全 文 ：Plant Regeneration in Eucalyptus pellita
Xie Xiangming
＊ Chen Xiaoyang Han Liebao
ABSTRACT In this study , two different approaches of regeneration systems for explants in Eucalyptus pellita
have been taken to establish an efficient differentiation and regeneration protocols.The somatic embryogenesis from
in vitro hypocotyls and cotyledons in Eucalyptus pellita has been investigated.The typical three kinds of callus
types were induced from hypocotyls and cotyledons cultured on the 2 ,4-D containing medium with BA or kinetin.
High frequency (82%)plantlet production from the friable , granular embryogenic callus derived from hypocotyls
has been obtained on the differentiation medium(1μM TDZ;1μM BA plus 0.1μMNAA)developed in this study.
The relationship between the callus types formed on different callus induction media and their corresponding differ-
entiation rate was further discussed.In addition , plant regeneration through organogenesis from in vitro hypocot-
yls , cotyledons and mature leaves in Eucalyptus pellita has also been conducted with the modified basal B5 medi-
um supplemented with a range of combinations of BA and NAA.The highest frequency of shoot formations for
three kinds of explants varied on the different regeneration medium , with the regeneration rate for leaves , cotyle-
dons and hypocotyls in ascending order:41.67%, 66.67% and 83.33%, respectively.The significance of the
two different plant regeneration systems was discussed.To our knowledge , this is the first report on the regenera-
tion of Eucalyptus pellita.
KEYWORDS embryogenesis , organogenesis , regeneration , Eucalyptus pellita
Xie Xiangming , Chen Xiaoyang.College of Biological
Science and Biotechnology , Beijing Forestry University ,
Beijing 100083 , P.R.China
Han Liebao.Institute of Turfgrass Science , Beijing For-
estry University , Beijing 100083 , P.R.China
＊
Author for correspondence.Tel.:86-10-62338130
Email:xiangmingx@hotmail.com
Received March 17 ,2001
1 Introduction
Eucalyptus is a very large genus of plants that in-
cludes more than 700 species occurring naturally
across wet and dry , and tropical , subtropical and
temperate environments (Boland et al.1984).
Though the majority of Eucalyptus species originate
from Australia , several species have been extensive-
ly planted as exotic for commercial reforestation in
many parts of the world , due to their characteristics
of fast-growth , superior wood quality , and wide
adaptability to a variety of environments.Today ,
they constitute the majority of the world s exotic
hardwood forest and one of the world s main sources
of biomass(Eldridge et al.1993).
Eucalyptus pellita F.Muell.occurs naturally in
northern Queensland , Australia , in south-west Pap-
ua New Guinea(PNG)and southeast Irian Jaya , a
province of Indonesia , respectively.Over several
decades , a number of Queensland provenance of E.
pellita have been widely tested in tropical and sub-
tropical environments in Australia and in south Chi-
na.Generally , their growth has been proved some-
what inferior to other eucalyptus species such as E.
grandis and E .urophylla which have been two of
the several major eucalyptus plantation species , but
the paper properties of Eucalyptus pellita and Euca-
lyptus urophylla were very similar , slightly inferior
to those of young plantations-grown Eucalyptus
globulous (Clark and Hicks 1996).There have
been the largest areas of plantations as a commercial
timber species in Brazil and Cuba (Harwood
1997).
7
Forestry Studies in China 3(1):7 ～ 14
Eucalyptus pellita has been identified as one of
Eucalyptus species with much promise for commer-
cial hardwood plantations in south China , where the
diseases have been found to be the major problem
threatening the plantation program , based on the
observation of the plantation trials for reforestation
purpose.The species appears widely adaptable ,
growing well on a wide range of site types.In par-
ticular , the species has shown good resistance to in-
sect pests and diseases , as compared to other euca-
lyptus species such as E.grandis and E.urophyl-
la (Harwood et al.1997).Though the breeding
program for the genetic improvement in Eucalyptus
pellita has been established through the conventional
approach , the genetic transformation which aims to
modify some genetic traits will be considered as an
option in the long term.To this end , an efficient
regeneration system for Eucalyptus pellita , a pre-
requisite for the gene introduction into , and stable
expression in the genome of plant cells , needs to be
established.
So far , a lot of papers have been published on
the regeneration through either organogenesis or so-
matic embryogenesis in a few Eucalyptus species ,
such as E.camaldulensis(Muralidharan and Mas-
carenhas 1987 , Yang Jeng-Chuan at al.1996 ,
Kawazu T.et al.1997 , Ho C K et al.1998),
E .citriodora(Muralidharan andMascarenhas 1987
, Muralidharan et al.1989), E.grandis hybrids
(Warrag et al.1991), E .grandis×E .urophyl-
la (Penchel and Ikmori 1994 , Yang Jeng-Chuan at
al.1996), E.grandis(Watt et al.1991 , War-
rag et al.1991 , Laine and David 1994 , Major G
et al.1997), E.leichou (Ouyang et al.1990
), E.grunnii(Arnould and Favre 1991), E.ter-
iticornis(Sabbaiah and Minocha 1990).But there
has not yet been any report on the regeneration sys-
tem for Eucalyptus pellita.In this paper , compre-
hensive studies on the regeneration in Eucalyptus
pellita through embryogenesis and organogenesis will
be presented.
2 Materials and methods
2.1 Plant materials and culture conditions
Seeds of Eucalyptus pellita (provenance:North-
west of Kuranda , Queensland)were purchased from
the Australian Tree Seed Center(CSIRO), Division
of Forestry and Forest Product , Canberra , Austra-
lia.Mature seeds were soaked in water for 2 hours ,
surface sterilized by immersing in 70% ethanol for
one minute , in a 75%(v v)dilution of commercial
bleach “Clorox” for 3 minutes , and in a 50%(v v)
dilution of the bleach for 5 minutes , respectively ,
followed by rinsing five times with sterile water.All
sterile seeds were shaken in sterile water for 2
hours , and placed on a half-strength MS basal me-
dium(Murashiege and Skoog 1962)solidified with
0.8% agar for one week at 26℃ in darkness , and
for subsequent 10 ～ 30 days in the growth room with
the condition of(26±2)℃ and 16 hrs photoperiod
for germination.Hypocotyls and cotyledons were re-
moved from two-week old seedlings , and mature
leaves of 30 days were excised as explant sources for
embryogenesis and organogenesis.
2.2 Somatic embryogenesis
Callus was induced from hypocotyls with 4 ～ 5
cm segments chopped and cotyledons on the follow-
ing primary medium:callus induction medium:
EC1:MS medium +0.25 mg·L-1 BA(6-Benzyl-
aminopurine)+1.5 mg·L-12 , 4-D(2 , 4-Dichloro-
phenoxyacetic acid)+4%sucrose+0.75% agar
EC2:EC1+10%CW(coconut water)
ET1:MS + 0.8 mg · L-1 KT(6-Furfurylamin-
opurine)+2.0 mg·L-1 2 , 4-D+4% sucrose +
0.75% agar
ET2:1 2MS+0.8 mg·L-1 KT+2.0 mg·L-1 2 , 4-
D+4% sucrose+0.75% agar
Embryogenic calli were cultured on the second-
ary medium:bud induction medium(EM:modified
MS + 0.22 mg · L-1 TDZ (Thidiazuron) +
0.18 mg·L-1 NAA(1-Naphthaleneacetic acid)+
8 Forestry Studies in China ,Vol.3 ,No.1 ,2001
3%sucrose+0.75%agar), subsequently on shoot
multiplication medium(X4:B5 +1.0 mg·L-1 BA
+3.00 mg·L-1 NAA+3% sucrose)and on elon-
gation medium(ES:MS+0.22 mg·L-1 BA+0.09
mg·L-1 NAA+3% sucrose +0.75%agar), re-
spectively.Regenerated plantlets with a height of
2.5cm were rooted on the rooting medium (half-
strength MS with a combination of NAA and IBA
(Indole-3-butyric acid)or NAA and IAA(Indole-3-
acetic acid)).
2.3 Organogenesis
Hypocotyls , cotyledons and mature leaves in in
vitro seedlings were induced for both callus induc-
tion and shoot proliferation on the modified B5 medi-
um(Gamborg et al.1968)solidified with 0.75%
agar and supplemented with a range of combinations
of BA and NAA.Regenerated plantlets of 2.5cm in
height are subjected to rooting on the optimal rooting
medium.
3 Results and discussion
3.1 Induction and differentiation of somatic
embryogenesis
Callus initiation was observed for the cultured
explants on the four different callus induction medi-
um after 10 ～ 14 days.After three subcultures
(generally , one subculture at intervals of three
weeks), there were a variety of callus types
formed , characteristically including three kinds of
calli , with the amount of each type callus varied ,
depending on callus induction and explant sources
used (Table 1), namely , (type I)soft , glassy-
transparent , and cream-white in appearance (Plate
I-1);(type II)predominantly friable and nodular
clusters mixed with dark red pigment (Plate I-2);
(type III)compact , solid and hard (Plate I-3).
There were also some intermediates between the
three callus types (Plate I-4&5).These callus
types have also been described in many publications
on embryogenesis of Eucalyptus species (Major et
al.1997 , and publications therein).
The explants cultured on the MS medium with
BA and 2 , 4-D with or without coconut water (EC1
or EC2)yielded glassier , friable callus , suggesting
the coconut water has not dramatic effect on the for-
mation of callus type.No substantial difference in
the formation of callus type between hycopotyls and
cotyledons was observed on the EC1 or EC2 medi-
um.
TABLE 1 The effect of different callus induction
medium on the formation of callus types＊
Explant
type
Predominant callus type formed on the callus induction medium
EC1 EC2 ET1 ET2
Type Ⅱ
Hypocotyls Type Ⅰ Type Ⅰ Type Ⅰ without
Type Ⅲ
Intermediate Type Ⅰ
Cotyldons Type Ⅰ between Type Ⅰ without Type Ⅲ
and Type Ⅱ Type Ⅲ
Note:＊The type of predominant callus accounts for more than 50% of the cal-
lus formed.
The hypocotyls placed onMS medium with kine-
tin and 2 , 4-D(ET1)produced predominantly soft ,
glassy-transparent callus , whereas the hypocotyls
cultured on 1 2MS medium with kinetin and 2 , 4-D
(ET2) produced predominantly friable , granular
callus type that presented embryogenic cell ag-
gregates , indicating the half mineral salts of MS is
more appropriate for the production of embryogenic
callus for hypocotyls.The cotyledons cultured on
the ET1 medium , however , produced nearly almost
all glassy , friable callus type , while the cotyledons
on the ET2 medium developed mostly compact cal-
lus type with a little proportion of glassy , friable
callus type.These findings relate to the difference
between hypocotyls and cotyledons in their regenera-
tion ability as shown below(see Table 1 and Figure
1).The regeneration rate varied according to differ-
ent callus types formed.Some researchers have also
emphasized the importance of early identification ,
selection , and culture of embryogenic callus devel-
oped in the primary medium(Warrag et al.1990).
In this study , all of the callus induction media
contained 2 , 4-D with BA or kinetin , which could
9Xie Xiangming et al.
induce three types of callus as described above.
There has been a similar report on the callus forma-
tion in the embryogenesis of Eucalyptus grandis
(Major et al.1997).During the culture of callus
induction , it was also found that callus induced in
the 2 , 4-D containing medium had difficulty in re-
generating shoots even after up to three or more sub-
cultures on the primary medium.This finding has
also been confirmed in the regeneration of Eucalyp-
tus grandis(Major et al.1997)and Eucalyptus ca-
madulensis(Ho C K et al.1998).
FIGURE 1 The differentiation rate of
callus types induced on the four different
callus induction media
After transferring these callus types to bud in-
duction medium (EM), a lot of discernible buds
were initiated from the type II callus that presented
embryogenic cells aggregates.In comparison , the
bud induction occurred less frequently from the type
I callus , where there were nodular structures min-
gled with the soft , glassy , and cream-like mass ,
from which a few shoots were proliferated (Plate I-
2).However , bud induction has rarely been ob-
served for the type III callus , the compact callus
(Plate I-3).As shown in Figure 1 , generally , the
differentiation rate of calli derived from hypocotyls
was much higher than that from cotyledons.In the
case of hypocotyls , the high regeneration rate was
attributable to the formation of predominantly type II
callus consisting of embryogenic cell aggregates in-
duced on the ET2 medium.
Based on our preliminary observation , the type
II calli were embryogenic clusters with the callus
and embryos , from which averaging 20 ～ 25 shoots
were proliferated on the bud induction and shoot
multiplication medium after being cultured for one
month (Plate I-6).These clusters were initially
bright and red in color , and turned to be soft , fria-
ble and green , mixed with pigmentation deposit as
the subculture underwent 2 ～ 3 stages.Though we
postulated based on their morphological aspects ,
that the shoots were putatively derived from the
“embryo-like” structures , sections of embryogenic
calli remain to be examined under microscope , con-
firming the presence of both root and shoot meris-
tems , the typical bipolar structure , in the embryo-
genic cells.
After the induced buds removed from embryo-
genic callus clusters were cultured on the shoot mul-
tiplication medium(B5 basal medium with 1.0 mg·
L
-1
BA and 3.0 mg·L-1NAA)for half a month , a
lot of shoots began to proliferate vigorously , fol-
lowed by rapid shoot elongation on the medium com-
prising MS with 0.22mg·L-1BA and 0.09 mg·L-1
NAA .
In our study , the regenerated plantlets have been
obtained from the callus clusters derived from 2 , 4-D
induction.The nodular cultures in Major s study
(Major et al.1997), however , though to be exam-
ined under microscope to confirm the presence of
the structure of “embryos” , failed to develop plant-
lets on the differentiation medium with 0.5 mg·L-1
BA and0.1 mg·L-1 NAA .While there have been
many reports on somatic embryogenesis in many Eu-
calyptus species , the high frequency of plantlet pro-
duction has been obtained only by Muralidhara E M
et al.(1989), and Ouyang Q et al.(1990).The
high frequency plantlet production from the embryo-
genic clusters derived from hypocotyls has been
achieved in this study.
In this experiment , the phytagel or agar was
10 Forestry Studies in China ,Vol.3 ,No.1 ,2001
used in some callus induction medium and multipli-
cation medium (data not shown).As a gelling
agent , phytagel was preferred to be used in the cal-
lus induction medium of embyogenesis in E.gran-
dis(Major et al.1997)or used in a maintaining
medium of embrogenic cultures in E.citriodora
(Muralidharan et al.1989).The phytagel is able
to prevent oxidation that caused the browning of cal-
lus , a phenomenon occurring in the regeneration of
Eucalyptus species.In our initial experiment (data
not shown), though it could prevent the oxidation at
early stage , the browning still emerged as the sub-
culture went on.In addition , based on our observa-
tion , the adverse effects of phytagel were the induc-
tion of vitrification and slow multiplication as well ,
compared to agar used in the multiplication medi-
um.Therefore , we concluded that phytagel was
preferably used as a gelling agent in the early stage
of the callus induction.
The rooting ability of elongated shoots on a range
of rooting media has also been investigated.It has
been found that the combination of 0.5 mg·L-1 IBA
without NAA or with 0.5 mg·L-1 NAA achieved
100%rooting effect within 14 days.Initiated on the
optimal rooting medium with 0.5 mg·L-1 NAA , ad-
ventitious roots appeared thick and short , white in
color , as compared to those thin and long roots de-
veloped on the same medium without NAA.A con-
siderably high rooting percentage has been obtained
on a wide range of IAA concentration combined with
NAA(data not shown), indicating that the rooting
for Eucalyptus pellita is not a major problem which
hindered the regeneration of some other Eucalyptus
species as has been reported (Teulieres et al.
1994).
3.2 Organogenesis
In the previous study , it has been found that the
X4 medium was very effective for the shoot multipli-
cation for the embryogenic callus derived from hypo-
cotyls of E.pellita.To further test organogenesis
through the direct approach of both callus and shoot
induction for hypocotyls , cotyledons and mature
leaves , respectively , a series of combinations of BA
and NAA were employed to explore the highest re-
generation efficiency.The result was presented in
Table 2.
TABLE 2 The shoot formation frequency(percentage)of explants
in Eucalyptus pellita on arange of combinations of BA mg·L-1 and NAA mg·L-1
NAA 0.1 0.2 0.5 1.0 2.0 3.0
BA H C L H C L H C L H C L H C L H C L
0.1 25.00 33.33 0.00 8.33 0.00 16.67 16.67 0.00 16.67 25.00 33.33 8.33 0.00 0.00 0.00 0.00 16.67 8.33
0.2 33.33 33.33 0.00 58.33 66.67 8.33 58.33 33.33 25.00 50.00 25.00 8.33 16.67 0.00 8.33 0.00 0.00 8.33
0.5 33.33 16.67 16.67 50.00 41.67 41.67 25.00 16.67 16.67 25.00 8.33 8.33 25.00 8.33 8.33 16.67 0.00 0.00
1.0 41.67 25.00 16.67 0.00 8.33 8.33 25.00 16.67 16.67 0.00 8.33 0.00 33.33 0.00 0.00 33.33 0.00 0.00
2.0 50.00 33.33 8.33 25.00 33.33 0.00 16.67 16.67 8.33 41.67 16.67 16.67 33.33 16.67 8.33 41.67 8.33 8.33
3.0 8.33 16.67 0.00 83.33 41.67 25.00 25.00 16.67 0.00 8.33 0.00 0.00 16.67 0.00 8.33 8.33 0.00 0.00
Notes:Data were collectedwithin 2 months from each t reatment consisting of 12 explant , H:hypocotyls , C:cotyledons , L:leaves .
As shown in the Table 2 , in general , the vari-
ous explants were able to regenerate adventitious
shoots on the B5 basal medium with a wide range of
combinations of BA and NAA (plate I-7 , 8 , 9),
with the shoot formation frequency in descending or-
der for hypocotyls , cotyledons and leaves.The
highest regeneration rate for hypocotyls , cotyledons
and leaves is 83.33%, 66.67%and 41.67%, re-
spectively.An interesting finding is the optimal
callus and shoot induction medium varied for these
three different types of explants.The use of combi-
nation of 3 mg·L-1 BA plus 0.2 mg·L-1NAA , and
0.2 mg·L-1 BA plus 0.2 mg·L-1 NAA has achie-
ved the highest regeneration rate for hypocotyls and
cotyledons , respectively , whereas the best combi-
nation for leaves organogenesis is 0.5 mg·L-1 BA
and 0.2 mg·L-1 NAA.While this experiment is
preliminary , the data obtained have revealed a fact
11Xie Xiangming et al.
that the usage of BA and NAA only resulted in a
low-to-moderate regeneration frequency for leaves ,
suggesting the other hormones should be explored to
achieve the potentially high differentiation rate.In
Eucalyptus camaldulensis , the shoot formation fre-
quency for leaves on the basal B5 medium with a
combination of 1 mg·L-1 BA and 3 mg·L-1 NAA
was up to 62.5% as reported (Ho C K et al.
1998).In this study , however , the 12 explants in-
duced on the same hormone combination for 2
months resulted in no shoot formation , indicating
the regeneration ability varied among Eucalyptus
species , even among different clones for the same
species(This phenomenon has also been confirmed
in the regeneration of different Acacia species).
The direct organogenesis system is different from
the previous somatic embryogenesis , in which the
embryogenic cluster was induced first on the 2 ,4-D
containing medium.The embryogenic cultures
could be propagated by repetitive subcultures and
its differentiation did not lose even after being ma-
intained for more than half a year on the callus re-
tention medium (data not shown in this study),
even for as long as 3 years(Muralidharan E M et
al .1989).While somatic embryogenesis is gener-
ally a fairly long process , this approach could offer
a large number of production of regenerated plants.
Such “embryos” could be encapsulated to form arti-
ficial seeds that could be stored until used(Major G
et al.1997 , Muralidharan E M et al.1989).In
stead , the organogenesis provides a direct , quick
approach to the regeneration of tree species , more
feasible for the genetic transformation.Though we
have obtained a very efficient regeneration system
for hypocotyls and cotyledons in Eucalyptus pellita ,
the current moderate regeneration frequency of ma-
ture leaves(41.67%)remains to be modified to a
high level , which can effectively be applied in the
gene transfer system for genetic improvement.
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FOR
粗皮桉再生系统的研究
谢响明 陈晓阳
(北京林业大学生物科学与技术学院 ,北京 100083)
韩烈宝
(北京林业大学草坪研究所 ,北京 100083)
摘要 该研究在国内外首次报导了粗皮桉的再生系统.利用粗皮桉的外植体通过两种不同的再生途
径建立了高效的再生系统.首先探讨了粗皮桉的下胚轴和子叶的体细胞胚胎发生.研究发现 ,下胚轴
和子叶在含有 2 ,4-D(添加有 BA或激动素)的培养基上可诱导三种典型不同的愈伤类型.其中 ,来源
于下胚轴的颗粒状的 、松脆的胚性愈伤在分化培养基上可获得高频率的再生小植株.讨论了不同类型
的愈伤组织和其对应的分化再生率之间的关系.此外 ,本研究探讨了来源于粗皮桉的下胚轴 、子叶及
成熟叶片在添加有一系列 BA和NAA的不同浓度组合的 B5 培养基上的器官发生途径 ,三种不同的外
植体在不同的再生培养基上的最高再生频率不同 ,其中成熟叶片 、子叶和下胚轴的最高再生率逐渐提
高 ,分别为 41.67%、66.67%和 83.33%.该文还讨论了两种不同再生系统的意义.
关键词 胚胎发生 ,器官发生 ,植物再生 ,粗皮桉
13Xie Xiangming et al.
14 Forestry Studies in China ,Vol.3 ,No.1 ,2001