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Induction of variations in certain tropical ornamental plants and selection of variants for large scale cultivation
Author
Majumdar, Kakali
Supervisor
Lee, Sing-Kong
Abstract
This study was undertaken to introduce morphological variations in Fagraea fragrans Roxb. (a way-side tree), Heliconia psittacorum cv. 'Golden Torch' (a landscape herb) and Peperomia magnoliaefolia (Jacq.) Diert. (a potted herb) through in vitro mutagenesis. These species are commonly grown in Singapore either for landscape beautification or for indoor displays at homes and offices. Introduction of any phenotypic variation in these plants would enhance their ornamental values.
A stock culture of each plant species under study was developed for in vitro mutagenesis experiments. Since there was no known protocol for tissue culture of P.magnoliaefolia, a regeneration protocol was developed through the culture of leaf blade and petiole explants on MS medium supplemented with both BAP and NAA. The optimum medium for shoot regeneration from petiole is the one supplemented with 5.2 M BAP and 2.6 M NAA and those from leaf blades 11 M BAP and 5.2 M NAA respectively. A combined application of cytokinin and auxin was essential to induce a high rate of normal shoot regeneration and growth on both petioles (50%) and leaf blades (47.6%). Shoot elongation and maturation was improved by providing regenerated shoots, excised from petiole surface with a brief flush of a high concentration of GA3 solution (280 M for 90 seconds).
A protocol for in vitro mutagenesis using two chemical mutagens namely N-methyl-N-nitrosourea (NMU) and ethyl methanesulfonate (EMS), was established for each plant species under study. The effects of different mutagens on in vitro survival and growth or regeneration of plant species were studied and compared with the control. A workable dose range and LD50 values were determined for each mutagen. Both mutagens had inhibitory effects on each plant species under study. These were very prominent from a threshold dose. The workable dose range of NMU established for F. fragrans, H. psittacorum and P. magnoliaefolia at 25o to 26oC were 1 to 10 mM (3 hours treatment), 0.5 to 5 mM (3 hours treatment) and 1 to 4 mM (1 hour treatment) respectively. Similarly, the dose range of EMS worked out for F. fragrans, H. psittacorum and P. magnoliaefolia at 25o to 26oC were 0.008 to 0.12M (3 hours treatment), 25 to 110mM (3 hours treatment) and 0.04 to 0.16M (1 hour treatment) respectively. It was observed that most of the aberrations were derived from dosages either close to or above LD50 values. The mutagen treated explants of F. fragrans and H. psittacorum were subcultured for at least 3 cycles on appropriate medium in order to allow regeneration and proliferation and to eliminate chimeras. In P. magnoliaefolia, adventitious shoots were regenerated on mutagen treated petioles and then allowed to multiply and develop fully so that the variants can be screened.
Mutants were identified through visual screening during in vitro culture and after transfer to soil, based on morphological changes that were absent among the control plants. Some of the interesting morphological variations observed were :
● A high frequency of variegated shoots (52.1% of total MV4 shoots screened) and albinos (17.2%) was derived from cultures of F. fragrans treated with 5 to 10mM NMU. Eight lines of mutants were obtained that consisted of variegated, albinos and light green shoots. These mutant lines differed from each other by leaf shape, size, variegation patterns, stem coloration and growth rate. The rooting ability of different mutants lines was tested. Lines 1, 2 and 3 hold great promise as commercial variants.
● A number of 'root variants' (23% of total plantlets) were derived from cultures of H. psittacorum initially treated with 1 to 3 mM NMU. The variants displayed about 86.2% increase in the number of roots per rooted plantlet over the control.
● One dwarf and one vigorously growing plant of P. magnoliaefolia were obtained from 1 and 4 mM NMU, respectively. They constituted about 1.9% of the total NMU treated shoots that survived after transfer to soil.
NMU induced a broad spectrum of aberrations in the three species (chlorophyll variations, changes in leaf shape, growth habit and rooting efficiency). Whereas, only chlorophyll variations were induced by different EMS treatments. Therefore, NMU appears to be more effective on the three species than EMS. A definite conclusion in this regard can only be drawn after long term field trial of the mutagen treated plants as variations can also be appeared at later stages of growth.
The effects of EMS and NMU on the rooting efficiency of the plant species were studied in terms of percentage of rooted plantlets and mean number of roots per plantlet as compared to the control. In F. fragrans and H. psittacorum EMS reduced the percentage of rooted plantlets to 35% and 65% of control respectively. Similarly, NMU treatments reduced rooting efficiency in F. fragrans to 80% of control. However, a stimulatory effect on root formation (150% of control) was observed in H. psittacorum in plantlets derived from higher concentrations of NMU (1 and 3 mM). In the case of P. magnoliaefolia the mutagen treatments did not have any effect on rooting efficiency of the shoots derived from the treated cultures as compared to the control.
A stock culture of each plant species under study was developed for in vitro mutagenesis experiments. Since there was no known protocol for tissue culture of P.magnoliaefolia, a regeneration protocol was developed through the culture of leaf blade and petiole explants on MS medium supplemented with both BAP and NAA. The optimum medium for shoot regeneration from petiole is the one supplemented with 5.2 M BAP and 2.6 M NAA and those from leaf blades 11 M BAP and 5.2 M NAA respectively. A combined application of cytokinin and auxin was essential to induce a high rate of normal shoot regeneration and growth on both petioles (50%) and leaf blades (47.6%). Shoot elongation and maturation was improved by providing regenerated shoots, excised from petiole surface with a brief flush of a high concentration of GA3 solution (280 M for 90 seconds).
A protocol for in vitro mutagenesis using two chemical mutagens namely N-methyl-N-nitrosourea (NMU) and ethyl methanesulfonate (EMS), was established for each plant species under study. The effects of different mutagens on in vitro survival and growth or regeneration of plant species were studied and compared with the control. A workable dose range and LD50 values were determined for each mutagen. Both mutagens had inhibitory effects on each plant species under study. These were very prominent from a threshold dose. The workable dose range of NMU established for F. fragrans, H. psittacorum and P. magnoliaefolia at 25o to 26oC were 1 to 10 mM (3 hours treatment), 0.5 to 5 mM (3 hours treatment) and 1 to 4 mM (1 hour treatment) respectively. Similarly, the dose range of EMS worked out for F. fragrans, H. psittacorum and P. magnoliaefolia at 25o to 26oC were 0.008 to 0.12M (3 hours treatment), 25 to 110mM (3 hours treatment) and 0.04 to 0.16M (1 hour treatment) respectively. It was observed that most of the aberrations were derived from dosages either close to or above LD50 values. The mutagen treated explants of F. fragrans and H. psittacorum were subcultured for at least 3 cycles on appropriate medium in order to allow regeneration and proliferation and to eliminate chimeras. In P. magnoliaefolia, adventitious shoots were regenerated on mutagen treated petioles and then allowed to multiply and develop fully so that the variants can be screened.
Mutants were identified through visual screening during in vitro culture and after transfer to soil, based on morphological changes that were absent among the control plants. Some of the interesting morphological variations observed were :
● A high frequency of variegated shoots (52.1% of total MV4 shoots screened) and albinos (17.2%) was derived from cultures of F. fragrans treated with 5 to 10mM NMU. Eight lines of mutants were obtained that consisted of variegated, albinos and light green shoots. These mutant lines differed from each other by leaf shape, size, variegation patterns, stem coloration and growth rate. The rooting ability of different mutants lines was tested. Lines 1, 2 and 3 hold great promise as commercial variants.
● A number of 'root variants' (23% of total plantlets) were derived from cultures of H. psittacorum initially treated with 1 to 3 mM NMU. The variants displayed about 86.2% increase in the number of roots per rooted plantlet over the control.
● One dwarf and one vigorously growing plant of P. magnoliaefolia were obtained from 1 and 4 mM NMU, respectively. They constituted about 1.9% of the total NMU treated shoots that survived after transfer to soil.
NMU induced a broad spectrum of aberrations in the three species (chlorophyll variations, changes in leaf shape, growth habit and rooting efficiency). Whereas, only chlorophyll variations were induced by different EMS treatments. Therefore, NMU appears to be more effective on the three species than EMS. A definite conclusion in this regard can only be drawn after long term field trial of the mutagen treated plants as variations can also be appeared at later stages of growth.
The effects of EMS and NMU on the rooting efficiency of the plant species were studied in terms of percentage of rooted plantlets and mean number of roots per plantlet as compared to the control. In F. fragrans and H. psittacorum EMS reduced the percentage of rooted plantlets to 35% and 65% of control respectively. Similarly, NMU treatments reduced rooting efficiency in F. fragrans to 80% of control. However, a stimulatory effect on root formation (150% of control) was observed in H. psittacorum in plantlets derived from higher concentrations of NMU (1 and 3 mM). In the case of P. magnoliaefolia the mutagen treatments did not have any effect on rooting efficiency of the shoots derived from the treated cultures as compared to the control.
Date Issued
1996
Call Number
QK725 Maj
Date Submitted
1996