Larix laricina (tamarack): Somatic embryogenesis and genetic transformation

Excised, immature zygotic embryos of Larix laricina (Du Roi) K. Koch (tamarack) gave rise to embryogenic cultures on modified Murashige and Skoog (MSG) medium supplemented with growth regulators. Three lines of embryonal masses were maintained for 1 year by biweekly subcultures prior to the maturation experiments. All of those lines showed the ability to produce mature somatic embryos. Both elevated medium osmolality (315.0-543.6 mmol.kg(-1)) and presence of abscisic acid (ABA) at 40 mu M stimulated the maturation process when applied simultaneously. Sucrose was most effective at 0.4 M, and polyethylene glycol (PEG) at 5 or 10% was effective only in combination with 0.2 or 0.4 M sucrose. The germination frequency of somatic embryos depended on both osmolality and ABA concentration in the maturation medium. Over 90%;, of mature somatic embryos were capable of secondary somatic embryogenesis when placed on the induction medium. This particular ability was exploited in order to achieve genetic transformation. Four vectors were delivered to the embryonal masses and somatic embryo cells via bombardment of DNA-coated Sold particles. The vectors pBI426 and pRT99gus carried a gene encoding resistance to kanamycin, pRT66gus to hygromycin, and pRT55gus to methotrexate. All vectors carried the gene coding for beta-glucuronidase (GUS) and were over 6 kilobases in size. Assays for both transient and stable transformation were carried out. The only vector that yielded two transgenic lines was pBI426. These lines of embryonal masses, designated as 2D1 and 2D2, were analysed by hybridization of the plasmid to the genomic Southern blots and revealed several insertions of the vector. Line 2D1 gave rise to young germinants that expressed the GUS gene uniformly throughout the root, hypocotyl, and cotyledons but failed to develop further. Line 2D2 gave rise to transgenic plants that displayed random and ''patchy'' expression of the GUS gene. Polymerase chain reaction (PCR) amplification of the GUS insert in 6-month-old 2D2 transgenic plants showed the presence of diagnostic fragment in all parts of the plants.