全 文 ：第 20 卷　第 4 期　　　　　　　木　　本　　植　　物　　研　　究 2000 年　10 月
Vol.20　No.4　　　　　　　BULLETIN OF BOTANICAL RESEARCH Oct., 　2000
荞麦愈伤组织分化与过氧化物酶活性
及其同工酶关系的研究
郝建平　裴雁曦　曲运波
(山西大学生命科学系 , 太原　030006)
摘　要　在附加不同外源激素的培养基中 ,龙山荞的子叶愈伤组织表现出不同的分
化状态:(1)不分化;(2)分化根;(3)分化芽;(4)分化全苗。四种愈伤组织的过氧化
物酶活性的相对比值为 1:1.47:5.95:7.78 ,并且过氧化物酶同工酶谱也存在明显
差异 。酶带 4 为分化愈伤组织所特有;酶带 2 为未分化和分化愈伤组织所共有;在
分化根和未分化愈伤组织中缺少酶带 1 ;未分化愈伤组织比分化愈伤组织多出酶
带 3 。
关键词　荞麦;组织培养;过氧化物酶;过氧化物酶同工酶
STUDY ON PEROXIDASE ACTIVITY
AND ITS ISOENZYMES IN CALLUS
DIFFERENTIATION OF COMMON BUCKWHEAT
HAO Jian-ping　PEI Yan-xi　QU Yun-bo
(Depar tment of Life Science , Shanxi University , Taiyuan 030006)
Abstract　On various mediums wi th different variety and level of phy toho rmone ,
cotyledon callus of Long shan common buckwheat showed dif ferent dif ferentiation
states:(1)no differentiat ion took place;(2)root dif ferentiation;(3)bud differentia-
tion and(4)plantlet dif ferent iation.The ratio of peroxidase activity in 4 kinds of calli
is 1:1.47:5.95:7.78.There appeared also significant difference among their zymo-
g ram of peroxidase isoenzyme.Band 4 in zymogram is characterist ic for the differenti-
ated callus;band 2 is common for the undifferentiated and the dif ferent iated;band 1
is of sho rtage fo r the differentiated root and the undif ferentiated;band 3 is an ex tra
for the undifferentiated.
Key words　Common buckwheat;Tissue culture;Peroxidase;Peroxidase isoenzyme
作者简介:郝建平(1959-),男 ,山西大学生命科学系副教授。研究方向为植物细胞工程。
The project is f inanced by Shanxi Natural Science Funds(961020)收稿日期:1999-2-4
1　Introduction
Yamane for the f irst time applied t issue culture technology in common buckwheat research
w ork in 1974 and since then the study has at tracted more and mo re attention.But a thorough
investigation and study is being seriously hindered by the problem of dif ferentiation and plant
regeneration in tissue culture.The external morphological change is resulted by internal phy sio-
logical and biochemical activi ty.The basic cause for dedif ferentiat ion and redifferentiation of
tissue is the specif ic character express of gene and the formation of special protein.In present
paper , it is studies that differences of peroxidase activities and their isoenzymes of calli show ing
different dif ferent iation states to provide scientific basis for further study on mechanism of cell
differentiat ion and org anogenesis.
2　Materials and Methods
2.1　Experimental materials
Af ter convention sterility coty ledon explant of Longshan common buckwheat w as inoculat-
ed on MS solid medium to induce callus.The calli showed different differentiation states in
medium with various phy tohormone levels.
2.2　Measurement of pero xidase activity
Calli of 1 gram of various differentiation states were sampled and 1ml sample ex tracting so-
lution w as added into each sample.Then the samples w ere abrades into uniform serous fluid at
0℃ and centrifuged for 15 min in 10000 rpm freezing centrifuge to get supernatant liquid(for
those w ith high activi ty , proper dilution needs to be done at first).After addi tion of 1.0 m l
pH5.0 acet ic acid buffer , 1.0ml 0.1%guaiacol and 1.0ml 0.08%H2O2 solution , the samples
w ere shaken quickly to uniformity.Then OD were measured at 470nm with spect-ropho tome-
ter.The enzyme activity w as measured based on light absorbancy in each minute(OD diversity
of 0.01 per minute is one unit of peroxidase activity).
2.3　Analy sis to peroxidase isoenzyme
Here the ex traction method for the applied enzyme solution w as the same w ith that in ac-
tivi ty measurement.After proper dilution the ex tracting solution w as separated in polyacry-
lamide gel vertical slab elect ropho resis and stained w ith benzidine method.Then the zymogram
from elect rophoresis w as taken photos w ith Gel Documentation and Analy sis System.
3　Results
3.1　States of calli differentiation
In M S differentiation medium with various phy tohormone , callus of Long shan buckwheat
cotyledon show ed different dif ferentiation states:(1)undifferentiat ion , (2)root differentia-
tion , (3)bud differentiation and(4)bud and root differentiation.
3.2　Comparison of peroxidase activi ties
The activities of peroxidase w ere listed in an order from low to high by undifferentiation ,
4173 期　　　　郝建平等:荞麦愈伤组织分化与过氧化物酶活性及其同工酶关系的研究
root differentiation , and bud differentiation and plant let dif ferentiation.Peroxidase activity w as
higher in adventitious bud than that in all calli(Table 1).
Table 1　Peroxidase activities in calli of different differentiation States and adventitious
bud
Type of materials Pero xidase activity(0.01 ΔA/min)
The undifferentiated callus 6.56
The callus of differentiated root 9.66
The callus of differentiated bud 39.05
The callus of differentiated plantlet 51.05
Adventitious bud 162.7
Note:the data was mean values from three measurements
3.3　Analy sis of peroxidase isoenzyme zymogram
a.the undifferentiated callus　　　　　　d.the callus of differentiated root
b.the callus of differentiated bud and root　　　　　　 e.adventitious bud
c.the callus of differentiated bud　　　　　　　　　　　　　　　　　　
Fig.1　Zymog ram of pero xidase isoenzymes of calli of different differentiation states
Figure 1 showed clearly zymogram of pero xidase isoenzymes for materials of 5 different
differentiat ion states w ere signif icantly dif ferent.In plant let , bud or root dif ferentiated callus
and adventitious bud there is an ex tra band 4 than in the undif ferentiated callus;in plantlet ,
bud differentiated callus and adventit ious bud there is an ex t ra band 1 than in root differentiat-
ed and undifferentiated callus.In undifferentiated callus there is an ext ra band 3 than in the
rest 4 kinds of materials.In adventit ious bud ext ra bands of 6 and 7 than in the rest 4 kinds
of calli.And the band 2 in zymogram for the undifferentiated callus is the deepest stained and
band 2 of root differentiated callus took second place.
4　Discussion
Peroxidase play s an important part in regulation of cell development and differentiation.
Many physiological metabolism process of plant has something to do wi th activity of peroxidase
418 木　本　植　物　研　究　　　　　　　　　　　　　　　20 卷
and pat tern of its isoenzyme.Peroxidase activity of Longshan buckwheat callus increased in an
o rder f rom the undif ferentiated to the root , bud and plantlet differentiated.The dif ferentiation
deg ree is positively related w ith the activity .The conclusion is agreeable w ith the result f rom
tissue culture of Cnidium monnieri.It might be caused by the participation of peroxidase to
lignin s synthesis and lignin is demanded in const ructure of vascular system in organogenesis.
Bajaj s study reported that the metabolism regulated by phy tohormone is related with
presence of perox idase isoenzyme and the effect of different variety and proportion of phy toho r-
mone are dif ferent to gene.Not to mention the inf luence f rom other factors like environment
result difference in gene expresses of callus and finally leads to product ion of isoenzyme.That is
w hy calli of dif ferent differentiation states have dif ferent pat terns of zymogram.Band 4 in zy-
mog ram is a specific for dif ferentiation materials.It suggests it s special function in differentia-
tion and could be applied a biochemical indicator fo r callus dif ferentiation in tissue culture of
common buckwheat.Band 2 appeared in the dif ferentiated , undif ferentiated calli and in ad-
vent itious bud and it suggests that it is a main band.Band 1 and 3 both has something to do
w ith bud differentiation and the undifferentiated respectively.
One of the final purposes for tissue culture is to produce regeneration plant for which cell
differentiat ion is an essential prerequisite.Since cell differentiation and morphogenesis di rection
is depended on physiological and biochemical change , i t is of great importance to understand
such properties of buckwheat callus to the application of tissue culture technolog y in buckwheat
scientific research and comprehensive utilizat ion.
References
1.Hao Jianping , Li Baoping and Zhou Xiaomei.S tudy on perox idase and its isoenzymes in tissue culture of Cni-
dium monnieri.Nor th Bo tanical Research , 1993 , 1:97～ 100.Nankai University P ress , China
2.Lin Rufa.Isoenzymes and chromosomes of buckwheat.Buckw heat in China , 1994 , 97 ～ 100.Agriculture
Publishing House , Beijing
3.Rumyantzeva N.I.et al.Morphogenesis and plant regeneration in buckw heat tissue culture.Proceeding of
the 4th International Symposium on Buckw heat , 1989
4.Yamance J.Induced differentiation o f buckw heat plants from subcultured callus in vitro.Jap.J.Geneti.
1974 , 49 ～ 139
5.Zhao Gang , Tong Yu.Study on pero xidase isoenzymes in buckw heat.Buckwheat Research T rend (in Chi-
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4193 期　　　　郝建平等:荞麦愈伤组织分化与过氧化物酶活性及其同工酶关系的研究