In Vitro Haploid Production—A Fast and Reliable Approach for Crop Improvement - Crop Improvement Under Adverse Conditions - page 195

established, it requires 7-8 repeated inbreeding cycles which in turn is time-con-
suming and labour intensive. Moreover, this approach is difficult in self-incom-
patible male sterile plants and tree species with long gestation period. Also, the
pure lines obtained after several generations of self-pollination may not be 100 %
homozygous (Germanà 2006 ). Production of fertile doubled haploid lines in S.
cereale (Immonen and Anttila 1996 ) and Festuca and Lolium (Nitzche 1970 ) are
of immense importance as these species suffer from inbreeding depression. One
of the most efficient techniques for production of homozygous diploid plants in a
single generation is through diploidization of the haploids. Doubled haploids have
a key feature in establishing chromosome maps in a number of species, notably
barley Hordeum vulgare , Oryza sativa, Brassica napus, Triticum aestivum (For-
ster and Thomas 2005 ), apart from providing a vast majority of mapped genetic
markers. Molecular marker maps provide a platform for trait mapping, which is of
particular interest to plant breeders. Doubled haploidy is also useful in rapid isola-
tion and purification of selected mutants in subsequent generations. It is possible
to create a population of homozygous mutant lines directly by targeting the muta-
tion treatment at single gametic cells and then inducing embryogenesis, followed
by subsequent chromosome doubling for homozygous diploid plant production
(Szarejko and Forster 2006 ).
Doubled haploidy can be significantly enhanced by artificial means using
chemicals such as colchicine, pronamide, trifluralin, oryzalin and amiprophos
methyl (APM) (Wan et al. 1991 ). Colchicine treatment is one of the most pre-
ferred techniques for chromosome doubling, which in turn is one of the most
critical steps in the doubled haploid breeding process. For Nicotiana tabacum,
a 0.4 % solution of colchicine is recommended to diploidize the pollen plants.
In practice, the young pollen derived plantlets are immersed in a filter sterilized
solution of colchicine for about 96 h and then transferred to a culture medium to
allow their further growth. Alternatively, the treatment is given in the form of a
lanolin paste (Bhojwani and Razdan 1996 ). It is applied to the axils of the upper
leaves and the main axis is decapitated to stimulate the axillary buds to grow
into diploid and fertile branches. Besides bringing about chromosome duplica-
tion, colchicine treatment may also result in chromosome and gene instabilities
(Burk 1970 ). Therefore, the frequent occurrence of spontaneous duplication of
chromosomes in differentiated plant cells like cortex and pith and callus cells
in long-term cultures has been exploited to raise homozygous, fertile diploids
from haploid plants. In Apiaceae, for diploidization the plantlets grown in Petri
dishes were taken out and the agar was removed from the roots. Subsequently,
the roots were submerged in a 0.34 % (w/v) solution of colchicine for 1½ h. The
roots were rinsed in water and the plantlets were transferred to a soil-less mix and
grown in the greenhouse (Ferrie et al. 2005 ). For Brassica species, the roots of
25-30 pollen-derived plantswere immersed in a bunch in 0.25 % (w/v) colchicine
solution for 5 h in light, for diploidization. After rinsing the treated roots with dis-
tilled water, the plants were transferred to a potting mix for hardening and further
growth (Bhojwani and Dantu 2010 ).