Molecular Characterization of Dg3, a cDNA that Encodes a Novel Lipid Transfer Protein in Brassica napus
David E. Hanke
In this study, we have analysed the sequence of Dg3
clone using bioinformatic tools, determined copy number of this transcript
in the genome of B. napus and expression levels at various tissues/organ.
The cDNA contained 307-base pair open reading frame encoding 102 amino
acid residues, 60-base pair 5`-untranslated region and 127-base pair 3`-untranslated
region. The predicted mature protein has a molecular weight of 9.2 kDa
and is acidic, with a predicted isoelectric point (pI) of 6.2. The Dg3
sequence has all the conserved structural characteristics of plant
LTPs and showed highest homology to LTPs from other plant species. The
transcripts of Dg3 were detected in all tested tissues but highest expression
was in siliques and in vitro embryogenic cultures. Possible roles
of Dg3 during somatic embryogenesis and normal plant development are discussed.
The process of acquisition of embryogenic competence by somatic cells
must involve reprogramming of gene expression patterns as well as changes
in the morphology, physiology and metabolism. These alterations reflect
dedifferentiation, activation of cell division and a change in cell fate
(Feher et al., 2003). Also, the changes are dependent on the down
regulation of some functioning genes in differentiated cells and up regulation
of genes which are required for the transition to happen. A logical first
step to understand the molecular mechanisms responsible for the induction
of somatic embryogenesis will be to isolate and identify genes that are
differentially regulated at the transcriptional level.
In recent years, various attempts have been made to unravel the molecular
mechanism involved in the acquisition of embryogenic competence. This
was achieved by profiling and comparing gene expression between somatic
embryos and callus cells (Sato et al., 1995; Kairong et al.,
1999; Dong and Dunstan, 1999) or between embryogenic cells and non-embryogenic
cells (Giroux and Pauls, 1997; Yasuda et al., 2001; Quiroz-Figueroa
et al., 2002; Bishop-Hurley et al., 2003). Most of the identified
genes were structural genes (Sterk et al., 1991; Sato et al.,
1995; Gyorgyey et al., 1997; Helleboid et al., 2000a; Bishop-Hurley
et al., 2003), early or late embryogenesis genes (Lin et al.,
1996; Dong and Dunstan, 1999; van Zyl et al., 2003), hormone responsive
genes (Kitamiya et al., 2000; Yasuda et al., 2001)and wound
or stress induced genes (Rojas-Herrera and Loyola-Vargas, 2002; Thibaud-Nissen
et al., 2003). These identified genes may not play a direct role
in the induction of somatic embryogenesis. The exception is the Serk gene
whose expression appears to mark the vegetative to embryogenic transition
clearly (Schmidt et al., 1996). In spite of now having Serk
as a molecular marker for competent cells, we are still far from understanding
the key events underlying the transition of differentiated somatic cells
to the totipotent and embryogenic cell state.
To address this problem, we have isolated and identified genes that
are up regulated specifically in the pre-embryogenic stage using the long
term Brassica napus embryogenic culture established in our laboratory
(Namasivayam and Hanke, 2006b). The pre-embryogenic stage, is important
as the phase in which the cells acquire embryogenic competence, the ability
to become an embryo at any time later. Unlike many other embryogenesis
systems, the B. napus secondary embryogenesis system requires no
exogenous hormone to stimulate embryo development (Loh et al.,
1983), hence it is an appropriate system for identification of genes truly
involved during induction of somatic embryogenesis and not the response
to an external stimulus.
In this study, we have attempted to characterise the expression pattern
of Dg3, a cDNA of a novel lipid transfer protein, one of the previously
isolated sequences from the pre-embryogenic tissue of B. napus
(Namasivayam and Hanke, 2006). Studies of gene expression were carried
out by Northern analysis and RT-PCR using various tissues of mature B.
napus plants and at different developmental stages of the embryogenic
tissue during tissue culture. To complement the gene expression analysis,
Southern analysis was performed to check gene copy number in the B.
napus genome. Based on the detailed gene expression pattern and nucleotide
sequence analysis, some hypotheses on its possible functions were made.
MATERIALS AND METHODS
Sources of plant materials: Plants of B. napus ssp. oleifera
cv. Primor were grown from seed, in pots with soil in the Botanic Garden,
Cambridge. Sources and preparation of plant materials for the pre-embryogenic,
mature embryogenic and non-embryogenic B. napus ssp. oleifera
cv. Primor culture were as described in Namasivayam et al., 2006;
Namasivayam and Hanke, 2006. The cytokinin-treated embryogenic tissue
(CK-EC) was generated from hypocotyls of embryoids grown for 20 days on
MS media containing 10-4M kinetin, 2% (w/v) sucrose and 0.8%
(w/v) agar. Various organs/tissues such as young leaves, stem, buds, flowers,
siliques, roots, stamens, carpels, petals and sepals from mature B.
napus plants were harvested, immediately frozen in liquid nitrogen
and stored at -80°C until isolation of DNA or total RNA.
Sequence analysis: The sequence of Dg3 has been submitted to the
GenBank and the database accession number is AY570248. Sequence analysis
was carried out using BLAST 2.0 (Basic Local Alignment Search Tool; Altschul
et al., 1997), accessible from the internet (http://www.ncbi.nlm.nih.gov/BLAST/).
Alignments of the protein sequence with several closely related genes
was carried out using the CLUSTAL W program from the Biology Workbench
version 3.2, accessible from the internet (http://biowb.sdsc.edu/CGI/BW.cgi).
Other sequence analyses were performed using Biology Workbench version
3.2 to compute molecular weight (MW), hydrophobicity and isoelectric point
(pI) determination, signal sequences were predicted using signalP (version
2.0, Nielsen et al. (1997), (http://www.cbs.dtu.dk/services/SignalP/)
and prediction of motifs using PROSITE program from ExPASy Molecular Biology
Total RNA isolation: Total RNA from various frozen tissues/organs
of the mature plant and tissue culture materials were extracted using
the acid guanidium thiocyanate-phenol-chloroform extraction method described
by Chomczynski and Sacchi (1987). The concentration of RNA in each sample
was determined spectrophotometrically (Sambrook et al., 1989).
Northern blot analysis: Equal amounts of total RNA (10 μg
per lane) were resolved on 1.3% (w/v) agarose-formaldehyde denaturing
gel and blotted onto HybondTM-XL nylon membrane (Amersham Biosciences).
Hybridisation was carried out at 65°C using standard techniques (Sambrook
et al., 1989). The entire Dg3 sequence was used as probe
labelled with [32P-α]-dCTP using the Prime-IT®
II Random Primer Labeling Kit (Stratagene). Washes were carried out at
room temperature in the first wash buffer (40 mM sodium phosphate pH 7.2,
1% (w/v) SDS and 1 mM EDTA) for 10 min and followed by second wash in
40 mM sodium phosphate pH 7.2, 5% (w/v) SDS and 1 mM EDTA at 65°C
for 15 min. The hybridisation signals were captured by a Phosphorimager
Typhoon 8600 (Amersham Pharmacia Biotech). After removal of probe, the
same blot was hybridised with radiolabelled Arabidopsis Actin2/7
cDNA probe as a loading control.
Extraction of plant genomic DNA: Genomic DNA of B. napus
was prepared from young leaves using a modified CTAB (cetyltrimethyl ammonium
bromide) method and is detailed below. Two to five grams of frozen young
leaves were ground into a fine powder, added with 25 mL of CTAB buffer
(140 mM sorbitol, 220 mM Tris-HCl pH 8.0, 22 mM EDTA, 800 mM NaCl, 1%
(w/v) N-lauryl sarcosine, 0.8% (w/v) CTAB) and then incubated at 65°C
for 20 min. Ten milliliter of chloroform/isoamylalcohol (24:1) was added,
inverted for 20 min at room temperature and centrifuged at 2,800 g for
5 min to recover the aqueous phase. The nucleic acid was then precipitated
from the aqueous phase by addition of 17 mL isopropanol, placed on ice
for 10 min and centrifuged again as described above. The drained pellet
was dissolved in 2 mL TE pH 8.0 and high molecular weight RNA was precipitated
with an equal volume of 4 M lithium acetate, incubated on ice for 20 min.
After centrifugation (as before), the DNA in the supernatant was precipitated
by adding 2 volumes of 100% (v/v) ethanol, left on ice for 20 min and
centrifugated as before. The pellet was dissolved in 1 mL of TE, 100 μL
of 3 M sodium acetate pH 5.2 added, was extracted with an equal volume
of Tris-saturated phenol and centrifuged (as before). The recovered aqueous
phase was extracted with an equal volume of phenol/chloroform/isoamylalcohol
(25:24:1), followed by final extraction of the aqueous phase with an equal
volume of chloroform/isoamylalcohol (24:1). The DNA was precipitated from
the aqueous phase by addition of 2 volumes of ethanol and dissolved in
250 μL of TE.
Southern analysis: Three microgram of genomic DNA was digested,
electrophoresed on a 0.8% (w/v) agarose gel and transferred to a positively
charged HybondTM-XL nylon membranes (Amersham Biosciences)
by the method of Southern (1975). Hybridization with 32P-labeled
DNA probes were carried out at 65°C according to the standard procedure
by Sambrook et al. (1989). Low stringency washes were performed
as follows: twice in 2xSSC, 0.1% (v/v) SDS at room temperature for 10
min and a final wash in 1xSSC, 0.1% (v/v) SDS at 55°C for 15 min.
For high stringency washes, additional washes with 1xSSC, 0.1% (v/v) SDS
at 55°C for 30 min and a final wash in 0.5xSSC, 0.1% (v/v) SDS at
55°C for 30 min were performed.
RT-PCR: Equal amounts of DNase-treated total RNA (200 ng) from
each tissue sample was added individually to a sterile 0.2 mL PCR tube
and the volume adjusted to 13.5 μL with diethylpyrocarbonate-treated
sterile deionised water. One microliter of 20 pmoles μL-1
oligo (dT18) was added to the tube and the reaction mix incubated
for 10 min at 70°C. Following brief centrifugation, the following
reagents were added: 4 μL 5xfirst strand buffer (Promega), 0.5 μL
‘RNase Out‘ ribonuclease inhibitor (40U μL-1)
(Invitrogen), 0.5 μL 10 mM Bioline dNTPs mix and 0.5 μL MMLV-RT
RNase H minus (200U μL-1) (Promega) and incubated at 37°C
for 1 h. Later, the reaction mix was heat deactivated before using for
PCR reactions. PCR reactions were performed in 12.5 μL reactions
with the following: 2 μL of the RT product, 1xBioline PCR buffer
(Mg2+- free), 1.5 mM MgCl2, 0.4 mM dNTP mix, 2.5
pmoles of forward primer (5´-GTTCCAATTAGAGCAACCAGGGTTGAAAG-3´)
and reverse primer (5´-CGAGCGTTTGG AGAAGTAACATACATACT-3´),
respectively and 1.5U BioTaq DNA polymerase. An internal control was prepared
using actin2 primers (forward primer: 5´-CCATTCTTGCTTCCCTCAG-3´
and reverse primer: 5´-GACGTAAGTAAAAACCCAG-3´) and containing
all the components as above to test for equal loading of the template.
Also, a negative control without template was included. Amplification
was as follows: 95°C for 3 min; followed by 35 cycles at 94°C
for 30 sec, 65°C for 1 min, 72°C for 1 min; and a final extension
at 72°C for 3 min. The annealing temperature used for Actin2
was 60°C. RT-PCR products were separated on a 2% (w/v) agarose gel
and the gel was photographed. The agarose gel containing the PCR amplified
products was blotted (Southern, 1975) and hybridised with a labelled specific
probe (Dg3 or Actin2 cDNAs).
Sequence analysis of Dg3: The cloned fragment of Dg3 presents
494 bp and encodes a predicted peptide of 102 amino acids (Fig.
1). The longest ORF has a start codon at position A61TG
and a stop codon at position T367AA as shown in Fig.
1. According to the alignment of the known plant LTPs with the amino
acid sequence of Dg3 (Fig. 2), confirmed that it contains
a full length coding region and is similar to the Arabidopsis LTP.
The deduced amino acid sequence of Dg3 showed greatest homology to two
putative lipid transfer proteins (LTP) from Arabidopsis (65% identical
to Arabidopsis At5g38195 and 50% identical to Arabidopsis
At5g38160). The lowest homologies (36%) are observed with the rice
LTP. The predicted mature protein has a molecular weight of 9.2 kDa and
is acidic, with a predicted isoelectric point (pI) of 6.2. Although the
homology detected to lipid transfer protein was not high, the deduced
amino acid sequence of Dg3 has all the conserved structural characteristics
of plant lipid transfer proteins (Kader, 1997) including a signal peptide
at the N terminal, 8 cysteine residues located at conserved positions
(shown in Fig. 2) and no tryptophan. Therefore, we concluded
that the Dg3 encodes an acidic lipid transfer-like protein in B.
napus. The PSORT program (Nakai and Horton, 1999) predicts the deduced
protein to be localised extracellularly. Using PROSITE program, two protein
kinase C phosphorylation sites were predicted in the mature BNPE DG3 protein,
at positions 25-28 and 29-32.
Expression analysis of Dg3: Figure 3A shows
the results of the tissue culture blot. Dg3 mRNA was detected as abundant
in the pre-embryogenic, moderately expressed in mature embryogenic, at
a low level in cytokinin-treated tissues and not detectable in non-embryogenic
tissue. By using RT-PCR, it was shown that Dg3 transcripts were present
in all tested tissues of a mature B. napus plant namely leaves,
stems, buds, flowers, siliques, roots and carpels. Expression level of
Dg3 was very low in all tissues except in silique (Fig.
3B) which the expression level was found to be elevated relative to
other tissues, suggesting expression of Dg3 gene is spatially controlled.
RT-PCR using Actin2 primers as internal control showed an amplified
PCR product of 300 bp in all lanes except lanes L, B and G (Fig.
3C). In lane G, a PCR product of 380 bp was observed when genomic
DNA was used as template. The Actin2 primers were designed to span
different exons so that the amplification product would be of different
length from any contaminating genomic DNA which would include introns.
The larger band size is due to the intron sequences and also shows that
the total RNA did not have any contaminating traces of DNA after the DNAse
treatment. However, when a blot of this gel was probed with radiolabelled
Actin2 cDNA in a Southern analysis, signals were detected in both
lanes L and B. Thus, the amount of PCR amplification product must have
been too low to be visualised on the gel. Although at the start all RT
samples had the same amount of RNA and cDNA for PCR, there were variations
in signal intensity between lanes for Actin2. This may be due to
variation of actin abundance in different tissues (McDowell et al.,
||Nucleotide and deduced amino acid sequence of Dg3 (AY570248).
The ORF is underlined. The (*) mark indicates the stop codon of the
||Alignment of the deduced amino acid of Dg3 with the
deduced amino acid sequences of other putative lipid transfer proteins.
Shaded sequences denote identical amino acids and gaps introduced
in the alignment are marked with dashes. The asterisk marks denote
8 cysteine residues present in conserved positions in most plant LTPs.
An arrow marks the putative cleavage site of the secretory signal
peptide in Dg3. The amino acid sequences were obtained from GenBank:
Triticum_aestivum (GenBank accession No.P82900), Rice (GenBank accession
No. 1L6H_A), Maize (GenBank accession No. P83506 ); Arabidopsis At5g38195
(GenBank accession No. NP_568555) and Arabidopsis At 5g38160 (Gen
Bank accession No. NP_198632)
||Expression analysis of Dg3 in Brassica napus
||Tissue culture RNA gel blots containing 10 μg of total RNA
per lane were first hybridised to 32P-labelled Dg3 and
then to an Arabidopsis Actin 2/7 cDNA (control). Lanes: NEC,
non-embryogenic tissue; PEC pre-embryogenic tissue; MEC, mature embryogenic
tissue; CK-EC, cytokinin-treated tissue
||Top panel show results of RT-PCR analysis of Dg3 gene expression
in various tissues of mature B. napus C, Top panel shows results
of RT-PCR analysis of Actin 2 gene expression. B, bottom panel is
southern blot analysis of the RT-PCR gel probed with 32P-labelled
||Bottom panel is Southern blot analysis of the RT-PCR gel probed
with 32P-labelled Actin 2 cDNA. Lanes: M, 1 kb Bioline
DNA ladder; L, leaves; S, stems; B, buds; F, flowers; Si, siliques;
R, roots; Ca, carpels; G, genomic DNA
Southern analysis: In search of sequences in the B. napus
genome related to the Dg3 gene, genomic DNA blot analysis was performed.
The entire ORF of the Dg3 sequence was used to probe the genomic
blot, which was then was subjected to low and high stringency washing
conditions. Under high stringency condition (Fig. 4),
only one fragment hybridised with the probe when genomic DNA was digested
with BamHI or NotI-there were no restriction sites within
the Dg3 sequence for either of those enzymes. The Dg3 sequence
has one restriction site for HindIII and none for RsaI.
The lane containing HindIII genomic digest had two strongly hybridising
bands as expected but there were also two additional faint bands in that
lane. For RsaI genomic digestion, two smaller bands were observed
and that was not expected. Under low stringency washing conditions (data
not shown), no additional hybridising bands were detected, only an increase
in background. The presence of two additional bands in the HindIII
and RsaI digestion suggests that the B. napus genome may
contain another gene with weak similarity to the Dg3 gene.
||Genomic Southern blot analysis of Dg3 in B. napus,
Southern blot of B. napus genomic DNA probed with 32P-labelled
Dg3, washed under high (A) and low (B) stringency conditions as described
in the materials and methods. DNA was digested with BamHI, HindIII,
RsaI and NotI. DNA Size markers on the left are in kb.
Dg3 encodes a putative acidic lipid transfer protein: The BNPE
DG3 clone encodes a protein with all the expected characteristics of a
plant lipid transfer protein. The LTPs are small, soluble basic or acidic
proteins ranging in size from 7 to 12 kDa (Kader, 1997; Blanckaert et
al., 2002). LTPs are synthesised as precursors with a putative signal
peptide (Bernhard et al., 1991) and are located extracellularly
or are associated with the cell wall (Thoma et al., 1994; Molina
et al., 1996). Many genes and cDNAs for plant LTPs have been isolated
and characterised (Kader, 1997). The plant LTPs are encoded by a multigene
family (Arondel et al., 2000) and different isoforms may correspond
to different functions (Kader, 1997). They are thought to be involved
in various biological roles including cutin biosynthesis (Sterk et
al., 1991; Meijer et al., 1993) or surface wax formation (Pyee
et al., 1994; Pyee and Katattukudy, 1995), pathogen defence reactions
(Molina et al., 1993; Garcia-Olmedo et al., 1995; Sohal
et al., 1999a,b), somatic embryo development (Sterk et al.,
1991; Soufleri et al., 1996; Sabala et al., 2000) and in
adaptations of plants to environmental changes (Dunn et al., 1998;
Yubero-Serano et al., 2003; Jung et al., 2003).
The Dg3 gene is a member of a small subfamily: In most plant species,
Ltp genes are present as a member of a multigene family, as in
maize (Tchang et al., 1988), tomato (Torres-Schumann et al.,
1992), broccoli (Pyee and Kollattukudy, 1995), barley (White et al.,
1994), rice (Vignols et al., 1997) and Arabidopsis (Arondel
et al., 2000). However, in Daucus carota (Sterk et al.,
1991) and Spinacia oleracea (Bernhard et al., 1991), only
one Ltp gene was detected. Arondel et al. (2000) have reported
that the Ltp genes in Arabidopsis exist in small subfamilies
of one to three genes which may or may not weakly cross hybridise at low
stringency conditions in Southern analysis. Therefore, they suggested
that Southern genomic DNA analyses are likely to underestimate the number
of Ltp genes in a plant genome. Since Dg3 presents an unusual pattern
of expression and has relatively low sequence homology with the previously
reported Ltp genes in B. napus (Foster et al., 1992;
Ostergaard et al., 1993), we conclude that the Dg3 gene may belong
to a new small Ltp gene subfamily comprising one or two genes and
distinct from the related genes reported to date.
Expression of Dg3 is developmentally controlled and correlates with
embryogenesis: The spatial expression of Dg3 (assuming DG3 is a LTP)
is consistent with a function in the secretion and deposition of extracellular
lipophilic material such as cutin and wax in the cuticle or epicuticular
layer found in aerial parts of the plant as reported in most previous
studies (Thoma et al., 1994; Pyee and Kollatukuddy, 1995; Canevascini
et al., 1996; Sohal et al., 1999a; Park et al.,
2000). Sterk et al. (1991) suggested that the LTPs may function
in transporting a variety of lipid molecules including cutin monomers
from their place of synthesis in the endoplasmic reticulum to various
extracellular locations such as the outer epidermal cell wall of leaves/stems,
developing ovary integuments and later in seed coat. The transported,
soluble cutin monomers would be available for polymerisation/cuticle formation
which is important for plant defence during pathogen attack and for prevention
of water loss (Chugh and Khurana, 2002).
Alternatively, LTPs have also been proposed to participate in the deposition
of lipophilic material in floral organs such as corolla (Kotilainen et
al., 1994), nectaries and stigma for adhesion (Thoma et al.,
1994; Park et al., 2000) and anther (including pollen), where sporollenin
biosynthesis occurs (Foster et al., 1992).
However, neither studies reported by Canevascini et al. (1996)
nor Sohal et al. (1999a) could establish a reason for localisation
of Ltp gene expression at root tips. In fact, Thoma et al.
(1994) suggested that the LTP expression in Arabidopsis lateral
roots could be artefactual based on the premise that roots do not have
a cuticular layer equivalent to the shoot. It is not impossible that LTPs
are being expressed in the roots because roots do secrete lipophilic material
in root mucilage. The surface of the root cap of growing roots is often
covered by a thick layer of mucilage (Rougier and Chaboud, 1985). The
root exudates play a major role in the interactions between roots, microorganisms
and soil such as root penetration, soil aggregate formation, microbial
dynamics and nutrient turnover (McCully, 1999). Previous microscopy studies
have shown that the root mucilage contained oily droplets in onion root
tips (Scott et al., 1958) and in radish root hairs (Dawes and Bowler,
1959). More recently, the lipid composition of maize, lupin and wheat
root mucilages was analysed by thin-layer chromatography and gas chromatography-mass
spectrometry. This study has shown that the lipid content was low and
mainly composed of saturated acyl groups (Read et al., 2003). The
low level of expression of Dg3 in B. napus roots may represent
a small proportion of root cells expressing Dg3 and low levels of lipid
secretion in the root mucilage.
Based on the expression analysis results, Dg3 was expressed at a basal
level in all tissues of the mature plant examined but upregulated during
somatic and zygotic embryogenesis. This is not surprising because some
important genes in embryogenesis have been shown to be expressed in vegetative
tissues too (Dodeman et al., 1997). Detection of Dg3 transcripts
in the pre-embryogenic but not the non-embryogenic tissue confirms the
association of LTPs with somatic embryogenesis reported in carrot (Sterk
et al., 1991; Meijer et al., 1993), grapevine (Coutos-Thevenot
et al., 1993), barley (Vrinten et al., 1999), Picea
abeis (Sabala et al., 2000) and Cichorium (Blanckaert et
al., 2002). Of the above-mentioned studies, the most relevant was
conducted by Blanckaert et al. (2002) who reported the presence
of 9 kDa acidic and basic LTPs in the culture medium during somatic embryogenesis
in Cichorium. They found that the acidic LTP was present during the induction
step whereas the basic LTP was only detected during globular embryo formation.
Therefore, it was suggested that the acidic LTP might have a role in the
initiation of somatic embryogenesis (Blanckaert et al., 2002) whereas
the basic LTP protein may be involved in the secretion or deposition of
extracellular lipophilic molecules on the protoderm layer of the globular
embryo (Sterk et al., 1991). This is intriguing because the Dg3
mature protein was predicted to encode an acidic LTP and the mRNA transcripts
were found to be associated with the pre-embryogenic tissue, a stage at
which cells are acquiring embryogenic competence. This is consistent with
Blanckaert et al. (2002) proposed role for acidic LTP as associated
with induction of somatic embryogenesis.
Another interesting observation in regards to gene regulation is that
the cytokinin-treated embryogenic tissues showed a low level of Dg3 gene
expression. It is intriguing that the transcription of the Dg3 gene appears
to be down-regulated in response to cytokinin since there are no previous
reports of effects of cytokinin on the regulation of Ltp gene expression.
Loh et al. (1983) reported that if embryoids were treated with
cytokinin during the early stages of culture and then transferred to growth
regulator-free medium, these embryoids developed directly into plantlets
and did not produce secondary embryoids i.e. on cytokinin treatment the
tissue lost its embryogenic competence. An important observation made
during our study and worth mentioning is that cytokinin-treated embryoids
still exhibited the secondary embryogenesis phenotype at a minimal level
compared to the untreated embryoid (mature embryogenic culture). Hence,
complete loss of embryogenic competence was not observed. As such, low
levels of Dg3 transcripts in cytokinin-treated embryogenic tissue may
not have been a direct effect of cytokinin treatment but could be related
to the low level of secondary embryogenesis in the in vitro culture.
This could explain why Dg3 transcripts were detectable by Northern analysis
in cytokinin-treated tissue but not in the non-embryogenic tissue which
does not have embryos at all. If this is true, Dg3 expression correlates
strongly with secondary embryogenesis. That raises the interesting question
of whether Dg3 expression correlates generally with embryogenesis, in
view of the fact that Dg3 expression is elevated in siliques and in
vitro embryogenic cultures. Future work should involve anti-sense
studies to give insights into the contribution of Dg3 to somatic embryogenesis.
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