Pradeep C. Deo

Improving taro (Colocasia esculenta var. esculenta) production using biotechnological approaches.

Taro (Colocasia esculenta L. Schott) is an important crop worldwide but is of particular significance in many Pacific Island countries where it forms part of the staple diet and serves as an export commodity. Escalating pest and disease problems are jeopardizing taro production with serious implications to food security and trade. Biotechnological approaches to addressing pest and disease problems, such as somatic embryogenesis and transgenesis, are potentially viable options. However, despite biotechnological advancements in higher profile agronomic crops, such progress in relation to Colocasia esculenta var. esculenta has been slow. This paper reviews taro biology, highlights the cultural and economic significance of taro in Pacific Island countries and discusses the progress made towards the molecular breeding of this important crop to date.

Seed germination and seedling development in Taro (Colocasia esculenta)

Two taro (Colocasia esculenta (L.) Schott var. esculenta) cultivars from Fiji and Papua New Guinea were grown at the University of the South Pacific, Laucala Campus, Fiji to produce seeds for seed storage experiments. Gibberellic acid at a 500ppm concentration was used to induce flowering. Very few flowering shoots (inflorescence) were observed in the Fiji cultivar and all pollinations were unsuccessful. However the PNG cultivar flowered well and was used to obtain seed after hand pollination. Hand pollination was carried out to ensure seed setting in developing fruits in the inflorescence. Seeds were extracted in the laboratory after harvesting mature inflorescences. Experiments were conducted on seed moisture content, desiccation, germination, seedling development and seed storage behaviour of taro (Colocasia esculenta) seeds. Seed moisture content was determined using oven methods and air-drying. Results demonstrated that taro seeds have a moisture content of 12-13% after air-drying for three to four weeks. Seeds were dried to desired moisture contents in a desiccator over silica gel. After drying to 5% moisture content seed viability was tested by germinating seeds on moist filter paper at room temperature with 65% relative humidity and seven to eight hours day length. Preliminary seed germination tests demonstrated up to 83% germination for seeds with 13% moisture content. Germination occurred within five to seven days. Maximum germination was achieved within 21 days. The highest germination (80%) was achieved with seeds with 12% moisture content. Results indicated there was no relationship between moisture content and seed germination. Normal seedling development and growth was recorded after germination.

In vitro micro propagation of Nicotiana benthamiana via axillary shoots

Axillary shoots of Nicotiana benthamiana were regenerated from nodal explants in two weeks using MS media supplemented with the cytokinin, kinetin (0.5 mg/L), and the auxin, indole-3-butyric acid (IBA) (0.1 mg/L). Ninety two percent of shoots were 2.1-20 mm tall, a size ideal for root induction. After transfer to hormone-free MS they readily produced roots within seven days, with phenotypically normal, fully developed plants being obtained within four weeks. Leaf chlorosis due to iron deficiency was observed in plants over time, however, this was overcome by doubling the concentration of inorganic iron. This rapid micro-propagation system is particularly useful for the in vitro mass production of N. benthamiana plants for various biotechnological applications.

Novel Gene Transfer Technologies

Gene transfer is the process by which a gene from any source can be introduced into plant cells or tissues. Gene transfer technologies are used to manipulate plant cells for scientific research as well as for commercial purposes like production of transgenics. Transgenic plants generated using these technologies are used either for field deployment or for identifying and evaluating gene and promoter function. In the last few decades, significant developments have been made in gene transfer technology since the discovery of Agrobacterium tumefaciens as a natural tool for plant transformation, and there is a long list of transgenic crop varieties that have now been released for commercial production. These advances are related to major improvements in Agrobacterium-mediated and direct DNA delivery techniques, along with modifications in tissue culture techniques for regenerating transgenic plants from transformed cells or tissues. Bananas are not lagging too far behind in this race, with many laboratories engaged in field trials of transgenic bananas carrying genes of interest. With efficient gene transfer technologies available for banana, banana transformation research is now more focussed on the problems associated with generating cell suspensions of recalcitrant cultivars, identifying useful gene/trait associations, promoters and problems associated with stable integration and reliable expression of the DNA once it has been integrated. This chapter focuses on the gene transfer technologies currently available for generating transgenic banana cultivars and summarises the various traits of interest for which genes have been transformed into bananas.

Production of human vitronectin in Nicotiana benthamiana using the INPACT hyperexpression platform

Summary Human vitronectin (hVN) is a glycoprotein that functions as a cell adhesion molecule and a regulator of coagulation in blood plasma and the extracellular matrix. In vitro, hVN is added to serum‐free media in order to promote the adhesion of animal cells to tissue culture surfaces and the proliferation of undifferentiated stem cells. Here, we report the production of hVN in Nicotiana benthamiana using the inducible In Plant ACTivation (INPACT) hyperexpression platform. N. benthamiana plants were transformed with an INPACT expression cassette encoding hVN, and both the Tobacco yellow dwarf virus Rep/RepA activator and Tomato bushy stunt virus p19 gene under the transcriptional control of the ethanol‐inducible AlcR:alcA gene switch. hVN expression was maximal 4–5 days postactivation of the INPACT platform with a dilute ethanol solution, and crude yields of the recombinant protein reached a maximum of 643 ± 78 mg/kg fresh weight. A three‐stage purification protocol was developed using heparin and polyhistidine tag affinity binding and size exclusion filtration, resulting in a plant‐made hVN product of >90% purity. Storage conditions for plant‐made hVN were identified that maximized the capacity of the recombinant protein to promote cell adhesion. Critically, plant‐made hVN was shown to be functionally equivalent to commercial, plasma‐derived hVN at promoting one‐half maximal attachment of murine fibroblast cells (BALB‐C/3T3) in serum‐free medium at <0.1 μg/cm2 to tissue culture plasticware. The INPACT platform represents an attractive means of producing large quantities of functional, animal‐free hVN for in vitro applications.