Production of transgenic pineapple (Ananas cosmos (L.) Merr.) plants via adventitious bud regeneration

Abstract

A new protocol for the production of transgenic pineapple plants was developed. Adventitious buds were induced directly from Agrobacterium-infected leaf bases and stem discs of in vitro plants, bypassing the establishment of callus cultures. Non-chimeric transgenic plants were obtained by multiple subculturing of primary transformants under increasing levels of selection. A total of 42 independent transgenic lines were produced from two cultivars with two different constructs: one containing a modified rice cystatin gene (Oc-IΔD86) and the other with the anti-sense gene to pineapple aminocyclopropane synthase (ACS). GUS histochemical staining provided the first evidence of the non-chimeric nature of the transformed plants. Their non-chimeric nature was further demonstrated by PCR analyses of the DNA extracted from individual leaves of a primary transformed plant and also from multiple plants propagated from a single transformation event. Southern hybridization confirmed random integration patterns of transgenes in the independent lines. For the Oc-IΔD86 gene, the expression at the mRNA level was detected via RT-PCR and its translation was detected by protein blot. Agronomic evaluation and bioassays of the transgenic plants will further validate the utility of this new tool for pineapple improvement.

Preservation and Faithful Expression of Transgene via Artificial Seeds in Alfalfa

“…Thus, the transgene integration in plant genomes remained stable and was not affected by the desiccation process. Different lines had different hybridization patterns as found in other studies [42], [43]. Although the hybridization patterns in P2 and P3 were alike, indeed, these were different and independent transgenic plants.…”

Direct shoot bud organogenesis and plant regeneration from pre-plasmolysed leaf explants in Catharanthus roseus

“…Direct shoot bud organogenesis also helps in avoiding the interference of somaclonal variation in the transformation process when an intervening callus interface also becomes involved in the regeneration process (Annapurna and Rathore 2010;Ghimire et al 2010;Liu et al 2010). Though such direct de novo regeneration of adventitious shoot buds and plants from cultured tissue may frequently follow a multicellular and multihistogenic pattern of origin, they normally allow the recovery of more stable and uniformly transformed plants in comparison to those arising from the cells of a preformed meristem like axillary/apical shoot buds or somatic embryos with pre-defined germ-lines (Newell 2000;Yan and Wang 2007;Zhu et al 2007;Wang et al 2009). In case of C. roseus, such an attempt is limited to just one report wherein petiole explants were tested for adventitious shoot bud regeneration (Lee et al 2003).…”

Effective Agrobacteriummediated transformation of pineapple with CYP1A1 by kanamycin selection technique

“…Pineapple is self-incompatible and its breeding is a lengthy process (Botella et al, 2000) owing to long generation time (about 4 years), and these traits lagged the improvement of physiological and horticultural traits. Although, extensive efforts have been made to produce improved varieties via hybridization of this self-incompatible monocot species, only a few new hybrids have been released worldwide (Wang et al, 2009). Due to high level of genomic heterozygosity and apparent genome instability, conventional *Corresponding author.…”