Evaluation on in vitro somatic embryogenesis and optimization of genetic transformation via transient expression for pepper (Piper nigrum L.)

Chen, Teck You (2012) Evaluation on in vitro somatic embryogenesis and optimization of genetic transformation via transient expression for pepper (Piper nigrum L.). Masters thesis, Universiti Malaysia Sarawak.

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Abstract

Black pepper (Piper nigrum L. ) is one of the important spices in the world. Pepper has numerous uses and functions to consumers however the occurrence of pests and diseases have become main problem to pepper cultivation. Conventional approach of pepper breeding is hampered by absence of pest and disease resistance genes in the species. Molecular biological approach of plant breeding could assist and complement the conventional approach of breeding for pest and disease resistance. In order to adopt molecular biological approach of pepper breeding, the present study was conducted to develop an efficient regeneration and genetic transformation system for pepper. A successful regeneration of somatic embryos was obtained using micropylar of seeds cultured on MS medium supplemented with 0.3% activated charcoal) When maintenance extended to four months, cyclic secondary somatic embryo formation on root pole explants enabled mass production of pepper plantlets. The in vitro generated somatic embryos were used as source of explants for developing transformation system whilst a number of somatic embryos were left to grow into plantlets to deter plants. For developing a transformation system for pepper, Agrobacterium-mediated and particle bombardment approaches were applied by initially monitoring the transient gene expression. However, due to gxplants browning problem, the GUS reporter gene was found unsuitable for use in Agrohacterium -mediated transformation technique. Consequently, GFP reporter gene was employed in particle bombardment method and transient green fluorescent spots were discovered on transformed explants. The p35SCaMV-sgfpS65T construct co-bombarded with pCambia 1300 showed the highest transient expression spots among all the GFP constructs examined. After optimization of bombardment parameters, 1.0 μm microcarriers, 900 psi pressure and 6 cm distance were found optimal for producing green transient fluorescent on explants. However, no single transgene was detected from in vitro regenerated putative transformed plantlets via PCR analysis. Based on the result from RAPD analysis, no genetic variation was detected among mother plants and somatic embryo plantlets themselves respectively. However, small polymorphism was found when compared mother plant and somatic embryo plantlets using OPD 05, OPM 04 and OPP 16 primers.

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