Efficient Biolistic Transformation and Regeneration Capacity of an EgTCTP Transgene in Protocorm-like Bodies of Phalaenopsis Orchid

Kantamaht Kanchanapoom,Amornrat Phongdara,Kamnoon Kanchanapoom,Alisa Nakkaew

doi:10.15835/nbha4017256

Kantamaht Kanchanapoom, Amornrat Phongdara + Show 2 more

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https://doi.org/10.15835/nbha4017256

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Abstract

An efficient genetic transformation system using the biolistic method and protocorm-like bodies (PLBs) of Phalaenopsis orchidwas established for introducing the EgTCTP gene obtained from oil palm leaves. A pCAMBIA 1302 vector containing the greenfluorescent protein (mgfp5) as reporter gene and the selectable marker hygromycin phosphotransferase (hpt) gene under the cauliflowermosaic virus (CaMV) 35S promoter were used in this experiment. The transformed PLBs were cultured on MS medium containing 20mg/L hygromycin for 2 months. The surviving PLBs with green fluorescence spots were used to calculate a transformation frequency(93.34%). PLBs containing the transformed EgTCTP gene had the highest percentage of regeneration frequency (95.66%) and numbersof regenerated shoots per explant (3.78Â±1.89 shoots) compared to the control. The time required for initiation of primordial shoots inthe transformed PLBs (55.22Â±26.56 days) was much shorter than for the control. Evaluation of the regeneration efficiency, determinedthat the status of the EgTCTP transformants was above average: score=4.04Â±0.88. The EgTCTP gene was detected in the PLBs overa period of at least 6 months with subculturing every 4 weeks. The stability of the transgenes within the PLBs was confirmed by PCRand this indicated that the transgenes had been integrated into the genome of the transformants. This is the first successful report tointroduce EgTCTP gene into PLBs of Phalaenopsis orchid.

Highlights

Phalaenopsis, a member in the family Orchidaceae, is one of the most popular epiphytic orchids with high economic value in the orchid trade all over the world
Plasmids The pCAMBIA 1302 vector (CAMBIA, Australia), containing an EgTCTP gene as the transgene together with the green fluorescent protein reporter gene and the selectable marker hygromycin phosphotransferase gene driven by the cauliflower mosaic virus (CaMV) 35S constitutive promoter was used in this experiment
Transformation frequency in the bombardment of Phalaenopsis protocormlike bodies (PLBs) In this experiment the transformation frequency in the survived Phalaenopsis PLBs was much higher when compared to that obtained by the bombardment of the PLBs of Phalaenopsis (‘Danse’ × ‘Happy Valentine’)

Summary

Introduction

Phalaenopsis, a member in the family Orchidaceae, is one of the most popular epiphytic orchids with high economic value in the orchid trade all over the world. It is noted for its good form, beauty, long-lasting flowers and long inflorescence which make it eminently suitable for ornamental plants. An Agrobacterium-mediated transformation method was used to produce orchid PLBs (Chai et al, 2002; Chan et al, 2005; Liau et al, 2003) since the origin of the orchid PLB is from a single somatic cell, that has a high capability of regenerating into a new plant, is easy to root, presumed to be genetically uniform and can be induced efficiently from various somatic tissues including young leaves and stem segments (Chen and Chen, 2007; Yee et al, 2008)

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