An SEM study of embryogenesis and seedling development in ABPHYL plants
--David Jackson and Sarah Hake

All grasses are characterized by a simple two-ranked or distichous phyllotaxy, where one leaf and corresponding lateral bud is initiated per node. However, there have been several reports of phyllotactic variants in maize (Greyson et al., Can. J. Bot. 56:1545-50, 1978, and references therein), some of which were heritable and were referred to as ABPHYL (for "aberrant phyllotaxy"). Plants from ABPHYL families showed a range of phyllotaxies including decussate, bijugate, spiral and distichous. We were recently provided with seed from families showing ABPHYL characteristics (a kind gift from M. Menzi, Swiss Federal Research Station). These families give rise to between 50 - 90% of plants having altered phyllotaxy, with the majority being decussate, i.e. having two leaves per node (and two ear shoots at ear nodes). These lines also give rise to a high proportion (up to 10%) of twin plants. The ABPHYL character behaves as a recessive and is penetrant in a number of inbred lines. In order to understand how organogenesis is occurring in ABPHYL plants, we undertook an SEM study of embryo development in ABPHYL and normal sibs. The material was generated by using decussate ABPHYL plants (which had two ears at one node) and pollinating one ear with pollen from an inbred (B73) and the other with pollen from a decussate sib. Developing kernels were sampled at different times after pollination and the embryos were dissected out, fixed in 4% glutaraldehyde in PBS and dehydrated, critical point dried, coated and viewed in the SEM using standard techniques. The seedling apex samples were prepared using the replica technique developed by Williams and Green (Protoplasma 147:77-79, 1988).

In normal embryos, the shoot apical meristem is first visible as a dome-shaped bulge on the face of the scutellum, which becomes surrounded by the coleoptilar ring (Fig. 1A). The first true leaf primordium is then initiated opposite to the scutellum (Fig. 1B). This pattern is reiterated such that leaves are always initiated opposite to the previous leaf, as seen in the seedling apex after removal of most of the leaves (Fig. 1C).

In ABPHYL plants of the same age, the initiation of the major embryonic structures is roughly synchronous with normal sibs. The most striking aspect of ABPHYL is that the newly initiated shoot apical meristem, and the surrounding coleoptilar ring, is much larger than normal (Fig. 1D). The first true leaves are initiated as a pair perpendicular to the normal leaf axis (Fig. 1E). Of many (>100) embryos from the ABPHYL sib cross which we dissected, all showed very similar early embryo development, leading us to suggest that a consistent defect in ABPHYL is the ability to control the size of the initiating shoot meristem, and this problem leads to a certain proportion of plants having decussate phyllotaxy. In decussate ABPHYL seedling apices, the two leaves in a pair are highly synchronized (Fig. 1F).

We are continuing to characterize ABPHYL by using molecular markers for organogenetic events in the shoot apical meristem (Jackson et al., Development 120:405-413, 1994), and are also setting up a clonal analysis to determine how the increase in meristem size compensates for the doubling in leaf initiation events per plastochron.

Fig. 1. SEM analysis of normal and ABPHYL embryos and seedling apices. A-C= normal, D-F=ABPHYL. A,D = coleoptilar stage, B,E = first leaf stage, C,F = 14 day seedling apex. S= scutellum, C= coleoptilar ring, M= shoot apical meristem, 1= first leaf (leaves), 2,3= plastochron 2, 3 leaves. Bar=50um.

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