全 文 ：第26卷 第4期 作　物　学　报 V ol. 26, N o. 4
2000 年7月 A CTA A GRONOM ICA S IN ICA July, 2000
Compar ison of Culture Procedures for Regenera tion of Plan ts from
Protopla st-der ived Ca lluses of R ice (O ryza sa tiva L. ) X
YAN G Yue2Sheng　J IAN Yu2Yu　CH EN Yuan2L ing
(College of B iotechnology , S outh China A g ricultural U niversity , Guangzhou, 510642)
Abstract 　 Four differen t culture p rocedures com bined w ith several treating m ethods w ere
compared fo r their effects on regeneration of p lan ts from p ro top last2derived calluses of rice. O n ly
a sm all num ber of w eak p lan ts could be regenerated w hen the calluses w ere transferred and
cultured directly from a p ro liferation m edium con tain ing 2, 42D to a p lan t regeneration m edium
con tain ing BA and NAA (p rocedure 1). A ddition of ABA to the p ro liferation m edium induced
the fo rm ation of nodular calluses w ith enhanced regeneration po ten tial (p rocedure 2) , w h ile
addition of ABA to the regeneration m edium resulted in the fo rm ation of compact calluses w ith
supp ressed adven titious buds, w h ich would grow fast upon transfer to a grow th m edium free of
ABA ( p rocedure 3 ). By Culturing of the calluses consequen tly on ABA supp lem en ted
p ro liferation m edium , ABA supp lem en ted regeneration m edium and the grow th m edium
(p rocedure 4) , large num ber of more healthy p lan ts w as obtained. Statistical test indicates that
p rocedure 2 and 3 w ere m uch more efficien t than p rocedure 1, w h ile p rocedure 4 w as the most
efficien t fo r p lan t regeneration.
Key words　P lan t regeneration; P ro top last; Callus; A bscisic acid; R ice
Several years ago, our labo rato ry succeeded in obtain ing p lan t regeneration of an indica rice
cultivar Q iuguiai 11[ 1 ] , how ever, the p lan t regeneration efficiency w as very low and the m ajo rity
of the regenerated p lan ts w ere very w eak. T h rough years of experim en t, w e have confirm ed the
effectiveness of several treating m ethods such as dehydration treatm en t of the calluses[ 2 ] , aeration
of the cultures[ 3, 4 ] and found that use of h igher concen tration of copper[ 5 ] w as conducive to p lan t
regeneration in rice calluses cultures.
R ecen tly, in o rder to imp rove further the p lan t regeneration of the p ro top last2derived
calluses w e have compared quan titatively and qualitatively four competen t culture p rocedures in
com bination w ith the above m en tioned treating m ethods. A t the sam e tim e w e have also
investigated the effect of various concen trations of ABA in two k inds of m edia invo lved in th ree
of the four culture p rocedures. D etails of the experim en ts are p resen ted in th is paper.
1　M ATER IAL S AND M ETHOD S
1. 1　Protopla st-der ived Ca lluses
P ro top lasts w ere iso lated from suspension cells o riginating from dehusked m ature seeds of
X This research w as supported by The Chinese Foundation for A gricultural Science and Education
Received on 1998211212, A ccep ted on 1999205214

the indica rice cultivar Q iuguiai 11 and cultured by the m ethod described befo re[ 1 ]. Co lon ies
recovered from the p ro top lastsw ere p ro liferated on N 6 m edium [ 6 ] supp lem en ted w ith 2 m g ö L 2,
42D , 1000 m g ö L p ro line, 3% (w ö v) sucrose and gelled w ith 0. 75% (w ö v) agar. Calluses
grow n from these co lon ies w ere used as m aterial fo r evaluation of the effectiveness of the four
culture p rocedures.
1. 2　Regenera tion of Plan ts from the Protopla st-der ived Ca lluses
For the conven ience of quan titative and qualitative comparisons among the four culture
p rocedures, sam e amoun t of 400±10 m g fresh calluses w as used as o riginal m aterial fo r every
treatm en t in each culture p rocedure. To insure the reliability and rep roducibility of the results all
the experim en ts w ere repeated at least tw ice and the data w ere subjected to statistical analysis
w hen possible. A ll cultures in the p resen t experim en ts w ere p laced under 10 ö 14 h ligh t ö dark
pho toperiod of about 20 LEm - 2 s- 1 p rovided by dayligh t fluo rescen t tubes at 24～ 26 ℃. N am es
and compositions of m edia fo r culture of the calluses are given in T able 1, and four culture
p rocedures are show n as fo llow s:
Table 1　　Supplements to media used for regeneration of plants from protoplast-der ived calluses1)
N am e of
m edium 2)
Supp lem ents(m g ö L )
2, 42D BA NAA ABA CuSO 4ı 5H 2O P roline Casein hydro lysate
PDA 2 - - 1, 3, 6, 9 - 1000 -
PBNA - 2 1 1, 3, 6, 9 1. 25 - 500
RBN - 2 1 - 1. 25 - 500
GBN - 0. 2 1 - - - 500
　1)N 6 basal elem ents w ere used for all the four m edia. A ll these m edia contained 3% sucrose and w ere gelled w ith 0. 75% agar.
　2) PDA , a p reculture m edium containing 2, 42D and ABA ; PBNA , a p reculture m edium containing BA , NAA and ABA;
RBN , a regeneration m edium containing BA and NAA ; and GBN , a grow th m edium containing BA and NAA.
P rocedure 1. Calluses w ere dehydrated (T sukahara and H irosaw a, 1992) → calluses w ere
cultured on RBN (30 days)→ regenerated p lan ts and shoo ts w ere transferred to GBN (20 days) ;
P rocedure 2. Calluses w ere cultured on PDA ( 20 days) → calluses w ere dehydrated →
calluses w ere cultured on RBN (30 days) → regenerated p lan ts and shoo ts w ere transferred to
GBN (20 days) ;
P rocedure 3. Calluses w ere dehydrated→ calluses w ere cultured on PBNA (30 days) →
calluses w ith o r w ithout shoo ts (buds) w ere transferred to GBN (20 days) ;
P rocedure 4. Calluses w ere cultured on PDA ( 20 days) → calluses w ere dehydrated →
calluses w ere cultured on PBNA ( 30 days) → calluses w ith o r w ithout shoo ts (buds) w ere
transferred to GBN (20 days).
1. 3　Eva lua tion of Plan t Regenera tion
N um ber of regenerated p lan tsw as coun ted at the end of each experim en t, and the quality of
the regenerated p lan ts w as evaluated by w eigh ing the regenerated p lan ts and by calculating the
survival rate of the regenerated p lan ts after a 10 days′liquid culture period. 1 ö 3 strength N 6 salts
so lution of m acroelem en ts w as used fo r the liquid culture.
1944期　　　　YAN G Yue2Sheng et al. : Comparison of Culture P rocedures fo r R egeneration of ⋯⋯　　　　

2　RESUL TS
2. 1　Plan t Regenera tion by Procedure1
Calluses cultured by p rocedure 1 regenerated on ly a very sm all num ber of p lan ts on RBN
m edium. Because most of these p lan ts did no t have sufficien t roo ts and w ere sm all and very
w eak, a subculture on GBN m edium w as necessary. U pon transfer to GBN m edium , there w as
on ly a low rate of the p lan ts could resum e sustained grow th.
Table 2　　Effect of var ious concentrations
of ABA in PDA medium 1)
Concentration
of ABA in
PDA (m g ö L )
Callus grow th
on PDA (m g
f. w t. ö flask)
No. p lants
regenerated
on RBN
0. 0 3324 a2) 23 c
1. 0 3355 a 25 c
3. 0 3008 a 46 b
6. 0 2116 b 89 a
9. 0 1523 c 83 a
　　1)A ll calluses on PAD w ere used for
culture on RBN m edium.
　　2)Data fo llow ed by the sam e letter are not significantly
different (p> 0. 05) according to the t2test.
The sam e as in Table 3 and4.
2. 2　Plan t Regenera tion by Procedure 2
T he effects of ABA at concen trations of 1, 3,
6 and 9 m g ö L in the PAD m edium on cultures
w ere compared and the m edium w ith no ABA
supp lem en t w as used as the con tro l. T he result of
th is experim en t is show n in T able 2.
Concen trations of ABA at 1 and 3 m g ö L had on ly
sligh t effect on p ro liferation of the calluses and on
p lan t regeneration in the subsequen t culture. O n
m edia w ith h igher concen trations of ABA , the
calluses becam e more compact and nodular in
appearance. P rom inen t effects of enhancem en t on
p lan t regeneration of the calluses w ere observed in
treatm en ts using ABA at concen trations of 6 and 9 m g ö L. M oreover, roo t developm en t of the
regenerated p lan ts w as also imp roved by the addition of ABA at the h igher levels in PAD
m edium.
Table 3　　Effect of var ious concentrations
of ABA in PBNA medium 1)
Concentration
of ABA (m g ö L )
in PBNA
Callus grow th
on PBNA (m g
f. w t. ö flask)
No. p lants
regenerated
on GBN
0. 0 8244 a 8 c
1. 0 7277 a 21 bc
3. 0 5715 b 51 a
6. 0 4114 c 62 a
9. 0 2156 d 36 b
　　1)A ll calluses w ith shoots and buds on
PBNA w ere used for culture on GBN.
2. 3　Plan t Regenera tion by Procedure 3
T he effects of ABA at concen trations of 1,
3, 6 and 9 m g ö L in the PBNA m edium on
culturesw ere compared and the m edium w ith no
ABA supp lem en t w as used as the con tro l. T he
result of th is experim en t is show n in T able 3. In
the con tro l culture the p lan t regeneration w as
just as that by the p rocedure 1: on ly a sm all
num ber of w eak p lan ts regenerated. Supp lem en t
of 1 m g ö L ABA to the m edium resulted in the
regeneration of som e stronger p lan ts. O n m edia
w ith ABA at 3 and 6 m g ö L , calluses regenerated supp ressed buds. U pon transfer to GBN
m edium , these buds burst in to fast grow th. T he concen tration of 9 m g ö L seem ed too h igh:
calluses on th is m edium p roduced apparen tly less amoun t of on ly heavily supp ressed buds. T he
effects of BA in GBN m edium w ere also tested. It w as found that BA concen tration of 0. 2 m g ö
L w as superio r to 0. 0 o r 1. 0 m g ö L : w ithout BA supp lem en t the num ber of regenerated p lan ts
294　　　　　　　　　　　　　　　　　作　　物 　　学　　报　　　　　　　　 　　　　　　　　26卷

w as sligh tly decreased, w h ile at 1. 0 m g ö L level inh ibition to the grow th of the roo ts becam e very
eviden t.
Table 4　　Effect of var ious concentrations of
ABA in PDA and PBNA media1)
Concentration
of ABA in
PBNA (m g ö L )
Concentration of ABA in PDA (m g ö L )
1. 0 3. 0 6. 0
1. 0 15 Bb2 19 Bb 49 Ca
3. 0 63 A c 97 A b 121 A a
6. 0 75 A a 80 A a 77 Ba
　　　1)A ll calluses on each PAD m edia w ere evenly divided into
three group s and cultured on the three respective PBNA
m edia.
　　　2) The cap ital letters in dicate the significance among row s and
the sm all letters among colum ns.
2. 4　Plan t Regenera tion by Procedure 4
T he effects of various concen trations of
ABA in PDA and PBNA m edia on p lan t
regeneration w ere comparatively in2
vestigated. Calluses cultured on PDA
m edium of each treatm en t w ere co llected and
then divided even ly in to th ree group s and
cultured respectively on to each of the th ree
k inds of PBNA m edia. D ata fo r num ber of
regenerated p lan ts of the experim en t are
show n in T able 4. T he best result of p lan t
regeneration w as obtained by culturing the
calluses first on PDA m edium w ith ABA at 6 m g ö L and then on PBNA m edium w ith ABA at 3
m g ö L. M oreover, w ith ABA at 3 o r 6 m g ö L in PBNA m edium , developm en t of the roo ts of the
regenerated p lan ts w as greatly enhanced in GBN m edium.
2. 5　Quan tita tive and Qua l ita tive Com par ison s of the Four Procedures
T he effects of the four culture p rocedures w ere compared quan titatively and qualitatively.
Table 5　　Compar ison of the effects on plant regeneration
of the four culture procedures1)
P rocedure
No. days
Required
No. p lants
regenerated
M ean f. w t.
ö p lant (m g)
No. p lants
survived2)
1 55 8 61 2 (25. 3)
2 75 89 97 36 (40. 5)
3 55 62 144 47 (75. 8)
4 75 363 157 291 (80. 2)
　　1)Data in th is table are taken from the most p roper treatm ent in each
culture p rocedure. The data for p rocedure 1 are taken from the cont2
ro l in Table 3, and the data for p rocedure 4 have been revised from
Table 4 by tim ing 3 to rep resent the amount of p lants originated
from 400±10 m g m aterial calluses as in the o ther treatm ent.
　　2)O btained after a 102day′s liquid culture period. Data in parentheses
are the percentages.
For quan titative comparison, data fo r
the best treatm en t in each culture
p rocedure w ere co llected. A nd fo r
qualitative comparison, the m ean
w eigh t of the regenerated p lan ts and
the num ber of survived p lan ts after the
liquid culture period w ere used.
R esults of the comparisons show n in
T able 5 clearly indicate that p rocedure
1 w as the most inefficien t, w h ile
p rocedure 3 is obviously more efficien t
than p rocedure 2 because p rocedure 3
had more survival p lan ts as w ell as
required sho rter tim e fo r the culture. P rocedure 4 w as no doubt the most efficien t one, by w h ich
the fresh w eigh t per regenerated p lan t w as doubled and the num ber of survival p lan ts w as
increased by nearly 150 tim es, respectively, in comparison w ith the con tro l(p rocedure 1).
3　D ISCUSSION
R egeneration of p lan ts from p ro top last2derived calluses is an indispensable step in a breeding
p rogram using p ro top lasts fo r up tak ing fo reign genes o r by p ro top last fusion. In rice, although
3944期　　　　YAN G Yue2Sheng et al. : Comparison of Culture P rocedures fo r R egeneration of ⋯⋯　　　　

h igh efficien t p lan t regeneration in p ro top last cultures has been repo rted [ 7, 8 ] , w e believed that
poo r regeneration ability of the calluses and poor quality of the regenerated p lan ts are still two
frequen t and m ain p roblem s to m any breeders. In the p resen t experim en ts by comparing four
competen t culture p rocedures, w e have screened a h igh ly rep roducible and efficien t culture
p rocedure fo r regeneration of p lan ts from p ro top last2derived calluses of rice.
Among the four culture p rocedures in the p resen t studies, p rocedure 1 (direct transfer of
calluses from a m edium con tain ing 2, 42D to a m edium con tain ing BA or k inetin) w as a simp le
and the most common culture p rocedure used in regeneration of p lan ts from rice calluses of
various o rigin including those derived from p ro top lasts[ 9～ 11 ]. P rocedure 2 w as almost the sam e as
the“two2step culture m ethod”described by Inoue and M aeda [ 12 ] , p rocedure 3 w as sim ilar to that
adop ted by Yin et al. [ 13 ] , andM ei et al. [ 14 ] , and p rocedure 4 w asmodified from a culture m ethod
designed by Yang et al. [ 15 ] , w ith m ain differences in using agar2gelled m edium instead of their
liquid m edium in the first culture step and by om itting po tato ex tract in the nex t step culture
m edium. For conven ience and in reference to the two2step culture m ethod, w e nam ed the
p rocedure 4 a“th ree2step culture p rocedure”.
Excep t p rocedure 1, the o ther th ree p rocedures emp loyed ABA. T he enhancing effect of
ABA on p lan t regeneration culture of rice callus has been described and som e of the possible
functions in the culture have been discussed ( Inoue and M aeda, 1981; Torrizo and Zapata,
1986). By the app lication of ABA in the culture m edia, Yang et al. [ 5 ] and Yin et al. [ 13 ] had
succeeded in obtain ing p lan ts w h ich could o therw ise no t be regenerated by the common culture
m ethod. R esults of the p resen t experim en ts no t on ly confirm ed the positive effect of ABA on
enhancing p lan t regeneration, but also clearly demonstrated the effect of ABA on imp roving the
quality of the regenerated p lan ts. T hus the th ree2step culture p rocedure had satisfacto ry results.
N evertheless, the great con tribution to the regeneration of p lan ts by the adop ting dehydration of
the calluses, aeration of the cultures and supp lem en t of larger amoun t of copper to the m edium ,
also deserved to be m en tioned.
Som e of the p lan ts regenerated w ere grow n to m aturity and the fertility w as investigated. It
w as found that the fertility of the regenerated p lan ts dropped considerably in comparison w ith
that of p lan ts grow ing from seeds, how ever, little difference in fertility w as recogn ized among
group s of p lan ts regenerated by the four culture p rocedures. T h is observation indicates that the
th ree2step culture p rocedure has h igher app lication po ten tial in p ractical breeding of rice.
Acknowledgements
W e thank D r. W u Xinrong for h is sk illful work on culture of the p ro top lasts, and thank M iss Zheng Yingdong for her
efficient assistance in the experim ents.
494　　　　　　　　　　　　　　　　　作　　物 　　学　　报　　　　　　　　 　　　　　　　　26卷

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7　A bdullah R , et al. B ioö T echnology , 1986, 4: 1087～ 1090
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11　Xue Q Z, E D Earle. P lant Cell R eports, 1995, 15: 76～ 81
12　 Inoue M , E M aeda. J apanese J C rop S cience, 1981, 50: 318～ 322
13　Yin Y H , et al. P lant Cell T issue O rgan Culture, 1993, 32: 61～ 68
14　M ei C S, et al. J A g r B iotech ( in Ch inese) , 1994, 2: 96～ 99
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水稻原生质体愈伤组织再生植株培养程序的比较
杨跃生　简玉瑜　陈远玲
(华南农业大学生物技术学院, 广东广州 510642)
提　要　在4种不同的培养程序中应用了几种处理方法, 并对其在诱导水稻原生质体起源的愈伤组织
再生植株中的效果进行了比较。直接将愈伤组织从含有2, 42D 的增殖培养基转移到含有BA 和NAA
的植株再生培养基上培养, 只能得到少量的弱苗 (第1种程序)。在增殖培养基中添加ABA 诱导了结节
状的愈伤组织形成, 使愈伤组织的植株再生能力明显加强 (第2种程序) ; 而在植株再生培养基中添加
ABA 则使愈伤组织变得紧结并形成生长受抑制的不定芽, 当这些愈伤组织被转移到不含ABA 的生长
培养基后, 不定芽开始快速生长 (第3种程序)。先将愈伤组织培养在含有ABA 的增殖培养基上, 然后
相继转移到含有ABA 的植株再生培养基和生长培养基上, 可以取得大量健壮的再生苗 (第4种程序)。
统计结果显示, 第2和第3种程序的培养效果比第1种程序要好, 而第4种程序的培养效果则比其他程序
好得多。
关键词　植株再生; 原生质体; 愈伤组织; 脱落酸; 水稻
5944期　　　　YAN G Yue2Sheng et al. : Comparison of Culture P rocedures fo r R egeneration of ⋯⋯