The Introduction of Transgenes to Control Blackheart in Pineapple: Biolistics Vs Agrobacterium Transformation, in 'Contributing to a Sustainable Future'

Techniques for the introduction of transgenes to control blackheart by particle bombardment and Agrobacterium co-transformation have been developed for pineapple cv. Smooth Cayenne. Polyphenol oxidase (PPO) is the enzyme responsible for blackheart development in pineapple fruit following chilling injury. Sense, anti-sense and hairpin constructs were used as a means to suppress PPO expression in plants. Average transformation efficiency for biolistics was approximately 1% and for Agrobacterium was approximately 1.5%. These results were considered acceptable given the high regeneration potential of between 80-90% from callus cultures. Southern blot analysis revealed stable integration of transgenes with lower copy number found in plants transformed with Agrobacterium compared to those transformed by biolistics. Over 5000 plants from 55 transgenic lines are now undergoing field evaluation in Australia

Morphological Characteristics of Genetically-Modified Pineapple Fruit

Genetic engineering is an attractive method for changing a single characteristic of ‘Smooth Cayenne’ pineapple, without altering its other desirable attributes. Techniques used in pineapple transformation, however, such as tissue culture and biolistic-mediated or Agrobacterium-mediated gene insertion are prone to somaclonal variation, resulting in the production of several morphological mutations (Smith et al., 2002). Fruit mutations can include distortion in fruit shape (round ball, conical, fan-shaped), reduced fruit size, multiple crowns, crownless fruit, fruitless crowns, and spiny crown leaves (Dalldorf, 1975; Sanewski et al., 1992). The present paper describes the variability in fruit-shape mutations between transgenic and non-transgenic fruit, and its subsequent impact on organoleptic characteristics.

Heterologous signals allow efficient targeting of a nuclear-encoded fusion protein to plastids and endoplasmic reticulum in diverse plant species

Approximately 30% of plant nuclear genes appear to encode proteins targeted to the plastids or endoplasmic reticulum (ER). The signals that direct proteins into these compartments are diverse in sequence, but, on the basis of a limited number of tests in heterologous systems, they appear to be functionally conserved across species. To further test the generality of this conclusion, we tested the ability of two plastid transit peptides and an ER signal peptide to target green fluorescent protein (GFP) in 12 crops, including three monocots (barley, sugarcane, wheat) and nine dicots (Arabidopsis, broccoli, cabbage, carrot, cauliflower, lettuce, radish, tobacco, turnip). In all species, transient assays following microprojectile bombardment or vacuum infiltration using Agrobacterium showed that the plastid transit peptides from tomato DCL (defective chloroplast and leaves) and tobacco RbcS [ribulose bisphosphate carboxylase (Rubisco) small subunit] genes were effective in targeting GFP to the leaf plastids. GFP engineered as a fusion to the N-terminal ER signal peptide from Arabidopsis basic chitinase and a C-terminal HDEL signal for protein retention in the ER was accumulated in the ER of all species. The results in tobacco were confirmed in stably transformed cells. These signal sequences should be useful to direct proteins to the plastid stroma or ER lumen in diverse plant species of biotechnological interest for the accumulation of particular recombinant proteins or for the modification of particular metabolic streams.

Efficient organogenesis of an Australian passionfruit hybrid (Passiflora edulis x Passiflora edulis var. flavicarpa) suitable for gene delivery

An efficient regeneration protocol based on organogenesis from cotyledon explants and suitable for gene delivery has been developed for an Australian passionfruit hybrid. Multiple shoots were regenerated from 30-day-old cotyledon explants on Murashige and Skoog (MS) medium containing 6-benzylvaminopurine (BAP) and coconut water. Media pulsing experiments were conducted to investigate the effect on organogenesis of exposure time of the explants to MS containing 10 mu M BAP and 10% (v/v) coconut water, i.e. passionfruit regeneration medium (PRM). Continuous exposure of these explants to PRM maximised the number of shoots produced to 12.1 per explant. However, periods on hormone-free medium improved the appearance of the shoots and increased the number of explants with shoots from 75 to 84.6%. Further, shoots exposed for 7 days to half-strength MS supplemented with 10 mu M NAA (1-napthalene acetic acid) produced twice as many plantlets than those on half-strength MS alone. Transient GUS histochemical assays indicated delivery of the uidA gene via Agrobacterium tumefaciens.