CrMSP1, a candidate gene involved in asexual reproduction in the fern Ceratopteris richardii

Linh T. Bui, Erin E. Irish, Chi-Lien Cheng

1. INTRODUCTION

All land plants complete the life cycle through an alternation between two different

generations: the haploid gametophyte and the diploid sporophyte. Sporophytes undergo meiosis to

produce haploid spores, which then develop into multicellular gametophytes. These haploid

gametophytes contain male and female gametes, which will fuse during fertilization to restore the

ploidy in the zygote, the first cell of the sporophyte generation. Both the spores and gametophytes

of angiosperms are ephemeral, embedded deep in the flower, and dependent on the sporophyte for

nutrients and support. In contrast, the two generations in ferns are free-living entities. Fern spores

once mature are shed from the sporophytes into the environment where, upon germination, will

develop into photosynthetic gametophytes, from which gametes are produced.

In addition to sexual reproduction pathway via meiosis and fertilization, approximately

400 species from 40 angiosperm families have evolved the ability to reproduce asexually

bypassing both meiosis and fertilization through multiple pathways collectively called apomixis

(Nogler, 1984; Carman, 1997). In ferns, some species can undergo asexual reproduction through

the two separate pathways: apogamy and aposopry (Scheme 1). In nature, approximately 10% of

fern species are obligatory apogamous; in these plants, sporophytes arise directly from

gametophytes lacking functional archegonia or antheridia (Steil, 1939; Bell, 1992). During

sporogenesis, a compensatory mechanism of forming restitution nuclei acts to give rise to

diplospores with the same chromosome numbers as the apogamous sporophytes (Walker, 1979).

Apospory can also occur in ferns, but rarely in nature (Walker, 1979). In apospory, diploid

Bui et al.

2

gametophytes are produced from somatic cells of sporophytes in the absence of meiosis. Both

processes can be induced in the laboratory with the sexual fern Ceratopteris richardii simply by

altering the level of sugar supplement in the growth media, and in the case of apogamy, by also

preventing fertilization (Cordle et al., 2007). Glucose at 2.5% (w/v) was optimal for the induction

of apogamy (Cordle et al., 2007) and medium with no sugar or 0.5% (w/v) glucose supplement

was used in apospory induction (Munroe and Sussex, 1969; De Young et al., 1997; Cordle et al.,

2011; Bui et al., 2012) in C. richardii.

Here, I use the model fern C. richardii to study how these two asexual reproduction

pathways are controlled. My long-term goal is to identify the key genes that play controlling roles

in determining the two fern asexual reproduction pathways, apogamy and apospory. The central

hypothesis for this research is that a group of conserved genes control sexual reproduction

in ferns and angiosperms, and an altered regulation of a subset of these genes will lead to

apogamy and apospory in C. richardii (Scheme2).

Currently, genes that control asexual reproduction in angiosperms have not been

identified, but seem to be restricted to one or a few dominant loci in the apomictic plant species

examined so far (Ozias-Akins and Van Dijk, 2007; Okada et al., 2011). Albertini et al. (2005)

proposed that specific genes are activated, modulated or silenced in the primary steps of sexual

reproduction to ensure the formation of functional embryo sac from meiotic spores or apomeiotic

cells. In contrast, genes involved in the development of both male and female gametophytes are

beginning to come to light, they include EXCESS MICROSPOROCYTES 1/ EXTRA

SPOROGENOUS CELLS (EMS1/EXS) and TAPETUM DETERMINANT (TPD1) genes which

function together as part of an intercellular signaling mechanism regulating sporogenic cell fate

(Jia et al., 2008); SPOROCYTELESS (SPL) and WUSCHEL (WUS) act on megasporemother cell

Bui et al.

3

differentiation (Sundaresan and Alandete-Saez, 2010); the DYAD/SWITCH1 (SWI1) genes are

required in the subsequent meiosis of the megasporemother cell (Mercier et al., 2001). There are

also genes that, when ectopically expressed will cause somatic embryogenesis from vegetative

tissues, similar to apomixis phenotypes, but it is still unknown if these genes actually involve in

apomixis. Among these genes, BABYBOOM (BBM) is a transcription factor that is expressed in

developing embryos and seeds (Boutilier et al., 2002); SOMATIC EMBRYOGENESIS

RECEPTOR KINASE 1 (SERK1) is expressed in nucellar tissue during meiosis, and in developing

embryo sac and zygotic embryo (Schmidt et al., 1997; Hecht et al., 2002). The LEAFY

COTYLEDON 1 (LEC1) regulates embryo development and is normally expressed in both

morphogenesis and maturation phases of embryogenesis in seeds (Braybrook and Harada, 2008).

Additionally, there are genes with recessive mutations have been identified, such as

FERTILIZATION INDEPENDENT SEED 1 and 2 (FIS1, FIS2), both are members of the PCR2

complex. These mutations cause the initiation of embryo development in the absence of

fertilization, hence comparable to asexual reproduction (Chaudhurry et al., 1997). Most

interestingly, a loss of function mutation in another gene member of the PCR2 complex, CURLY

LEAF (CLF), has been shown to cause apogamy in the moss P. patens (Okano et al., 2009),

suggesting that apogamy in moss and apomixis in angiosperms share genetic components, even

though these plants are evolutionarily distant (Cordle, University of Iowa PhD dissertation, 2011).

The rice MULTIPLE SPOROCYTE 1 (MSP1) was characterized by Nonomura et al.

(2003) as a master regulator that controls early sporogenic development. This gene encodes a

Leu-rich repeat receptor-like protein kinase (fig1. a), and loss of function mutation results in an

excessive number of both male and female sporocytes (Nonomura et al., 2003). In situ

hybridization showed that MSP1 is expressed in surrounding cells of both male and female

Bui et al.

4

sporocytes. The Arabidopsis homolog of this gene, EXS/EMS1, plays similar role in anther

development and also in embryo development (Zhao et al., 2002; Canales et al., 2002). It has

been shown in Arabidopsis that EXS/EMS1 acts together with another gene (TPD1) in restricting

surrounding cells undergoing meiosis and becoming the microsporocytes (Jia et al., 2008).

However, whether MSP1/ EXS/EMS1 is involved in asexual reproduction in angiosperms and

whether its homolog exists and plays a similar role in ferns and other lower plants are not yet

known. In this talk, I will focus on presenting the preliminary results on MSP1, one of the

candidate genes that I have cloned from C. richardii. Hereafter, the fern MSP1 will be called

CrMSP1. In situ hybridization and RT-PCR analysis showed that fern MSP1 expresses in both

sporogenesis and embryo development, similar to its homologs in Arabidopsis and rice.

Therefore, this gene is a promising candidate gene to study both sexual and asexual reproduction

pathways in C. richardii.

2. METHODOLOGY

Plant Material and Growth Condition

C. richardii plants used in these experiments were of the wild-type genotypes Rn3 and Hnn

(Carolina Biological Supply, Burlington, NC). Spore germination and gametophyte culture

conditions were as described by Cordle et al. (2007). Spores were inoculated at a density of 150-

300 spores per plate containing a basal medium (BM) that is half-strength Murashige and Skoog

salts (MS) at pH 6.0 with 0.8% agarose. Gametophyte culture plates were maintained at 28°C in

humidity domes under 16-h light/8-h dark cycle. Light source was provided with Philips Agro-

Lite fluorescent bulbs (Philips Lightning Company, Somerset, NJ) at 90-100 M m-2

s-1

.

Bui et al.

5

Degenerate primer design, cloning and sequencing

NCBI BLAST was used to find homologous sequences of EMS1/EXS in the land plant database.

Then, CODEHOP program (Rose et al., 1998) was used to design degenerated primers from

CLUSTALW multiple sequence alignment results. Gradient PCR was used to amplify fern

sequences using appropriate degenerate primers (Forward primer: GAGAGAACCTCTGTC-

TATTAATGTTGCNAYNTTYGA, Reverse primer: GTTCCAGCATAATAACTCCATAAGAATANACRTCNCC)

with the following program: 5min at 950C, (30sec at 95

0C, 30sec at 50-60

0C, 30sec at 72

0C) for 39

cycles, 5min at 720C. The PCR product was cloned into a TOPOII® vector (Invitrogen) and

transformed into TOP10 F’ E. Coli cells (Invitrogen). Minipreps were performed with a Qiaogen

Plasmid Kit and the resulting plasmids were sequenced with T7 promoter primers on an ABI 3730

DNA Analyzer (Applied Biosystems) at the Carver Center for Genomics (University of Iowa).

Synchronized Fertilization

Nine days after germination, plates with gametophytes were inverted before the gametophytes are

sexually mature to prevent fertilization. When the gametophytes are sexually mature (12d after

germination), water was added into the culture plates to facilitate fertilization and the

gametophytes were collected at various time points (see the result section), flash frozen in liquid

nitrogen then stored at -700C.

RNA extraction and RT-PCR analysis

Total RNA extraction was performed as described in Corlde et al., 2012. Briefly, frozen tissues

were ground to a fine powder and total RNA was extracted using the guanidinium thiocyanate and

acid phenol method. The RNA pellet was dissolved in fresh, deionized formamide, heated to 700C

for 10min in the presence of 0.7M NaCl and 1% CTAB, and then extracted twice with

Bui et al.

6

chloroform. The RNA pellet was dissolved in DEPC treated water and 500ng of total RNA was

used for first strand cDNA synthesis using Superscript III Reverse Transcriptase (Invitrogen) and

random hexamer primers. RT-PCR was performed with the following program: 5min at 950C,

(30sec at 950C, 30sec at 58

0C, 30sec at 72

0C) for 35 cycles, 5min at 72

0C.

RT-PCR quantification

RT-PCR quantification was done using ImageJ 1.43r software (http://rsb.info.nih.gov/ij/)

(Abramoff et al., 2004). Briefly, after calibration, gel lanes were selected and plots were created

for each gel lane. Plot peaks, which represent each distinct PCR band within a gel lane, were

specified and the area beneath each peak was calculated. For each gel lane, the ratio of control

(UBQ11) area to experimental (CrMSP1) area was calculated. Graphs were created using

Microsoft Excel for Windows 2007 (Microsoft Corporation).

In-situ hybridization analysis

Probe synthesis

Plasmids were linearized overnight with either BamHI (NEB) or XbaI (NEB). Gel electrophoresis

was performed to verify complete linearization. 1µg of purified, linearized plasmids were used in

probe synthesis with T7 or SP6 RNA polymerases (Stratagene) and DIG RNA labeling mix

(Roche) to generate antisense and sense RNA probes. RNA probes were precipitated with 3M

LiCl to remove enzymes and DNA, and then resuspended in DEPC-treated water, then quantified

with the NanoDrop 1000 (Thermo Scientific).

Whole mount in situ hybridization

C. richardii gametophytes were fixed under vacuum for 10min in ice cold formaldehyde, acetic

acid and alcohol (FAA), and then fixed overnight at 40C in fresh FAA, stored in 75% Ethanol at -

Bui et al.

7

200C. For prehybridization, gametophytes were moved through a 3-step ethanol series in PBS pH

7.5, 20mins for each wash, permeabilized in 0.2M HCl for 20mins, neutralized with 2x washes in

PBS, and treated with 0.1mM Proteinase K for 10min at room temperature. Proteinase K

digestion was stopped with washes in PBS and glycine , then gametophytes were post-fixed in 4%

paraformaldehyde for 20min at room temperature. After a 90min prehybridization at 550C in

hybridization solution (6x SSC, 3% SDS, 50% formamide, 0.1mg/ml tRNA), probe hybridization

was performed at 550C for 15-18 hours with 1ng/µl denatured probe. Post hybridization washes

were performed with 0.2x SSC and 2x SSC, then RNase digestion was performed with 10µg/ml

RNase A in 2x SSC at 370C for 30min. Gametophytes were then washed with SSC and Tris-

buffered saline (TBS pH 7.5), incubated for 2 hours in 1% blocking solution (Roche), then 2

hours in a 1/1000 dilution of anti-DIG AP fab fragments (Roche) in 1% blocking solution.

Detection was performed with 1x color substrate solution (NBT/BCIP, Roche) in detection buffer

(100mM Tris-base pH 9.5 and 150mM NaCl) overnight. Gametophytes were mounted in 50%

glycerol and photos were taken using a Leica DM LB microscope.

Section in-situ hybridization

Samples were fixed in ice-cold FAA at 40C overnight, then dehydrated through a series of

ethanol, and embedded in paraplast in molds. The paraplast blocks were mounted on the

microtome and sections of 6-12μm thick were made and placed on poly-L-lysine coated slides.

Sections can be stores at 40C for several months. For in situ hybridization, sections were

transferred to a xylene to remove the wax and rehydrated in a series of ethanol at room

temperature. Hybridization was performed on slides at 550C for 16-20 hours, with the same

hybridization buffer as in whole mount, and the appropriate sense and antisense probes. Post

hybridization and detection were done as described in whole mount in situ hybridization above.

Bui et al.

8

3. RESULTS AND DISCUSSION

CrMSP1 is the homolog of Arabidopsis EMS1

Since the C. richardii genome is not sequenced, candidate gene approach has been used to

identify genes of interests. Using this approach, I was able to clone the fern MSP1 gene,

specifically the kinase domain of this gene, with degenerate primers. NCBI BLAST showed that

the CrMSP1 sequence is 78% identical to Arabidopsis EXS/EMS1, with an Evalue = 1e-108 (fig1.b).

Phylogram created by CLUSTALW program shows the close relationship between the fern MSP1

and the moss Physcomitrella homolog, as well as the evolution of this gene among the plant

kingdom (fig1c). However, since the CrMSP1 I have cloned is not complete (lacking the 5’

sequence upstream of the kinase domain), a longer sequence will obtain a more precise

phylogram, and further confirm the identity of this fern MSP1.

CrMSP1 is expressed during sporogenesis

Using Genevestigator program (www.genevestigator.com), a web-based database that contains

annotated database of public Affymetrix microarray experiments, the Arabidopsis EMS1/EXS

expression was examined in different plant tissues. AtEMS1/EXS expresses highly in the

inflorescence, especially in the ovules where the embryo sac is (fig2). This gene is also present in

the stamen where the anther is produced, although at a much lower level than in the ovules. This

result is consistent with previous studies of EMS1/EXS in Arabidopsis, as it was shown to regulate

anther formation (Zhao et al., 2002; Canales et al., 2002).

Compare to the expression pattern of EMS1/EXS in Arabidopsis, CrMSP1 is significantly induced

in the tip of the leaves, where sporogenesis occurs (fig3.e). This result is interesting because

Bui et al.

9

unlike angiosperms whose sporogenesis (and gametogenesis) occurs inside the flower, ferns form

the spores at the underside of the mature leaves. Fern sporogenesis occurs at the young part (the

tip) of a mature fern leaf, but not in young leaves, or in the base of the leaves where sporogenesis

is over. Therefore, the RT-PCR result indicates that CrMSP1 is involved in sporogenesis.

Tissues were also embedded and sectioned for in situ hybridization analysis to examine the

temporal and spatial expression of CrMSP1. It is clear that CrMSP1 is expressed in the spore

mother cells in the developing sporangia (fig3. a-d) suggesting a special role in these cells. To

understand the role it plays, it is necessary to study the biological function of the CrMSP1. I will

do this by knocking down or overexpressing the gene and then examine the effects in

sporogenesis, gamete formation, and embryogenesis (explained below) in C. richardii.

CrMSP1 expression is induced in the embryo and gametophyte after fertilization

EMS1/EXS is expressed in early developing embryos and homozygous ems1/exs embryos are

lethal (Canales et al., 2002). These findings indicate that this gene is also required for embryo

development in Arabidopsis. However, it is unclear whether EMS1/EXS acts alone or with other

genes to regulate embryo development.

Using whole mount in situ hybridization analysis (developed by me and another graduate student

in the lab) for C. richardii gametophytes, I detected the expression of CrMSP1 before and after

fertilization. For this experiment, it is necessary to obtain embryos at the same developing stages.

To achieve, I synchronized the fertilization in a population of mature gametophytes (described in

the Methodology). CrMSP1 is highly induced not only in the embryo (fig.4d) but also in the

whole gametophyte (fig.4b) at 72h post fertilization, but not in the gametophyte (fig.4a) or the

archegonia (harboring the egg cell) (fig.4c) before fertilization, or when sense probe was used

(data not show). RT-PCR using RNA extracted from gametophytes at various time points after

Bui et al.

10

fertilization further confirms the in situ hybridization result. High level of CrMSP1 expression

was detected at 24h post fertilization but not before fertilization (fig5). These results are consistent

with the expression pattern of the Arabidopsis homolog, EMS1/EXS, and indicate a role of

CrMSP1 in fern embryo development.

Is CrMSP1 involved in asexual reproduction in C. richardii?

The central question of my research is how asexual reproduction in C. richardii is controlled. As

described above, the expression of CrMSP1 increases in both sporogenesis and embryo

development, and this expression pattern is consistent with the Arabidopsis and rice homologs.

Loss of function mutations in this gene causes abnormal gamete formation in Arabidopsis and

rice. The next obvious experiment is to alter the expression of CrMSP1 in C. richarii and see

whether sporogenesis and embryo development will be affected. In order to do this I have

developed an Agrobacterium-mediated transformation protocol for knock-down and

overexpression of CrMSP1 in C. richardii. In addition to abnormality in sexual reproduction, we

will also carefully examine an increased rate of apogamy (the asexual reproduction of embryo

without fertilization).

Bui et al.

11

4. FIGURES

Scheme 1. Alternation of generations and

asexual reproductions in the fern

Ceratopteris richardii

Scheme 2. Candidate genes. All genes are involved in

Arabidopsis sexual reproduction steps as indicated except

CLF, BBM (also LEC1) who are involved in somatic

embryogenesis. Green represents sporophyte and blue

gametophyte. Genes with asterisks have not been cloned

from C. richardii.

Fig 1. C. richardii MSP1 (CrMSP1) is the fern homolog of the Arabidopsis MSP1 gene. a) Structure of the

AtMSP1 protein with the three different domains: leucine-rich repeat, transmembrane and kinase domains. b)

Sequence alignment by NCBI BLAST program shows the similarity between CrMSP1 and AtMSP1 in the

kinase domain (Evalue = 1e-108). c) Phylogram of MSP1 in the plant kingdom created by CLUSTALW

a

) c

) b

)

Bui et al.

12

Fig 5. CrMSP1expression is significantly induced in the

gametophytes after fertilization. a) RT-PCR result using CrMSP1

or UBQ11 (positive control) primers for gametophytes at various

time points after fertilization. b) Quantification of three separate

RT-PCR replicates using ImageJ program, bars indicate standard

errors.

0 12 24 48 72 96

Time after fertilization (h)

Rel

ativ

e m

RN

A l

evel

Time after fertilization (h)

0 12 24 48 72 96

CrMSP1

UBQ11

a

b

Fig2. In-silico analysis of microarray

database from

www.genesvestigator.com shows the

expression of AtMSP1 in the female

gametophyte (red box), numbers on the

right indicate the numbers of

microarrays analyzed.

Fig 3. CrMSP1 is expressed during sporogenesis in C. richardii. a-d) fern

leaf cross section in situ hybridization with CrMSP1 antisense probes (a &

c), or sense probe (b & d); c & d are higher magnification of insert in a &

b, respectively; e) RT-PCR result using CrMSP1 or UBQ11 (positive

control) primers for different tissue types (young leaves, tip of the leaves

and base of the leaves). Bars

Fig 4. Whole-mount in situ of CrMSP1. 12-d-

old gametophyte before (a) and 72 h after (b)

fertilization. (c), (d) are archegonia in (a) and

(b). Red arrows point to archegonia, green

arrows point to the egg in (c) and developing

embryo in (d). Note lack of staining in

archegonia. Bar=0.5 mm in a,b. Bar=0.05 mm

in c,d.

Bui et al.

13

REFERENCES

1. Albertini E, Marconi G, Reale L, Barcaccia G, Porceddu A, Ferranti F and Falcinelli M (2005). SERK and APOSTART.

Candidate Genes for Apomixis in Poa pratensis Plant Physiol. August 2005 138: 2185-2199.

2. Bell PR (1992). Apospory and apogamy: implications for understanding the plant life cycle. Int J of Plant Sciences. 153:S123-

S136.

3. Boutilier K, Offringa R, Sharma VK, Ouellet T, Zhang L, Hattori J, Liu CM, Lammeren AAM, Miki BLA, Custers JBM and

Lookeren Campagne MM (2002). Ectopic Expression of BABY BOOM Triggers a Conversion from Vegetative to Embryonic

Growth Plant Cell 2002 14: 1737-1749.

4. Braybrook SA, Harada JJ. (2008) LECs go crazy in embryo development. Trends Plant Sci.13:624–630

5. Bui LT, Hurst A, Erish EE, Cheng CL (2012). The effects of sugars and ethylene on apospory and regeneration in Ceratopteris

richardii. AJPS.

6. Canales C, Bhatt AM, Scott R, Dickinson H (2002). EXS, a putative LRR receptor kinase, regulates male germline cell number

and tapetal identity and promotes seed development in Arabidopsis. Curr Biol.12(20):1718-27.

7. Carman, J. G. 1997. Asynchronous expression of duplicate genes in angiosperms may cause apomixis, bispory, tetraspory, and

polyembryony. Biol. J. Linn. Soc.61: 51–94.

8. Chaudhury, AM, Ming L, Miller C, Craig S, Dennis ES and Peacock, WJ (1997). Fertilization-independent seed development in

Arabidopsis thaliana. PNAS. 94:4223-4228.

9. Cordle AR, Erish EE, Cheng CL (2007). Apogamy induction in Ceratopteris richardii. International Journal of Plant Sciences.

168:361-369.

10. Cordle AR, Bui LT, Erish EE, Cheng CL (2011). Laboratory-Induced Apogamy and Apospory in Ceratopteris richardii.

Springer New York. 25-36.

11. DeYoung, B., Weber, T., Hass., T. and Banks, J. A. (1997). Generating autotetraploid sporophytes and their use in analyzing

mutations affecting gametophyte development in the fern Ceratopteris. Genetics. 147(2): 809–814.

12. Hecht V, Vielle-Calzada JP, Hartog MV, Schmidt EDL, Boutilier K, Grossniklaus U, De Vries SC (2001). The Arabidopsis

SOMATIC EMBRYOGENESIS RECEPTOR KINASE 1 gene is expressed in developing ovules and embryos and enhances

embryogenic competence in culture. Plant Physio. 127:803-816.

13. Jia G, Liu X, Owen HA, Zhao D.(2008). Signaling of cell fate determination by the TPD1 small protein and EMS1 receptor

kinase. PNAS. 105(6):2220-5. doi: 10.1073/pnas.0708795105.

14. Lotan T, Ohto MA, Yee KM, West MAL, Lo R, Kwong RW, Yamagishi K, Fischer RL, Goldberg RB, Harada JJ (1998).

LEAFY COTYLEDON 1 is sufficient to induce embryo development in vegetative cells. Cell. 93:1195-1205.

15. Mayer KF, Schoof H, Haecker A, Lenhard M, Jurgens G, Laux T (1998). Role of WUSCHEL in regulating stem cell fate in the

Arabidopsis shoot meristem. Cell. 95:805-815.

16. Mercier, R., Vezon, D., Bullier, E., Motamayor, J. C., Sellier, A., Lefevre,F., Pelletier, G. and Horlow, C. (2001). SWITCH1

(SWI1): a novel protein required for the establishment of sister chromatid cohesion and for bivalent formation at meiosis. Genes

Dev. 15, 1859-1871.

17. Munroe, M. H. and Sussex, I. M. (1969). Gametophyte formation in bracken fern root cultures. Can. J. Botany. 47: 617–621.

18. Nogler, G.A. (1984). Gametophytic apomixis. in Embryology of Angiosperms, B.M. Johri, ed (Berlin: Springer-Verlag). 475–

518.

19. Nonomura K, Miyoshi K, Eiguchi M, Suzuki T, Miyao A, Hirochika H and Kurata N (2003). The MSP1 Gene Is Necessary to

Restrict the Number of Cells Entering into Male and Female Sporogenesis and to Initiate Anther Wall Formation in Rice Plant

Cell. 15: 1728-1739.

20. Okada T, Ito K, Johnson SD et al. (2011) Chromosomes carrying meiotic avoidance loci in three apomictic eudicot Hieracium

subgenus Pilosella species share structural features with two monocot apomicts. Plant Physiology 157: 1327–1341.

21.

17. Okano Y, Aono N, Hiwatashi Y, Murata T, Nishiyama T, Ishkawa T, Kubo M, Hasebe M (2009). A polycomb repressive

complex 2 gene regulates apogamy and gives evolutionary insights into early land plant evolution. PNAS. 106:16321-16326.

18. Ozias-Akins P, Dijk van PJ (2007). Mendelian genetics of apomixis in plants. Annual Review of Genetics. 41:509-537.

19. Schmidt EDL, Guzzo F, Toonen MAJ, de Vries SC. (1997). A leucine-rich repeat containing receptor-like kinase marks somatic

plant cells competent to form embryos. Development.124:2049–2062

20. Steil WN (1939). Apogamy, apospory, and parthenogenesis in the pteridophytes. Botanical Review. 5:433-453.

21. Sundaresan V, Alandete-Saez M.(2010). Pattern formation in miniature: the female gametophyte of flowering

plants.Development. 2010 Jan;137(2):179-89. doi: 10.1242/dev.030346.

22. Walker, T. G. (1979). The cytogenetics of ferns. In the Experimental Biology of Ferns, ed. A. F. Dyer. Academic Press. 87–123.

23. Zhao DZ, Wang GF, Speal B, Ma H. (2002). The excess microsporocytes1 gene encodes a putative leucine-rich repeat receptor

protein kinase that controls somatic and reproductive cell fates in the Arabidopsis anther.Genes Dev. 16(15):2021-31.