全 文 ：文章编号:1001 － 4829(2011)04 － 1472 － 08
Received date:2011 － 01 － 27
Foundation item:Supported by Key Technologies Research and Develop-
ment Program of Guangxi(1099063-5) ;Basic Research Fund of Guangxi
Academy of Sciences (11YJ24ZW01)
Author introduction:ZHAO Zhi-guo(1964 －) ，male，born in Guiping
of Guangxi，mainly engages in the biotechnological researches，(E-mail)
kfc@ gxib． cn，* author for correspondence，E-mail:zc@ gxib，cn．
Rapid Propagation of Mesona chinensis
Benth． in vitro and Its Optimization
ZHAO Zhi-guo，SHI Yun-ping，HUANG Ning-zhen，FU Zhuan-ming，TANG Feng-luan，JIANG Qiao-yuan*
(Guangxi Institute of Botany，Guangxi Zhuang Autonomous Region and the Chinese Academy of Sciences，Guangxi Guilin 541006，China)
Abstract:Aseptic nodal stem segments being used as explants，three factors(6-BA，ZT，NAA)affecting the in vitro rapid propagation of Mes-
ona chinensis Benth． were optimized under three concentration levels by L9(34)orthogonal design． The results showed that the optimal media
for primary induction，subculture and rooting of M． chinensis Benth． in vitro were respectively as follows:MS + 6-BA 0． 5 mg /L + NAA 0． 1
mg /L (no vitrification) ，MS +6-BA 0． 5 mg /L + ZT 0． 5 mg /L + NAA 0． 02 mg /L + PVA 1000 mg /L (resulted with high multiplication co-
efficient by 9． 1，strong buds and no vitrification) ，1 /2MS + NAA0． 1 mg /L + KT0． 01 mg /L． The suitable concentration of PVA used to a-
void effectively the vitrification of tube-seedlings in rapid propagation of M． chinensis Benth． was 1000 mg /L． By using this technique，theo-
retically，90 000 young individuals of M． chinensis Benth． would be produced from only 1 tube-seedling in one year． All experimental data
were analyzed using SPSS 13． 0． In addition，seedling vitrification during in vitro propagation was discussed also．
Key words:Mesona chinensis Benth．;Tissue culture;Orthogonal experimental design;Rapid propagation;Optimization
凉粉草组织培养快繁技术及优化研究
赵志国，石云平，黄宁珍，付传明，唐凤鸾，蒋巧媛*
(广西壮族自治区、中国科学院广西植物研究所，广西 桂林 541006 )
摘 要:以凉粉草带茎节的茎段为外植体，采用 L9(34)正交设计，对影响凉粉草组培快繁过程的 3 个因素(6-BA、ZT、NAA)的 3 个
浓度水平进行优化试验，试验结果采用 SPSS13． 0 软件统计分析，并针对凉粉草在组培快繁过程中极易发生玻璃化等问题进行研
究。结果表明:凉粉草的最适初代诱导培养基为 MS +6-BA 0． 5 mg /L + NAA 0． 1 mg /L，无玻璃化;最适继代增殖培养基为 MS + 6-
BA 0． 5 mg /L + ZT 0． 5 mg /L + NAA 0． 02 mg /L + PVA 1000 mg /L，无玻璃化，增殖系数达 9． 1，芽壮;最适生根培养基为 1 /2MS +
NAA 0． 1 mg /L + KT 0． 01 mg /L。1000 mg /L PVA为有效防治凉粉草组培快繁过程中玻璃苗发生的最佳浓度。采用这一技术，理
论上 1 株试管苗一年可以生产凉粉草种苗约为 9 万株。
关键词:凉粉草;组织培养;正交设计;快繁技术;优化
中图分类号:S567． 2 文献标识码:A
Mesona chinensis Benth．，also named as fairy
grass and belongs to genus Mesona in Labiatae，distrib-
utes in the southern provinces such as Guangdong，
Guangxi，Fujian and mainly occurs in gully brook or
tussock［1］． It is an important medicinal and edible
plant with the function of summer thirst quenching and
heat-toxin clearing，hence is also used as the material
for producing‘Wanglaoji’drinks and‘Tortoise Herb
Jelly’herbal sauce［2］． Moreover，mesona polysaccha-
rides and some other active extractions from the plant
can be applied in various healthcare foods or functional
foods，which makes it a food material source with great
development potential［3］． In old method，the leaves
were boiled in water to make cool jelly and now it has
been the best green food or popular drink for appeasing
thirst in summer of South China，that is why it is named
as‘Liangfencao’in Chinese． Obviously，the plant has
a good prospect either as a medicine or a healthcare
food． However and unfortunately，with the developing
2741
西 南 农 业 学 报
Southwest China Journal of Agricultural Sciences
2011 年 24 卷 4 期
Vol. 24 No. 4
DOI:10.16213/j.cnki.scjas.2011.04.036
of cool tea and cool jelly industry，the need for M．
chinensis Benth． increased sharply，so wild resources
were short in supplying and the traditional method
propagating seedlings by asexual reproduction was de-
generated． And tradition method also leads to some
problems such as shorter growing period，low yield，eas-
ily lodging and more insect pests and diseases，which
seriously affected the yield and quality of M． chinensis
Benth． Thus，the problem of propagating the seedlings
becomes an important factor hindering the production．
Plant tissue culture can not only provide more pla-
ntlets to meet the market，but also ensure the plantlets’
quality through purification and rejuvenation［4 － 5］． In
the past years，some researches on M． chinensis Benth．
were reported at home and abroad，but they mainly fo-
cused on cultivation and processing，chemical composi-
tions and pharmacological research［3］． While few
works had been done about its tissue culture，except
Yan Sheng-Qi’s preliminary study［6］． And during the
rapid propagation of M． chinensis Benth．，it is found
that vitrification popularly appeared in the subculture
and this seriously affected the quality and production
efficiency of plantlets． So it is necessary to conduct the
research work on in vitro rapid propagation of M．
chinensis Benth． In this experiment，orthogonal design
method was used under multi-factors with different lev-
els，and the culture media with optimal multiplication
coefficient were obtained by optimization． The culture
media were then added with different concentrations of
polyvinyl alcohol (PVA) so as to select an optimal
technical scheme that would effectively avoid the vitrifi-
cation of tube-seedlings． This study will provide scien-
tific basis for its rapid propagation，and it is in great
significance to the industrialized production of M．
chinensi Benth． seedlings．
1 Materials and Methods
1. 1 Materials
The fresh and young branches of M． chinensi
Benth． without any harm by insect pests and diseases
were collected from the herbal garden of Guangxi Insti-
tute of Botany，their leaves were removed and cut into 1
－ 2 cm sections with 1 stem node．
Reagents used in the experiment:NAA (Naphtha-
leneacetic acid) ，6-BA (6-Benzylaminopurine) ，ZT
(Trans zeatin) ，IBA (Indolyl butyric acid) ，IAA (In-
dolyl acetic acid) ，KT (Kinetin)and PVA (Polyvinyl
alcohol)． All of them are analysis degree and buy from
Shanghai Sigma Company．
1. 2 Methods
1． 2． 1 Material sterilization Stem sections were
cleaned under the running water for 20 － 30 min，then
the adhering water was absorbed up by filter papers．
On an aseptic table，after soaked in 75 % ethyl hydrate
15 s foring water were placed under gation of pid prop-
agation in vitro of l propagated seedlings disinfection
and washed by aseptic water，these sections were steri-
lized by 0． 1 % HgCl2 for 5 min and then washed again
by aseptic water for 5 times． Slices hurted by steriliza-
tion liquid at two ends of the stem sections were ex-
cised． Finally，these aseptic stem sections were inocu-
lated into the initial induction medium．
1． 2． 2 The preparation of media and the conditions of
cultivation MS or 1 /2 MS was used as basic medium
and were added with 6 g agar，20 or 30 g sucrose per
litre． Plant growth regulators and their concentrations
were added according to the purposes of experiments．
The media were adjusted to pH value 5． 8 and sterilized
under 121 ℃ and 0． 11 kPa for 25 min． Explants were
inoculated and cultured under 1500 － 2000 μmol·
m －2·s － 1，12 h /d，26 － 28 ℃ ．
Initial induction． After sterilization，the explants
were inoculated into MS，MS +6-BA 0． 5 mg /L + NAA
0． 1 mg /L，MS + 6-BA 1． 0 mg /L + NAA 0． 1 mg /L，
20 stem sections per treatment，and they were observed
every 7 days． The number of buds and the bud poten-
tial were recorded on the 30th day．
Subculture multiplication． In the conducted or-
thogonal test method，three plant growth regulators (6-
BA，ZT and NAA)were set as experimental factors，
three levels for each factor (Table 1)． Using MS as
basic medium，induction buds from the initial culture
were cut into single ones and inoculated onto different
culture media，respectively，20 buds for each treatmen
and duplicating for 2 times．
Table 1 Orthogonal experimental design for factors and levels
Level
Factors (mg /L)
A (6-BA) B (ZT) C (NAA)
1 0 0 0
2 0． 2 0． 2 0． 02
3 0． 5 0． 5 0． 05
37414 期 赵志国等:凉粉草组织培养快繁技术及优化研究
Table 2 Effect of different culture media on bud induction and buds growth
Medium(mg /L) Inoculation number No． of budsinduced
No． of shoots at
each axillary bud Growth of buds
MS 20 40 1 Ordinary
MS + 6-BA 0． 5 + NAA 0． 1 20 172 4． 3 Stronger
MS + 6-BA 1． 0 + NAA 0． 1 20 205 5． 1 Weak，and glass transition
Note:the Mesona belongs to leaf plant，axillary bud of each stem sections had two points．
Young shoots sprouted from the bottom of the cul-
tured buds after 7 days later． They were observed every
7 d，and the number of total buds and vitrification buds
were recorded on the 30th day． Through intuition analy-
zing on the effects of the factors，and analysis of vari-
ance on experimental data，media with optimal multipli-
cation coefficient were screened．
PVA concentration． On the media with optimal
multiplication coefficient，0，500，1000，1500 and 2000
mg /L PVA were added，repectively，and 20 clustered
buds were inoculated for each treatment，duplicating for
2 times． The results were treated by statistic software
and analyzed by multiple comparison so as to screen
the optimal PVA concentration．
Rooting culture． Taking 1 /2 MS as basic medium
and CK，20 g sucrose was respectively added and af-
fixed with 0． 1 and 0． 3 mg /L NAA，IBA and IAA． 20
buds were inoculated on each medium，duplicating for 2
times． They were observed every 7 days，the number of
rooting buds and the plantlet potential were recorded on
the 30th day．
1． 2． 3 The transplantation of testtube plantlets
When the aseptic buds grew up to 3 cm，they were sin-
gly cut and inoculated into rooting media． Roots sprou-
ted on the 7th day and they became stronger on the 30th
day with the length of 3 － 5 cm． After rooting，testtube
plantlets were transplanted into media with burned soil
and fine sand (1 ︰ 1)． Survival rates were investiga-
ted on the 20th day．
2 Results and Analysis
2. 1 Effects of different concentrations of 6-BA on
the initial induction of M． chinensis Benth．
The initial induction on MS，MS +6-BA 0． 5 mg /L
+ NAA 0． 1 mg /L，MS + 6-BA 1． 0 mg /L + NAA 0． 1
mg /L were compared (Table 2)．
It showed that on MS medium (Table 2) ，each ax-
illary node under MS medium with 0 mg /L 6-BA had
only 1 bud which grew in normal condition without the
induction of clustered buds． On MS + 6-BA 0． 5 mg /L
+ NAA 0． 1 mg /L medium，clustered buds were in-
duced with averaged in 4． 3 buds per node，the buds
showed strong growth． On MS + 6-BA 1． 0 mg /L +
NAA 0． 1 mg /L medium，more clustered buds were in-
duced with averaged 5． 1 buds per node，but more weak
and vitrified buds appeared with low induction rate． By
conctrast，MS +6-BA 0． 5 mg /L + NAA 0． 1 mg /L was
more suitable for the initial induction of M． chinensis
Benth． This indicated that higher concentrations of 6-
BA would induce more clustered buds，it enhanced the
induction and multiplication of buds． However，vitrifi-
cation appeared if this concentration exceeded a certain
range．
2. 2 Effect of different plant growth regulators on
subculture and vitrification of M． chinensis Benth．
2． 2． 1 Effect of different combination of plant growth
regulators and concentrations on subculture and vitrifi-
cation of M． chinensis Benth． By using L9(3
4)or-
thogonal method，a culture scheme containing three
factors and three levels was designed for the multiplica-
tion culture of M． chinensis Benth． In the experiment，
there were 9 treatments representing for different com-
binations between factors and levels． The experimental
data were converted into multiplication coefficient and
vitrification rate (Table 3)．
By comparing the effect of 6-BA，ZT and NAA on
the multiplication coefficient and vitrification rate in
Table 3，it showed that in combinations between single
cytokinin factor(6-BA or ZT)and NAA，multiplication
coefficient increased with the increasing of cytokinin
concentration，while the vitrification became more seri-
ous． This phenomena could be distinctly exhibited on
6-BA，during the change of its concentration from 0． 2
to 0． 5 mg /L，multiplication coefficient increased 1． 7
4741 西 南 农 业 学 报 23 卷
Table 3 The orthogonal design and the experimental results
No．
Factor (mg /L) Multiplication coefficient Vitrification rate (%)
A(6-BA) B(ZT) C(NAA) Ⅰ Ⅱ Mean Ⅰ Ⅱ Mean
1 1(0) 1(0) 1(0) 1． 2 1． 4 1． 3 0 0 0
2 1(0) 2(0． 2) 2(0． 02) 4． 0 3． 1 3． 55 7． 5 12． 9 10． 2
3 1(0) 3(0． 5) 3(0． 05) 3． 8 3． 5 3． 65 26． 32 21． 43 23． 88
4 2(0． 2) 1(0) 2(0． 02) 5． 8 4． 6 5． 2 16． 38 19． 57 17． 98
5 2(0． 2) 2(0． 2) 3(0． 05) 5． 3 7． 2 6． 25 14． 15 9． 03 11． 59
6 2(0． 2) 3(0． 5) 1(0) 6． 4 5． 5 5． 95 34． 38 43． 64 39． 01
7 3(0． 5) 1(0) 3(0． 05) 7． 0 6． 8 6． 9 72． 86 69． 85 71． 36
8 3(0． 5) 2(0． 2) 1(0) 6． 0 6． 4 6． 2 62． 5 55． 47 58． 99
9 3(0． 5) 3(0． 5) 2(0． 02) 7． 8 9． 1 8． 45 56． 41 43． 41 49． 91
Note:induced by K-factor = multiplication buds number /number of inoculated buds，vitrification rate(%)= glass of bud new definitions × 100．
while the vitrification rate increased 53． 38 % ． In con-
trast，ZT had little effect on it，during the change of its
concentration from 0． 2 to 0． 5 mg /L，the multiplication
coefficient and vitrification rate increased 0． 1 and 13．
68 %，respectively． Obviously，the changing of ZT
concentrations had weaker effect on multiplication coef-
ficient and vitrification rate of clustered buds than 6-
BA．
The combinations of multiple cytokinin factors
could be divided into two groups:6-BA with ZT，6-BA
and ZT with NAA． They had distinctly different effect
on the experimental results． Firstly，in combinations of
6-BA and ZT，the multiplication coeficient of treatment
6 was higher than that of combinations between single
cytokinin factor(6-BA or ZT)and NAA (treatment 2-
4) ，the same as to vitrification rate． Treatment 8 had
lower multiplication coefficient than treatment 7． This
demonstrated that ZT might enhance the effect of low
concentration (0． 2 mg /L)of 6-BA，while performed
antagonistic action to higher concentration (0． 5 mg /L)
of 6-BA． Secondly，in combinations of 6-BA and ZT
with NAA，treatment 5 and 9 had higher multiplication
coefficient than others，but the vitrification rates were
greatly decreased，the largest decreasing amplitude
reached 27． 42 %，the plantlets showed strong and good
growth．
Based on single factor and multiple factor analysis
mentioned above，it’s found that multiple cytokinins
combined with NAA would enhance the multiplication
of clustered buds of M． chinensis Benth．，and simulta-
neously decreased the vitrification． Therefore，the com-
bination of multiple plant growth regulators would con-
duce to the induction and growth of clustered buds of
M． chinensis Benth．
2． 2． 2 Intuitive analysis on the multiplication and vit-
rification of clustered buds of M． chinensis Benth．
The results were intuitively analyzed． According to R
value，the effect of different factors on the multiplica-
tion coefficient and vitrification rate of clustered buds
of M． chinensis Benth． were investigated (Table 4)．
It found that factor A (6-BA)had the highest R
value in both multiplication coefficient and vitrification
rate (Table 4) ，followed by factor B (ZT) ，and factor
C (NAA)had the lowest． This suggested that，during
the proliferaton of clustered buds，6-BA performed the
main role，ZT and NAA were subsidiary． The effects of
different factors on both multiplication coefficient and
vitrification rate obeyed the following ranking:A(6-
Table 4 Intuitive analysis of multiplication coefficient and vitrification rate of M． chinensis Benth． in the orthogonal design
Factors
Average multiplication coefficient
k1 k2 k3
R
Average vitrification rate(%)
k1 k2 k3
R
A(6-BA) 2． 83 5． 6 7． 18 4． 35 11． 36 22． 86 60． 09 48． 73
B(ZT) 4． 47 5． 33 6． 02 1． 55 29． 78 26． 93 37． 60 10． 67
C(NAA) 4． 48 5． 74 5． 6 1． 26 32． 67 26． 03 35． 61 9． 58
57414 期 赵志国等:凉粉草组织培养快繁技术及优化研究
Table 5 Variance analysis on multiplication coefficient of clustered buds of M． chinensis Benth．
Source SS DF MS F value Sig．
Area group 500． 334 1 500． 334 922． 274 0． 000
A (6-BA) 59． 274 2 29． 637 54． 631 0． 000
B (ZT) 7． 241 2 3． 621 6． 674 0． 020
C (NAA) 5． 654 2 2． 827 5． 211 0． 036
Deviation 4． 981 10 0． 864
Total variation 77． 156 17
BA)＞ B(ZT)＞ C(NAA)．
2． 2． 3 Screening optimal media for clustered buds of
M． chinensis Benth． It could be seen from the result
of orthogonal test that，different combinations of plant
growth regulators and concentrations would exhibite dif-
ferent effects on multiplication of clustered buds，but
combinations of different cytokinins and concentrations
performed greater effects． To screen the optimal multi-
plication medium，the experimental data was analyzed
using variance analysis．
Significant differences among treatment levels
were analyzed using F-test． The results in Table 5
showed that，there were significant differences among
different levels of factor A (P = 0． 000 ＜ 0． 01) ，sug-
gesting that different concentrations of 6-BA had very
significant effects on the multiplication of clustered
buds of M． chinensis Benth． Moreover，differences a-
mong levels of factor B (ZT)and C (NAA)were sig-
nificant (P ＜ 0． 05) ，indicating that different concen-
trations of ZT and NAA had distinct effect on the the
multiplication of clustered buds，respectively． Because
the differences among different levels of all factors were
significant，S-N-K method was applied in multiple com-
parison analysis，and the optimal combination of these
three factors was as follows:A3B3C2(6-BA 0． 5 mg /L
+ ZT 0． 5 mg /L + NAA 0． 02 mg /L)．
2. 3 Effect of different concentrations of PVA on
vitrification
After inoculating buds into media of all treatments
for 7 d，clustered buds were induced under 0 mg /L，
while no buds appeared under 500 － 2000 mg /L． On
the 15th day，clustered buds successively sprouted，but
they grew neatly on the 30th day when the numbers of
total buds and vitrified buds were recorded and conver-
ted (Table 6)． The result showed that vitrification rate
and multiplication coeficient of the clustered buds de-
creased with the increasing of PVA concentration，sug-
gesting that PVA alleviate the vitrification and simulta-
neously perform adverse effect on multiplication．
According to the variance analysis of experimental
data，there were significant differences among treat-
ments，indicating that different concentrations of PVA
had great effects on the vitrification and multiplication
of M． chinensis Benth． S-N-K method was applied in
the multiple analysis (Table 6)and the result showed
that，vitrification rate under 1000 mg /L PVA had sig-
nificant differences with that under 0 and 500 mg /L
PVA，while there was no significant difference among
the multiplication coefficients of them． On the contra-
ry，it had no significant difference with that under 1500
and 2000 mg /L，while the differences among their coef-
ficients were significant． Obviously，1000 mg /L PVA
Table 6 Effect of PVA concentrations on the vitrification of clustered buds of M． chinensis Benth．
PVA Concentrations
(mg /L)
Inoculation
number
Vitrification rate(%) Multiplication coefficient
Ⅰ Ⅱ Mean Ⅰ Ⅱ Mean
0 20 52． 7 50． 0 51． 4a 9． 0 9． 3 9． 2a
500 20 16． 5 17． 4 17． 0b 9． 1 8． 9 9． 0a
1000 20 3． 4 1． 1 2． 3c 8． 9 9． 0 9． 0a
1500 20 0． 6 1． 2 0． 9c 8． 2 8． 5 8． 4b
2000 20 0 0 0c 7． 6 7． 3 7． 5c
Note:a，b，c represent 22 deal with the significant difference between comparison，respectively．
6741 西 南 农 业 学 报 23 卷
Table 7 Effects of NAA，IBA and IAA on rooting and transplantation of M． chinensis Benth．
Medium
(mg /L)
Inoculation
number
Number of
rooting segments
The condition
of roots
The growth
of seedlings
Survival rate of
transplantation (%)
1 /2MS 40 12． 5 Slenderer Ordinary 77． 5
1 /2MS + NAA 0． 1 40 24． 5 Robust，long Good 82． 5
1 /2MS + NAA 0． 3 40 16． 5 Thick，short Partly Vitrified 50． 0
1 /2MS + IBA 0． 1 40 18 Thick，longer Good 80． 0
1 /2MS + IBA 0． 3 40 11 Thicker，long Partly Vitrified 47． 5
1 /2MS + IAA 0． 1 40 19． 5 Slender Good 87． 5
1 /2MS + IAA 0． 3 40 9 Thick，long Ordinary 67． 5
Note:The transplant survival rate = number of seedlings transplanting seedling transplant survival．
could not only alleviate the vitrification of clustered
buds，but also had little effect on multiplication coeffi-
cient． So this concentration would be optimal for avoi-
ding vitrification．
2. 4 Effects of plant growth regulators on rooting
and survival rate of testtube plantlets
2． 4． 1 Effects of NAA，IBA and IAA on rooting and
survival rate The results showing in Table 7 indicated
that，the roots and seedlings grew well under 0． 1 mg /L
NAA，IBA and IAA，respectively，NAA and IAA per-
formed the best effect，with more roots and higher sur-
vival rate． Under 0． 3 mg /L NAA，IBA and IAA，the
growth of roots were inhibited，vitrification appeared a-
mong testtube plantlets，and the survival rate became
lower in transplantation． The contrast effect between
IAA and NAA was done，in which IAA had better root
quality and survival rate than NAA under 0． 1 mg /L，
but the costs during the industrial production must be
considered． Therefore，0． 1 mg /L NAA would be more
suitable for root induction of testtube plantlets of M．
chinensis Benth．
2． 4． 2 Effects of 6-BA and KT on rooting In order
to improve the root system and survival rate of testtube
plantlets，0． 01 mg /L 6-BA and KT were added to 1 /
2MS + NAA 0． 1 mg /L medium，respectively． The re-
sults showed that，on the medium added with 6-BA，the
number of rooting buds sharply decreased (3 － 8 roots
per individual) ，although the seedlings were stronger，
the survival rate was lowered to 45 % due to the wea-
ker root system． On the medium added with KT，the
number of rooting buds also decreased (21 roots per
individual) ，but the plantlets were strong and the sur-
vival rate could reach up to 98 % due to the mid-sized
and flexible roots． So the optimal medium for the roo-
ting of aseptic buds should be 1 /2MS + NAA 0． 1 mg /
L + KT 0． 01 mg /L．
2. 5 Production efficiency for tissue culture plant-
lets of Mesona
In order to avoid unnecessary waste of blind pro-
duction，it is need to understand that one seedling can
reproduce quantity of plantlets annually before practice
of tissue culture plantlets of Mesona，which will provide
scientific evidence for practice．
Calculated basis:produced seedlings are multipli-
cation of the n (n is multiplication generations annual-
ly)．
n = 2 +(365 － 120)/30≈6
In which 2 means two generations of plantlets slow
growth，for 120 days，and one tissue culture plantlet is
produced for 120 days:primary induction 30 days，mul-
tiplication 30 days，rooting culture 30 days，transplant
30 days．
Practically multiplication rate = 9 － 9 × 0． 25 = 6．
75
In which，9 means calculated multiplication of every
generation，25 % loss of plantlets due to contamina-
tion． Therefore，the number of plantlets produced an-
nually = 6． 756≈94600．
3 Conclusions and Discussion
3. 1 The application of orthogonal design test a-
nalysis on plant tissue culture
Orthogonal design method had been widely used
for studying plant tissue culture，it is helpful to effec-
tively screen the optimal medium combination in an ex-
periment with multiple factors and levels，through less
tests． Furthermore，it could reveal the effect of different
inorganic nutrition，plant growth regulators and their
concentrations on the induction and differentiation of
callus［7 － 8］． Due to the serious vitrification during mul-
77414 期 赵志国等:凉粉草组织培养快繁技术及优化研究
tiplication culture，the present study was conducted to
discover the effect of different plant growth regulators
on the multiplication and vitrification of M． chinensis
Benth．，so as to screen the optimal medium for multi-
plication and finally raise the producing efficiency of
tube-seedlings．
3. 2 Effect of different types and combinations of
plant growth regulators on tissue culture of M．
chinensis Benth．
In tissue culture，the types and concentrations of
plant growth regulators would influence cultural out-
comes，their combinations were the key factors inducing
buds，roots and callus from plants［9］． It found that dur-
ing the tissue culture of M． chinensis Benth． that，un-
der the combinations of 6-BA or ZT with NAA，multi-
plication coefficient was increased with the increasing
of 6-BA or ZT in the treatment of 6-BA + NAA or ZT +
NAA． Concentration change of 6-BA had more obvi-
ously influence than ZT on multiplication coefficient
and vitrification． In the treatments of 6-BA + ZT and 6-
BA + ZT + NAA，it showed that cluster buds multiplica-
tion of Mesona was improved and vitrification was light-
er by adding NAA． The optimal combination for the
three factors was A3B3C2(6-BA 0． 5 mg /L + ZT 0． 5
mg /L + NAA 0． 02 mg /L)． This suggested that the
combination of multiple plant growth regulators would
conduce to the induction and growth of clustered buds
of M． chinensis Benth．
In Yan Sheng Qi’s result［6］，the optimal media for
initial induction，multiplication and rooting were as fol-
lows:MS +6-BA 1． 0 mg /L + NAA 0． 1 mg /L，MS +6-
BA 0． 5 mg /L + NAA 0． 1 mg /L and MS + NAA 0． 5
mg /L，respectively． But our result showed that，the
optimal medium for initial induction was MS + 6-BA 0．
5 mg /L + NAA0． 1 mg /L，without vitrification;the opti-
mal medium for multiplication was MS + 6-BA 0． 5 mg /
L + ZT 0． 5 mg /L + NAA 0． 02 mg /L + PVA1000 mg /
L，the vitrification was alleviated with high multiplica-
tion coefficient (reaching 9． 1) ，strong buds with good
growth;the optimal medium for rooting was 1 /2MS +
NAA 0． 1 mg /L + KT 0． 01 mg /L． It easily could be
seen that using plant growth regulators in our study was
quite different from that in Yan Sheng Qi’s，indicating
that different results would be gained if different types
and concentrations of plant growth regulators were
used． This difference might be caused by the different
sensitivity of M． chinensis Benth． cultivars to plant
growth regulators．
3. 3 Influence of PVA concentration on the vitrifi-
cation during tissue culture of M． chinensis Benth．
PVA is a permeation regulator，it can regulate the
permeability of testtube plantlets and improve the
breathability of medium，which is well for the restora-
tion of cell membrane and therefore decreases the vitri-
fication rate． It had been applied in the tissue culture
of Brassica oleracea［10］，strawberry［11］ and Lorpetalum
chinense var． rubrum［12］， and ideal result was ob-
tained． Vitrified seedlings appeared with transparent
glass leaves and stems［13］． 6-BA was one of the key
factors that caused vitrification［14］． During the tissue
culture of M． chinensis Benth．，plantlets were easily vit-
rified． In this study，the results showed that，on MS +
6-BA 1． 0 mg /L + NAA 0． 1 mg /L medium，more clus-
tered buds were induced，but the buds were slim and
weak and seriously vitrified． This indicated that if 6-
BA was singly used and its concentration was too high，
the vitrification would be more serious． Based on the
result of orthogonal test，plant growth regulators were
indispensable in promoting the multiplication of clus-
tered buds，but their adding in the media simultaneous-
ly brought vitrification to buds． Although it’s impossi-
ble to avoid vitrification by adjusting the types and con-
centrations of plant growth regulators，this study added
different concentrations of PVA to the optimal multipli-
cation medium and optimized the rapid propagation sys-
tem of M． chinensis Benth．，aiming to solve the contra-
dictions between multiplication coefficient and vitrifica-
tion． The result showed that，1000 mg /L PVA could
not only improve the vitrification of clustered buds of
M． chinensis Benth．，but also had little effect on multi-
plication coefficient，suggesting that this would be the
suitable PVA concentration to alleviate vitrification dur-
ing the tissue culture of M． chinensis Benth．
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(Edited by WEN Guo-quan and LI Xiao-hong)
97414 期 赵志国等:凉粉草组织培养快繁技术及优化研究