Induced mutation of in vitro aquatic plant, Cryptocoryne xwillisii reitz by using gamma irradiation and IRAP analyses to distinguish the sports (clonal mutation) / Norhanizan Sahidin
One part of ornamental fish industry is the aquatic plants. Ornamental fish industry is identifying as one of National Key Economic Area (NKEA) for Malaysia, under business opportunity. Cryptocoryne xwillisii Engler ex Baum is one of the highly demanded aquatic plants in international market. Unf...
Saved in:
| Summary: | One part of ornamental fish industry is the aquatic plants. Ornamental fish industry
is identifying as one of National Key Economic Area (NKEA) for Malaysia, under
business opportunity. Cryptocoryne xwillisii Engler ex Baum is one of the highly
demanded aquatic plants in international market. Unfortunately, the plants take months to
grow, seldom flowering and no viable seeds. The studies were done to mass-produce the
plants by tissue culture and to develop new variety by mutation.
Tissue culture of water trumpet, Cryptocoryne xwillisii Engler ex Baum, was
developed using Murashige and Skoog 1962 (MS) medium, which contained two different
plant growth regulators, namely 6-benzyladenine purine (BAP) and α-naphthalene acetic
acid (NAA). Seven different concentrations of BAP (0, 0.5, 1.0, 5.0, 10.0, 20.0 and 40.0
μM) and NAA (0, 0.5, 1.0, 5.0, 10.0, 20.0 and 40.0 μM) were investigated using a twofactor
factorial design with 10 replicates. Results of the experiments were collected and
analysed after 40 days of culture.
The results showed that the effects of plant growth regulators on increasing the
number of shoots were highly significant (p<0.01). The MS medium containing 1.0 μM
BAP alone was the optimum concentration producing 6.8 ± 1.75 shoots per explant. This
was the minimum formula concentration of BAP used that produced the highest number of
shoots. Results showed that there were twelve hormone combinations giving high mean
values of between 4 to 6 shoots per explant. All of the explants cultured in these media
produced shoot (100 %).
Although all the explants gave a positive response in terms of regeneration, they
however differed in the number and size of shoots produced. Analysis of the data using ANOVA indicated that the number of shoots produced was significantly influenced by
both BAP and NAA concentrations simultaneously. This was suggested by the significance
of the interactions between BAP and NAA which showed that BAP concentrations
affected the number of shoots differently for each concentrations of NAA tested and vice
versa.
Two new varieties of C. xwillisii have been developed through mutagenesis in this
work. Shoot tips explants of C. xwillisii were subjected to a range of 60Co gamma ray
irradiation of 0, 100, 200, 300, 400, 500, 600, 700 and 800 Gray. Results from experiments
showed that the LD50 for the tissue culture plants of C. xwillisii was at 32 Gy dose. And
was therefore, less than 32 Gray was used as the appropriate dosage for induced mutations
of the plants.
About two thousand of the shoot tips explants were irradiated using the 25 Gy and
variants from the M1, M2, M3 and M4 generations were screened for morphological
differences. The variants shoots were subcultured repeatedly until the 4th generation (M4)
to ensure stability of mutants. Although initially many regenerants with different
morphological traits were produced, only two mutants were shown to remain stable. The
mutants obtained were dwarf plants (D1) and plants of taller stature with pigmented leaves
(G1) than the controls. This was verified from the significant value of the F test in the
ANOVA where P<0.05.
The Inter-Retrotransposon Amplified Polymorphism (IRAP) markers distinguished
the D1 and G1 genomes from normal C. xwillisii genomes. The analysis revealed two
specific bands 325 bp and 420 bp using Nikita primer for the D1 mutant and 240 bp and
300 bp using combination of 3’LTR primer and LTR 6149 primer for the G1 mutant. |
|---|