全 文 ：植 物 学 报
Acta Botanica Sinica 2003, 45 (12): 1453-1464
Received: 2003-05-20 Accepted: 2003-07-30
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Control of In Vitro Regeneration of Individual Reproductive and
Vegetative Organs in Dracaena fragrans cv. massangeana Hort.
— Regularities of the Direct Regeneration
of Individual Organs In Vitro
LU Wen-Liang
(Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany,
The Chinese Academy of Sciences, Beijing 100093, China)
Abstract: Various individual organs (tepal, flower bud, inflorescence branch, inflorescence, adult
vegetative bud and juvenile vegetative bud) were directly regenerated respectively by callus in Dracaena
fragrans cv. massangeana Hort. During the regeneration of these individual organs some regularity
phenomena were observed. Firstly, the kind range of the individual organs, which are directly regenerated
in vitro, is in close relationship to the differentiated stages of the organs used for explant excision during
plant ontogeny. The explants excised from the epigeous organ that is differentiated at some stage (stage
A) during plant ontogeny must be able to separately regenerate all of those individual epigeous organs:
ones differentiated slightly later than the stage A, ones differentiated at the stage A and all ones
differentiated earlier than the stage A. Secondly, within this range which kind of organ is regenerated
depends on the exogenous auxin concentrations in medium. With the gradual increase of 2,4-D
concentration from 0.005 mg/L to 0.5 mg/L, the kinds of regenerated organs will change by the order as
follows: vegetative bud, inflorescence, inflorescence branch, flower bud, tepal. These regularities will be
able to be used for inducing the direct regeneration of a given epigeous organ in angiosperms.
Key words:irect regeneration of a given organ; regular patterns for the direct regeneration of
individual organs; reversed development of cells in vitro ; cell t tipo ency xpression;
Dracaena fragrans
Up to now, it has been not known yet whether or not
each kind of individual organ in animals and plants can be
directly regenerated by the cells or tissues in vitro. But its
importance is beyond doubt either in applications or in
mechanism studies. In the applications, the individual or-
gans regenerated in vitro can be directly used for replacing
those ill organs of human or animals (Caneedda and DeLyca,
1993; Langer and Vacanti, 1993; Pollok and Vacanti, 1996).
In the mechanism researches, the in vitro direct regenera-
tion of individual organs can be used as the in vitro experi-
mental system to understand the necessary conditions for
the initiation and development of organs (Lu et al., 1990;
Lu, 1994), their molecular mechanism (Zhang et al., 2000; Li
et al., 2002) and the control of the male or female sterility of
plants (Lu, 1996a; 1996b).
The studies of the in vitro direct regeneration of indi-
vidual organs in the botanical field are more in the lead than
those in zoological and medical fields (Lu, 2002). So far,
almost each kind of organs in angiosperms can be alone
regenerated in vitro (Lu, 2002). However, the regeneration
of a designated organ was still very difficult. On the one
hand, this shows that the regularities for inducing the re-
generation of individual organs were not known yet, on the
other hand, this also indicates that there is the great possi-
bility to reveal these regularities first in the botanical field.
We have studied the direct regeneration of individual
organs for a long time and induced the direct regeneration
of almost all kinds of individual epigeous organs in
angiosperms. These organs are respectively embryoid (Lu,
1978), vegetative bud (Lu et al., 1986), inflorescence (Lu,
2002), inflorescence branch (see this paper), spikelet (Lu,
1992), flower bud (Lu et al., 1986), tepal (Lu et al., 1999),
stamen (Lu, 1990), pistil (Lu, 1992), style-stigma-like struc-
ture (Lu et al., 1992), ovule (Lu et al., 1986) and fluid-like
structure (Lu and Liang, 1994). Normally, it should be pos-
sible to find out the regularities through comparing the dif-
ferences among the conditions for the regeneration of these
organs, and actually we have noticed that there seems to
植物学报 Acta Botanica Sinica Vol.45 No.12 20031454
exist some regularity among the kinds of directly regener-
ated organs, the differentiated stages of the organs used
for explant separation during the plant ontogeny and the
concentrations of exogenous hormones in medium. But to
reveal it used to be disturbed by the differences among
genotypes because the direct regeneration of the above
organs was completed separately in different species. To
find out the regularities among these three things, therefore,
it is imperative to establish a new experimental system by
using a unitary species. Thereupon, following these de-
mands has to be satisfied in this system: explants must be
excised respectively from those organs that are differenti-
ated at the different stages during plant ontogeny and can
be induced to form callus; the callus of different origins has
to be able to regenerate the individual organs in different
kinds through the induction of the exogenous hormones in
different concentrations. The present paper is especially
for this purpose. The experiments achieved the anticipate
results and some important regularities for inducing the
direct regeneration of individual epigeous organs were suc-
cessfully revealed.
1 Materials and Methods
1.1 Materials
Dracaena fragrans cv. massangena Hort. was used as
experimental material. The plants were individually grown
in pots in the experimental field of this institute. When the
plants producted 30 or more leaves they were used in
experiments.
1.2 Tissue culture
1.2.1 Sterilization and excision of explants The whole
inflorescences were excised respectively from plants when
the pollen grains in the anthers from the flower bud at the
middle parts of the inflorescences were at the early unicel-
lular to early bicellular stages. They were sterilized in 0.1%
aqueous mercuric chloride for 10 min, and then rinsed three
times with sterile distilled water. After that, perianth tubes,
inflorescence branch axes and rachises were excised sepa-
rately from the inflorescence and cut respectively into the
segments in about 2 mm (for perianth tube), 3 mm (for inflo-
rescence branch axis) and 5 mm (for rachis) thickness as
explants. The young stems were also used as explants;
they were excised from germ-free test-tube plantlets with
10-12 leaves and cut into the segments in 2-3 mm
thickness. The induction of the test-tube plantlet is the
same as that of adult vegetative bud in this paper.
1.2.2 Culture The base medium is that of MS (Murashige
and Skoog, 1962), supplemented with 6-benzylaminopurine
(6-BA) and 2,4-dichlorophenoxy acetic acid (2,4-D) in dif-
ferent concentrations as designed. The cultures were grown
at (25±1) ℃ in daily cycle of 9 h of light from Toshiba
(FLR40S·WM) cool-white fluorescent lamps (5 W/m2) and
15 h of darkness.
2 Results and Analyses
2.1 The induction of inflorescence differentiation in vivo
To observe the effects of the explants excised from dif-
ferent kinds of organs on the kinds of regenerated organs,
various reproductive and vegetative organs have to be used
in this experiment. But the plants of Dracaena fragrans
used to carry on vegetative growth only in Beijing. This
may be because the wintering of plants in glasshouse leads
to the lack of a low temperature stage in its ontogeny. To
induce the inflorescence differentiation, a simple test was
first done that the plants were grown at 12 ℃ and 15 ℃ for
20 d and 30 d, respectively. The results show that the low
temperature treatment really plays a crucial role in the in-
duction of inflorescence differentiation of plants. Treated
for 30 d at 12 ℃ or 15 ℃, the frequency of the inflores-
cence differentiation could reach 100%. Temperatures higher
than 15 ℃ or treated time shorter than 20 d made the fre-
quency go down quickly.
2.2 Morphological features of reproductive and vegeta-
tive organs in vivo
Morphological features of reproductive and vegetative
organs in vivo had to be first observed because which
could be used for identifying the kinds of various regener-
ated organs. The observation shows that the inflorescence
of Dracaena fragrans belongs to the panicle, and consists
of a longer axis (rachis) and some inflorescence branches
that come out separately from the axils of each bract at-
tached on the longer axis (Fig.1A). The inflorescence branch
is composed of a shorter axis and many flower buds at-
tached on the upper half of the axis (Fig.1B), the flower bud
consists of five tepals, five stamens and one pistil. Once
the inflorescence is cut out from the plant, a dormant veg-
etative bud at the leaf axil the nearest from rachis will sprout
to develop into a shoot (Fig.1C). We call this bud as adult
vegetative bud. An obvious trait of this bud distinct from
juvenile vegetative bud is that there are some shorter and
unspread leaves at its base.
2.3 The induction of callus
In a previous study (Lu, 2002) it has been proved that
the combination of 6-BA and 2,4-D is efficacious for the
callus formation of the explants excised from different or-
gans in Dracaena fragrans. This combination, therefore,
was used still in this experiment for inducing callus
formation of the explants excised from perianth tube,
1455LU Wen-Liang: In Vitro Regeneration of Individual Reproductive and Vegetative Organs in Dracaena fragrans
植物学报 Acta Botanica Sinica Vol.45 No.12 20031456
inflorescence branch axis, rachis and stem respectively. As
a result (Table 1), on three groups of media (included 6-BA
1.0 mg/L and 2,4-D 1.0 mg/L, 6-BA 1.0 mg/L and 2,4-D 0.8
mg/L, and 6-BA 1.0 mg/L and 2,4-D 0.5 mg/L separately)
each kind of the explants could form the callus with a higher
frequency (Fig.1D), showing that the effect of 2,4-D con-
centration changes in a narrower range on the induced fre-
quencies of the callus is not very distinct. The differences
of the explant origins had a certain influence on the callus
frequencies. The highest frequency (about 91.3%-93.9%)
was observed in the young stem explants; the next (about
90.2%-92.6%) was in the perianth tube explants; the third
(about 70.4%-81.6%) was in the inflorescence branch axis
explants; and the lowest (about 70.8%-79.2%) was in the
rachis explants. The effect of cut-segment thickness of ra-
chis on the callus frequencies was observed (Table 2). The
thicker (over 10 mm) and thinner (about 1-2 mm) cut-seg-
ments were difficult to form the callus on the media with 6-
BA 1.0 mg/L, 2,4-D 0.5 mg/L or 1.0 mg/L. The thickness in
3-5 mm had better effect, among them the 5 mm thickness
had the highest induced frequency ((82.1 ± 8.2)%) on me-
dium with 6-BA 1.0 mg/L and 2,4-D 0.5 mg/L. We noticed
that the initiation of the callus had different ways. For the
explants that are separately excised from perianth tube, in-
florescence branch axis and rachis, the callus was sepa-
rately initiated from their cortical tissues and pith tissues.
The former was observed to be a main way, in which the
fiber-like structures were first formed through the swelling
of cortical cells, and then callus was formed through the
dedifferentiation of the fiber-like structure. The latter was
observed sometimes, in which some granular structures
were first formed by the pith cells at the cut end of the
segments, then the callus was formed in the further devel-
opment of the granular structures. The way that cut-seg-
ments of the young stem formed the callus was rather simple,
in which callus was directly formed through the swelling of
the whole cut-segments of the stem.
2.4 Induction of organ regeneration
To describe the regeneration of organs in different kinds
easily, the identification of the kinds of regenerated organs
has to be first done through comparing the morphological
characteristics between organs in vitro and in vivo.
2.4.1 Identification of the kinds of directly regenerated
organs (DR organ) These following organs (Fig. 1 E-L)
are directly regenerated by the callus. A. DR tepal (Fig.1E).
The basis of identifying this organ is that it has the same
Fig.1. The inflorescence in vivo and the various individual organs regenerated by callus in vitro in Dracaena fragrans. A. A young
terminal inflorescence in vivo (I). It is composed of a longer rachis with some bracts (B) and inflorescence branches (IB) that separately
come out from the axils of each bract. Bar = 7.7 cm. B. An inflorescence branch in vivo (IB). It comes out from the axil of bract (B) attached
on rachis (R) and consists of a shorter axis and many flower buds (FB) attached on the upper half of the axis. The flower bud can be
distinguished into two parts: tepal (the part with purplish red color) and perianth tube (P). Bar = 0.6 cm. C. A shoot that comes from the
development of an axillary bud in vivo (AB). After inflorescence was excised from the plant an axillary bud at the axil of the leaf the nearest
from rachis (now only a remains of the rachis (R) can be seen) can grow up, it is called as adult vegetative bud and characterized by some
shorter and unspread leaves (SL) at the its base. Bar = 7.7 cm. D. Callus. It is formed by the cut-segment of rachis on MS medium with
6-BA 1.0 mg/L and 2,4-D 0.5 mg/L. Bar = 1.26 mm E. Regenerated tepal. It is directly regenerated by the perianth tube callus cultured on
the medium with 6-BA 0.5 mg/L and 2,4-D 0.5 mg/L. Bar = 0.96 mm. F. Regenerated flower bud. It is directly regenerated by the perianth
tube callus cultured on the medium with 6-BA 0.5 mg/L and 2,4-D 0.5 mg/L. Bar = 2.0 mm. G. Regenerated inflorescence branch. It is
regenerated directly from the inflorescence branch axis callus cultured on the medium with 6-BA 0.5 mg/L and 2,4-D 0.5 mg/L , and
consists of an axis and some flower buds (FB) attached on the upper half of the axis. Bar = 2.80 mm. H. Regenerated inflorescence. It is
regenerated directly by the inflorescence branch axis callus cultured on the medium included 6-BA 0.5 mg/L and 2,4-D 0.1 mg/L, and
composed of a rachis (R) with some bracts (B) and some inflorescence branches (IB) come out from the axils of each bract separately. FB:
flower buds. Bar = 2.88 mm. I. Regenerated transforming bud. This bud looks like a flower bud by its external form and the external tepal-
like structure with purplish red color, but only leaves can be initiated at the places where sexual organs should be initiated. It was directly
regenerated by inflorescence branch axis callus that was first cultured on the medium with 6-BA 0.5 mg/L and 2,4-D 0.5 mg/L for inducing
flower bud initiation then transferred onto the medium with 6-BA 0.5 mg/L and 2,4-D 0.005 mg/L prior to initiation of its stamen. Bar =
1.92 mm. J. Regenerated adult vegetative bud. It is regenerated directly by the perianth tube callus cultured on the medium supplemented
with 6-BA 0.5 mg/L and 2,4-D 0.005 mg/L, some shorter and unspread leaves (SL) can be seen at the base. Bar = 1.81 mm. K. Regenerated
juvenile vegetative bud. It is regenerated directly by the rachis callus cultured on the medium with 6-BA 0.5 mg/L and 2,4-D 0.005 mg/L
for over 60 d, and no shorter and unspread leaves can be seen at the its base. Bar = 0.96 mm. L. Flowering of the regenerated flower bud
in vitro. Before flowering the flower bud is directly regenerated by the perianth tube callus cultured on the medium with 6-BA 0.5 mg/L
and 2,4-D 0.5 mg/L at 25 ℃ in daily cycle of 9 h of light and 15 h of darkness, then was immediately transferred onto the medium with
6-BA 0.5 mg/L and 2,4-D 0.005 mg/L soon after the initiation of its sexual organs and grown at 15 ℃ in the daily cycle of 8 h of light and
16 h of darkness. Several weeks late, the flowering happened and five tepals, five stamens and one pistil could be clearly seen. Bar =
2.58 mm.
←
1457LU Wen-Liang: In Vitro Regeneration of Individual Reproductive and Vegetative Organs in Dracaena fragrans
form as the tepal in vivo, its continual initiation from callus
brings on the formation of a fasciculated structure. B. DR
flower bud (Fig.1F). This organ has the same morphologi-
cal features as the flower bud in vivo. C. DR inflorescence
branch (Fig.1G). This organ consists of a shorter axis and
some flower buds attached on its upper half, showing that
its basal morphological characteristics are in accordance
with that of inflorescence branch in vivo, so it was identi-
fied as regenerated inflorescence branch. But some smaller
differences are existent, for example, the number of the
flower buds is always less than that of flower bud in vivo
and the axis became so short that sometimes its lower half
without flower buds is observed hardly. D. DR inflores-
cence (Fig.1H). Based on this organ is composed of a longer
axis with some bracts and several inflorescence branches
that come out from the axils of each bract respectively, it
was identified as regenerated inflorescence. But as a organ
regenerated in vitro it always has some differences that
differed from the inflorescence in vivo, for example, the
numbers of both its inflorescence branch and the flower
bud are always less than those on the inflorescence in
vivo. E. DR transforming bud (Fig.1I). This bud looks like
the flower bud in vivo according to its external form and the
perianth-like structure with the purplish red color, but only
leaves are initiated at the places where sexual organs should
be initiated, showing that a transformation from flower bud
to vegetative bud occurs during the regeneration of this
bud, so it was identified as transforming bud. F. DR adult
vegetative bud (Fig.1J). The basic characteristics of this
organ conform to that of the adult vegetative bud in vivo
because there are some shorter and unspread leaves at its
base. G. DR juvenile vegetative bud (Fig.1K). This vegeta-
tive bud has no shorter and unopened leaves at the base.
2.4.2 Induction of individual regenerated organs The
calli used for organ regeneration were formed by the
explants that were excised from perianth tube, inflorescence
branch axis, rachis and young stem respectively on the
media with 6-BA 1.0 mg/L and 2,4-D 0.5 mg/L. When the
size of the callus reached just about 3 mm in direction, they
were separately transferred onto four groups of media used
for organ regeneration (Tables 3-6) to observe the relation
among the developmental stages of the organs used for
explant excision during plant ontogeny, the concentrations
of exogenous hormones and the kinds of regenerated
organs. The data in Tables 3-6 was scored at the 75th day
post-transfer.
2.4.2.1 Organ regeneration of the perianth tube callus
In the presence of 6-BA 0.5 mg/L, supplementation of the
medium with 2,4-D in different concentrations could induce
the perianth tube callus to regenerate various individual
reproductive and vegetative organs (Table 3). When 2,4-D
0.5 mg/L three kinds of reproductive organs DR tepals
(frequency (2.2 ± 1.4)%), DR flower buds (frequency
(2.4 ± 1.3)%), and DR inflorescence branch (frequency
(12.3 ± 2.5)%) were initiated. The slight reduction of the
2,4-D concentration (0.1 mg/L) inhibited the initiation of
the DR tepal, but raised the frequencies of the DR flower
buds (frequency (4.6 ± 2.4)%) and the DR inflorescence
branches (frequency (16.7 ± 3.6) %), meanwhile, act i-
va te d t he in i t iat io n o f t he DR i n fl o r esc en ce
(frequency (6.4 ± 2.8)%). Further reducing the 2,4-D con-
centrations (0.01 mg/L and 0.005 mg/L), the initiation of all
three kinds of reproductive organs (DR tepal, DR flower
bud, DR inflorescence branch) were inhibited, but the ini-
tiation of DR adult vegetative bud were activated
(frequencies (35.5 ± 6.2)% and (43.6 ± 7.1)%). In this experi-
ment the perianth tube callus regenerated five kinds of in-
dividual organs, showing that the perianth tube cells in
vitro have the higher potential ability in regenerating the
individual organs in different kinds. Meanwhile, it
Table 1 Effect of exogenous hormones in different concentrations on callus formation by the explants excised from different organs
Exogenous hormones (mg/L)Frequencies of callus formation by different explants (%)(1)
6-BA 2,4-D PTE IBE RE SE
1.0 1.0 90.2 ?5.6 81.6 ? .4 70.8 ? .1 91.3 ?4.7
1.0 0.8 91.0 ?6.4 76.5 ? .8 78.7 ?9.9 92.6 ?5.6
1.0 0.5 92.6 ?4.8 70.4 ? .2 79.2 ?7.4 93.9 ?4.4
(1), each data is an average repeated twice. IBE, inflorescence branch explant; PTE, perianth tube explant; RE, rachis explant; SE, stem
explant.
Table 2 Effect of exogenous hormones in different concentrations on callus formation by the rachis cut-segments in different thickness
Exogenous hormones (mg/L) Percentage of callus formation by the cut-segments in different thickness (%)(1)
6-BA 2,4-D 10 mm 5 mm 3 mm 1-2 mm
1.0 1.0 0 74.4 ?8.6 48.5 ?14.3 0
1.0 0.5 0 82.1 ?8.2 52.6 ?12.4 0
(1), each data is an average repeated twice.
植物学报 Acta Botanica Sinica Vol.45 No.12 20031458
was noticed that there was a regularized relationship
between the kinds of regenerated organs and the exog-
enous auxin concentrations. When 6-BA was fixed at 0.5
mg/L, with the gradual increase of 2,4-D concentrations
from 0.005 mg/L to 0.5 m/L, the kinds of individual regener-
ated organs changed successively from formed at the early
stage to late stage of plant ontogeny. From this we infered
(inference Ⅰ) that the gradient change of auxin concentra-
tions in this hormonal combination can lead to that the
regeneration of the individual organs is in the order that
they are differentiated during plant ontogeny by.
2.4.2.2 Organ regeneration of the inflorescence branch
axis callus On the four groups of media the same as the
above experiment the inflorescence branch axis callus could
directly regenerate various reproductive and vegetative
organs also (Table 4). The combination with 2,4-D 0.5 mg/L
induced the direct initiation of the DR flower bud (frequency
(4.4±2.3)%) and DR inflorescence branch (frequency
(48.6±6.8)%), respectively. The slight reduction of 2,4-D
concentration in the combination (2,4-D 0.1 mg/L) resulted
in the reduction of the frequencies of the DR flower bud
(frequency (1.6±0.6)%) and the DR inflorescence branch
(frequency (38.1±8.5)%), but activated the initiation of the
DR inflorescence initiation (frequency (10.7±2.5)%). Fur-
ther reduction of the 2,4-D concentration (0.01 mg/L or
0.005 mg/L) inhibited the regeneration of all reproductive
organs, but activated the initiation of the DR adult vegeta-
tive buds (frequency (45.3±7.6)% and (46.2±8.7)%
separately). In this experiment the inflorescence branch axis
callus regenerated four kinds of individual organs under
the same condi tions of the exogenous hormones,
indicating that the potential ability of the inflorescence
branch axis cells in regenerating the individual organs is
lower than that of the perianth tube cells. Since the inflo-
rescence branch axis is differentiated slightly earlier than
the perianth tube during plant ontogeny, it may be able to
be considered that the earlier the differentiated stage of
organs during plant ontogeny, the lower the potential abil-
ity of their cells; the later, the higher (inference Ⅱ). As for
the relationship between the kinds of regenerated organs
and auxin concentrations, the regularity same as observed
in the above experiment was observed still in this
experiment, showing the correctness of the inference Ⅰ
preliminarily.
2.4.2.3 Organ regeneration of the rachis callus Also,
the rachis callus could directly regenerate various repro-
ductive and vegetative organs on the four groups of me-
dia (Table 5). When 6-BA 0.5 mg/L and 2,4-D 0.5 mg/L
only DR inflorescence branches were initiated (frequency
(41.6±8.4)%). When 6-BA 0.5 mg/L, 2,4-D 0.1 mg/L the DR
inflorescence (frequency (11.1±1.8)%) and the DR inflores-
cence branch (frequency (19.2±2.5)%) were initiated. When
6-BA 0.5 mg/L and 2,4-D 0.01 mg/L or 0.005 mg/L, only the
DR adult vegetative buds (frequencies (41.4±9.7)% and
(48.5±8.3)% respectively) was initiated. The experiment re-
vealed that under the same exogenous hormonal condition
the rachis callus could directly regenerate three kinds of
organs only, showing that the potential ability of rachis
cells in regenerating the individual organs in different kinds
is lower than that of the inflorescence branch cells. Since
the rachis is earlier differentiated than the inflorescence
branch during plant ontogeny, the correctness of the
Table 3 Effect of exogenous hormones in different concentrations on regenerating the individual organs in different kinds by the perianth
tube callus
Exogenous hormones (mg/L) Frequencies of regenerating different organs (%) (1)
6-BA 2,4-D T FB IB I VB
0.5 0.5 2.2 ?1.4 2.4 ?1.3 12.3 ?2.5 0 0
0.5 0.1 0 4.6 ?2.4 16.7 ?3.6 6.4 ?2.8 0
0.5 0.01 0 0 0 0 35.5 ?6.2
0.5 0.005 0 0 0 0 43.6 ?7.1
(1), each data is an average repeated twice. FB, flower bud; IB, inflorescence branch; I, inflorescence; T, tepal; VB, vegetative bud.
Table 4 Effect of exogenous hormones in different concentrations on regenerating the individual organs in different kinds by the
inflorescence branch callus
Exogenous hormones (mg/L) Frequencies of regenerating different organs (%) (1)
6-BA2,4-D T FB IB I VB
0.5 0.5 0 4.4?.3 48.6?.8 0 0
0.5 0.1 0 1.6?.6 38.1?.5 10.7?.5 0
0.50.01 0 0 0 0 45.3?.6
0.50.005 0 0 0 0 46.2 ?.7
(1), each data is an average repeated twice. FB, flower bud; IB, inflorescence branch; I, inflorescence; T, tepal; VB, vegetative bud.
1459LU Wen-Liang: In Vitro Regeneration of Individual Reproductive and Vegetative Organs in Dracaena fragrans
inference Ⅱ was verified preliminarily. As to the relation
between exogenous auxin concentration and the kinds of
regenerated organs the result of this experiment was still
that with the gradual increase of 2,4-D concentration from
0.005 mg/L to 0.5 mg/L the kinds of regenerated organs
changed orderly from differentiated at earlier stages to later
stages in plant ontogeny, showing the correctness of the
inference Ⅰ further.
2.4.2.4 Organ regeneration of the stem callus Under
the same exogenous hormonal conditions the stem callus
could not regenerate any reproductive organs, only the DR
adult vegetative organs were initiated on two groups of
media included 6-BA 0.5 mg/L and 2,4-D 0.01 mg/L or 0.005
mg/L (frequencies (24 .8±9.7 )% and (26 .5±8.4 )%
respectively) (Table 6). This shows clearly that which kind
of individual organs can be directly regenerated is not in-
dependently determined by the concentrations of exog-
enous hormones, but jointly determined by both the exog-
enous hormone concentrations (external causes) and the
differentiated stages of the organs which are used for ex-
plant excision during plant ontogeny (internal causes). The
stem callus only regenerated one kind of organs, showing
that the potential ability of the stem cells in organ regenera-
tion is lower than that of the rachis cells. Undoubtedly,
stem is earlier differentiated than inflorescence during plant
ontogeny, so the correctness of the inference Ⅱ was veri-
fied once again in the organ regeneration of stem explants.
In this experiment the relation between the kinds of regen-
erated organs and the exogenous auxin concentrations
appears to be hard to find regularity. But we considered
that the result of this experiment is still in accordance with
the inference Ⅰ because the adult vegetative bud is the
earliest differentiated in the all regenerated organs in plant
ontogeny and the auxin concentration for its regeneration
is the lowest in these four combinations.
2.4.2.5 Other phenomena observed in the above experi-
ments After each kind of individual organ was directly
initiated, gradually reducing the exogenous hormone con-
centrations to 0 mg/L in medium was of advantage to their
normal development. Initiation of the DR juvenile vegeta-
tive bud and roots (Fig.1K) were observed when the calli of
different origins were continuously subcultured on the me-
dium including 6-BA 0.5 mg/L and 2,4-D 0.005 mg/L over 60
d. The all regenerated flower buds, which are directly re-
generated or attached on the inflorescence, could not nor-
mally develop to blooming under the conditions given in
the above experiments, but inducing them to flower in vitro
is possible (Fig.1L), if only they were transferred onto the
medium with 6-BA 0.5 mg/L and 2,4-D 0.005 mg/L and grown
at 15 °C in the daily cycle of 8 h of light and 16 h of darkness
soon after the initiation of their stamens and pistil. If the
DR flower buds were immediately transferred onto the me-
dium with 6-BA 0.5 mg/L and 2,4-D 0.005 mg/L before the
initiation of their sexual organs, the DR transforming bud
could be formed (Fig.1I).
2.4.2.6 A summary of the above regularity phenomena
To explain the direct regeneration of individual organs in
this paper using a picture, a schematic diagram was drawn
(Fig.2). From this picture the regularities to induce the di-
rect regeneration of various individual organs can be clearly
seen. They were summarized as follows:
(1) The regularity on the relationship between the
Table 5 Effect of exogenous hormones in different concentrations on regenerating the individual organs in different kinds by the rachis
callus
Exogenous hormones (mg/L) Frequencies of regenerating different organs (%) (1)
6-BA2,4-D T FB IB I VB
0.5 0.5 0 0 41.6 ?8.4 0
0.5 0.1 0 0 19.2 ? .5 11.1 ? .8 0
0.50.01 0 0 0 0 41.4 ?9.7
0.50.005 0 0 0 0 48.5 ?8.3
(1), each data is an average repeated twice. FB, flower bud; IB, inflorescence branch; I, inflorescence; T, tepal; VB, vegetative bud.
Table 6 Effect of exogenous hormones in different concentrations on regenerating the individual organs in different kinds by the stem
callus
Exogenous hormones (mg/L) Frequencies of regenerating different organs (%) (1)
6-BA2,4-D T FB IB I VB
0.5 0.5 0 0 0 0 0
0.5 0.1 0 0 0 0 0
0.5 0.01 0 0 0 0 24.8 ?9.7
0.5 0.005 0 0 0 0 26.5 ?8.4
(1), each data is an average repeated twice. FB, flower bud; IB, inflorescence branch; I, inflorescence; T, tepal; VB, vegetative bud.
植物学报 Acta Botanica Sinica Vol.45 No.12 20031460
- A schematic diagram for explaining the mutual relations among the kinds of regenerated organs, the differentiated stages of
organs used for explant excision during the plant ontogeny and the concentrations of exogenous hormones. DRFB Directly regenerated
flower bud; DRIB. Directly regenerated inflorescence branch; DRI. Directly regenerated inflorescence; DRT. Directly regenerated tepal;
DRVB. Directly regenerated vegetative bud; IBC. Inflorescence branch axis callus; PTC. Perianth tube callus; RC. Rachis callus; SC. Stem
callus. A schematic diagram for explaining the idea on the cycle of endogenous auxin concentrations in apical meristem during the cycle
from seed to seed in angiosperms. A schematic diagram for explaining the relations between the different levels of development
inversion in cultured cells and the direct regeneration of individual epigean organs by these cells. . Explants are excised from fruit. .
Explants are excised from flower bud. . Explants are excised from vegetative bud. . Explants are excised from young embryo.
1461LU Wen-Liang: In Vitro Regeneration of Individual Reproductive and Vegetative Organs in Dracaena fragrans
differentiated stages of organs during plant ontogeny and
the potential abilities of their cells in organ regeneration
The earlier the differentiated stages of the organ dur-
ing plant ontogeny, the lower the potential ability; the
later, the higher. Exactly, the cells in the epigeous organ
differentiated at some stage (stage A) during plant ontog-
eny can directly regenerate those epigeous organs: ones
differentiated slightly later than the stage A, ones differ-
entiated at the stage A and all ones differentiated earlier
than the stage A. As shown in Fig.2, the perianth tube
callus (PTC) can directly regenerate the tepal (DRT) (which
is slightly later differentiated than the perianth tube), the
flower bud (DRFB) (which is almost simultaneously differ-
entiated with the perianth tube) and the inflorescence
branch (DRIB), the inflorescence (DRI), vegetative bud
(DRVB) (which are earlier differentiated than the perianth
tube) respectively; the inflorescence branch axis callus
(IBC) can directly regenerate the flower bud (DRFB) (slightly
later differentiated than the inflorescence branch axis), the
inflorescence branch (DRIB) (simultaneously differentiated)
and the inflorescence (DRI), the vegetative bud (DRVB)
(earlier differentiated) separately; the rachis callus (RC) can
directly regenerate the inflorescence branch (DRIB) (slightly
later differentiated than rachis), the inflorescence (DRI)
(simultaneously differentiated) and the vegetative bud
(DRVB) (earlier differentiated); the stem callus (SC) can di-
rectly regenerate the vegetative bud (DRVB) only.
(2) Regularity on the relation between the kinds of di-
rectly regenerated organs and the concentrations of exog-
enous hormones
When cytokinin concentration is fixed at some level in
the combination of cytokinin and 2,4-D, with the gradual
increase of auxin concentration, the regeneration of indi-
vidual organs in different kinds is in the order that they
are differentiated in plant ontogeny by. In Fig.2 the combi-
nation included lower concentration of auxin (6-BA 0.5
mg/L and 2,4-D 0.01-0.005 mg/L) (green arrow) is advanta-
geous to the regeneration of the vegetative bud; the slighter
increase of the auxin concentration in the combination (red
arrow) (6-BA 0.5 mg/L and 2,4-D 0.1 mg/L) is beneficial to
regeneration of the inflorescence, the further increase of
the auxin concentration in the combination (violet arrow)
(6-BA 0.5 mg/L and 2,4-D 0.1-0.5 mg/L) leads to the regen-
eration of the inflorescence branch; the 6-BA 0.5 mg/L and
2,4-D 0.5 mg/L (black arrow) induces the regeneration of
the flower bud and tepal.
(3) Regularity on the determination of the kinds of indi-
vidual regenerated organs
Which kind of organ can be directly regenerated in
vitro is jointly determined by two factors, differentiated
stages of the organs used for explant excision in plant
ontogeny and used exogenous hormonal concentrations.
The former determines the limits of kinds of individual
regenerated organs (internal causes), and the latter de-
termines which kind of organ can be regenerated within
this limits (external causes).
3 Discussion
Plant tissue culture has had its history over 100 years.
As a research means it has been applied in almost all
branches of learning in botany. This technique, however,
was still hard to be grasped, especially the induction of the
direct regeneration of an assigned individual organ. One of
the important reasons is that only a few of the regular pat-
terns for inducing the direct regeneration of individual or-
gans were so far known. On the basis of summarizing the
experiences about inducing the direct regeneration of indi-
vidual reproductive organs, we designed this paper and
revealed some regularities phenomena in the induction of
the direct regeneration of individual organs in vitro. To
discuss the internal mechanism of these regularities
phenomena, we daringly put forward here some new ideas
and wish them able to open up some new thinking.
3.1 Whether can the regularities revealed by this paper
be used for inducing the direct regeneration of all epigeous
organs in angiosperms?
As shown in the introduction of this paper we have
successfully induced the direct regeneration of each kind
of floral organ in angiosperms. The regeneration of various
individual floral organs was found to have the regularities
similar to those observed in this experiment. For example,
the concentrations of exogenous auxin for regenerating the
individual floral organs according to the following order:
tepal (Lu et al., 1999), stamen (Lu, 1990; Li et al., 2002),
ovule (Lu et al., 1988), and fruit-like structure (Lu et al.,
1994) are of gradual reduction (Lu et al., 2000), showing a
similar gradient effect of the auxin concentrations as ob-
served in this paper; in addition, the perianth explants in
different development stages can directly regenerate vari-
ous individual organs: sexual organs, tepal, flower bud and
vegetative bud respectively (Lu et al., 1986; 1988), indicat-
ing the similar potential ability of the explant cells as ob-
served in the present experiment. Therefrom, we infered
that the regularities observed in this paper may be able to
be used for inducing the direct regeneration of all epigean
organs in angiosperms so as making a bit of supplement to
the regularity about the effect of concentration change of
exogenous auxin on the kinds of regenerated organs. This
植物学报 Acta Botanica Sinica Vol.45 No.12 20031462
regularity, thereupon, was changed as that the concentra-
tions of exogenous auxin for the organ regeneration from
vegetative bud to flower bud are gradually raised, while
from tepal to fruit like structure are gradually reduced.
3.2 Endogenous auxin concentrations in the meristem of
shoot apex may be in a cycle change during the cycle from
seed to seed
In general, the experiments in vitro were used for under-
standing the various changes in vivo. The role of the gradi-
ent change of the exogenous auxin concentrations on the
regeneration of individual organs suggests whether in vivo
organ initiation from the meristem of shoot apex in order is
also to result from the gradient change of concentrations
of endogenous auxin synthesized by the meristem self. If
so, the gradient change must be in a cycle during the cycle
from seed to seed (Fig.3), namely from the embryogenesis
to the initiation of flower bud the concentration is increased
gradually, then from the initiation of perianth to the fruit
formation is decreased gradually.
3.3 Exogenous hormones in different concentrations can
finally lead to the activation of each class of epigean organ
identity genes that will soon been expressed and has been
expressed before in cultured cells
The regeneration of an organ in vitro should be under-
stood as that the identity genes of this organ are activated.
The results of the present experiment show that the cells in
the epigeous organ differentiated at some stage (stage A)
during plant ontogeny can directly regenerate those
epigeous organs that are differentiated from the earliest
stage during plant ontogeny to slightly later stage than
stage A (Fig.2). That is to say, the exogenous hormones in
different concentrations can finally result in the activation
of each class of epigeous organ identity genes that will
soon been expressed and has been expressed before in
cultured cells. As for why exogenous hormones can acti-
vate these genes and how about its molecular mechanism,
this will remain to be further studied.
3.4 Reversion of development in cells in vitro
A marked characteristic of development in plant is that
all organs are initiated in order one after another, namely
the all organ identity genes are expressed successively.
For the organ that is initiated at the intermediate stage of
plant ontogeny, of all organ identity genes in the its cells a
part must have been expressed because some organs have
been initiated prior to this stage, the others are not ex-
pressed yet because the organs that will be initiated post
this stage are not initiated yet. If these having been ex-
pressed genes are reactivated in vitro, the development
must be reversed in cultured cells. It has been discussed
above that the exogenous hormones in different concen-
trations can result in the activation of each class of epigean
organ identity genes that have been expressed before in
cultured cells. The reversion of development, therefore,
must occur in the cultured cell.
3.5 Characteristics of the revered development in cul-
tured cell
Firstly, the reversed development of cells in vitro must
be accompanied by the dedifferentiation of this cell just as
development in vivo must be accompan ied by
differentiation. As above, that these having been expressed
genes are reactivated in cultured cells brings on organ
initiation. It was well known that any organ must be initi-
ated from the aggregate of embryonic cells called meristem.
These cultured cells, therefore, must first transform into
embryonic cells through dedifferentiation to form the
meristem. Thus, once the reversed development happens
in vitro, the gene expression in the cultured cells must re-
turn to some stage that has been expressed before,
meanwhile, the cultured cells must become embryonic ones
to form callus or meristem. We consider there may be such
a relationship between the two things, namely the dediffer-
entiation results in the formation of the meristem similar to
that of shoot apex, while the reversed development deter-
mines the stages of the shoot apex-like meristem in plant
ontogeny. Secondly, differing from the development, the
reversed development moves forward in a jumping way,
while the former is in an evolutionary way. For example, in
the developmental course from seed germination to flower
bud formation various organs such as vegetative bud, in-
florescence and inflorescence branch can be formed one
by one and which needs a longer time, but in the course of
the reversed development from the pollen grains cultured
in vitro to pollen embryoids any one of above these or-
gans is not formed and this needs a shorter time only.
Finally, the reversed development in vitro possesses dif-
ferent levels. As shown in Tables 3-6 the lower the exog-
enous auxin concentrations, the more advantageous the
regeneration of the organs differentiated at the early stage
of plant ontogeny (namely the organ identity genes that
are separately expressed at the early stage of plant ontog-
eny are activated); the higher, the more suitable the regen-
eration of the organs differentiated at the late stage of plant
ontogeny (namely the organ identity genes that are ex-
pressed at the late stage of plant ontogeny are separately
activated). This shows that the exogenous auxin in differ-
ent concentrations can make the reversion of development
reach to the different stages of plant ontogeny, namely the
reversed development has different levels (Fig.4).
1463LU Wen-Liang: In Vitro Regeneration of Individual Reproductive and Vegetative Organs in Dracaena fragrans
3.6 Why do the cells in the explants excised from differ-
ent organs have different potential ability to regenerate
the individual organs?
On the basis of the above discussion we now can come
to discuss this question. Undoubtedly, this is in close rela-
tionship with development and reversed development.
Thereupon, a schematic diagram (Fig.4) was made to ex-
plain this question through development and reversed
development. On the light of the diagram the green arrows
represent development and differentiation, in which the
various epigean organs: embryo, vegetative bud, flower
bud and fruit are initiated successively. On the left of the
diagram the red arrows stand for reversed development
and dedifferentiation to show that under the action of ex-
ogenous hormones in different concentrations the devel-
opment of cultured cells is reversed to the different stages
of plant ontogeny: fruit initiation stage (red point), flower
bud initiation stage (green point), vegetative bud initiation
stage (yellow point), embryoid initiation stage (grey point).
The violet arrows stand for the organ regeneration at the
each level of the reversed development. The a, b, c, d ar-
rows show separately the culture of the explants excised
from different organs. Following these a, b, c, d arrows, it
can be seen that the later the differentiated stage of organ
during plant ontogeny, the more the kinds of regenerated
organs by the explant excised from the organ, oppositely,
the earlier, the less. This is because the former has a longer
course of the reversed development, while the latter has a
shorter course of the reversed development.
3.7 Why can cell totipotency expression do complete
expression and partial expression?
To discuss this question, it is necessary to explain here
the meaning of cell totipotency expression. This concep-
tion was advanced in 1994 (Lu and Liang, 1994). At that
time various individual reproductive organs have been suc-
cessfully regenerated, but it was still very difficult to ex-
plain these phenomena by the theory of cell totipotency.
This new conception, thereupon, was put forward, and it
includes the meaning as follows. The cell totipotency
should be understood as a potential characteristic of cells
because it can be induced to express only if the cells are
cultured in vitro. This expression is called as cell totipo-
tency expression, and it can be divided into two patterns:
complete expression and partial expression. By the com-
plete expression is meant that in vitro cell can be induced
to regenerate the embryoid then to develop into a plant
exactly same as its mother plant. The meaning of the partial
expression is that in vitro cells can be induced to regener-
ate the various individual organs same as ones of its mother
plant. When this conception was proposed we knew noth-
ing about why the cell totipotency can do both complete
expression and partial expression. Now it can be explained
using the conception of the reversion of cell development.
As discussed above, under the action of exogenous hor-
mones in different concentrations the inversion of devel-
opment in cultured cells can reach different levels (Fig.4).
When it reaches the beginning point of development (grey
point), the regeneration of embryoid leads to the formation
of plant exactly same as its mother plant. Under this level of
the reversed development the cell totipotency is completely
expressed because the genes in the cultured cell are ex-
pressed once again from beginning to end by the program.
When it reaches the other stages behind the beginning
point (yellow, blue and red color points), the individual
organs in different kinds are regenerated respectively. Such
levels of the reversed development make the cell totipo-
tency do partial expression because the genes in the cul-
tured cell are partially expressed by the program from the
stages behind the beginning point to end only.
Acknowledgements: The author thanks Professor S. N.
BAI for his encouragement and technical assistance in mak-
ing the figure and plate.
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花叶千年木单个生殖器官和营养器官的体外直接再生的控制
——单个器官直接再生的规律性
陆文木梁
（中国科学院植物研究所光合作用与环境分子生理学重点实验室，北京 100093）
摘要： 花叶千年木(Dracaena fragrans cv. m s angeana Hort.)的各种单个器官（花被片、花芽、花序分枝、
花序、成年态营养芽和幼态营养芽）在离体培养中被愈伤组织直接再生了。在这些单个器官的再生期间，一些规律
性现象被观察到了。首先，单个再生器官种类的范围与分离外植体的器官在植物个体发育中被分化的时期有密切关
系。从植株个体发育某个时期（时期A）分化的地上部分器官上分离的外植体能够分别再生下面这些地上部分器官：
稍晚于时期A分化的器官，与时期A同期分化的器官和早于时期A分化的所有器官。其次，在这个范围内，究竟再生
哪一种器官被再生取决于培养基中外源生长素的浓度。随着2,4-D浓度从0.005 mg/L逐渐升高到0.5 mg/L，单个再
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给定的被子植物地上部分器官的直接再生。
关键词： 给定器官的直接再生；单个器官再生的规律；离体培养中细胞的逆发育；细胞全能性表达；花叶千年木
中图分类号: Q945 文献标识码: A 文章编号: 0577-7496(2003)12-1453-12
收稿日期:2003- 5-20 接受日期:2003- 7-30
(责任编辑: 王 葳)
(Managing editor: WANG Wei)
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