Bulletin USAMV-CN. 63 - 64/2007
PLANT SOMATIC EMBRYOGENESIS – AN USEFUL TOOL FOR THE
RESEARCHES IN EMBRYOLOGY
Palada-Nicolau Magdalena
Forest Research and Management Institute. Research Station Simeria. Biscaria str. no. 1. 335900
Simeria. county Hunedoara. Romania. tel./fax: ++40 254 261254;
e-mail: [email protected]
Key words: in vitro cultures. somatic embryogenesis. embryology. embryo biochemistry
Abstract. There are various reasons to consider plant somatic embryogenesis as an useful tool in the
researches of plant embryology: the similarity of zygotic and somatic embryogenesis. the visibility of somatic
embryos. the availability of a big amount of somatic embryos for biochemical analyses. The aim of this paper is
to present a comparison between the zygotic and the somatic embryogenesis. to show a method for the
investigation of the in vitro embryo maturation. and to discuss the functionality of synchronous and sequential
embryogenic systems.
INTRODUCTION
The embryogenic ability is a special quality of the totally or partially dedifferentiated
tissues. expressing the totipotency. Only few cells of such tissues are really embryogenic.
Nevertheless. during the process of embryogenesis in a stable embryogenic culture. a
big amount of embryos in divers stages are accumulated. These masses of embryos can be
studied much more easier than the zygotic embryos extracted from immature seeds and offer
reliable results. due to the deep similarity of zygotic and somatic embryogenesis processes.
The comparison between the zygotic and somatic embryogenesis was the subject of
some studies ( A l e m a n n o e t a l . . 1 9 9 7 ; P a l a d a a n d H a u s m a n . 1 9 9 9 . 2 0 0 0 . 2 0 0 1 ) .
Despite of the multiple similarities between the zygotic and the somatic embryogenesis.
we cannot ignore an essential difference: the origin of totipotency. that in the case of zygote is
naturally due to the fecundation. but in the case of the somatic cells is due to the embryogenic
competence resulted from a special gene expression stimulated by the in vitro growth
conditions. The possibility of controlling in vitro embryogenesis and the analysis of factors
affecting the embryogenic ability helps. using somatic embryos. to elucidate the genetic
background of embryo formation.
In the case of zygotic embryogenesis. each zygote being a unique combination of
maternal and paternal genetic factors. as consequence. each zygotic embryo is unique and the
clonal propagation is not possible by seeds. In spite. in the somatic embryogenesis. each
embryogenic cell can produce a multitude of somatic embryos that form a clone and the
regenerated plants from one initial embryogenic cell belong to the same somaclone. The
somaclones of monoembryonal origin can be either analyzed from genetical and molecular
point of view. or manipulated.
Using the masses of somatic embryos. biochemical analyses can be performed along the
embryo development and the embryo maturation can be investigated.
MATERIAL AND METHOD
Various embryogenic cultures were studied in order to establish models for embryology
researches. The oak (Quercus robur) embryogenic cultures are based on direct embryogenesis
and sequential evolution by serial. recurrent. adventitious embryogenesis.
The plant material was represented. from genetical point of view. by maternal
progenies. As explants. immature zygotic embryos were used.
For the comparisons. somatic embryos from stable. long-term cultured embryogenic
lines were isolated and grouped in defined developmental stages.
Somatic embryogenesis experiments
he oak somatic embryos used in these experiments were produced by the neo-formation
of adventitious somatic embryos. mostly at the basal region of cotyledonary explants. by
epithelial organization processes.
Stable. embryogenic somaclones of cotyledonary origin. composed of somatic embryos
and embryo clusters. in a young cotyledonary stage of development. were maintained in
permanent culture. for more than 7 years (Fig. 1).
Direct
embryo
induction
Proliferation by serial adventitious
embryogenesis – partial synchronisation
Germination
and
conversion
Fig. 1 – Oak somatic embryogenesis: direct embryo induction. serial adventitious embryogenesis. partial
synchronisatio
An incomplete maturation of somatic embryos was obtained (about 5-15% of
embryos). and only occasionally. embryo germination.
The “maturing” embryos have opaque. consistent cotyledons of 5-9 mm in the length.
some times with an irregular shape. Cotyledonar asymmetry. as well as polycotyledony. were
rather frequent.
Carbohydrate content determination: by liquid cromatography. in cotyledons of zygotic
and somatic embryos in similar developmental stages. using an anion exchange
chromatography coupled with a pulsed amperometry detection (Wilson et al.. 1995). The
amounts of carbohydrates were expressed in nM/g fresh weight.
Lignin content determination: using a colorimetrical method (Morrison. 1972) and
expressed as percentiles of the fresh weight of plant material.
RESULTS AND DISCUSSIONS
1. Comparison between oak zygotic and somatic embryos in similar stages of development
1.1. Morphology
In order to compare zygotic and somatic embryos in different developmental stages
during embryo maturation. the young cotyledonary embryos were classified in 7 stages.
according to their morphological traits (dimension. degree of development. color and
consistence of cotyledons). The first 5 of them were found in both zygotic and somatic
embryos. while the stages 6 and 7 were not usually reached by the somatic embryos.
1.2. Carbohydrate content evolution
Glycerol. inositol. mannitol. trehalose. arabinose. glucose. xylose. sucrose and fructose
are present in both zygotic and somatic embryos. in almost all stages. but in different amounts
Accumulation of glycerol. inositol. glucose and sucrose was about 4 to 10 times higher
in the zygotic embryos than in somatic ones. especially in the stages 1-3 for glycerol and
inositol and in the stages 2-5 for glucose and sucrose (Fig. 2. 3).
However. fructose content was constantly higher in somatic embryos than in zygotic ones.
The level of glycerol and inositol decreased continuously in the zygotic embryos.
beginning with the stage 4. approaching to their relatively stable level in the somatic ones
1600
3500
1400
3000
1200
2500
sucrose component amount
glycerol component amount
1000
800
600
2000
1500
1000
400
500
200
0
0
st.1
st.2
st.3
st.4
st.5
embryo developmental stages
z.e.
s.e. - 1.2.1
st.1
st.2
st.3
st.4
st.5
st.6
st.7
embryo developmental stages
s.e. - 3.1.1
s.e. - 3.2.1.
Fig. 2
Variation of glycerol content (HPAEC analysed) during the
development of oak zygotic and somatic embryos in early
cotyledonary stages
z.e.
s.e. - 1.2.1
s.e. - 3.1.1
s.e. - 3.2.1.
Fig. 3
Variation of sucrose content (HPAEC analysed) during the
development of oak zygotic and somatic embryos in early
cotyledonary stages
The glucose and sucrose content in the somatic embryos. compared with that of zygotic
ones. is small (150-700 nM/g. FW) and relatively stable during the embryo development
Similar results for sucrose were obtained by Chanprame et al. (1998) in soybean.
1.3. Lignin content evolution
The lignin content increased continuously during the embryo development. Its evolution
was similar in both zygotic and somatic embryos. During the early stages of development (1
to3). the lignin content was in the range of 0.5 - 0.9 % of FW. and in the stages 4 and 5. it rise
only to values around 1.5 %.
However. in the more advanced zygotic embryos. able to germinate (stage 6). the lignin
content of cotyledons reached 5.8% of FW. In the cotyledons of somatic embryos able to
germinate (stage 5c’) it was also obviously superior to that of nongerminating embryos in the
same stage. reaching the value of 2.6 % of FW Fig. 2).
2. Experiments of in vitro maturation of zygotic embryos in the absence of endosperm
Zygotic embryos were excised in defined developmental stages (1 to 5) and
cultivated for 60 days on MS-0 medium (without growth regulators). as well as on MSABA medium (ABA being known as embryo maturation regulator). After this period. the
embryos were assessed for morphological development and for carbohydrate and lignin
content.
2.1. Morphology
Morphologically. a progression of embryo maturation can be observed. especially on
MS-0 medium. from stages 1. 2 and 3 to stage 5. from 4 to 6 and from 5 to 7.
On MS-ABA medium. the maturation seems to be inhibited. the embryos developed
only from stage 2 to 3. from 3 to 4. and from 4 and 5 to 5.
t=60 d.
Stage 7
Stage 6
Stage 5
Stage 4
Stage 3
Stage 2
Stage 1
MS0
MSCB
MSM
MSABA
t0
Fig. 4 – The evolution of oak in vitro matured zygotic embryos on different culture media
2.2. Carbohydrate content
Glycerol content was drastically reduced in all stages. during the maturation on MS-0.
as well as on MS-ABA medium. even beyond the amounts found in the somatic embryos.
The inositol content evolution showed a big difference between the maturation on MS-0
and on MS-ABA medium. On MS-0. the inositol content of embryos excised in all stages was
reduced to the level of the most advanced naturally developed zygotic ones (the effect upon
the younger embryos was stronger). but in the case of the embryos matured on MS-ABA
medium. the inositol level was similar or even higher than that of the naturally developed
zygotic embryos in corresponding developmental stages (Fig. 5)
Similar. while somehow reduced effects were found in the case of glucose and sucrose.
2.3. Lignin content evolution
Lignin synthesis in the cell wall of the embryos matured on MS-0 medium was similar
to the one observed in naturally matured embryos in corresponding developmental stages.
In the presence of ABA. lignin accumulation was superior to that of somatic embryos
and even to that of naturally matured zygotic embryos in corresponding stages (Fig. 6).
900
7
800
6
700
5
lignin content (% of FW)
inositol component amount
600
500
400
4
3
300
2
200
1
100
0
st.1
st.2
st.3
st.4
st.5
st.6
st.7
0
st.1
embryo developmental stages
e.z. mat. in vitro, MS-0
e.z. mat. in vitro, MS-ABA
z.e.
s.e..
Fig. 5
Comparison of inositol content (HPAEC analysed) evolution in
oak zygotic embryos, somatic embryos and in vitro maturated
zygotic embryos
st.2
st.3
st.4
embryo developmental stages
st.5
st.6
e.z. mat. in vitro, MS-0
e.z. mat. in vitro, MS-ABA
z.e.
s.e..
Fig. 6
Comparison of lignin content evolution
in oak zygotic embryos, somatic embryos
and in vitro maturated zygotic embryos
CONCLUSIONS
1.
We succeeded to produce a high amount of oak somatic cotyledonary embryos and to
maintain the embryogenic ability of the selected cell lines for more than 7 years.
2.
It was also possible to isolate individual embryos and to group them according to
developmental stages. similar in the zygotic and somatic embryogenesis.
3.
The masses of somatic embryos synchronized in defined developmental stages proved to
be an useful tool for the investigation of carbohydrate and lignin accumulation during the
embryo maturation
4.
A study of the accumulation processes during the embryo maturation was imagined. since
it was hypothesized that the low rate of germination was due to an incomplete or deficient
maturation of somatic embryos. and that the possible deficient maturation is the consequence
of either different nature of somatic and zygotic embryos. or in vitro maturation conditions. or
both of them.
5.
Significant differences between somatic and zygotic embryos in similar developmental
stages were found. especially at the level of soluble carbohydrate content evolution during the
embryo maturation.
6.
The in vitro zygotic embryo maturation in the absence of the endosperm was shown to be
possible on MS medium without growth regulator. However. the presence of ABA seemed to
inhibit the embryo maturation.
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