全 文 ：广 西 植 物 Guihaia 23(4)：334— 338 2003年 7月
影响决明无菌苗子叶原生质体
分离和培养 因素 的研 究
周延清 2，张根发3，贾敬芬
(1．西北大学生命科学学院，陕西西安 710069；2．河南师范大学生命科 学学 院
河南新乡 453002；3．北京师范大学生命科学学院 ，北京 100875)
摘 要 ：以决明(Cassia obtusifolia)无菌苗子叶为材料 ，对酶组合 、无菌苗 日龄 ，植物激素 组合 和培养方法对
其原生质体的分离和培养的影响进行了研究。结果表明：用3 的纤维素酶和0．2 Pectinase Y一23的酶组合
处理决明无菌苗子叶块 8小时可 以高效分离出有活力的原生质体 ；约 14日龄 的决明无菌 苗子叶 比较适 合于
原生质体的分离 ；适 当浓度 的 2，4一D有利 于原生质体 的分离 。促进 原生质 体分裂 的理 想的植 物激素组合 为
0．4 mg／L 2，4一D，1．0 mg／L NAA and 0．1 mg／L KT；漂浮培养法最有利于原生质体 的分 裂和发育 。找出了
适合于决明无菌苗子叶原生质体的分离和培养的酶组合、植物激素组合、有效培养方法和决明无菌苗子叶日
龄。这为有效地从决 明无菌苗子叶原生质体再生植株奠定 了基础 。
关键词 ：决明 ；原生质体培养 ；酶组合 ；日龄；培养方法 ；植物激素
中图分类号 ：Q942．5 文献标识码 ：A 文章编号 ：1000—3142(2003)04—0334—05
Factors influencing protoplast culture of
the seedlings of Cassia obtusifolia
(Leguminosae)
ZHOU Yan—qing 一，ZHANG Gen—fa。，JIA Jing—fenI*
(1．College of Life Sciences，Northzt,est University，Xi an 710069，China：2．College of
L fe Sciences，Henan Normal University，Xinxiang 453002，China：3．College of
L fe Sciences，Beijing Normal University，Beijing 100875，China)
Abstract：In this work，the cotyledons of Cassia obtusifolia seedlings were used as materials．The effects of
some factors such as different combinations of enzyme mixture，day—age of C
． obtusifolia seedlings，culture
methods and phytohormones in medium ，on protoplast isolation and culture were investigated
． The resuIts
demonstrated that protoplasts with high yield and quality were obtained by treating the cotyledon pieces with a
mixture of 3 cellulase(Onozuka R一10)and 0．2％ Pectinase Y一23 for 8h．The cotyledons from 14 day-old
seedlings were appropriate for isolating protoplasts
．
The floating culture method~vas more suitable for proto—
plast division and the division frequency was 9
． 7 ．The proper phytohormone combination was 0
． 4 mg／L 2．
4-D，1．0 mg／L NAA and 0．1 mg／L KT 2，4-D，in which the division frequency 2l_2 was gotten
． The opti—
mum combination of enzyme mixture，phytohormone combination，culture method and proper day-age of C
． 0b—
tusifolia seedlings were determined．This lald the foundation of efficient plant regeneration from cotyledonarv
收稿 Et期 ：2002—09—09 修订 日期 ：2003—02—2O
Author introduction：ZHOU Yan-qing(1963一)，Male，born in Dengzhou city，Henan Province，Doctoral student
， Ass。ciate Profes
SOr，WOrking in plant ceil engineering and cr。p genetic breeding． *Author for COrrespondence：E-mail：wangxl@nwu
． edu．cn
一 ’ ’ 。 ‘ — 。 。 。 ’ ’ ’ ’ ’ 。 _ 。 — ’ 。 ’ 。 。 。 — —
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4期 周延清等 ：影响决明无菌苗子叶原生质体分离和培养因素的研究 335
protoplasts of C．obtusi‘folia seedlings．
Key words：Cassia obtusifolia；protoplast culture~enzyme combination)day-age；culture method)phytohor—
m one
1 Introduction
The higher frequency of plant regeneration
from protoplasts is not only the foundation of
plant genetic m anipulation and plant improvement
by means of cell engineering manip ulation but also
fl good experimental system for the study of gene
expression and in vitro cell differentiation． There—
fore，scientists all over the word have been paying
great attention to it． C． obtusifolia L．(Legu—
minosae)is fl kind of common traditional Chinese
herbs． Its seed is known as“false phaseolus radi—
atus’or“coffee bean”．used for treating liver dis—
eases，purging，reducing blood pressure，contrac—
ting the uterus，and dispelling rheumatism (Feng，
1 9 9 3；Lian，1 986)． So far，traditional breeding
has been used in improving this species，but the
limitations of traditional breeding method，such as
the limited gene pool and the necessity for succes—
sive back crossing，could not be overcome． Ac—
cordingly，it requires im provement by biotechnolo-
gy． In previous study，we had got the regenerated
plants from its protoplasts(Zhou et al，1 9 98)and
cotyledon cultures of C．obtusifolia(Zhou et al，
2 0 0 1)． In addition，because genetic m anipulation
and somaclonal variation are efficient ways to im—
prove wild plant，they will be put to good use in
C．obtusifolia production by increasing its plant
regeneration frequency from protoplasts． In this
work，some factors influencing its protoplast iso—
lation and culture were investigated．
2 M aterials and M ethods
c．obtusifolia is fl cross—pollinated species．
Its seeds provided by Professor Yuan Baoj un of
Zhoukou Prefecture Institute of Agricultural Sci—
ences，P．R．China were treated in concentrated su1一
furic acid for 40 minutes and in 0．1 HgC12 for 5
minutes for sterilization，respectively，then washed
4～ 5 times with sterilized double distilled water
and placed on agar—-solidified M S medium(M urash-。
ige and Skoog，1962)without any plant growth reg—
ulator in 100ml flasks(Zhou et al，2001)．Dry seeds
were allowed to germinate for proper days under
light conditions and then the cotyledons of its seed—
lings were harvested．
These cotyledons were cut into about 1 mm—
long pieces．Firstly，the pieces were put in CPW 一
1 3M solution containing 1 3 mannitol(W ei and
Xu，1990)for preplasmolysis for 1．5 h．Secondly，
about 1 g pieces were placed in eight milliliter an—
zyme solution(Table 1)and incubated at 26 ℃ in
the dark while vibrated at forty r／pro．Third，the i—
solated protoplasts during the enzyme digestion
were observed by microscope every other one
hour．The adopted isolation time was determined
when mean protoplast density did not increase any
longer in the last hour． In the best isolation time，
the isolation condition was shown by percentage of
protoplasts in enzyme—protoplast suspension．
Floating，collection，purification and harvest of
protoplasts were carried out using th the method
described by Zhang et al(1994)． The cotyledons
from different day—age seedlings of C．obtusifolia
were used to study the effect of day—-age on the iso-·
lation of protoplasts． The effect of several phyto—
hormones such as NAA ，2，4-D and KT on proto—
plast culture was investigated．The protoplasts re—
leased from the cotyledons were cultured in CK8P
thin layer liquid medium (Reinert and Yeoman，
1989)at fl density of 5．1×10 numbers／ml(1 mL
protoplasts suspension in fl 3．3 cm × 1 cm dish)．
Percentage of survival protoplasts was calculated
after fl culture of two days
． The effect of phyto—
hormones on the division frequency of protoplasts
was studied as stated by Zhang et al(1999)．
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336 广 西 植 物 23卷
3 Results and Discussion
3．1 Effect of different combinations of enzyme m ix—
ture on the protoplast yield from the cotyledons of
C．obtusifolia seedlings
After the cotyledon pieces were digested for 8
～ 24 h in the enzyme solution(Table 1)，1．8× 10
～ 1．6× 1 0 protoplasts／g．fresh weight were sue—
cessfully produced (Fig．1)． The results showed
that the proper duration of protoplast isolation and
protoplast yield and quality in different enzyme
compositions were different．It was very clear that
protoplasts with high yield and quality were ob—
tained by treating the cotyledon pieces for 8h in a
mixture of 3％ cellulase(Onozuka R一10)and 0．2
Pectinase Y一23．This result was consistent with
Johnson ￡Ⅱf(1982)．However，the enzyme comb卜
nations of cellulase(3 Onozuka R一10)with 0．2
M acerozyme R一10 and 1 Hemi cellulase(Sigma)
or 0．5 Pectinase(Serva)and 0．5 Hemi cellu一
1ase (Sigma)gave poor protoplast yield in longer
d1glration．
Fig． 1 Protoplasts i：；olated from cotyledon segments
Table 1 Effect of different combinations of enzyme mixture on the protoplast yield
from the cotyledons of C．obtusifolia seedlings
N。te：Enzymes was dissolved in CPW一9：9 o,4 Mannit。1 CPW (Reinert andYeoinan．1989)；+ ：2～5 Clusters，high isolati。n f equency；+ +
6-10 Clusters，1ow isolation frequency；+ ：11～ 15 Clusters，very low isolation frequency
3．2 Effect of the age of seedlings on protoplast isola—
tion of C．obtusifolia
The age of C obtusi{olia seedlings also af—
fected the protoplast isolation． It was more diffi—
cuIt to isolate protoplasts from older cotyledons
than that from younger ones． However，very
young cotyledons were not suitable for the proto-,
plast isolation，because their protoplasm mem—
branes were easily damaged． The experimental re—
suIts showed that the cotyledons from 14 day—age
seedlings were appropriate for the isolation of pro—
toplasts(Table 2)
Table 2 Elfect of the age of seedlings on protoplast isolation of C．obtusifolia
3．3 Effect of different culture methods on protoplast
division and development
The effect of different culture methods on pro—
toplast division and development was seen in table
3．It was obvious that the culture methods indeed
influenced protoplast division and further develop—
ment；floating culture was the best suitable for pro—
toplast division(Fig．1～ 2)． The frequency was
9．7 ．As a result，small calli formed and brown—
ing decreased；agarose—embedded culture was more
suitable for protoplast division than thin layer liq—
uid culture．
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周延 清等 ：影 响决 明无菌苗子叶原生质体分离 和培 养因素 的研究 337
Fig．2 The first protoplast division
3．4 Effect of different phytohormones on the divi_
sion frequency of protoplasts
In this experiment，the effect of several plant
phytohormones on cell division frequency of proto—
plasts was summarized in Table 4．
Table 3 Effect of different culture methods on
protoplast division and development
As shown In Fable 4，(1)W hen only 2，4-D
was used，high concentration of 2，4-D was better
than the low one for the protoplasts division．
Higher division frequency(18．8 )of protoplasts
＼vas obtained in composition Ⅱ at the higher con—
centration of 1．0 mg／L 2，4-D than that(12．6 )in
composition I at the low one of 0．4 mg／L 2，4-D．
(2)The results from composition m to composition
IV revealed that the division frequency of proto～
plasts was improved from 11．7 to 21
． 2 with
1 he increase of tl1e concentration of 2，4-D from 0
． 2
to 0．4 mg／I in the compositions plus the same
concentration of NAA and KT
． To put(1) and
(2)together，it was concluded that the higher the
concentration of 2，4-D，the higher the division fre～
quency of protoplasts． After that，by comparing
composition 1 with compositionIV and composition
1 with composition V ，it was found that NAA and
KT could also inlprove the frequency
． The positivc
effect of 2，4-D，NAA and KT was stronger than
that of 2，4-D alone on the protoplast division．
However，NAA and KT im proved less protoplast
division frequency than 2，4-D did． This was more
obviously shown by comparing composition T
with composition m ，because high concentration of
2，4-D(0．4 mg／L)could produce higher protoplast
division frequency than low one of 2，4-D(0．2 mg／
L)，together with 1．0 mg／L NAA and 0．1 mg／L
KT，could． In addition，it could be known from
compositionⅣ and composition V that the higher
division frequency ( 2 1．2 0 o ) of protoplasts was
gotten in the composition of 0．4 mg／L 2，4-D and
1．0 rag／L NAA and 0．1 mg／L KT than that(20．
5 )in the one of 1．0 mg／L 2，4-D and 1．0 mg／L
AA and 0．1 mg／L KT at the same concentration
of NAA and KT．The possible reason was that the
ratio of auxin to cytokinin affected the frequency．
This ratio in composition Ⅳ was more suitable for
the frequency than that in composition V ． M ean—
while，the frequency(21．2 )in composition IV was
the highest among al’the 5 compositions．Hence，it
was believed from the above analyses that composi—
tion IV was the best suitable for the improvement
of the frequency in this work． In a word．different
phytohormones had a positive impact on the divi—
sion frequency of protoplasts；phytohormone com—
position was very important for the cell wall regen—
eration of protoplasts and cell division at the initial
stage of protoplast culture；2，4-D was indispensa—
ble to cell division under its proper concentration
．
Table 4 Effect of several phytohormones on the
frequency of protoplasts
Phytohormone composition(mg／I ) Division frequency( )
In conclusion， the optim um combination of
enzyme mixture (3 cellulase(Onozuka R一10)and
0．2 Pectinase Y一23)，phytohormone combination
(O．4 mg／L 2，4-D，1．0 mg／L NAA and 0．1 mg／L
KT)，culture methoc(floating culture) and better
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338 广 西 植 物 23卷
day—age(1 4 to 1 5 d)of C．obtusifolia seedlings
were determined． This laid the foundation of effi—
cient plant regeneration from cotyledonary proto—
plasts of C．obtusifolia seedlings and will greatly
contribute to its breeding via somaclonal variation，
germ line improvement and genetic transforma—
tion
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