Alan C. Cassells

Variation in potato microplant morphology in vitro and DNA methylation

Heterotrophic and autotrophic culture in agar and in polyurethane foam, the latter used as an alternative tissue support to agar, resulted in potato microplants with different in vitro morphologies. The microplants were visually characterised in terms of their relative developmental maturity, by comparing the respective leaf shapes in vitro with ontogenetic differences in leaf shape in glasshouse-grown potato plants. Cytosine methylation in the DNA of microplants of the different morphologies was determined using a method based on the AFLP technique but employing methylation-sensitive restriction enzymes (MSAP analysis) to test the hypothesis that DNA methylation could be used to characterise differences in microplant development in vitro. In three of the four treatments there was a good correlation between the visual assessment of relative morphological maturity and DNA base methylation levels. In these microplants there was increased DNA methylation in the leaves with mature leaf morphology represented by a decreased number of restriction fragments. The fourth in vitro morphology had the most juvenile leaf shape but did not have the predicted level of DNA methylation, having a relatively low number of restriction fragments. Subtraction analysis was used to discriminate the fragments that were unique to the juvenile and mature in vivo leaf morphologies. Comparison of the fragment patterns from the microplants with the latter reference profiles, confirmed the relationship with the total DNA methylation as detected by MSAP analysis, that is, the number of common fragments with the juvenile or mature in vivo leaf profiles, respectively. However, none of the fragment profiles, while sharing some common bands at random, was identical to any other; or to that of either the juvenile or mature in vivo leaf. The anomalous relationship of the microplants with most juvenile leaf shape and highest DNA methylation was confirmed. The measurement of DNA methylation in in vitro plants is discussed in the context of the development of a method to assess the quality of microplants produced by different in vitro protocols.

The influence of gas permeability of the culture lid on calcium uptake and stomatal function in Dianthus microplants

The establishment of microplants has been shown to be related to the moisture vapour transmission rate of the culture vessel lid. Lids allowing high moisture vapour loss from the container give increased microplant establishment rates. Further, it has been shown that calcium accumulation in the leaves of microplants is correlated with the moisture vapour transmission rate of the vessel lid and this is related to stomatal function in vitro and ex vitrum and to decreased desiccation in the microplants. It is hypothesized that the increased moisture vapour transmission rate of the lid results in increased transpiration in vitro, which in turn causes increased calcium uptake into the leaves of the microplants with consequential increased stomatal function and reduced losses due to desiccation ex vitrum.

The effects of arbuscular mycorrhizal fungi and chitosan sprays on yield and late blight resistance in potato crops from microplants

The established microplants, mycorrhized and non-mycorrhized controls, were planted in the field and unsprayed (controls), sprayed with a conventional fungicide (Ridomil), with Ridomil alternating with N,O-carboxymethyl chitosan (NOCC — a soluble chitosan) or sprayed only with NOCC. The effects of the treatments on late blight progression in the crop were monitored throughout the season and yield measured. In a preliminary study on the inoculation of detached leaves from unsprayed plants and plants sprayed with Ridomil and NOCC, respectively, NOCC was shown to delay disease development. In the field, late blight development was most rapid in the unsprayed controls, delayed in the Ridomil-treated plants and disease progression was slowed in the other treatments (AMF-inoculation, AMF-NOCC sprayed, NOCC-sprayed and Ridomil-NOCC sprayed). It was shown that plant chitinase activity increased in the AMF, AMF-NOCC and NOCC treatments. At harvest, yields for the Ridomil-NOCC, NOCC and AMF-NOCC treatments were significantly higher than the un-sprayed, non-mycorrhizal control and were not significantly different to the Ridomil-sprayed control. The results are discussed in relation to the production of seed potatoes for organic (ecological) growers.

The effect of mycorrhizal fungi on the hatch of potato cyst nematodes

In the presence of potato plants cv. Golden Wonder, Globodera pallidaexhibited delayed in-soil hatch compared to that of G. rostochiensis, with significantly fewer G. pallida second-stage juvenile nematodes hatching in the first two weeks, though the difference disappeared after four weeks. Inoculation of potato plants with arbuscular mycorrhizal fungi eliminated this delay in G. pallida hatch, so that the two potato cyst nematode (PCN) species exhibited similar in-soil hatch rates. When the corresponding in vitro hatching activities of root leachate from uninoculated and mycorrhiza-inoculated plants were compared, similar effects were revealed. G. pallida hatch in root leachates from uninoculated plants increased significantly from one-week-old to two-week-old plants, but this increase was not significant in the mycorrhizal-inoculated plants. When the in-soil experiment was repeated using the potato cyst nematode non-host plant strawberry, mycorrhizal inoculation induced no significant increase in G. pallidahatch. The results indicate that mycorrhizal inoculation of potato plants stimulates production of G. pallida-selective hatching chemicals, either hatching factors or hatching factor stimulants.

An ontology-driven system architecture for precision agriculture applications

Our research has been performed in the context of the EU-funded R&D project PLANTS. In this paper, we describe an ontology-driven architecture for developing systems that can be used in precision agriculture applications. Central to our approach is the use of an ontology, which views plants and associated computation as an integral part and allows the interaction of plants and artefacts in the form of synergistic mixed societies. PLANTS ontology sets up a conceptual framework that combines the knowledge about sensors, actuators and other domain concepts available, on the one hand, and the biological studies about plant stressing and sensing mechanisms and consequent plant behaviour, on the other hand, to make plants a proactive component of agricultural systems.

Establishment of a plantation from micropropagated Arnica chamissonis a pharmaceutical substitute for the endangered A. montana

Arnica montana L., like many endangered species, does not give stable yields in commercial plantations. Other Arnica species were investigated for their pharmaceutical activity and A. chamissonis Less., was confirmed as a pharmaceutical substitute for A. montana, extracts having on average 40% higher inhibition of lipoxygenase activity and reduction of superoxide and hydroxyl free radicles on a tissue dry weight basis. A micropropagation protocol was established for A. chammisonis and a plantation of 10,050 plants was established with 0.9% microplant loss at the weaning stage. The plantation is now in commercial production.

Pathogen and Biological Contamination Management

Multiplication of certified pathogen-free stock plants in vitro makes an important contribution to the production of disease-free planting material for vegetatively propagated crops. Meristem culture is extensively used to eliminate pathogens and contaminants from microbially contaminated plants prior to micropropagation. The approach to pathogen and contamination management differs. It is essential to avoid the release of pathogen-contaminated microplants and to ensure this the plant pathogen-testing guidelines and protocols issued by the Food and Agriculture Organisation (FAO) and its regional representative organizations should be followed. Where in vitro methods are used to eliminate pathogens, the progeny plants should be established in vivo under quarantine conditions and tested under FAO guidelines before being used as stock plants for in vitro multiplication. At establishment of microplants in vitro (stage 1), cultures should be culture-indexed for the presence of microbial contaminants. If pathogen-and contaminant-free cultures are established, then the risk is that of managing laboratory contamination by common environmental microorganisms based on Hazard Analysis Critical Control Points (HACCP) principles. International plant health certification organizations are conservative and rely on established pathogen indexing protocols. They are reluctant to accept DNA-based tests and do not accept testing of in vitro cultures. Given that in time both of these restrictions may be relaxed, micropropagators may look forward to availing more of diagnostic service providers using polymerase chain reaction-based multiplex assays for pathogen-indexing and advances in diagnostic kits for environmental microorganisms in support of laboratory contamination management; with the caveat that molecular tests for pathogens may continue to require confirmation by inoculation of indicator plant species.

Influence of in vitro factors on titre and elimination of model fruit tree viruses

The titre of apple chlorotic leaf spot virus (ACLSV) was higher in microplants of Malus domestica cv. Jonagold than in 2-year-old grafted scions. Cytokinin concentration in the medium increased the titre of prunus necrotic ringspot virus (PNRSV) in the apex of microplants of Prunus insititia cv. Kozlienka but did not affect the titre of ACLSV in M .domestica.Virus titre of ACLSVwas higher in the haulms of autotrophically-grown compared with heterotrophically-grown microplants whereas as for PNRSV the results were the reverse. For both viruses, however, titre of the virus in the roots of autotrophically-grown plants was significantly higher than in haulm tissue from heterotrophic cultures. Ribavirin incorporation resulted in elimination of both viruses. Negative ELISA results were confirmed independently by PCR. The efficacy of Ribavirin in elimination of ACLSV was increased by increasing the concentration of cytokinin in the medium in parallel with decreasing the concentration of Ribavirin. These results are discussed in the context of the reliability of in vitro virus testing.

The Use of AMF and PGPR Inoculants Singly and Combined to Promote Microplant Establishment, Growth and Health

Upon transplanting, microplants are exposed to biotic and abiotic stresses. Here, the potential of biological inoculants to improve microplant establishment and to add value to micropropagules by improving post-establishment growth and health, is discussed. Both AMF and PGPR have been used successfully to improve the establishment and growth of microplants. Recently, higher benefits have been reported using combinations of AMF and PGPR. The latter results, however, have been variable. Holistic biocontrol strategies, involving the use of inoculants and elicitors to bioprime plant defences, are proposed here to promote plant health and to protect against root and haulm diseases.

Detection of Economically Important Variability in Micropropagation

Variability in micropropagated plants includes random phenotypic variation (‘somaclonal variation’) and developmental variation. In the former, the progeny may require grading and if the variants are above an arbitrary percentage, the client may reject the batch. In the latter, plant maturation may be altered in the population with consequent economic losses (George, 1993). While the presence, elimination or partial elimination of intracellular biotic contaminants, chimeral breakdown and, or rearrangement and unstable loci may explain some of the variability encountered, these problems should be avoided by testing of the plants prior to clonal propagation (Stage 0 in micropropagation; see Cassells, 1997). A generalization is that the appropriate choice of cloning strategy will also avoid somaclonal variation (George, 1993) However, even using nodal cloning, economically important variability in the form of epigenetic variation can be encountered (Cassells et al. 1997; 1998). Since the whole batch may be uniformly affected, e.g. ‘hyperjuvenile’ (developmentally retarded), this represents economically important variability for the industry.