Biologia Plantarum

, Volume 49, Issue 3, pp 361–370 | Cite as

Agrobacterium-mediated transformation of Pisum sativum in vitro and in vivo

Article

Abstract

Six pea (Pisum sativum L.) cultivars (Adept, Komet, Lantra, Olivin, Oskar, Tyrkys) were transformed via Agrobacterium tumefaciens strain EHA105 with pBIN19 plasmid carrying reporter uidA (β-glucuronidase, GUS, containing potato ST-LS1 intron) gene under the CaMV 35S promoter, and selectable marker gene nptII (neomycin phosphotransferase II) under the nos promoter. Two regeneration systems were used: continual shoot proliferation from axillary buds of cotyledonary node in vitro, and in vivo plant regeneration from imbibed germinating seed with removed testa and one cotyledon. The penetration of Agrobacterium into explants during co-cultivation was supported by sonication or vacuum infiltration treatment. The selection of putative transformants in both regeneration systems carried out on media with 100 mg dm−3 kanamycin. The presence of introduced genes was verified histochemically (GUS assay) and by means of PCR and Southern blot analysis in T0 putative transformants and their seed progenies (T1 to T3 generations). Both methods, but largely in vivo approach showed to be genotype independent, resulting in efficient and reliable transformation system for pea. The in vivo approach has in addition also benefit of time and money saving, since transgenic plants are obtained in much shorter time. All tested T0 – T3 plants were morphologically normal and fertile.

Additional key words

grain legumes pea transgene integration transgene stability 

Abbreviations

BAP

6-benzylaminopurine

CN

cotyledonary node

GUS

β-glucuronidase

LK medium

medium according to Langley and Kado (1972)

MSB medium

medium with mineral salts according to Murashige and Skoog (1962), vitamins according to Gamborg et al. (1968)

MS-salts

mineral salts according to Murashige and Skoog (1962)

NAA

α-naphthaleneacetic acid

PCR

polymerase chain reaction

S

seed

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Copyright information

© Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Praha 2005

Authors and Affiliations

  1. 1.AGRITEC Ltd., Plant Biotechnology DepartmentSumperkCzech Republic

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