It is a common phenomenon that the p lasma
membranes a re prone to be broken in the course of the
pro top lasts preparation
[ 2] , and thus ce ll organs, for
example the nucle i and ch lo roplasts and so on, w ill o-
ve rflow from the broken protop lasts. But so far, the re
is no report about subsultu ring these ce ll organs such
as nuclei, ch lo roplasts escap ing from the broken pro-
toplasts by the artificial means, and no pape rs focus
on the p ro toplasts recombina tion. In our research, we
broke the pro top lasts by ultrasonic trea tmen t, and
then subcu ltured the ce ll organs for the regene ra tion
of pro toplasts. Th is p robab ly gave us some hints about
organs recomb ination and even for the ce lls recon-
struction
[ 3]
.
A fte r having iso lated the protop lasts of A. thali-
ana , we used u ltrasonic trea tmen t to break pro to-
plasts, and then filte red to get rid of the p lasmam em-
branes, then w e subcu ltured the filtrate. It’ s w e ll
known that the nucleus is the centre of the ce lls’ he-
red ity and metabo lism
[ 4] ;the chlo roplast is the m ain
site fo r plan t pho tosyn thesis
[ 5] ;and the endoplasm ic
re ticulum is the centre for communication, and it’ s
responsible for the transpo rta tion o f prote ins and lip-
ids
[ 6]
. In the conclusion tha t a new protoplast won’ t
come in to being if there are no a lo t o f ce ll o rgans.
1　Materials and methods
1. 1　Preparation forA. tha liana protoplasts
W e used the leaves of the in-v itro plantle ts cul-
ture o f A. tha liana to prepare the protoplasts. The
ce llw all d igestive so lu tion is made up o f buffer solu-
tion and enzyme so lu tion. The buffer solution includ-
ed 400mmo l L- 1 Mannitol, 8 mmo l L - 1 calcium
ch lo ride, 5 mmo l L- 1MES (a k ind o f pharmaceu ti-
ca l pro tection membrane), and adding KOH to regu-
late pH to 5. 6. The enzyme liquid is made up o f
0. 1% BSA , 1% ce llu lose and 0. 25% macerozyme.
A fte r the cell digestive so lu tion disso lved, we filte red
it by filtermembrane ( 0. 22 μm) in the supe r-clean
bench.
1. 2　Filtra te cu ltures of the broken protoplasts
A fte r gathering the pro top lasts by centrifugation,
we broke them using ultrasonic fo r 1 h (100 W ,
25℃). Then w e filte red the protoplasts by filter
membrane (10μm) unde r the aseptic condition and
then dropped the filtrate into the tissue cu lture plate
(30μL perw e ll) to subcu lture unde r thew eak ligh.t
The subcu lture solution component includesM S medi-
um supplemen ted w ith 1 mg L -1 2, 4-D , 0. 5
mg L -1 BA , 0. 1 mg L -1 NAA , 72 g L -1 g lu-
cose and 0. 5mmo l L -1 ATP.
1. 3　The obse rvation of filtrate by fluorescen tm icro-
scope
W e dyed the filtra te w ith Hoechst 33258 (5
μg mL -1) and D ioC5 (3) ( 2. 5 μmo l L -1)[ 7] ,
they w ere mo lecular probe ma rking the nuc leus and
endoplasm reticu lum respectively. A fter being dyed
w ith Hoechst, the filtrate need to be incubated in the
darkness for 20 m inutes;and after being dyed w ith
DioC5(3), the filtrate need to be incubated in the
darkness for 30 m inutes. Then w e observed the fil-
trate using the inverted fluo rescentm icroscope (N icon
TE 2000—U).
2　Results and analysis
2. 1　Changes of the chlorop last in the filtrate cu l-
tured in the cu lture pla te
A t first the ch loroplasts in the filtrate mostly e-
m itted the green light under the na tura l light (Pla te
Ι:1), and they em itted brigh t red light under b lue
excitated light(Pla teΙ:2);A sma llpart of the chlo-
roplasts have no green light o r brigh t red ligh.t This
indicated that u ltrasonic destroyed the ch lo roplasts,
some of them deg raded and fo rmed lamellar structu re.
A fter be ing cultured fo r 115 h , the chlorop lasts began
to gathe r (Plate Ι:6).
2. 2　Changes of the nucleus in the filtra te cultured
in the cu lture p late
In the beg inning, in the filtrate the re w ere few
nucleiw ith irregu lar shape (Plate Ι:3), fo r the rea-
son pe rhaps they w ere destroyed by the ultrasonic.
A fter be ing cultu red fo r 69 h , the number of the nu-
c le i increased and they become much brighter (Pla te
Ι:4). The nuclei increased obv iously and the chloro-
plasts assemb led around the nuclei after be ing cu l-
tured for 115 h (Plate Ι:6).
2. 3　Detection o f the endop lasm re ticulum in the fil-
tra te
A t first the endoplasm re ticu lum did be ob-
served, bu t after being cu ltured for 115 h , the re w ere
very strong bright fluo rescent spo ts in some a reas
(Plate Ι:5). It indicated there existed endop lasm
re ticulum.
3　Discussion
Our present research is a base for the organs
recomb inance and regeneratinon of pro toplas.t The
mechanism is very comp licated and not clear so far,
we need do much w ork in the fu ture.
Reference
1. Fo rd K G. P lant regene ra tion from Arabidopsis thaliana pro-
top lasts. P lan tC ell Rep, 1990, 8:534 ～ 537
2. Koop H U. Som a tic em bryogene sis in cu ltu red imm ature zy-
go tic em bryo s and leaf pro top la sts o fArabidopsis tha liana e-
co types. P lanta, 1997, 202:387 ～ 396
442 　　　　　　植　　物　　研　　究　　　　　　　　　　　　　　　　　　25卷
3. RonM cKay. M amm a lian deconstruction fo r stem ce ll recon-
struc tion. NatureM edicine, 2000, 6:747 ～ 748
4. Zha i ZH , W angX Z, DingM X. Ce ll B io logy. Be ijing:H igh-
e rEduca tion P ress, 2002
5. Zhang X S, H e X L. P lan ta. Beijing:Chine se Ag riculture
P ress, 2003
6. W ang J F. Ce ll bio logy. Beijing:Sc ience P re ss, 2003
7. R icha rd P. H aug land. H andbook o f fluorescent P robes and
Research P roduc ts(9th). M o lecu la r P robe, 2002
P la teⅠ 　1 ～ 3:P ic ture s o f observation o fArabidopsis tha liana pro top la sts which we re b roken by ultra sonic trea tm en t and
filte red by memb rane;4 ～ 6:P ic tures o fA. tha liana p ro toplasts wh ich w ere b roken by u ltrasonic trea tm en t and filtered by
m em brane.
1. C u lture for 0 h under the natural ligh t;2. C u lture for 0 h under th e b lue excitated ligh t, arrow show s ch lorop last;3. C u lture for 0 h under
th e ultra excitated ligh t, arrow show nucleus, nucleus appears irregu lar shap e;4. Cu ltu re for 69 h under the u ltra exci tated light, arrow show
nucleus;5. C u ltu re for 115 h und er the b lu e excitated light, arrow show endop lasm reticu lum;6. Cu lture for 115 h under the u ltra excitated
ligh t, arrow show nucleus, nucleus increases obviou sly.
4434期 祖元刚等:超声破碎拟南芥原生质体后的再生培养初报