Flax and Linseed - Industrial Crops: Breeding for BioEnergy and Bioproducts - page 245

new biotechnological methods are increasingly used resulting to the stabilization of
genotypes via the process of homozygosity - haploid, double haploid production, or
increasing genetic variability of genotypes using other DNA molecular methods
resulting in GMO flax/linseed development.
Using double haploids is a very effective method since completely homozygous
genotypes, non-segregating in any trait, are obtained in one generation. The oldest
method of haploid production is polyembryonic method [ 111 ]. Some genotypes are
able to produce double embryos in one seed, from which mostly one is diploid and
one is haploid, but mostly in a very low frequency. Moreover, induction of
polyembryos into breeding materials is very lengthy. There are several methods
to produce double haploids in flax/linseed which include microspore culture [ 112 ,
113 ], ovule culture [ 114 - 120 ], and anther culture [ 121 - 123 ]. The details of these
methods have been previously described by Pavelek et al. [ 59 ].
In order to evaluate the level of genetic variability of genotypes, breeding lines,
or developed varieties, molecular methods play a very important role, and com-
pared to the traditional methods of evaluation based on morphological traits,
characterization is considered more effective and accurate. The abundance of
DNA markers, their environmental insensitivity, and non-tissue-specific character-
istic are some of their advantages. Markers are useful for varietal identification and
evaluation of genetic variation. Among the different marker systems include ran-
dom amplified polymorphic DNA (RAPD), restriction fragment length polymor-
phism (RFLP), amplified fragment length polymorphism (AFLP), or simple
sequence repeat (SSR). Previous applications of these methods in the crop have
been described [ 79 , 80 , 124 ].
To incorporate various genes into the flax/linseed genome, different genetic
transformation methods have been developed. Flax, like most dicotyledonous
crop species, is amenable to gene transfer via Agrobacterium [ 125 ]. Flax cells are
easily transformed with Agrobacterium tumefaciens , and these can be easily grown
but require an elaborate inoculation/selection/regeneration procedure [ 126 ]. The
success of these methods is very strongly influenced by the chosen part of the
plants. It has been reported that the hypocotyl is the most regenerable part of flax/
linseed plant [ 127 , 128 ]. To enhance transformation efficiency, an improved pro-
cedure for the production of flax plant was developed [ 129 ].
Next to Agrobacterium transformation, other methods are developed like parti-
cle bombardment. Wijayanto and McHughen [ 130 ] documented a successful
biolistic process for producing transgenic linseed flax. Successful plant regenera-
tion is closely influenced by the respective chosen selection medium (kanamycin,
hygromycin B, spectinomycin) [ 131 , 132 ] and protocols [ 30 ].
For monitoring gene expression in transgenic tissues, markers such as

β -galactosidase (LazC) are used.

The GUS assay is the most useful assay in flax transformation [ 129 ] but destructive.
As an alternative, green fluorescent protein (GFP) is used as a visual marker during
the establishment, evaluation, and improvement of transformation procedures for
flax plants. GFP allows nondestructive evaluation and enables plant growth and
development without damage to transgenic tissues [ 133 ].

-glucuronidase (GUS), luciferase (LUC), or β