全 文 ：广 西 植 物 Guihaia 24(3)：266—269 2004年 5月
石竹细胞悬浮培养研究
李宗艳
(西南林学院园林学院，云南昆明 650224)
摘 要：石竹细胞继代周期为7 d时，悬浮细胞培养系生长最快，生长率最高，而且培养物中胚性细胞较多，并
能保持较快的分裂和生长，能促进已形成的大细胞团的生长和分化。转代时接种物与新鲜培养基的体积比以
1：2较好，悬浮系细胞生长最快，生长率最高，以 1：2和 1：3的高倍稀释接种有利于胚性细胞的形成及产生
小的胚性细胞团，对悬浮系添加椰乳和水解乳蛋白的混合物，可较大幅度地提高悬浮细胞系的生长速率，单独
添加上述两种物质的效果均不如二者的综合效应好。在 6种不同激素组合中，配方 2(2，4-D 1．5 mg／L+
NAA 0．5 mg／L+6-BA 0．5 mg／L)最好，生长率最高。配方 5(2，4-D 1．5 mg／L+NAA 0．5 mg／L+6-BA 1．0
mg／L)其次；配方 1(2，4-D 1．0 mg／L+NAA 0．5 mg／L+6-BA 0．5 mg／L)次之。
关键词：石竹；细胞悬浮培养；生长
中图分类号：Q949．72 文献标识码：A 文章编号：1000-3142(2OO4)O3一O266一O4
Study on cell suspension culture
of DianthUS chinensis
LI Zong—yan
(Faculty of Landscape Architecture，Southwest Forestry College，Kunming，650224，China)
Abstract：In the present research，the suspension culture clones of Dianthus chinensis can keep cell muhiplica—
tion and growth-rate at high speed when the secondary cycle is arranged for 7 days．Moreover，there exist lots
of embryonic cels in the culture fluid that could keep growth and division fast and promote growth and differ-
entiation of the formed big cell groups．W hen conducting trans—generation，it is better if volume rate between
inocula and fresh culture media is 1；2．Because the cell multiplication and cells·growth keep the fastest un-
der this condition．It is the high dilution rate of 1；2 or 1：3 that will contribute tO form the embryonic cells
and produce the smal embryonic cell groups．Provided that a mixture of coconut milk(CK)and lacotalbumin
hydrolysate(LH)is added，the growth-rate of suspension clones could be greatly improved．Adding both is bet-
ter than adding only one of them．The concentration of hormone plays a key role in cels，growth and division．
Compared tO six different formulas，formula 2(2，4-D 1．5 mg／L+NAA 0．5 mg／L+6-BA 0．5 mg／L)is the
best．Formula 5 comes second(2，4-D 1．5 mg／L+NAA 0．5 mg／L+6-BA 1．0 mg／L)and formula 1(2，4-D
1．0 mg／L+NAA 0．5 mg／L+6-BA 0．5．mg／L)comes third．
Key words：Dianthus chinensis；eel suspension culture；growth
Dianthus chinensis L．is widely distributed in
China and an important economic plant．This spe—
cies is cultivated in the garden not only as an orna—
mental plant，but also as a medical plant．This pe—
rennial herb contains lots of useful chemical COrn—
positions in its leaves，stems and roots，such as eu—
Received date：2003-06·—23 Accepted date；2003‘_09‘_24
作者简介：李宗艳(1974一)，女，云南文山人，硕士，讲师，园林植物专业，主要从事园林植物方向的教学和科研工作。
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3期 李宗艳：石竹细胞悬浮培养研究 267
genol，benzyl benzoate，methyl salicylatc，saponins
and so on．(Huang TK P￡nZ．，2001)
The fluid suspension culture of plant cells is
applied for the virus。-free and high quality seed—-
lings，the secondary metabolites，producing artifi—
cial seeds and building the efficient reproduction
system(Tan WD P￡nZ．，2001)．The present re—
search is carried out to find the best formula by
testing the secondary cycle，the proper dilution rate
of inocula，additives and concentration of hormone
that will improve cells’growth and division rapid—
ly，keep high growth—rate and frequency of the em—
bryonic cells．
I Materials and Methods
1．1 Materials
Stem apex about 0．4～ 0．6 mm is cut from a
strong branch in greenhouse to be cultured for the
test—tub seedlings．
1．2 Inducement of calli
The stem apex taken from test-tub seedling is
used as an explantation to be inoculated on the cul—
ture medium for the wound callus at 25℃ under
dark conditions(formula；MS+2，4一D O．5 mg／L+
NAA 1．0 mg／L+6一BA 0．6 mg／L+LH 1 000 mg／
L+ Cane Sugar 3 )．After producing calli，they
are transferred to the same culture medium without
2，4一D for extension of propagation．The secondary
cycle is 10 to 15 days．
1．3 Establishing suspension culture clones
The calli about 3 or 6 gram are put into a cul—
ture bottle with 1 00 mL MS fluid culture medium
when the secondary cells have grown for 5 to 6
days．They are cultured for establishing the SUS—
pension lines with shakers functioning at 1 20
rounds per minute at 25～ 27 ℃ under the light
(Chen W et a1．，2002；Gan FY et a1．，1997；Zhang
QW nZ．，1995)．
1．4 Secondary culture of suspens ion cells
The secondary culture will be carried out peri-
odically after the calli become dispersed to form a
suspension line．When transferred，there are three
settings of dilution volume ratio between the inoc-
ula and fresh culture fluid，respectively 1：1，1
2，1：3．
1．5 Growth determination
1．5．1 Fresh weight determination The culture ma—
terials are filtrated through a previously weighed
paper when the periodical culture have ended，then
weigh it．
1．5．2 Dry weight determination The freshly
weighed cells，together with the filtrating paper，
will be baked for 1 2 hours at 60℃ in the incubator
before weigh．The result is the dry weight．
1．5．3 Relative volume ratio determination W hen
the suspension period is over，suspension materials
are put into the scaled—tube and centrifuged for 5
minutes under 800 rounds per minute，then the rel-
ative volume ratio is determined(Yu SW et a1．，
2001)．
1．5．4 Increasing rate determination The increas—
ing rate is the ratio between fresh weight and inoc—
ula weight．
1．5．5 Net growth rate determ ination Net growth
rate is the net gaining in fresh weight every day．
1．5．6 Relative water content determ ination Rela-
tive water content(RWC)js the ratio between wa—
ter content and fresh weight．Fresh weight minus
dry weight is the water content．
1．6 Suspension cells observation
The microscopic observation is used to analyze
the types of suspension cells．
2 Results and Discussion
2．1 Composition and Characters of suspension cul-
ture clones
The present study has revealed that there are
two types of cells constituting the suspension cul—
ture clones by the microscopic observation．One is
the embryonic cells，whose volume is small，ovate
and elliptical with a thin cell—wall，non-apparently
big vacuoles or central vacuoles，dense cytoplasm，a
apparent cell nucleus and nucleus．The embryomic
cells display in the suspension fluid usually in sin—
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268 广 西 植 物 24卷
gle dispersing or two to several cells which cluster
closely together into small embryonic cell groups．
The other type is the non-embryonic cell，whose
volume is big，stripe-oblong with an apparently big
vacuole or central vacuole．These non-embryonic
cells gather together and form the sparse，apparent
intercellular space and disorderly crumb structure
(Zhang LY et a1．，1997)．
3d 7d 15d
Fig．1 Effect of three secondary cycle
on suspension cells·growth
口 Increasing rate( )} Net growth rate(g·d． )．
2．2 Effects of the secondary cycle on suspension cul·
ture clones
The result(Fig．1)shows that it is fl good
time for the secondary cells to grow when the cycle
of secondary culture is arranged for 7 days．Its in—
creasing rate reaches to 172％，which is 18 or
more higher than those of formula 2． Its net
growth—rate is 0．543 g／per day，which exceeds
0．076 than that of 3 days，0．339 than that of 15
days respectively．Meanwhile，in the culture fluid，
the suspension clones can not only keep the growth
and division of embryonic cells rapid，but also pro—
mote the formed cell groups to grow and differenti—
ate．The reason of the decreases in growth-rate by
the 15 day might be that there is not enough nutri—
tion for cells·growth and division，which is resul—
ted from the nutrition of culture medium consumed
with the extension of culture time，and is that the
accumulations of metabolic products produced by
the continuous growth and division of cells re—
strained the continued growth of cells．However,
Table 1 Effects of volume rate between inocula and culture fluid on suspension cells‘growth
N0te：1．The sec0ndary cyclel7 days}2．Culture formula：MS+2，4一D 1．5 mg／L+NAA 0．5 mg／L+6-BA 0．5 mg／L+CM 5 ．
Table 2 Effects of different additive on
suspension cells·growth
Note：1．The secondary cycle：7 days；2．Culture formula：MS+2，
4-D 1．5 mg／L+NAA 0．5 rag／L+6一BA 0．5 mg／L’3．Dilution rate r
1：2．
the reason why the growth—rate is low by the third
day might be that the suspension clones can not
reach the top of growth and division because it is fl
short time before the cels j ust started to divide．
2．3 Effects of different volume rate on suspension
culture clones
According to the results，the increasing rate
and the cells’net growth rate of suspension culture
clones keep the highest under rate 1 ：2． Its in—
creasing rate is 17 1％ of the increasing rate and
cells·net growth is 0．370 gram of fresh weight per
day(Fw ·g· )in net growth rate under this
condition．The increasing—rate of rate 1：1 is simi—
lar to that of rate 1：3．At the same time，the mi—
croscopic observation shows that high dilution rate
of 1：2 and 1：3 will be beneficial to form embry—
onic cell groups and produce the small embryonic
cels．However，low rate of 1 t 1 will be good to
(_ 等 ．I c一盘 ucH
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3期 李宗艳：石竹细胞悬浮培养研究 269
form many non—embryonic and big cell groups．Re—
suhs of relative water content(RWC)have proved
that．
2．4 Effects of coconut milk and lacotalbumin hy-
drolysate on suspension culture clones
The net growth rate and increasing rate will
be apparently improved if either of two additives is
added in the fluid culture medium． Using fl mix—
ture of CM and LK is better than using only one of
them．Its increasing rate and net growth rate keep
the highest．Its increasing rate is 198 of the in—
creasing rate，which is 45％ higher than that of
LH，2 3 higher than CM respectively． Its net
growth rate reaches 0．641 unit(Fw ·g· 。)．Mo—
reover，there is fl higher ratio of embryonic cells in
the fluid culture medium．Compared to lacotalbu—
min hydrolysate(LH)，coconut milk(CM)will be
better．
2．5 Effec ts of hormone on suspension culture clones
The variety and concentration of hormone
have fl key effect on the growth and division of SUS—
pension cells．According to Table 3，when NAA
Table 3 Effects of hormone on suspension cells·growth
Note：1．The secondary cyclet 7 days~2．Culture[ormula；MS+CM 5％+LH 1 000 rag／L,3．Dilution rate：1：2．
and 6-BA have the same
concentration of 2。4一D is
concentration，the proper
1．5 mg／L，whose increas—
ing rate can reach 256 and growth rate is 0．922．
The proper concentration of 6-BA is 0．5 mg／L．
Formula 2 is the best for promoting the growth and
division of suspension cels(2，4-D 1．5 mg／L+
NAA 0．5 mg／L．1—6-BA 0．5 mg／L)．Its growth-
rate is more than 6 9 higher than that of other 5
formula．Formula 5 comes second，follow by for-
mula 1(2。4一D 1．0 mg／L+NAA 0．5 mg／L+6一BA
0．5 mg／L)．
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