Bruce G. Baldi

Dissolution of Pollen Intine and Release of Sporoplasts

Methods for preparation of plant protoplasts are well established but the extension of such methods to the release of exine-free gametophytes from pollen appears to be limited (Bajaj 1974; Bajaj and Davey 1974; Bhojwani and Cocking 1972; Power 1973; Takegami and Ito 1975; Zhu et al. 1984). We have discovered that 4-methylmorpholine N-oxide monohydrate (MMNO·H2O) is an effective solvent of the intine layer when pollen grains of Lilium longiflorum Thunb. (trumpet lily) are dispersed in MMNO·H2O at its melting point, 75°C (Loewus et al. 1985). Exine-free gametophytes, which we term sporoplasts, are quickly released from their exine enclosures. With time, MMNO also disperses the empty exine ‘shells’ into immiscible droplets. Prolonged heating ruptures the sporoplasts to produce empty sporoplast envelopes or ‘ghosts’ which remain intact in the MMNO·H2O melt.

Development of Genetic and Analytical Systems for Studies of Auxin Metabolism

Early investigations of auxin conjugates concerned the general “release” of auxin in vivo or in situ [e.g., 8]. More recent studies have examined either IAA released by hydrolysis of extracts of plant tissue, or have studied specific conjugates formed by the covalent attachment of IAA to other molecules. Conjugated forms of IAA can be classified by size, type of covalent linkage, or the molecule to which the IAA is attached. Low molecular weight conjugates include esters such as IAA-glucose and IAA-myo-inositol and amides such as IAA-aspartate and IAA-glutamate. The higher molecular weight conjugates include esters where the IAA is linked to the carbohydrate portion of a glycoprotein [17], or is linked to a glucan [18]. Higher molecular weight amide conjugates are also known, where IAA is linked directly to a peptide or protein [3]. These higher molecular weight conjugates have been difficult to study due to the lack of suitable methods for macromolecular separations and structure determination. Improvements in available methods for studies of macromolecules now make it practical to examine these types of compounds in more detail and begin to ask questions as to their role in the hormonal relationships within the plant.

Stable isotope techniques for the analysis of indole auxin metabolism in normal and mutant plants

Techniques for the analysis of indole-3-acetic acid (IAA) using stable isotope dilution are now well established. This basic technology, designed for the measurement of levels of phytohormone, can be extended to study the metabolic relationship of IAA to other indolic compounds and thus provide a more complete picture of hormone metabolism. We have developed methods for the analysis of the biosynthesis of IAA in plants and have applied these techniques to study IAA metabolism in normal and mutant plants as well as during embryogenesis, seed germination and root formation. The approaches we have recently used in our laboratory are: 1) To expand our analytical techniques to encompass additional indolic compounds and precursors. 2) To use D2O as a totally invasive label which can be incorporated into very early precursors in the aromatic biosynthetic pathway. Such experiments are able to answer the question “Is IAA being made de novo in this plant tissue?” 3) To use stable isotope labelled precursors to measure pool sizes and turnover of compounds important for the biosynthesis of tryptophan (Trp) and IAA, resulting in a quantitative approach to understanding carbon flow in these pathways. 4) To use stable isotope labelled compounds to track the interconversion of indolic compounds. 5) To develop techniques for the production and selection of mutant plants well suited for measurement of auxin metabolism in situ. In this paper we will discuss the methodology we use to study several plant systems which have been chosen to illustrate how to answer specific questions with regard to auxin metabolism. The plant systems we will discuss include carrot embryogenic cultures, seed germination in Phaseolus, precursor studies using normal and mutant Lemna and measurement of IAA and IBA levels in carrot transformed with Agrobacterium rhizo genes.