Spatially quantitative control of the number of cotyledons in a clonal population of somatic embryos of hybrid larch Larix x leptoeuropaea.

Abstract

Many conifer embryos, both in natural seeds and in clonal populations of somatic embryos, display variability in the number of cotyledons. In hybrid larch, Larix x leptoeuropaea (synonymous with L. x marschlinsii Coaz), such variability has previously been reported in somatic embryos, together with a decrease in the average cotyledon number when benzyladenine (BA) is applied exogenously. Described here is a spatially quantitative study with the aim of throwing some light on the way cotyledon number is determined, and hence the mechanism of cotyledon formation. Stock cultures of embryogenic tissue were maintained and later made embryogenically active by standard methods. Development through cotyledon formation was followed by optical microscopy with quantitative measurement of embryo diameter and number of cotyledons. SEMs of representative stages and cotyledon numbers were done for purposes of illustration in this account. Existing mathematics of waveforms on a disc were cast into a form suitable to compare with the quantitative data. The number of cotyledons is linearly related to the diameter of the apical surface of the embryo (which approximates a circular disc) at the time of first appearance of the cotyledon primordia. This linearity is a constant-spacing phenomenon between adjacent primordia. Addition of BA to the medium restricts the range of apical diameters without changing inter-cotyledon spacing. Slope/intercept ratio of the linear plot matches expectation for initiation of cotyledon pattern as a harmonic waveform on a circular disc. The entire pattern of cotyledon primordia arises as a single entity coordinated by a mechanism with wave-forming properties. This is explicable by diverse mechanisms, especially either mechanical buckling ('biophysical') or reaction-diffusion kinetics ('physicochemical').