The transcription factor MtSERF1 of the ERF subfamily identified by transcriptional profiling is required for somatic embryogenesis induced by auxin plus cytokinin in Medicago truncatula.

Transcriptional profiling of embryogenic callus produced from Medicago truncatula mesophyll protoplasts indicated up-regulation of ethylene biosynthesis and ethylene response genes. Using inhibitors of ethylene biosynthesis and perception, it was shown that ethylene was necessary for somatic embryogenesis (SE) in this model legume. We chose several genes involved in ethylene biosynthesis and response for subsequent molecular analyses. One of these genes is a gene encoding a transcription factor that belongs to the AP2/ERF superfamily and ERF subfamily of transcription factors. We demonstrate that this gene, designated M. truncatula SOMATIC EMBRYO RELATED FACTOR1 (MtSERF1), is induced by ethylene and is expressed in embryogenic calli. MtSERF1 is strongly expressed in the globular somatic embryo and there is high expression in a small group of cells in the developing shoot meristem of the heart-stage embryo. RNA interference knockdown of this gene causes strong inhibition of SE. We also provide evidence that MtSERF1 is expressed in zygotic embryos. MtSERF1 appears to be essential for SE and may enable a connection between stress and development.


INTRODUCTION
There have been numerous studies concerning the hormonal induction of somatic embryogenesis (SE) in a wide range of species. In almost all cases auxin has a critical role and cytokinins are frequently involved (Fehér et al., 2003;Rose 2004). Stress is also a factor that has been increasingly recognised as having an important role in the induction of somatic embryogenesis (Touraev et al., 1997;Fehér et al., 2003, Nolan et al., 2006. Hormones and stress collectively induce dedifferentiation of differentiated cells and the initiation of an embryogenic program (Fehér et al. 2003;Ikeda-Iwai et al., 2003;Rose and Nolan, 2006).
The molecular mechanisms involved in the induction of SE from cultured tissue are not well understood. There has, however, been progress in identifying the involvement of the SOMATIC EMBRYO RECEPTOR KINASE (SERK) and a number of transcription factors.
Arabidopsis transformed with the AtSERK1 gene under the control of the cauliflower mosaic virus 35S promoter showed a marked increase in SE compared to wild-type cultures (Hecht et al., 2001). Ectopic expression of the transcription factors LEAFY COTYLEDON1 (Lotan et al., 1998), LEAFY COTYLEDON 2 (Stone et al., 2001), BABY BOOM (Boutilier et al., 2002), and WUSCHEL (Zuo et al., 2002) in Arabidopsis cause spontaneous formation of somatic embryos on intact plants or explants. AGL15 is another transcription factor that promotes SE in Arabidopsis (Harding et al., 2003). In addition, many other genes are specifically expressed in somatic embryogenesis (Imin et al., 2005).
In Medicago truncatula high rates of somatic embryo formation can be induced in the Jemalong genotype 2HA (Rose et al., 1999) by application of the hormones auxin and cytokinin (Nolan et al., 2003). The 2HA genotype is a "super embryogenic" mutant that is 500-fold more embryogenic than wild-type Jemalong (Nolan et al., 1989;Rose et al., 1999, Rose andNolan 2006). M. truncatula is a model legume (Cook, 1999) with the sequencing of the gene-rich euchromatin nearing completion (Young and Shoemaker, 2006). Mutant resources (Tadege et al., 2005), numerous expressed sequence tags, microarray chips, proteomic tools, physical and genetic maps are available for M. truncatula (VandenBosch and Stacey, 2003). The 2HA genotype coupled with the genomic and molecular genetics tools makes M. truncatula an attractive system to investigate the molecular genetics of somatic embryogenesis (Nolan et al., 2003;Imin et al., 2005;Rose and Nolan, 2006).
In addition to the application of hormones to induce somatic embryogenesis there is the stress component, induced by the excision and culture of the explant, to consider (Nolan et al., 2006). In M. truncatula there are many stress-related proteins associated with SE (Imin et al., 2004). A number of these proteins are differentially expressed between 2HA and Jemalong (Imin et al., 2005). The synthesis of the growth regulator ethylene can be rapidly evoked in response to a variety of biotic and abiotic stresses including wounding (Kende and Zeevart,1997;Wang et al., 2002). Here, microarray studies on the induction of SE in M.
truncatula identified genes predicted to encode ethylene biosynthesis and ethylene response proteins that are differentially expressed in SE. More detailed analysis of the role of ethylene in SE showed that a transcription factor of the AP2/ERF superfamily and ERF gene subfamily designated SOMATIC EMBRYO RELATED FACTOR 1 (MtSERF1), that is dependent on ethylene biosynthesis and perception for its expression, is required for SE in M. truncatula.
MtSERF1 may enable a connection between stress and development.

Microarray Analysis
The use of mesophyll protoplasts was valuable for the microarray analysis as cultures are derived from one cell type and should identify critical gene expression changes more clearly than leaf explants. Leaf explants in addition to mesophyll cells contain cells of the vasculature, stomates and epidermis. Trends in gene expression from 40 to 80 day old 2HA cultures were profiled using a 16 K oligonucleotide array and Cy3 and Cy5 fluorescent labels.
At 40 days the cultures are at the cell proliferation stage, at 60 days globular embryos are forming and at 80 days heart and later stage embryos are forming ( Fig.1 and Supplementary   Fig.1). We made direct comparisons between 40 and 60 day old cultures, 60 and 80 day old cultures and 40 and 80 day cultures. The determination of up and down-regulated genes was determined statistically using the strategies described in the methods. The statistical test is very important as the developing embryos are diluted amongst the proliferating cells and the fold change may be relatively small. Further, while there is a degree of synchrony in the production of embryos from protoplasts, embryo development is not perfectly synchronized.
At 80 d of culture embryo development in many cases has reached the heart stage but synchronicity starts to be lost. Vascular tissue has also started to form in the callus at 80 d.

7
We have grouped genes into functional classes to assist in the interpretation. These are the first transcriptional profiling data obtained from differentiating single protoplasts using large scale microarrays.
In Fig. 2 we show the distribution of the number of genes associated with different functional classes that are up-or down-regulated for 60 d compared to 40 d and 80 d compared to 60 d.
By including all genes that show statistically significant changes in expression, transcriptional changes occurring in only small numbers of cells will be included (see Supplementary Table   1). Our main interest is the time point where the cell culture (Suplementary Fig.1) switches to SE formation (60 d) from proliferation (40 d). Statistically significant changes in expression were found for more than 1500 genes at 60 d compared to 40 d: 883 and 823 genes were upor down-regulated respectively. Comparison of 80 d and 60 d of culture revealed about 2000 genes differentially expressed from which 889 were up-regulated and 1089 down-regulated. Figure 2 shows the number of genes whose expression were up-or down-regulated within 27 functional groups. There is down-regulation of cell proliferation and protein synthesis genes (histones, DNA replication factors, ribosomal and a number of other translation associated proteins) as cells switch into SE. Two cyclin dependent kinases, cdc2Ms1 and cdcMsF, which are actively expressed during the G2-to-M phase in alfalfa cells (Magyar et al., 1997) are down-regulated at 60 days. These data are consistent with that of Thibaud-Nissen et al. (2003) in soybean where the most rapid cell division occurs in early callus formation. Our data also demonstrate changes in expression of a number of cell wall modifying enzymes as well as cell wall proteins.

Development-Related Genes
It is known that cells undergoing SE as well as zygotic embryogenesis show changes in cell wall polysaccharides and proteoglycans (Majewska-Sawka and Nothnagel, 2000). There is up-regulation of embryo specific genes as the somatic embryos within the callus develop. There is also increased expression of chloroplast-and photosynthesis-related genes, reflecting plastid changes associated with the development of the embryogenic callus in low light. The developmental changes are consistent with the morphological development of the embryogenic callus and support the reliability of the arrays.

Stress-Related Genes
There is up-regulation of genes involved in biosynthesis of flavonoids, redox, P450 and other stress related genes that could be related to general stress that is a part of cell culture and an important component in the induction of SE (Nishiwaki et al., 2000;Ikeda-Iwai et al., 2003;Belmonte and Yeung 2004;Stassola et al., 2004 ). Most of the enzymes from the isoflavonoid biosynthetic pathway are upregulated at 60 days (chalcone reductase and chalcone synthase, chalcone reductase, isoflavone 2'-hydroxylase, isoflavone reductase) and their product can be involved in defense or nodulation processes.

Hormone-and Regulatory-Related Genes
We categorised a number of functional groups that were likely to have regulatory roles and provide a useful overview of the potential contributors to the regulatory networks involved in somatic embryo induction and development. Transcriptional regulators, signal transduction and hormone biosynthesis and hormone response genes are represented. Auxin and cytokinin are the hormones supplied so it might be expected that there would be changes in gene expression for many genes directly related to these hormones. This was the case for the auxin response genes, but less so for the cytokinin response genes. What was of particular interest was the up-regulation of ethylene biosynthesis genes at both time points and ethylene response genes in the SE transition period.
To obtain a view of the major transcriptional changes involved in the induction of somatic embryogenesis we focused on a selection of genes from Supplementary We were interested in the contribution of stress responses to successful somatic embryogenesis. Therefore we focused on the ethylene biosynthesis genes and an ethylene response transcription factor, the AP2/EREBP homolog TC102138. The AP2/EREBP homolog was of more interest than other ethylene response genes because of its pattern of expression in qRT-PCR studies (detailed below), it showed a near 2 (1.94) fold increase and was a transcription factor. In a separate protoplast experiment the increase in expression in AP2/EREBP occurred in the highly embryogenic 2HA at 60d but not in the near nonembryogenic Jemalong (Fig. 3). We designated the ethylene responsive AP2/EREBP homolog SOMATIC EMBRYO RELATED FACTOR 1 (MtSERF1).

Gene Expression Analysis Using qRT-PCR
Measurements of gene expression using qRT-PCR were carried out for both the ethylene biosynthesis genes and the ethylene response gene on leaf explants to see if these genes were similarly up-regulated as they were using mesophyll protoplasts. Leaf explants are experimentally simpler than using isolated single protoplasts and are commonly used to produce embryogenic callus for legume transformation experiments (Wang et al., 1996, Chabaud et al., 2003. Experiments can be turned over more quickly using leaf explants as they produce embryos about 40 days earlier than protoplasts These experiments were carried out with both the highly embryogenic 2HA and the near non-embryogenic wild-type Jemalong. The As MtSERF1 is a member of the AP2/ERF family of transcription factors, the promoter region was examined for an ethylene response element (ERE). A 1,758 bp region upstream from the transcription start site was isolated, cloned and sequenced. In addition to the TATA and CAAT boxes, in silico analysis indicated that the promoter region contained a number of potential regulatory elements (Fig.5). Two ERE elements were present, as well as two WUSCHEL binding sites, three Arabidopsis Response Regulator 1 (ARRI) elements that are associated with cytokinin signalling, an Auxin Response Factor (ARF) element and a tobacco EIN3-like (TEIL) element.

Somatic Embryo Induction
In Given that embryogenic cultures are a mixture of embryos and callus cells, in order to establish a stronger connection with ethylene biosynthesis and embryo formation, we directly compared non-embryogenic callus with embryogenic callus, somatic embryos and ovules.
The ACS and ACO genes are consistently expressed at higher levels in embryogenic tissue, somatic embryos and ovules with globular stage embryos compared to non-embryogenic callus (Fig. 8 ).

Localisation of MtSERF1 Expression and Requirement for Somatic Embryogenesis
To localise MtSERF1expression we carried out in situ hybridisation. The MtSERF1 specific probe was a 376 bp fragment from the 3 ' region. As shown in Fig  showed expression was present in the embryo but not in the ovule wall (unpublished data).
In order to examine whether MtSERF1 expression is required for SE we used an RNAi approach. As shown in Fig 10,  We also obtained transformed plants using an inducible vector containing RNAi and produced calli in the presence and absence of dexamethasone. The induction of RNAi by dexamethasone reduced the number of calli producing embryos by 90%. The empty vector control showed no change in the presence or absence of dexamethasone.

Sequence and Phylogenetic Analyses of the Transcription Factor MtSERF1
MtSERF1 is a protein of molecular mass 23 kDa and contains 201 amino acids. The amino acid sequence of MtSERF1 contains a single AP2/ERF domain as shown by position specific iterated and pattern hit initiated BLAST. As indicated by Nakano et al. (2006) this domain is characteristic of the AP2/ERF superfamily and the ERF subfamily contains a single domain.
An alignment of this domain with other proteins containing a single AP2/ERF domain shows high similarity ( Fig 11A).
In which was essential for somatic embryogenesis.
Some of the major changes in the microarray data relate to stress, reflected in a range of genes connected to abiotic, biotic and oxidative stresses. This may have been predicted given that protoplast isolation (Pasternak et al. 2002) and tissue excision (Nolan et al., 2006) associated with the induction of SE is a very stressful, wound-related procedure. Transcriptional profiling in response to mechanical wounding has been carried out in Arabidopsis (Cheong et al., 2002;Delessert et al., 2004) and a diverse groups of genes previously related more specifically to wounding, pathogen attack, abiotic stress and plant hormones are up-regulated.
In this study M. truncatula flavonoid biosynthesis genes were also up-regulated and have also been related to stress protection (Winkel-Shirly, 2002 (Domoki et al., 2006). In soybean, somatic embryogenesis is induced by 2,4-D in cotyledons and is associated with up-regulation of oxidative stress and defense genes (Thibaud-Nissen et al., 2003). Studies by Che et al. (2006) involving microarray analysis of shoot, root and callus development in Arabidopsis tissue culture also noted an increased expression of specific stress-related genes.
Amongst the most highly induced genes in our study was an ethylene biosynthesis gene (Table 1). Up-regulation of transcripts of ethylene biosynthesis genes has also been seen in wounding (Cheong et al., 2002;Delessert et al., 2004) and somatic embryogenesis in soybean cotyledons (Thibaud-Nissen et al., 2003). ACC synthase was up-regulated on an auxin-rich callus induction medium (Che et al., 2006) in Arabidopsis. We also noted an up-regulation of ethylene-response genes and this contributed to ethylene becoming a focus of our studies. In addition to the suite of up-regulated genes related to ethylene, it was of interest to note that it might be expected that as auxin and cytokinin were present in the medium auxin and cytokinin response genes would be the only prominently featured hormone-related genes.
However, this was not the case. Genes related to ABA, gibberellin and brassinosteroids were also featured. We have recently discussed the possible roles of these hormones in somatic embryogenesis (Rose and Nolan, 2006 straightforward. Leaf explants also produce embryos more quickly, about 40 days earlier than protoplasts. We were able to show that ethylene biosynthesis and ethylene response genes in leaf explants were also up-regulated. The first question that arises is whether The lack of MtSERF1 expression in Jemalong, rarely embryogenic and near isogenic with respect to 2HA, provides a focus for further analysis. There is a small inhibition of ACC SYNTHASE expression and a more strongly reduced ACC OXIDASE expression in Jemalong. This could ultimately lead to reduced signaling and reduced MtSERF1 expression. We also know that Jemalong and 2HA respond to auxin by producing roots but when cytokinin is added to the auxin only 2HA forms embryos (Nolan et al., 2003) and Jemalong usually forms callus only. The significance of the localisation of MtSERF1 expression to the early embryo and to a localized region of the shoot pole of the heart-stage embryo also requires further investigation. We also note that the MtSERF1 promoter contains WUSCHEL binding sites and WUSCHEL is implicated in the induction of somatic embryogenesis as well as stem cell maintenance in apical meristems (Zuo et al., 2002, Rose andNolan, 2006). it has two repeated AP2/ERF domains (Boutilier et al., 2002;Nakano et al., 2006). Recently the ERF transcription factor ERN, required for nodulation, has been identified in Medicago tuncatula (Middleton et al., 2007). MtSERF1 is in group 9 of the ERF subfamily while ERN is in group 5 (Fig. 11B). The AP2/ERF superfamily has a mix of transcription factors which relate to growth and development, abiotic and biotic stressors, and ethylene response (Alonso et al., 2003;Nakano et al., 2006;). This may relate to the need to link growth and stress in the evolution of sessile plants. Protoplasts were isolated from leaves of the highly embryogenic 2HA genotype of Medicago truncatula cv. Jemalong. A wild-type Jemalong plant frequently produces no embryos. The highest embryo producing plant we have ever recorded was one embryo per 6 explants (Nolan et al., 1989). The 2HA genotype was derived from a rare regenerated plant obtained by a single cycle of tissue culture from wild type Jemalong. This regenerated Jemalong showed enhanced somatic embryogenesis and the seed progeny segregated into types with and without the capacity to produce somatic embryos. Seed from the regenerated Jemalong was used to continue to select for high embryogenicity over four seed generations (Rose et al. 1999). The 2HA genotype can be considered to be a near isogenic, highly embryogenic mutant of Jemalong. Plants were grown in controlled environment rooms at low light intensity, as described by Tian and Rose (1999). The flotation procedure utilised for protoplast isolation yields almost exclusively mesophyll protoplasts. Isolated protoplasts were grown in 1% low melting point agarose droplets and then transferred to agar plates on P4 10:4 (10 µM NAA and 4 µM BAP) medium for culture as described by Rose and Nolan (1995). For microarray analysis calli derived from these isolated single protoplasts were taken at the following stages of development ( Fig.1and supplementary Fig.1) -the cell proliferation stage (40 days of culture), the early globular stage (60 days of culture), and the heart and later stages (80 days of culture).

Cultured Leaf Explants
Cultured Medicago truncatula leaf explants were obtained from glasshouse grown 2HA or wild type Jemalong. Seeds of wild type Jemalong were originally obtained from the National

Medicago Collection, South Australian Research and Development Institute (SARDI),
Adelaide. The standard leaf culture procedure was as described by Nolan et al. (2003).

The 16K Oligo Microarray Slides
The Medicago 16 K microarray was utilised and has a probe set mapped to the Medicago Gene Index Release 8.0 (http//www.tigr.org/docs/tigr/scripts/medicago/ARRAYS/array.TC mapping). The 70-mer oligos were synthesised by Qiagen-Operon and the slides printed at The University of Arizona in the laboratory of Dr. David Galbraith. After printing, the slides were baked for 80 min at 80 0 C. The oligonucleotide array elements were immobilised by UV cross-linking at 300 mJ then washed twice with gentle rocking, for 2 min each wash, in 2X SSC + 0.2% SDS. The slides were then immersed in boiling hot water for 2 min, blotted briefly and transferred to ice cold ethanol for 2-5 min. Slides were then dried by centrifugation at 1500 rpm for 2-5 min and finally stored in a light proof box under cool dry conditions.

RNA Preparation, cDNA Synthesis, and Hybridization of Microarrays
Calli grown from individual protoplasts in an isolation that produced thousands of embryogenic microcalli, consistent with high protoplast quality, were collected at 40, 60 and 80 days after initiation of culture. The calli were frozen in liquid nitrogen and stored at -80 0 C until RNA was isolated. RNA was isolated as described by Lohar et al. (2006) and stored at - µg of RNA was resuspended in 8 µL of nuclease free double distilled water and used for cDNA synthesis with an RT primer for labelling with either Cy3 or Cy5 dyes using a 3DNA Array50 Kit (Genisphere) as previously described (Lohar et al., 2006).
Experiments were conducted using a regular dye-swap design as described earlier in Lohar et al (2006 repeats of each dye combination to control for dye effects (Lohar et al., 2006).

Microarray Analysis
Methods for array analysis were as described for a 6K microarray (Lohar et al., 2006).
Briefly, microarray slides were scanned using an Axon two-laser scanner and image analysis was performed using GenePix (Axon) software. Background-subtracted mean intensities for both tissues were log transformed and normalized before further analysis. Normalization of microarray data was performed using a statistical module developed as a part of Lab Normalization steps included i) within-slide normalization using local linear regression (LOWESS function) (Yang et al., 2000); ii) between-slide normalization using four-way ANOVA with replications for multi-slide dye-swap experiments (Kerr et al., 2000). More detailed description of pre-processing steps, such as log2 transformation of backgroundsubtracted Cy5 and Cy3 intensities are described in Lohar et al. (2006). Identification of differentially expressed genes was done using SAM software (Stanford), which allows flexible monitoring of the False Discovery Rate (Tusher et al., 2001). We applied a False Discovery Rate of < 0.1 % and the highest q value was <0.06%.
All genes of statistical significance with predicted or known function or that showed significant homology to characterised genes (annotated in the TIGR database at http://compbio.dfci.harvard.edu/tgi/) have been manually divided into 27 classes. Genes that did not fit readily into one of these classes have been classified as "other genes with defined function" and "genes with unknown function". Supplementary Table 1 lists all the genes incorporated into these classes. To obtain a subset of genes that passed a statistical significance test we have also imposed a fixed ratio threshold of 2.

In Situ Hybridisation
To generate the RNA probes, a 376 bp fragment specific to MtSERF1 was first amplified by PCR with the primers 5'-CTGTGAAATTGATGCTGCAAA-3' (forward) and 5'-TGACATAATTGTTGAGCTCACTCC-3' (reverse). Then, the promoter sequences of T7 and SP6 RNA polymerase were introduced to this fragment by a two-step PCR. The first primers used were 5'-GAGGCCGCGTCTGTGAAATTGATGCTGCAAA-3' (forward) and 5'-ACCCGGGGCTTGACATAATTGTTGAGCTCACTCC-3' (reverse). The second set of primers used were 5'-TTATGTAATACGACTCACTATAGGGAGGCCGCGT-3' (forward) and 5'-CCAATTTAGGTGACACTATAGAAGTACCCGGGGCT-3' (reverse). This PCR product was subsequently used as a template for in vitro transcription employing T7 and SP6 RNA polymerase to synthesize digoxigenin-labelled sense and anti-sense single-stranded RNA probes respectively using a DIG RNA Labeling Kit (Roche Diagnostics GmbH, Penzberg, Germany; Cat. 11 093 274 910). Two different cytological procedures were used; paraffin embedding and fresh tissue sectioned with a vibratome. For the paraffin procedure four to five week old 2HA calli from leaf explants were fixed in 4% formaldehyde in 0.025 M phosphate buffer at pH 7.2, dehydrated through an ethanol and ethanol: histolene (Fronine lab supplies) series, embedded in paraffin, sectioned (8 µM), and hybridized with the digoxigenin-labelled sense and anti-sense probes according to the manufacturer's instructions. For the fresh tissue procedure, the 2HA embryogenic tissue from leaf explants tissue was embedded in agar and 40 µM sections cut with a vibratome. In both cases hybridisation was detected using a Fluorescent Antibody Enhancer Set for DIG detection (Boehringer Cat. 176756) and was visualized as a red/purple color after the NBT/BCIP color reaction (Roche Diagnostics). In all cases, no signal over background was observed using control sense-strand probes.

Construction of Constitutive and Inducible RNAi Plasmids
For MtSERF1 RNAi construction, specific sequences in the 3' end of MtSERF1 mRNA were selected for construction of RNAi fragments. A cDNA fragment of MtSERF1 was amplified by PCR with the primers, 5'-CTGTGAAATTGATGCTGCAAA-3' (forward) and 5'-TGACATAATTGTTGAGCTCACTCC-3' (reverse). The MtSERF1 specific PCR products were cloned into the vector pCR®8/GW/TOPO® (Invitrogen, Carlsbad, USA). After linearization of the plasmids, the Gateway LR recombination reaction (Invitrogen, Carlsbad, USA) was conducted according to the manufacturer's protocol to incorporate the MtSERF1 specific fragment into the binary T-DNA destination vector pH7GW1WG2(II) (Karimi et al., 2002) and pOpOff2(hyg) (Wielopolska et al., 2005) for constitutive and inducible RNAi constructs, respectively. The resulting constructs were introduced into Agrobacterium tumefaciens strain AGL1 by electroporation.

Transformation of Medicago truncatula
Transformation of M. truncatula 2HA leaf explants was carried out as described by Wang et al. (1996) with some modifications. In brief, leaf pieces were prepared and sterilized according to the method described by Nolan et al (2003) and were dipped into bacterial solution, followed by co-cultivation for 2-5 days. After co-cultivation, the explants were decontaminated by dipping in a solution containing 750 mg/L Augmentin (5 parts amoxicillin/L part clavulanic acid; Beecham Laboratories, France) before plating onto solid media as described previously in the section on cultured leaf explants. Transformed calli were screened for Hygromycin resistance by including Hygromycin at 15 mg/L in the media. 500 mg/L Augmentin was also added in the media to eliminate the Agrobacterium. The explants were subcultured on fresh media every 4 weeks. RNAi contructs were induced by 2.5 uM dexamethasone.

Sequence Analysis and Construction of Phylogenetic Trees
Multiple alignment analyses were performed with ClustalW using a Clustal X 1.8 software package. Phylogenetic trees were constructed using the neighbour-joining method (Saitou and Nei, 1987) included in the Clustal X 1.8 software. Phylogenetic trees were drawn using TreeView (Win32) 1.6.0 software (Page, 1996).       treatments. This confirms that wild type Jemalong rarely produces embryos.