Induction of secondary somatic embryogenesis was studied with hybridlarch (Larix x leptoeuropaea)cotyledonary somatic embryos obtained after 3, 4, 5 and 6 weeks of culture on amaturation medium supplemented with abscisic acid. Almost all 3-week maturedcotyledonary somatic embryos can develop embryonal masses whereas only 78, 27and 12% of them are able to do so after 4, 5 and 6 weeks of maturation,respectively. During the first week of culture on the induction medium, somaticembryos with high embryogenic potential (i.e. 3-weekmatured) release little ethylene (less than 1.5 nL h−1g−1 FW), whereas those which have almost completelylosttheir ability to induce embryonal masses (i.e. 6-weekmatured) produce much more ethylene. Thereafter, ethylene production by bothtypes of embryos is very similar at around 5–6 nLh−1 g−1 FW. Enrichment of theatmosphere with ethylene, or addition of 2-chloroethylphosphonic acid(ethephon)or ACC in the induction medium strongly reduced the induction of secondarysomatic embryogenesis. Moreover, inhibitors of ethylene action(AgNO3and 2,5-norbornadiene) improved the development of embryonal masses fromsomaticembryos, particularly from the 6-week maturated ones. The results obtainedclearly suggest that ethylene is involved in the regulation of somaticembryogenesis in hybrid larch. The possible relationship between somaticembryogenic potential and ethylene biosynthesis by the explants or sensitivityof the latter to ethylene is discussed.

Larches are deciduous needle-leaf gymnosperms family Pinaceae in the genus Larix which is composed of ten species, with numerous varieties and hybrids. Larches are well adapted to regions where the climate is typically cold with a short growing season: some larch species are native to the lowlands of boreal zones (Larix laricina in Northern America, L. russica and L. gmelinii in Siberia), whereas others are localized at the subalpine or montane level in more temperate areas (L. decidua in Europe, L. occidentalis and L. lyalli in the Rocky Mountains, L. kaempferi in Japan, L. griffithiana in Himalayas, L. potaninii and L. mastersiana in China; Schmidt 1992).

Conifers such as hybrid larch (Larix x leptoeuropaea) are wanted for reforestation programmes. To improve productivity in somatic embryogenesis, we need a better understanding of the physiology of growth of hybrid larch embryogenic masses cultured in vitro, This paper compares the hormonal status of growing embryogenic masses and nonembryogenic callus. Hormonal analyses were done using an HPLC purification and fractionation of the samples coupled to an ELISA method for hormonal quantitation involving several antibodies elicited against each hormonal family. Hormones analysed were abscisic acid, indole-3-acetic acid, zeatin, isopentenyladenine and their main metabolite as well as 2,4-dichlorophenoxyacetic acid (2,4-D) and benzyladenine (BA) used in the culture medium. Growing embryogenic masses of hybrid larch are distinguishable by their high 2,4-D, indole-3-acetylaspartic acid, abscisic acid glucose ester and isopentenyladenosine level, and low BA level. This report constitutes an original description of such hormonal status during somatic embryo proliferation in hybrid larch and generally for gymnosperms.

A transformation procedure was developed for hybrid larch embryogenic tissue using Agrobacterium tumefaciens. The cocultivation procedure yielded one to two transformation events per 100 cocultivated masses. The addition of 100 µM coniferyl alcohol increased the yield. This improved procedure was successfully applied to three other genotypes. After 3 months on selective medium, the transgenic tissue remained embryogenic, which allowed production of transgenic plants in the greenhouse. Stable integration of the transgene was confirmed by PCR and Southern hybridisation on transformed tissues and acclimatised plants.

Cotyledonary somatic embryos ofLarix × leptoeuropaea that developed after various maturation times on media containing abscisic acid showed different frequencies of conversion into plants. Drying of these somatic embryos under high relative humidity (RH) before germination improved plantlet recovery and eliminated differences in the performance of somatic embryos matured for different times. However, dehydration of somatic embryos under 98% RH to a water content below that of zygotic embryos excised from mature seeds (0.97 and 1.36 g H2O/g dry weight, respectively) showed a strong positive correlation between longer maturation time and desiccation tolerance. Drying somatic embryos at 4° C under 59% RH for 1 wk resulted in desiccation to a water content of 0.30 g H2O/g dry weight, which was the closest to the hydration state of zygotic embryos in dried, stored seeds (0.20 g H2O/g dry weight). Under this condition, only somatic embryos matured for 5 wk germinated and produced plantlets at a relatively high frequency (73 and 41%, respectively).

Germination and plantlet development in somatic embryos of Larix x leptoeuropaea were affected by the duration of the maturation treatment and the concentrations of sucrose and abscisic acid in the maturation media. Extension of the maturation period from 3 weeks to 4 weeks resulted in a significant decrease in germination and plantlet development frequencies. There was no significant effect of abscisic acid concentration on either the number of somatic embryos germinated or the number of plantlets obtained, but it affected the rapidity of the epicotyl development. Sucrose at 0.2 M, applied during maturation, was significantly more beneficial in attaining high germination rates than at 0.1 M. High germination rates (92 and 93%) and plantlet development rates (74 and 80%) were achieved when somatic embryos were matured for a 3-week period on media with either 40 or 60 μM abscisic acid, respectively, and 0.2 M sucrose prior to transfer to the growth regulator-free germination medium. Two acclimatization methods were applied: the first required 10 to 12 weeks and ensured 97% plantlet survival under greenhouse conditions; the second required 2–3 weeks and ensured 86% plantlet survival. This represents the first detailed study of the effects of maturation regimes on the recovery of somatic embryo-derived plants of Larix.