Tissa Senaratna

Acetyl salicylic acid (Aspirin) and salicylic acid induce multiple stress tolerance in bean and tomato plants

The hypothesis that physiologically activeconcentrations of salicylic acid (SA) and itsderivatives can confer stress tolerance in plants wasevaluated using bean (Phaseolus vulgaris L.) andtomato (Lycopersicon esculentum L.). Plantsgrown from seeds imbibed in aqueous solutions (0.1--0.5 mM) of salicylic acid or acetyl salicylic acid(ASA) displayed enhanced tolerance to heat, chillingand drought stresses. Seedlings acquired similarstress tolerance when SA or ASA treatments wereapplied as soil drenches. The fact that seedimbibition with SA or ASA confers stress tolerance inplants is more consistent with a signaling role ofthese molecules, leading to the expression oftolerance rather than a direct effect. Induction ofmultiple stress tolerance in plants by exogenousapplication of SA and its derivatives may have asignificant practical application in agriculture,horticulture and forestry.

Tissue Culture Studies of Tomato (Lycopersicon esculentum)

Tomato is a major vegetable crop that has achieved tremendous popularity over the last century. It is grown in almost every country of the world. Development of protocols for in vitro selection can provide new advances for the production of stress tolerant cultivars. Techniques have been optimised for the production of haploids and somatic hybrids. Attempts have also been made to transfer the higher regenerative ability of wild varieties to cultivated tomatoes. Although, some information is available on the morphogenesis of tomato, the techniques have not been developed to a level at which they can be utilised in large-scale multiplication of commercially important cultivars. The morphogenesis response seems to be highly dependent PGRs used in the media, which is again cultivar and genotypic specific. Somatic embryogenesis in tomato is still at its infancy, and efficient procedures for large-scale production via somatic embryogenesis are yet to be developed. Genetic stability of the tissue culture raised tomato plants also needs to be addressed. The use of a combination of molecular and conventional breeding techniques could be the option for the development of cultivars resistant to biotic and abiotic stresses. This paper reviews the advances made in various aspects of tissue culture in tomato. It also discusses the issues that still need to be addressed to utilise the full potential of plant tissue culture techniques in genetic improvement and mass propagation of tomato.

Benzoic acid may act as the functional group in salicylic acid and derivatives in the induction of multiple stress tolerance in plants

Benzoic acid, sulfosalicylic acid and methyl salicylic acid wereevaluated for their regulatory role in inducing multiple stress tolerance inbean (Phaseolus vulgaris cv Brown Beauty) and tomato(Lycopersicum esculentum cv Romano) plants. All threemolecules were effective in inducing tolerance to heat, drought and chillingstress similar to that reported previously for salicylic and acetylsalicylicacids. Benzoic acid is effective at lower concentrations than salicylic acid orits derivatives. The benzoic acid structural portion is common to all fivemolecules and is the most likely basic functional molecular structure impartingstress tolerance in plants.

Smoke-saturated water promotes somatic embryogenesis in geranium

The effect of smoke saturated-water (SSW) on somatic embryogenesis was studied using geranium hypocotyl culture as a model system. Treatment of explants with 10% SSW or the inclusion of SSW with thidiazuron, a compound which induces somatic embryogenesis, enhanced the embryogenic potential of the geranium hypocotyl culture. Prolonged exposure to SSW was detrimental to embryogenesis. The SSW treatment also accelerated the rate of embryo development suggesting a growth regulatory role of SSW.

Genetic fidelity and viability of Anigozanthos viridis following tissue culture, cold storage and cryopreservation

Abstract The effects of long-term storage conditions on the viability and genetic fidelity of plant somatic tissues are poorly known. In this study, the effects of three storage methods (tissue culture, cold storage and cryostorage) on genetic fidelity and shoot apex viability were evaluated for Anigozanthos viridis subspp. terraspectans (Haemodoraceae), a threatened plant from south west Australia. Genetic fidelity was assessed following 12 months of storage using the PCR-based multi-locus DNA fingerprinting technique Amplified Fragment Length Polymorphism (AFLP). Shoot apex viability was evaluated at 0, 3, 6 and 12 months for cryogenically stored material. The AFLP technique generated a total of 95 fragments for three primer pairs, and no differences were detected across treatments. Post-cryostorage viability was high (mean=85%) and not significantly different across storage times. These results show that genetic fidelity and shoot apex viability (for cryopreserved material) were maintained following tissue culture, cold storage and cryostorage of A. viridis subspp. terraspectans for up to 12 months.

Stereochemical arrangement of hydroxyl groups in sugar and polyalcohol molecules as an important factor in effective cryopreservation

The efficacy of several sugars and polyalcohols in preculture medium was investigated using Anigozanthos viridis ssp terraspectans Hopper (Haemodoraceae), a threatened plant species endemic to the south west of Western Australia. A vitrification protocol involving preculturing of shoot apices for 3 days on different concentrations of sugars and polyalcohols, followed by incubation in plant vitrification solution 2 (PVS2) for 25 min, prior to immersion in liquid nitrogen (LN) and warming resulted in shoot tip survival ranging from 34 to 84%. High levels of survival were obtained with polyalcohols, compared to sucrose, glucose, trehalose and raffinose when used at the same molarity (0.4 M) or at the equivalent concentration of total hydroxyl (OH) groups present in molecules. In both cases glycerol proved more effective. When polyalcohols (ribitol and erythritol) with similar stereochemical arrangement of OH groups as glycerol were examined, at the same molarity (0.4 M) and with equivalent OH numbers, higher survival was achieved when the total number of OH groups present was the same as glycerol. Additionally, when the structural isomers mannitol/sorbitol and ribitol/xylitol were compared at the same molarity (0.4 M), the isomer with the higher number of OH groups along the same side resulted in significantly higher levels of post-LN survival. We propose that the mode of action of polyalcohols is based not on molarity, but on the total number of OH groups present in the medium. Furthermore, based on these results we propose that the orientation of OH groups is a determining factor in effective cryopreservation.

Seed ageing of four Western Australian species in relation to storage environment and seed antioxidant activity

The influence of the storage environment on seed viability and antioxidant potential was examined for four species native to Western Australia: Acacia bivenosa DC., Anigozanthos manglesii D. Don, Banksia ashbyi E.G. Baker, and Mesomelaena tetragona (R. Br.) Benth. Seeds were stored at four water contents (at c . 5%, 11–15%, 20–23% and 50% relative humidity) at each of five temperatures (–196, –18, 5, 23 and 50°C), and seed germination and seedling vigour monitored over an 18-month period. Deterioration was apparent in all species (except A. bivenosa ) stored at 50°C, with 11% RH maximizing longevity for B. ashbyi and M. tetragona seeds, and 5% or 11% RH preventing deterioration for A. manglesii seeds. Seed viability generally remained high for all species stored at 23°C or less. Notably, however, germination and seedling vigour of A. manglesii and M. tetragona seeds gradually declined when stored at –18°C, suggesting that storage at this temperature was detrimental. The antioxidant activity of lipid extracts of seeds after 18 months storage at 5, 23 and 50°C was also examined to determine whether the seed viability decline was associated with a loss of antioxidants. Antioxidant activity varied between storage treatments and was not related to seed viability.

Micropropagation of the critically endangered Western Australian species, Symonanthus bancroftii (F. Muell.) L. Haegi (Solanaceae)

A micropropagation protocol was developed for the conservation of the critically endangered Western Australian shrub,Symonanthus bancroftii. It was necessary to screen antioxidant treatments to prevent the occurrence of lethal browning of explants upon excision. Potassium citrate and citric acid (0.1% w/v in a 4:1 ratio) prevented oxidative browning and was superior to the untreated control or other antioxidant treatments tested. Half strength Murashige and Skoog (MS) medium containing 0.5 μM kinetin and 0.25 μM benzyladenine produced three-fold multiplication compared to 1.75×, 1.5×, 1.8× and 1× multiplication for 2.5 μM kinetin + 0.25 μM benzyladenine, 0.5 μM kinetin + 5 μM gibberellic acid, 1 μM kinetin + 3 μM gibberellic acid and half strength MS with no plant growth regulators, over 4 weeks. Root production was achieved with indole-3-butyric acid (IBA) and α-naphthaleneacetic acid (NAA) at 0.5/0.5 μM (31% rooting) and 1.0/1.0 μM (36% rooting), after four weeks. Paclobutrazol (PBZ) at 0, 3.4 (1 mg 1−1), 10.2 (3 mg 1−1), or 17 μM (5 mg 1−1) improved tolerance to desiccation after transfer ofin vitro rooted shoots to soil. PBZ at 10.2 μM increased survival to 90% compared to 50% for those plantlets not treated with PBZ. The acclimatisation period from the glasshouse to the shadehouse was 1 week for plantlets treated with PBZ compared to 4 weeks for plantlets without any PBZ. PBZ at 3.4 μM increased the number of roots per shoot compared to untreated controls.

IN VITRO PROPAGATION OF EUCALYPTUS PHYLACIS L. JOHNSON AND K. HILL., A CRITICALLY ENDANGERED RELICT FROM WESTERN AUSTRALIA

SummaryIn vitro methods were applied to the only remaining plant of the Meelup Mallee (Eucalyptus phylacis), a critically endangered species from the southwest of Western Australia. Shoot explants were initiated into culture using a 1/2 MS [Murashige and Skoog basal medium (BM) for all experiments] liquid medium supplemented with 1% (w/v) activated charcoal, which was replenished twice daily, followed by transfer of explants to agar medium supplemented with 0.5 μM zeatin. Explants were cultured under low intensity lighting (PPFD of 5–10 μmol m−2s−1) to minimize blackening of tissues, and some explants were induced to produce nodular green calluses in response to BM supplemented with 5 μM thidiazuron. Nodular green calluses were induced to form adventitious shoots following transfer to medium supplemented with 0.5 μM zeatin and 1 μM gibberellic acid, A4 isomer (GA4). Development of shoots was completed on 1 μM zeatin + 0.1 μM 6-benzylaminopurine (BA) in vented culture tubes. Regenerated shoots were sequentially cultured on medium containing 0.5 μM zeatin + 0.2 μM indoleacetic acid (IAA) followed by either 0.5 μM zeatin + 1μM GA4 for shoot elongation or 1 μM zeatin + 0.5 μM IAA to optimize shoot growth. Rooted microshoots were produced after 4 weeks on 5 μM indolebutyric acid (IBA) and survived acclimatization and transfer to potting mixture.

Somatic Embryogenesis for Mass Propagation of Ericaceae – A Case Study with Leucopogon verticillatus

An efficient three-phase culture has been developed for plant regeneration of Leucopogon verticillatus (R. Br.) (Ericaceae formerly Epacridaceae [Ann. Missouri Bot. Gard. 85 (1998): 531–553]) via somatic embryogenesis as indicative of likely culture scenarios for other Ericaceae. The Ericaceae, particularly many Australian species, are often difficult to propagate by conventional forms of nursery propagation. Initiation of somatic embryos was best achieved using Gamborg’s B5 medium, pH 6, 4% maltose, 0.7% agar with the plant growth regulators 10 µM TDZ and 5 µM IAA. Somatic embryos were removed from the parent tissue and transferred to half strength basal GB5 medium for elongation. Root development did not occur unless specific treatments were used, a 2–5 day pulse treatment of 100 µM IBA significantly increased root production. All roots produced in agar-medium were fine and easily damaged when removed from culture. The most successful rooting medium (>60%) was sand on oat medium, which facilitated easy removal from the substrate and improved the survival of plants when transferred to soil.