In Vitro Haploid Production—A Fast and Reliable Approach for Crop Improvement - Crop Improvement Under Adverse Conditions - page 194

slight modifications. The pH of the buffer was adjusted to 7.5, filtered through
0.22 µm Polyvinylidene fluoride (PVDF) membrane filter and stored at 4 °C.
• The suspension containing the nuclei was mixed gently by pipetting up and
down, softly, several times, followed by filtering of the homogenate through a
30 µm nylon mesh.
• The nuclear suspension was stained with Propidium iodide at a concentration of
50 µg/ml.
• Simultaneously RNAse at a concentration of 50 µg/ml was also added to the
nuclei.
Ploidy analysis:
• The ploidy level was determined using a FACs Calibur cytometer (Becton-Dick-
inson, USA).
• All measurements were carried out in triplicate using fresh tea leaves as an ex-
ternal standard. Using instrument gain (photomultiplier voltage and amplitude
gain), the position of peak G 1 nuclei of the reference sample was established on
channel 200 on a 1,024 scale following which the instrument settings were kept
constant and the unknown samples were run under the same parameters. The
mean channel number of the unknown sample G 1 peak was determined and the
DNA Ploidy was calculated according to the relationship:
Sample Ploidy (integer) = Reference Ploidy X (mean position of the G 1 sample
peak/mean position of the G 1 reference peak)
6 Diploidization of Haploids
Doubled haploids are of immense importance for genetic studies and in crop breed-
ing programmes. Selected doubled haploid lines are used for production of com-
mercial hybrids (Chase 1974 ). To obtain fertile, homozygous diploids for analyzing
the progenies and the breeding behaviour of the pollen plants, the chromosome
complement of the haploids must be duplicated. This is because haploids may grow
normally up to the flowering stage, but in the absence of homologous chromo-
somes, meiosis is abnormal and consequently, viable gametes will not be formed
and, hence, they are sterile. Spontaneous duplication of chromosomes in pollen-
derived plants has been observed, but its frequency is very low.
In vegetable crops, doubled haploids are used prominently as parents for F 1
hybrid seed production. Similarly, in medicinal and aromatic plants, doubled
haploids for F 1 production have the potential to make significant advances in
providing high, stable and predictive yields of raw biochemicals to be processed
by pharmaceutical and neutraceutical industries (Ferrie 2007 ). In case of cross
pollinating species and hybrids, pure homozygous lines are highly desirable. Al-
though conventional breeding method to produce homozygous diploids is well