全 文 ：第 25卷　第 3期　　　　　　　　　　　　　植　　　物　　　研　　　究 2005年　7月
Vo .l 25　No. 3　　　　　　　　　　　　BULLETIN OF BOTAN ICAL RESEARCH July, 　2005
Foundat ion item:Supported by the S tate Key Basic Research and Developm ent P lan of Ch ina (G19990160)
Au thor in troduction:Zu Yuangang (1954— ), Ph. D. , Professor, m ajor in p lan t science. E-m ail:zygorl@ pub lic. h r. h.l cn
Received date:2004 - 01 - 07
组织培养条件下喜树叶片细胞壁酸性降解的 pH值观察
祖元刚　于景华　唐中华　郭晓瑞　孟庆焕　张宇亮
(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　喜树叶片外植体经组织培养第 13 d和23 d后进行解剖学观察 ,同时比较正常叶片的解剖
学特征 ,发现在 pH值为 5. 8的酸性培养基中 ,喜树叶片外植体中的海绵组织和栅栏组织等薄壁细
胞相继发生明显的细胞壁降解 ,而表皮细胞发生较微弱的细胞壁降解现象;进行组织培养前 ,正常
叶片的各类细胞未观察到细胞壁降解现象发生。应用 BCECF-AM pH荧光探标记并采用激光共
聚焦在 480 nm波长下进行 pH值测定发现 ,喜树叶片外植体经组织培养第 13 d和第23 d后的海绵
组织和栅栏组织等薄壁细胞部位的 pH值均为 5. 2 ,但表皮细胞部位的 pH值则为 5. 7 ～ 5. 8,而正
常叶片各类细胞的 pH值平均为5. 7。这说明 , pH值为 5. 8的酸性培养基和喜树叶片薄壁细胞内
的酸性成分自泌可能共同诱导了其细胞壁的酸性降解。
关键词　组织培养;喜树叶片;细胞壁酸性降解;pH值
Observation on pH variation dur ing cellwall ac id degradation in
Campto theca acum inata leaf under the tissue culture condition
ZU Yuan-G ang　YU Jing-Hua　TANG Zhong-Hua　GUO X iao-Rui　MENG Q ing-Huan　ZHANG Yu-Liang
(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　The explant of Camptotheca acum inata leafw as obse rved on anatomy after cu ltivation o f 13
day and 23 day, contrasting w ith norm al lea.f In pH 5. 8medium , we observed that the cellw all degra-
dation g radually arised in explan ts of pa renchym a ce ll inc luding the spongy tissue and palisade tissue in
C. acum ina ta lea f obv iously, whereas unconsp icuously in epide rm is. But before the tissue cu lture, vari-
ous types of ce ll in norm al leaf cannot be de tected. Using the labeled fluorescence BCECF-AM and laser
scanning m icroscopes mensura ted the pH under 480 nm , we found the pH of parenchyma cell bo th in
spongy tissue and pa lisade tissue are 5. 2 in exp lants o fC. acum ina ta leaf, afte r tissue cu lture 13 day s
and 23 days, and the pH of the epiderm is is 5. 7 ～ 5. 8, while the average pH is 5. 7 in norm al lea.f
Th is indicated themedium o f pH 5. 8 and the secretory acid component in parenchyma ce ll inC. acum i-
nata lea f bo th induced the ce llw a ll degradation.
Key words　 tissue culture;Camptotheca acum inata lea f;ce llw all acid degradation;pH values
Plant tissue culture is a sub ject that use the
m ixed cu lturemed ium by artificial to induce p lant cell
ded ifferen tiation and redifferentiation
[ 1]
. But it’ s still
unc lear the mechanism o f culture medium inducing
the p lant ce ll dedifferentia tion
[ 2 ～ 4]
. W e supposed
tha t, acid substances in cu lture medium prescrip tion
can ho ld the leaf in the ac id env ironmen t and bring
the acid degradation of the no rmal plant cell w all at
first, then present pro toplast breaking o r rebuil-
ding
[ 5]
. W e inve stiga ted the influence o f acid culture
medium on ce ll w all deg radation in C. acum inata
leaves, and observed that ce ll w a ll of C. acum inata
leaves has ana tomy characte ristic o f sligh tly degrada-
tion, obviously deg rada tion, abso lutely degrad ing and
ce ll w all d isappearanced unde r tissue cultu re condi-
tions
[ 6]
. In this a rticle, we observed the chang ing o f
pH in parenchyma ce ll and epiderm is ce ll in diffe rent
time in tissue cu ltures and compa red w ith the norm al
lea fw ith C. acum inata leaf to understand the d iffe r-
ent source of the ac id compositions during ce ll w all
acid deg rada tion.
1　Materials and methods
1. 1　Experimen tmaterials
TheC. acum inata Leaf is come from Key Labo-
ra to ry o f Forest P lantE co logy, M in istry of Education.
The young leaf w as chosen and cu t the slice o f 1 cm
×1 cm , and then the uppe r epide rm isw as pu t inWP
cu lturemedium w ith pH 5. 8 wh ich con tainsα-naph-
thy l ace tate (NAA) 0. 2 mg L - 1 , 6-Benzy lam ino
purine(6-BA) 1. 0 mg L- 1. And become into cal-
lus in explants after 23 day, cultu red in 28℃.
The norma l leaf and exp lants callusw ere cut into
th inner than 10μm and trea ted w ith w ax.
1. 2　Themeasurement of the C ell pH
W e measured intracellular pH (ph i) in 2′, 7′-
bis ( carboxye thy l)-5 ( 6 ) carboxyfluo resce in
(BCECF)-loaded leavesmonitored w ith aN ikon con-
focal m icroscope. Leaves w ere loaded w ith BCECF-
AM (20 μmol L- 1) for 30 m in in the dark and
w ashed in 10 mmo l L -1 MES-TRIS buffer, pH
6. 2. G roups o f leaves w ere put on slides and the ir
fluo rescence signa ls at 520 nm and 640 w ere meas-
u red fo r exc ita tion w ave leng ths set at 488 nm. F luo-
rescence in tensity ratios (520 /640, F lRs)were thus
monito red as indicators of pH.
Prepare a DAPI (4′, 6-d iam ino-2-pheny lindo le
dihydrochloride) stock so lu tion o f 1mg mL- 1. D i-
lu te th is stock so lution w ith distilled w ater to a DAPI
w orking so lu tion o f 10μg mL -1. W ith a m icropi-
pette, add 20 μL o f the DAPIw orking so lution to the
samp le ( final concentration:1μg mL -1). Incubate
the sample fo r 10m in in the dark. Then the samples
are observed w ith epifluorescence m icroscopy, using
an UV filte r(excitation 365 nm).
2　Results
2. 1　pH o f norma l leaf ce ll inC. acum inata
The comp le te leaf inC. acum inata inc lud ing ep-
iderm is, the pa lisades tissue, the spongy tissue and
conducting parenchyma did no t discover any ce llw a ll
degradation phenomenon(Plate Ⅰ:1). Through the
measuremen t, w ithout the condition o f tissue culture,
the pH of epiderm is o fC. acum inata leaf cell is 5. 3
～ 5. 8 , the ave rage is 5. 48, the pH of palisades tis-
sue cell to be w o rth to 5. 4 ～ 6. 9 , the average is
6. 13, the pH of spongy tissue ce ll is 5. 5 ～ 6. 3, the
ave rage is 5. 75(P lateⅠ:2). The re fo re, the signifi-
cant characte ristics o f pH in no rmal leaf ofC. acum i-
nata is that the pH of the palisades ce ll is the h igh-
est, the secondly is spongy tissue cell and the epide r-
m is ce ll is the low es.t
2. 2　The pH o f leaf cell o fC. acum inata after cu l-
tured 13 days
A fte rC. acum inata leaf exp lantw as cultured 13
days, the pa lisade tissue and spongy tissue appeared
different degrees of cell w all deg rada tion phenome-
non, but it isn’ t obv ious in the epiderm is ce ll(Pla te
Ⅰ:3). Through the measurement, the pH o f its epi-
derm is cell is 5. 2 ～ 5. 8, the ave rage is 5. 46. The
pH o f the pa lisade tissue and spongy tissue is 5. 2
(PlateⅠ :4). Therefo re, afte r C. acum inata leaf
cu ltured 13 days, the significant characteristics is that
the pH o f its epide rm is ce ll is the highest, bu t epide r-
m is ce lls have already deg raded partly, so the pH is
a lso 5. 2 ～ 5. 3, pH of the epiderm is ce ll wh ich did
no t deg rade is 5. 8. The ce ll w all o f palisade tissue
and spongy tissue deg raded obv iously, its pH is 5. 8
tha t is lowe r than the acid env ironment, also obv ious-
ly low er than the same k ind o f the cell of norm al lea.f
2. 3　The pH inC. acum inata leaf ce ll afte r cultured
23 day s
The pa lisades tissue and spongy tissue had the
further ce ll w all degrada tion, after the C. acum inata
leaf explan ts cu ltured 23 day s, the degradation of epi-
derm is slightly(PlateⅠ:5). Through themeasurement,
2873期 祖元刚等:组织培养条件下喜树叶片细胞壁酸性降解的 pH 值观察
the pH of epiderm is is 5. 7, pa lisades tissue is 5. 2 ～
5. 4, and sponge tissue is 5. 2(PlateⅠ:6). There fore,
the significant characte ristic of epiderm is ce ll is the pH
still highest, after the leaf cu ltured 23 days, bu t due to
sligh tly degrade of ce ll w all, so the pH is 5. 7;The
palisade tissue and sponge tissue ce ll w a ll deg rades
complete ly, so its pH maintains about 5. 2.
Reference
1. DeP ie rre J W , E rnaster L. Enzym e hopo logy of intace llula
m embranes. Ann Rev Biochem , 1977, 46:201～ 262
2. Danie l J Cosgrove. Expansive grow th o f p lant cellw alls. P lant
physio logy and biochem istry, 2000, 38(1, 2):109 ～ 124
3. Ray le C leland. The ac id g row th theory of auxin-induced ce ll
elongation is a live and w el.l P lant Physio logy, 1992, 4:1271
～ 1274
4. B rownlee C, Berger F. Ex trace llular m a trix and patte rn in
plant em bryos:on the lookout for deve lopm en ta l inform ation.
T rends Gene t, 1995, 11:344 ～ 348
5. Zu Yuangang, Yu Jinghua, Tang Zhonghua, et a l. Po ssible
m echanism invo lved w ith the form ation of lea f ca llus. Bulletin
of bo tanica l research, 2005, 25(1):1 ～ 2
6. Zu Yuangang, Yu Jinghua, Tang Zhonghuaet al. Influence of
acid culture medium on ce ll wa ll degradation o fCamptotheca
acum ina ta leaves. Bulle tin of Botanica l Research, 2005, 25
(2):129 ～ 131
P lateⅠ 　1, 3, 5. Im aged by using inverted m ic roscope;2, 4, 6. W e re sta ined w ith pH labe led before im ag ing using laser scanning
m ic roscope s(×400)　　　P t:Palisade tissue;Sp:Spongy tissue;Eu:Uppe r epide rm is
1. The norm alC. acum ina ta leaf do noth ave the phenom enon of the degradation;2. The pH of d ifferen tparts inC. acum ina ta leaf has great d iversi-
ty, and th e average is h igh er;3. The cellw all of p arenchym a cell appears degradation in d ifferent degree, af ter t issue cu ltu re C. acum ina ta leaf 13
days, bu t it’ s unconsp icuous in the ep iderm is;4. The pH of the w hole parenchym a cel l are a ll5. 2;wh ile the ep iderm is cellwh ich th e cellw all has
slight ly degradation are 5. 2～ 5. 3, bu tw h ich the cellw all undeg raded are 5. 8;5. Af ter 23 days, the cel lwa ll of parenchym a cell in C. acum ina ta
leaf has fu rther degraded, w hi le the epiderm is cellw allh as slight ly degraded;6. The pH of palisade tissue inC. acum ina ta leaf is 5. 2 ～ 5. 4 after tis-
sue cu ltures 23 day, and in palisade t issue is5. 2, bu t in ep iderm is cell is 5. 7.
288 　　　　　　植　　物　　研　　究　　　　　　　　　　　　　　　　　　25卷