Important methods to artificially induce mutations are the use of chemical and physical agents. Most chemical mutagens are alkylating agents and azides. Physical mutagens include electromagnetic radiation, such as gamma rays, X rays, and UV light, and particle radiation, such as fast and thermal neutrons, beta and alpha particles. Mutagenic treatment of seeds is the most convenient and, therefore, the standard method in seed propagated crops. Seeds can be treated in large quantities and are easily handled, stored, and shipped. It is fairly easy to repeat the conditions of mutagenic treatment, pre- and post-treatment, and hence, to obtain reproducible results within practical limits. Besides seed treatment, whole plants, cuttings, tubers, pollen, bulbs, corms, or in vitro plants or tissues can be treated. This chapter is restricted to the commonly applied techniques of mutation induction in seeds by ethyl methanesulfonate (EMS) treatment and by gamma and fast neutron irradiation.
This chapter presents the standard procedures for inducing mutations in plants using both physical and chemical mutagens. The sensitivity of plant materials to various mutagens is described for a number of plant species (including barley, Phaseolus vulgaris and soyabean). Factors influencing mutagenic effects (genetic factors, environmental factors, and radio-sensitivity of plant species) are outlined.
The aim of this protocol was to develop an alternative in vitro propagation system for
Cannabis sativa
L. by mimicking nursery-based vegetative propagation. Photoautotrophic micropropagation (PAM) was achieved on rockwool blocks as substrate combined with commercially available fertilizer suitable for cannabis cultivation. Stock plants were initiated after sterilisation in forced-ventilated glass jars which then provided a continuous supply of shoot tip and nodal cuttings. A 97.5% rooting rate of in vitro shoot tip cuttings and successful acclimatisation were achieved within 3 weeks in glass vessels with passive ventilation.
Field studies of fruit production from Lepidosperma concavum R.Br., L. laterale R.Br. and L. longitudinale Labill. showed that large proportions (21–77%) of fruits were unfilled and that filled and unfilled fruits looked alike. Bagging of inflorescences demonstrated that filled fruits tended to be shed, while empty fruits remained within the inflorescence. Time of collection was critical for obtaining viable seeds, with successful harvesting limited to a short period (weeks) after maturation. The timing of flowering and fruit maturation were fairly consistent between species, populations and years in our study area. In L. concavum fruit production was increased in cultivation compared with wild populations. In all three species, very little or no germination of fruits occurred under nursery conditions. In vitro culture initiation was attempted using intact fruits, nicked fruits and seeds on 1/2MS (Murashige and Skoog) medium with 1 µM zeatin and 0.5 µM gibberellic acid in darkness. Culture of intact fruit resulted in no germination, while nicked fruit showed some germination response. Best results were achieved from seeds with germination occurring as early as 7 to 18 days depending on the species. Germination of L. concavum, L. laterale and L. longitudinale was 86%, 64% and 83% respectively within 5 weeks. Germination response was strongly influenced by seed maturity. Mature seeds germinated significantly faster than immature seeds. On a small proportion of cultured seeds, calli formed and differentiated into numerous plantlets on growth regulator-free medium. Given the promising results observed in this study, in vitro culture appears to be a practical means of mass propagating Lepidosperma species.
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