Michael E. Kane

Biodiversity conservation and conservation biotechnology tools

This special issue is dedicated to the in vitro tools and methods used to conserve the genetic diversity of rare and threatened plant species from around the world. Species that are on the brink of extinction because of the rapid loss of genetic diversity and habitat come mainly from resource-poor areas of the world and from global biodiversity hotspots and island countries. These species are unique because they are endemic, and only a few small populations or sometimes only a few individuals remain in the wild. Therefore, the challenges to support conservation by in vitro measures are many and varied. The editors of this invited issue solicited papers from experts from Asia, Africa, Europe, Australia, and North and South America. This compilation of articles describes the efforts in these diverse regions toward saving plants from extinction, and details the direct application of in vitro and cryopreservation methods. In addition, these contributions provide guidance on propagation of rare plants, including techniques for large-scale propagation, storage, and reintroduction. The in vitro techniques for conserving plant biodiversity include shoot apical or axillary-meristem-based micropropagation, somatic embryogenesis, cell culture technologies and embryo rescue techniques, as well as a range of in vitro cold storage and cryopreservation protocols, and they are discussed in depth in this issue.

The Reliability and Validity of Weighted Composite Scores

The scores on 2 distinct tests (e.g., essay and objective) are often combined to create a composite score, which is used to make decisions. The validity of the observed composite can sometimes be evaluated relative to an external criterion. However, in cases where no criterion is available, the observed composite has generally been evaluated in terms of its reliability. The analyses in this article are based on a simple, content-based model for the validity of the observed composite as an estimate of a target composite, based on a priori weights for the 2 tests. The results suggest that giving extra weight to the more reliable of the 2 observed scores tends to improve the reliability of the composite, and up to a point tends to improve its validity. Giving too much weight to the more reliable score can decrease the validity of the observed composite as a measure of the target composite.

Compatible host/mycorrhizal fungus combinations for micropropagated sea oats

Micropropagation technology promises to improve the supply of sea oats for restoring Floridas eroded beaches, but concerns about genetic diversity need to be addressed. These dune plants are colonized by a wide array of arbuscular mycorrhizal (AM) fungi, yet little is know of the diversity of these fungal communities. Our goal was to test the level of functional diversity that exists among communities of AM fungi that are present in divergent Florida dunes. Community pot cultures were established from samples collected from ten transects in two Gulf coast and two Atlantic coast locations in Florida, and these were used to conduct two greenhouse studies. The objective of the first study was to evaluate within-location variance in the mycorrhizal function of different AM fungal communities associated with endemic sea oats. The objective of the second study was to evaluate among-location responses of plant and fungal ecotypes using selected combinations obtained from the first experiment. Within locations, the AM fungal community had significant impacts on shoot mass and shoot-P contents, confirming a range of symbiotic effectiveness exists within the beach-dune system. Among locations, there was a tendency for greater root colonization between host clones and fungal communities from the same location, indicating a degree of specificity between host ecotypes and their symbiotic fungi. Relative to plant growth response, one fungal community was superior across plant genotypes from all locations, while one plant genotype tended to have the best response across all fungal communities. These data suggest that while it is possible to select effective AM fungal-host combinations for outplanting, origin of host and AM fungi have little predictive value in screening these combinations.

The Precision of Change Scores Under Absolute and Relative Interpretations

Although some progress has been made in rehabilitating change scores by showing that they are not necessarily unreliable, they are still generally thought to be less reliable than the measures from which they are derived. However, the focus on reliability or generalizability as a measure of the precision of change scores may be misguided because both reliability and generalizability coefficients assume a norm-referenced interpretation, whereas many applications of change scores rely on an absolute interpretation of the change. We examined the precision of change scores in terms of error-tolerance (E/T) ratios for both relative and absolute interpretations of change scores. E/T ratios are used to evaluate the error in estimating the change relative to the tolerance for error in a particular context. Achievement test data in which people are crossed with items within categories are used to illustrate the results.

Shoot organogenesis from petiole explants in the aquatic plant Nymphoides indica.

A protocol for rapid shoot organogenesis from petiole explants of the ornamental aquatic plantNymphoides indica L. Thwaites O. Kuntze was developed for use in future mutation breeding and cultivar selection studies. Optimum culture conditions for shoot organogenesis were determined. Effects of factorial combinations of 2-iP, BA or kinetin (0–25 μM) in factorial combination with IAA or NAA (0–25 μM) were examined. On the basis of regeneration frequency (80%) and adventitious shoot number (11.5 shoots per explant), most efficient shoot organogenesis occurred on petiole explants cultured on a basal medium consisting of full-strength MS inorganic salts, 0.56 mM myo-inositol, 1.2 μM thiamine-HCl, 116.8 mM sucrose supplemented with 10 μM BA and 20 μM IAA and solidified with 0.8% TC agar. Formation of adventitious shoots by direct and indirect shoot organogenesis from the same explant was verified by histological sectioning. With the exception of variegated leaf production on a single adventitious shoot produced in the presence of 25 μM kinetin and 15 μM NAA, no visible phenotypic abnormalities were observedin vitro in any of the shoots generated. Solid achlorophyllous adventitious shoots were recovered following culture of this variegated leaf tissue. Plantlets were easily acclimatized toex vitro conditions.

Physiological and developmental problems encountered by in vitro cultured plants

After the realization of Haberlandts’ theory of totipotency (Krikorian and Berquam 1969 English translation), in vitro culture of plants has become an integral part of advances in plant science research. Plant tissue culture techniques allow for close monitoring and precise manipulation of plant growth and development, the presence of a controlled environment in vitro to analyze specific source(s) of variation and the possibility of manipulating growth at the cell, tissue and organ levels. These benefits, and others, have given tissue culture and its application paramount importance. It is now possible to understand plant growth and its underlying phenomena in vitro. Aspects of plant growth that were barely understood before the advancement of the science of tissue culture, such as the metabolism and interaction of plant hormones, as well as their physiological effects are now well documented. In addition to its role in the advancement of plant science research, plant tissue culture is also contributing enormously in food production, crop improvement, production of secondary products and conservation of endangered species. The development of tissue culture protocols is a rigorous procedure that involves optimizing the various chemical, physical and environmental factors of growth. It involves growing plants outside their natural environment and growth conditions. The artificial growth conditions are by no means the perfect substitutes for the natural ones. Furthermore, the natural variation in physiology and growth requirement among plant species warrants the need to optimize protocols for individual species. Despite the meticulous efforts involved in growing plants in vitro and the advances made in plant tissue culture, the application of this technique is still hampered by various physiological and developmental problems. The problems range from abnormal growth to increased genetic variability/instability. Papers in this Special Issue report on micropropagation problems related to developmental and physiological aspects such as shoot-tip necrosis (Bairu et al. 2011a), fasciation (Iliev and Kitin 2011), tissue proliferation (Brand 2011), epigenetic changes (Smulders and De Klerk 2011), somaclonal variation (Bairu et al. 2011b), effect of phenolics on rooting (De Klerk et al. 2011), alleviating problems related to caulogenic and rhizogenic processes (Moyo et al. 2011) and assessment of the role of metatopolins in micropropagation and the control of culture abnormalities (Amoo et al. 2011).

Asymbiotic Germination Response to Photoperiod and Nutritional Media in Six Populations of Calopogon tuberosus var. tuberosus (Orchidaceae): Evidence for Ecotypic Differentiation

BACKGROUND AND AIMS Ecotypic differentiation has been explored in numerous plant species, but has been largely ignored in the Orchidaceae. Applying a specific germination protocol for widespread seed sources may be unreliable due to inherent physiological or genetic differences in localized populations. It is crucial to determine whether ecotypic differentiation exists for restoration and conservation programmes. Calopogon tuberosus var. tuberosus, a widespread terrestrial orchid of eastern North America, is a model species to explore ecotypic differences in germination requirements, as this species occupies diverse habitats spanning a wide geographical range. METHODS Mature seeds were collected from south Florida, north central Florida, three locations in South Carolina, and the upper Michigan peninsula. Effects of three photoperiods (8/16, 12/12, 16/8 h L/D) were examined on asymbiotic in vitro seed germination and seedling development of C. tuberosus. Germination and early development was monitored for 8 weeks, while advanced development was monitored for an additional 8 weeks. In an additional experiment, asymbiotic seed germination and development was monitored for 8 weeks on six culture media (BM-1 terrestrial orchid medium, Knudson C, Malmgrem, half-strength MS, P723, and Vacin and Went). A tetrazolium test for embryo viability was performed. KEY RESULTS Short days promoted the highest germination among Florida populations, but few differences among photoperiods in other seed sources existed. Different media had little effect on the germination of Michigan and Florida populations, but germination of South Carolina seeds was higher on media with higher calcium and magnesium. Tetrazolium testing confirmed that South Carolina seeds exhibited low viability while viability was higher in Florida seeds. Seed germination and corm formation was rapid in Michigan seeds across all treatments. Michigan seedlings allocated more biomass to corms compared with other seed sources. CONCLUSIONS Rapid germination and corm formation may be a survival mechanism in response to a compressed growing season in northern populations. Ecotypic differentiation may be occurring based on seed germination and corm formation data.

Examining the interaction of light, nutrients and carbohydrates on seed germination and early seedling development of Bletia purpurea (Orchidaceae)

The effects of carbohydrate availability, carbohydrate source, nutrient availability and illumination on germination and early development of Bletia purpurea (Orchidaceae) seeds were investigated using asymbiotic seed germination. Of special interest was determining the minimum nutritional and light requirements for the completion of germination. Germination and development was limited when seeds were cultured in darkness without sucrose. Seeds were able to germinate under illuminated conditions even in the absence of sucrose and this effect was enhanced when mineral nutrients were incorporated into media. Sucrose, fructose, glucose and trehalose enhanced germination and seedling development while mannitol and sorbitol did not. These data suggest that carbohydrates, either as products of photosynthesis, from symbiotic fungi in situ or as exogenously supplied sugars in vitro, play an important role in regulating seed germination by fulfilling an energy requirement. This hypothesis has been often expressed but rarely satisfactorily tested. Mineral nutrients appear to be less important for germination than carbohydrates. The differential effect of sucrose, fructose, glucose and trehalose at two different concentrations on rhizoid production indicates carbohydrates may play a role in regulating rhizoid production.

Comparative in vitro germination ecology of Calopogon tuberosus var. tuberosus (Orchidaceae) across its geographic range

Seed responses to temperature are often essential to the study of germination ecology, but the ecological role of temperature in orchid seed germination remains uncertain. The response of orchid seeds to cold stratification have been studied, but the exact physiological role remains unclear. No studies exist that compare the effects of either cold stratification or temperature on germination among distant populations of the same species. In two separate experiments, the role of temperature (25, 22/11, 27/15, 29/19, 33/24°C) and chilling at 10°C on in vitro seed germination were investigated using distant populations of Calopogon tuberosus var. tuberosus. Cooler temperatures promoted germination of Michigan seeds; warmer temperatures promoted germination of South Carolina and north central Florida seeds. South Florida seed germination was highest under both warm and cool temperatures. More advanced seedling development generally occurred at higher temperatures with the exception of south Florida seedlings, in which the warmest temperature suppressed development. Fluctuating diurnal temperatures were more beneficial for germination compared to constant temperatures. Cold stratification had a positive effect on germination among all populations, but South Carolina seeds required the longest chilling treatments to obtain maximum germination. Results from the cold stratification experiment indicate that a physiological dormancy is present, but the degree of dormancy varies across the species range. The variable responses among populations may indicate ecotypic differentiation.

PHOTOSYNTHETIC AND CARBOHYDRATE STATUS OF EASY- AND DIFFICULT-TO-ACCLIMATIZE SEA OATS (UNIOLA PANICULATA L.) GENOTYPES DURING IN VITRO CULTURE AND EX VITRO ACCLIMATIZATION

SummaryThe photosynthetic and carbohydrate status of an easy-to-acclimatize (EK 16-3) and a difficult-to-acclimatize (EK 11-1) genotype of Uniola paniculata L. (sea oats), a native dune species of the southeastern US, were evaluated during in vitro culture and ex vitro acclimatization. Net photosynthetic rate was eight times greater for EK 16-3 than EK 11-1 plantlets after ex vitro transfer. In vitro-produced leaves were morphologically similar to ex vitro-produced leaves and exhibited similar photosynthetic competence. EK 11-1 plantlets exhibited greater transpiration rates at the time of ex vitro transfer than EK 16-3 plantlets. However, the small magnitude of this difference, although significant, indicated that control of water loss was probably not the main cause for poor acclimatization of EK 11-1 plantlets. Carbohydrate analysis in vitro revealed that EK 16-3 plantlets utilized leaf starch reserves more rapidly than EK 11-1 plantlets. Starch utilization correlated with the development of leaves with expanded leaf blades during in vitro rooting in EK 16-3 plantlets. After ex vitro transfer, both genotypes exhibited significant decreases of starch and soluble sugar content in shoots and roots. However, the higher photosynthetic ability of shoots in EK 16-3 resulted in greater accumulation of shoot soluble sugars than EK 11-1 after 2-wk ex vitro culture. After 6-wk in vitro rooting, there were significantly higher chlorophyll and soluble protein contents, ribulose 1,5-bisphosphate carboxylase (rubisco) and phosphoenolpyruvate carboxylase activities in EK 16-3 than EK 11-1 shoots. These differences also correlated with the development of anatomical and morphological leaf features in EK 16-3 similar to those of greenhouse-produced leaves.