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5. ORIGIN OF SEEDLING ROOT FLUORESCENCE
IN L. MULTIFLORUM AND L. PERENNE
5.1. INTRODUCTION
Different uses of L. multiflorum and L. perenne have significant effect on selection criteria
and result in phenotypic differences between their cultivars. L. multiflorum cultivars are
primarily used for forage purposes while the majority of L. perenne is sold to turfgrass market.
Therefore, Italian ryegrass cultivars often exhibit rapid vertical growth and lighter leaf colour
than perennial ryegrass. The latter when used for permanent turf and lawns grows slowly,
has dark and narrow leaves. If individuals of L. multiflorum get into L. perenne it results in
undesirable turf quality. Hence, the main goal of breeders is to protect from contamination of
one species by another. The overlapping range of morphological variation (Chapter 3) makes
difficult separation of Italian ryegrass cultivars from perennial ones on easily scored characters
especially in younger stages of development. The difficulties in species differentiation
create a serious problem for breeders and seed certification agencies that remains to be
solved. Methods to distinguish between annual and perennial types have long been sought.
Seedling root fluorescence as a phenotypic marker has been used to separate L. multiflorum
from L. perenne since it discovery in 1929 by Gentner. Generally, seedling roots of annual
ryegrass fluoresce when placed under ultraviolet light, and those of perennial ryegrass
do not fluoresce (Niemyski and Budzyńska 1972; Barker and Warnke 1999). This brilliant
blue glow of annual ryegrass roots is caused by an oxazole alkaloid which is leaked from the
roots of developing seedlings onto a white substrate during germination. For the first time,
the alkaloid was isolated from L. multiflorum roots in 1958 and named annuloline for “annual
Lolium”. The tentative empirical formula is C 20
H 19
NO 4
and there are three methoxyl groups
(Figure 5.1). It is a week base, sparingly soluble in water and strongly fluorescent. As little
as 0.01 µg of the alkaloid may be detected, when applied to a 2-mm diameter area on Whatman
N o 1 (Axelrod and Belzile 1958). A precursor of annuloline (Figure 5.1) is derived from
ß-hydroxyphenylethylamine and a substituted cinnamic acid (Hardwick and Axelrod 1969).
Seedling root fluorescence has been used widely as a supplement to morphological data but
also, in the USA and Canada - as the only accepted method for detecting the presence of
annual ryegrass in seed lots of perennial ryegrass (Niemyski and Budzyńska 1980). In 1953
the test was officially approved and adopted by the International Seed Testing Association
(Jones 1983). In 1990, the Federal Seed Act rules for testing seeds allowed the seedling
fluorescence test to be used as a cultivar descriptor in ryegrasses in the USA (Barker and
Warnke 1999).