Detection of Somaclonal Variation in Aquatic Plant Pearl Grass (Hemianthus micranthemoides)

Hemianthus micranthemoides is an ornamental plan used for foreground plant in aquarium decorations. In Malaysia, the plant available on market is regenerated through tissue culture techniques which expose the risk for somaclonal variation.  The objectives of this study are to identify suitable Random Amplified Polymorphic DNA (RAPD) markers for determining somaclonal variation rate in micropropagated plantlets of H. micranthemoides and to observe the effects of somaclonal variation on the plantlet growth (area of spreadability, shoot and root length). Out of 20 RAPD primers screened by polymerase chain reaction (PCR) using mother plant, one was selected as a suitable marker to detect somaclonal variation of H. micranthemoides which was P15. Somaclonal variation rate of the H. micranthemoides was determined by analyzing the RAPD-PCR bands. The results showed somaclonal variation was observed at the fifth subculture (S5) with the percentage of polymorphic bands of 50 % when compared to mother plant. Repeated subclutures has affected the growth of the regenerates negatively. It was shown that as the number of subculture increased, the growth of plantlet decreased in terms of decreasing shoot and root length as well as area of spreadability. This findings serves as a guide especially for tissue culturist to minimize subculture numbers when micropropagating H. micranthemoides

In Vitro Micropropagation of Aquarium Plants Pearl Grass Hemianthus micranthemoides (Nuttall) and Micro Sword Grass Lilaeopsis brasiliensis (Glaziou) Affolter (Apiaceae)

Hemianthus micranthemoides and Lileaopsis brasiliensis are nowadays sold in the form of tissue culture production by aquaria and ornamental trade in Malaysia. However, there is no to limited report about in vitro micropropagation of both aquarium plants. In this study, an experimental procedure for micropropagation of both plants was documented. The propagation were done in Murashige and Skoog (MS) medium with eight combination of different concentration of 6-Benzylaminopurine with Naphteneacetic acid (BAP-NAA) and 1-phenyl-3-1,2,3- thiadizol-5-yl urea with Naphteneacetic acid (TDZ-NAA) following the rooting technique for 42 days. All treatments for 1 L MS medium were regulated to pH 5.7 to 5.8 and added with sucrose (30 g/L) and phytagel (2.5 g/L). All combination treatments had induced rooting. There was a significance different in the shoot regeneration of H. micranthemoides in all treatment (95% confidence level, F = 13.907, n = 240, p = 0.000), on the contrary there was no different were observed in shoot regeneration of L. brasiliensis treatment. Overall, all treatment of TDZ- NAA were significantly higher in the shoot regeneration than those in BAP-NAA treatment. Particularly the T7 (TDZ-NAA, 0.075: 0.1 mg/L) given highest result in diameter (1.626±0.213 cm) and area (1.724±0.308 cm2) of shoot clumps in H. micranthemoides, and formation of shoots (51.5±4.95), nodes (20.0±2.83) and roots (45.5±13.4) in L. brasiliensis. Hardening of in vitro cultured plantlets was done in aquatic laboratory. All of the plants survived and transferred into aquarium. This study has provided beneficial information in further micropropagation protocol for the mass production of both aquarium plants.   Keywords: Aquarium plants, Hemianthus micranthemoides, Lilaeopsis brasiliensis, Micropropagatio

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. 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

Assessment of Bacopa caroliniana to sequester landfill heavy metals contaminant via in vitro hybrid model system

Most of developing countries used to develop various technologies to decrease the percentage of heavy metals contaminant from landfill site such as incineration, solvent extraction, soil washing by using physical separation methods or chemical agents, solidification or stabilization technology, indirect thermal treatment, soil venting, membrane technology and industrial water treatment. However, these approaches are expensive, difficult to apply in huge scale area and also may cause great environmental impact as it affects soil structure and fertility and biological activity. Therefore, one of the effective approach to control this matter is by using natural treatment. Phytoremediation is one of the natural ways for landfill treatment by using plants and is proven to be an economically competitive and eco-friendly to the environment as it does not require additional energy or substantial material just a small mechanical apparatus to operate it. Therefore, the study aimed to evaluate heavy metals sequestration rate of Bacopa caroliniana at three different concentrations ranging from low, medium and high (0.5 mol, 1.0 mol and 2.0 mol) of hybrids heavy metals from Al, Fe, Mn, and Mg at four different period of time (week 1 until week 4) through in vitro model system. The results showed that there were significant differences between the sequestration rates of different heavy metal hybrids and B. caroliniana in removing the contaminants. Thus, this species is strongly supported the phytoremediation approach in remediating the landfill heavy metals contaminant

The capability of Glossostigma elatinoides to accumulate Al, Mn, Fe and Mg from contaminated landfill leachate through in vitro landscape ecological model system

The growth of solid waste generation in Malaysia from year 2005 to 2013 statistically has risen from 19,000 tonne to 33,000 tonne per day. This rapid increase mainly affected the number of landfill which is the final disposal destination of solid waste. Landfill is very crucial site which need to be managed properly or it can be hazardous to the living things and also a silent killer. Thus, the landfill treatment to prevent water and soil pollution is widely renowned. Phytoremediation is one of the environmental friendly and economically approaches which using plants to treat soil contamination. Aquatic plants are known to have capability in sequestering heavy metals in phytoremediation. This is because it contains variety in species, high multiplication rate, global distribution and faster in growth. Therefore, this research aimed to assess heavy metals sequestration rate of Glossostigma elatinoides at three different concentrations ranging from low, medium and high (0.5 mol, 1.0 mol and 2.0 mol) of four types of heavy metal (Al, Mn, Fe, Mg) at four different periods of time (week 1 until week 4) through in vitro landscape ecological model system. The results established that there were significant differences between the sequestration rates of the plant, type of heavy metals, level of concentration, period of time and their interactions. This aquatic plant species has capability and resistance over 4 types of heavy metal toxicity. Hence, the high correlation between metal removal in soil and this aquatic plant species indicates that this species can be used effectively for the removal of heavy metals from polluted or contaminated landfill soil of different concentrations