Embryology of Onobrychis persica Sirj. and Rech.f. (Fabaceae) and its systematic implications

Figures & data

Figures 1-15. Longitudinal sections showing embryo sac development under bright field and fluorescence microscopes and different magnifications. 1: Young ovary containing a young ovule (right) showing dyad stage (right and left) (black arrowhead). 2: Two different magnifications of a T-shaped tetrad (black arrowheads). The outer integument (oi) grows faster than inner one (ii). 3: Three nuclei of four-nucleated embryo sac (black arrowheads). 4: Anatropous, crassinucellar and bitegmic ovules with nucellus and provascular bundle (PVB). The cone-shaped embryo sac with egg apparatus, two polar nuclei, antipodal cells and zigzag micropyle. 5: One of the synergids (SY) and oospher with its polarity are visible under bright field (black arrowheads) and fluorescence microscopes. 6: Synergids (black arrowheads), egg cell (EC), two polar nuclei before fusion and degeneration of micropylar end is visible. 7: Two polar nuclei and their finger-like tubers. 8: Egg apparatus and secondary nucleus (black arrowhead) formation after fusion of polar nuclei under bright field (left) and fluorescence (right) microscopes. 9: Mature embryo sac with differentiated egg (white arrowhead) and its polarity. 10: Sagittal section through the embryo sac under bright field and fluorescence microscopes with two different magnifications. Nucellar cells are degraded in the middle of embryo sac but persistent adjacent to the chalazal areae. Endothelium layer (En) is distinguishable at the inner integument. 11: Longitudinal section of zygote (Z) and synergids. 12: The primary endosperm nucleus (PEN), apical and basal cells (black arrowheads). 13: Basal cell and 2-celled proembryo (black arrowheads). 14: The U-shaped embryo sac and proembryo (Pe) under bright field (right) and fluorescence microscopes (left). The ovary with trichomes (in different magnifications; black arrowheads), long style (black arrow). 15: Globular embryo (Em) with young suspensor (S) surrounded by coenocytic endosperm. Staining with lugol indicates accumulation of starch grains at the base of embryo (black arrow).

Figures 16-25. Different developmental stages of seed and embryo in Onobrychis persica under bright field and fluorescence microscopes with different magnifications. 16: Early heart-shaped embryo (EM) with suspensor (S) consisting of one column of cells. 17: The heart-shaped embryo (EM) and suspensor (S). 18: Onobrychis mature legume contained just one seed but the legumes with two seeds were also observed. 19: Longitudinal section of seed stained with methylene blue shows the distinguishable layers in seed. 20: Transverse section of seed stained with Sudan black B and polarizing microscopy shows lipid reserves in cuticle layer (C) and endosperm (EN) specially in aleurone layer (black arrowhead). 21: Staining of seed coat and endosperm with toluidine blue indicates starch grains (black arrowheads); positive reaction for phenolic compounds gives a green color. Starch grains are absent in macrosclereids (MS) but there are high accumulation of starch grains in osteosclereids (OS) and parenchyma cells (P). Cuticle layer (C) is thick. 22: Staining with lugol indicates starch grains (black arrows). Storage of starch grains during embryogenesis is visible in the seeds. The tracheid bar is distinguishable (white arrowhead). 23: At the hilar pole, double palisade layer (white arrowheads), subhilar parenchyma, and tracheid bar is distinguishable. The aleurone layer is also noticeable (black arrowhead) in the larger figure. 24: A part of testa showing macrosclereids (MS), osteosclereids (OS) and parenchyma cells (P), and accumulation of starch grains. 25: Cotyledon (CO) stage which accumulate starch. In this stage suspensor shows signs of degeneration (black arrow). Note: In Figures 19–25 seed sections were stained with different staining techniques and different microscopes were used (bright field, polarizing and fluorescence).

Figures 26-33. Anther structure under bright field and fluorescence microscopes with different magnifications. 26: Microspore mother cells surrounding by a callose coat (white arrow) in a young anther with undifferentiated wall (black arrow). The vascular bundles are distinguishable (white arrowhead). 27: Microspore mother cells are surrounded by a callose coat (black arrow). A big nucleus is visible at the center. 28: The arrangement of tetrads inside the callose walls is tetrahedral (white arrow) and tetragonal (white arrowhead). 29: Released microspores in polar and equatorial view (white arrowheads). The tapetal cells are uni-nucleate (white arrow). 30: Microspores undergoes mitotic division and produces a vegetative cell (black arrowhead) and a generative cell (black arrow). The anther wall consists of four layers: epidermis (E), endothecium (EN), the middle layer (ML) and a secretory tapetum (T). 31: Fibrous thickenings are developed in the endothecium (white arrowhead). 32: The anther is tetrasporangiate with thick exine schahuensis are T-shaped microspores (black arrow). The anther wall layers are distinguishable with fluorescence microscope (white arrow). 33: Two-celled mature pollen with generative and vegetative nuclei (black arrowheads) in a mature anther.

Figures 34-37. 34: Degeneration of micropylar end during fertilization (white arrow). Pollen tube growth with fluorescence microscopy and aniline blue staining: 35: Pollen tubes stained with aniline blue (white arrowheads) shows growing and branching on the tube in the space of between integuments. 36: Longitudinal section of the pistil showing pollen tube growth (white arrowheads) through the short open stylar canal that leads to an ovule. 37: Pollen tube growing through the micropyle (white arrowheads).

Table 1. Embryological comparison among five species of Onobrychis and Fabaceae.

Features	O. persica	O. altissima	O. melanotricha	O. andalanica	O. schahuensis	Fabaceae	References
Anther wall formation	Dicotyledonous	Dicotyledonous	Dicotyledonous	Dicotyledonous	Dicotyledonous	Dicotyledonous and basic	Teixeira et al. 2002; Liu et al. 2003; Rezanejad 2007
Middle layer	1	1	1	1	1	variable	Teixeira et al. 2002; Liu et al. 2003; Rezanejad 2007
Tapetum	Secretory	Secretory	Secretory	Secretory	Secretory	Often secretory	Suzuki et al. 2001; Wilson 2001; Teixeira et al. 2002; Liu et al. 2003; Feng et al. 2006; Rezanejad 2007; Chehregani et al. 2008; Hashemi and Rezanejad 2013
Number of tapetal cell nuclei	1	1	1	1	1-2	Often 1	Suzuki et al. 2001; Teixeira et al. 2002; Liu et al. 2003; Galati et al. 2006; Rezanejad 2007
Cytokinesis in meiosis	Simultaneous and successive	Simultaneous and successive	Simultaneous and successive	Simultaneous and successive	Simultaneous and successive	Often successive	Suzuki et al. 2001; Wilson 2001; Teixeira et al. 2002; Liu et al. 2003; Feng et al. 2006; Rezanejad 2007; Chehregani et al. 2008; Hashemi and Rezanejad 2013
Microspore tetrads	Tetragonal and tetrahedral	Tetragonal and tetrahedral	Tetragonal and tetrahedral	Tetragonal and tetrahedral	Tetragonal and tetrahedral	Often tetrahedral	Suzuki et al. 2001; Wilson 2001; Teixeira et al. 2002; Liu et al. 2003;Galati et al. 2006; Rezanejad 2007
Type of aperture	Tricolpate	Tricolpate	Tricolpate	Tricolpate	Tricolpate	Often tricolpate	Pavlova and Manova 2000; Liu et al. 2003
Mature pollen	Two-celled	Two celled	Two celled	Two celled	Two celled	Variable	Liu et al. 2003; Galati et al. 2006
Ovule number	1–2	1	1	1	1–2	Variable	Hashemi and Rezanejad 2013
Curvature of ovules	Hemianatropous and anatropous	Anatropous	Anatropous	Anatropous	Anatropous	Hemianatropous and anatropous	Moço and Mariath 2003; Faigo´n-Soverna et al. 2003; Galati et al. 2006; Rezanejad 2006; Riahi and Zarre 2009; ; Rodriguez-Pontes 2007
Nucellus type	Crassinucellate	Crassinucellate	Crassinucellate	Crassinucellate	Crassinucellate	Crassinucellate	Moco and Mariath 2003; Faigo´n-Soverna et al. 2003; Galati et al. 2006; Rezanejad 2006; Riahi and Zarre 2009; ; Rodriguez-Pontes 2007
Number of integument	Bitegmic	Bitegmic	Bitegmic	Bitegmic	Bitegmic	Bitegmic	Moco and Mariath 2003; Faigo´n-Soverna et al. 2003; Galati et al. 2006; Rezanejad 2006; Riahi and Zarre 2009; ; Rodriguez-Pontes 2007
Number of layers in inner integument	2	2	2	2	2	Often 2	Ashrafunnisa and Pullaiah 1999; Galati et al. 2006; Rodriguez-Pontes 2008
Number of layers in outer integument	4–5	4–5	3	3	4–5	Several layers	Galati et al. 2006; Rodriguez-Pontes 2008
Megaspore tetrads	T-shaped	T-shaped	T-shaped	T-shaped	Linear shaped	Linear shaped and T-shaped	Moco and Mariath 2003; Galati et al. 2006; Rodriguez-Pontes 2008
Embryo sac	Polygonum	Polygonum	Polygonum	Polygonum	Polygonum	Often polygonum	Faigo´n-Soverna et al. 2003; Moco and Mariath 2004; Galati et al. 2006; Rezanejad 2006; Rodriguez-Pontes 2007
Antipodal cells	Persistent	Persistent	Persistent	Persistent	Persistent	Variable	Johansson and Walles 1994
Seed number	1–2	1	1	1	1	Variable	Riahi and Zarre2009; Chehregani et al. 2011
Amyloplast accumulation in embryo sac	Too much	Too much	Too much	Much	Few	Variable	Chehregani et al. 2011
Ovary peduncle and hair	+	+	+	+	–	Variable	Chehregani et al. 2011
Selected references	Present study	Chehregani et al. 2011	Chehregani et al. 2011	Chehregani et al. 2011	Chehregani et al. 2011	*

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