Production of Homozygous Diploid Plants
Haploids plants derived from either anther culture or pollen culture are sterile. These plants contain only one set of chromosomes. By doubling their chromosomes number, the plants can be made fertile and resultant plants will be homozygous diploid or isogenic diploid. These homozygous diploid plants show the normal meiotic separation. The fertile homozygous diploid plants are more important than the sterile haploid plants and can be used as pure line lines in breeding programme. Haploids plants can be diplodized by following methods.
i) Colchicine Treatment:
Colchicine has been utilized widely as spindle inhibitor to induce chromosome duplication and to produce polyploid plants. The young plantlets while still enclosed within the anther are treated with 0.5% colchicine solution for 24-48 hrs. Treated plantlets are planted in the medium after through washing. In case of mature haploid plantlets, 4% colchicine- lanoline pasts may be applied to the axil of the leaves.
ii) Endomitosis:
Haploids cells are unstable in culture and have tendency to undergo Endomitosis. i.e chromosome duplication without nuclear division. This property can be used for obtaining homozygous diploid plants. In this process, a small explant of stem from a haploid plant is cultured on auxin-cytokinin added medium where the segment forms the callus tissue. During callus growth, diploid homozygous cells are produced by endomitosis. Now large number of isogenic diploid plants can be obtained by organogenesis.
iii) Fusion of Pollen Nuclei:
Homozygous diploid callus or embryoids may form by the spontaneous fusion of two similar nuclei of the cultured pollen after first division. In Brassica, the frequency of spontaneous nuclear fusion in microspore is high in culture.