Francine M. Tremblay

Frequency of somaclonal variation in plants of black spruce (Picea mariana, Pinaceae) and white spruce (P. glauca, Pinaceae) derived from somatic embryogenesis and identification of some factors involved in genetic instability

Plants of black spruce (Picea mariana, N = 7047 individuals) and white spruce (P. glauca, N = 3995 individuals) were regenerated from a total of 87 clones over a 5-yr period by somatic embryogenesis to study factors that might be associated with the appearance of variant phenotypes. Morphological evaluation of the plants showed several types of variation. These variations were grouped into nine types: dwarfism (type A), reduced height with various form anomalies (types B, C, and D), needle fasciation (type E), abnormality in tree architecture (type F), variegata phenotype (type G), and plants with an overall regular morphology but smaller than normal plants (type H). Plagiotropic plants were also observed (type I). Each plant from types A to H (except type C where no plants survived more than 6 mo) had retained its phenotype over 4-5 yr of growth. Some of the variant types could be related to chromosomic instability: chromosome counts showed aneuploid cells for type-A and type-D plants. The type I (plagiotropism) was not related to genetic instability but rather to physiological disorders. In total, spruce variants of types A-H were obtained at relatively low frequencies, i.e., 1.0% (39/3995) for white spruce and 1.6% (110/7047) for black spruce. Statistical analyses, conducted with family, clone, and time in maintenance as variables, showed that clone was the most important source of genetic instability followed by time in maintenance.

Carbohydrate requirements for the development of black spruce (Picea mariana (Mill.) B.S.P.) and red spruce (P. rubens Sarg.) somatic embryos

Different carbohydrates were investigated for somatic embryo development of black spruce and red spruce. They were tested in a basal maturation medium consisting of Litvays salts at half-strength containing 1 g l-1 glutamine, 1 g l-1 casein hydrolysate, 7.5 μM abscisic acid, and 0.9% Difco Bacto-agar. A comparison of different sucrose concentrations showed that 6% was optimal for embryo development. Among the nine carbohydrates tested, sucrose, fructose, glucose, maltose, and cellobiose supported embryo development while arabinose, mannitol, myo-inositol, and sorbitol did not. A comparison of sucrose, glucose, and fructose at three concentrations showed that the general pattern of response for both species followed concentration expressed as a percentage, independent of the molarity of carbohydrate in the medium. Interspecific differences were observed concerning carbohydrate requirements. For red spruce, 6% fructose was found best for embryo development, while no such preference was observed for black spruce. No significant difference was observed in the number of embryos produced with 6% sucrose or 3% sucrose plus an equimolar concentration of either mannitol, sorbitol, or myo-inositol in the maturation medium, suggesting that the effect of the carbohydrate on the maturation was partly osmotic.

Maturation of black spruce somatic embryos: Sucrose hydrolysis and resulting osmotic pressure of the medium

The physiological and osmotic roles of sucrose during black spruce (Picea mariana (Mill.) B.S.P.) embryo maturation were investigated. The results showed that when both sucrose and mannitol were present in the medium, the optimum sucrose concentration varied between 4% and 6%. From these data, mannitol does not apparently replace sucrose during the maturation of somatic embryos and therefore it might not be a suitable osmoticum. For the media supplemented with 4% to 12% sucrose and various concentrations of mannitol, the osmotic pressure of the medium rose during maturation, particularly for the highest sucrose concentrations (7% to 12%). Medium containing 3% each of fructose and glucose produced fewer mature embryos compared to the medium with 6% sucrose. An increment in the osmotic potential was observed in medium with 6% sucrose in contrast to that containing 3% each of fructose and glucose. Sugar analysis revealed that the sucrose hydrolysis in the medium was detectable within 1 week of incubation and continued throughout the maturation period. Moreover, no significant uptake of the sugars was detected, since the total amount of fructose, glucose and sucrose remained constant. Our results indicate that the action of sucrose on embryo maturation is mostly achieved through an osmotic control.

Requirements for in vitro propagation of seven nitrogen-fixing Alnus species

Studies on the in vitro propagation of Alnus crispa, A. glutinosa, A. incana, A. japonica, A. rubra, A. sinuata and A. viridis indicated interspecific as well as intraspecific variations in their requirements for in vitro culture. The WPM and Blaydes media supported, respectively, growth of A. glutinosa and A. crispa but not that of both species, while the MS medium induced equal or significantly better growth than WPM and Blaydes media for both species. The optimum type and concentration of sugar to be used in the multiplication medium varied with species. Only A. glutinosa showed good growth on sucrose while glucose was optimum for all other species but at different concentrations. All species rooted in 3 weeks on half-strength MS medium including 1 μM IBA. All clones of A. glutinosa and A. rubra rooted 100%, whereas “easy-to-root” and “difficult-to-root” clones were observed in the other species. In the rooting medium, glucose promoted rooting of the “difficult-to-root” clones better than sucrose. Survival following transfer to an artificial substrate was 100% for all species. Nodulation tests using pure cultures of two Frankia strains showed 100% nodulation on all Alnus clones.

Maturation of black spruce somatic embryos. Part I. Effect of l-glutamine on the number and germinability of somatic embryos

Different concentrations of l-glutamine and different nitrogen sources in the medium were compared during maturation of black spruce (Picea mariana (Mill.) B.S.P.) somatic embryos. l-glutamine can be used as the sole nitrogen source for the maturation of Picea mariana somatic embryos at 2 to 3 gl-1. A significantly lower number of somatic embryos was obtained on a medium prepared with only inorganic nitrogen. Compared with a medium supplement to inorganic nitrogen resulted in a twofold increase in the number of embryos for six genotypes. The nitrogen source and concentration in the maturation medium significantly affected the germination sensus stricto of somatic embryos (radicle appearance), but not their development into plantlets; at the time of epicotyl appearance, an effect of the nitrogen source was no longer found. A comparison of the development of somatic embryos into plantlets from seven genotypes showed that the genotype had more effect in terms of epicotyl appearance and in conversion rate than the nitrogen source present in the maturation medium.

Effect of desiccation to low moisture content on germination, synchronization of root emergence, and plantlet regeneration of black spruce somatic embryos

A desiccation protocol was developed to evaluate the effect of different levels of desiccation on germination and plantlet regeneration of black spruce somatic embryos. Large desiccation chambers (80 l) with four liters of saturated salt solutions provided constant relative humidities (RH) of 63, 79, 88, and 97% (± 2%). Under these conditions, an embryo mass of 10 mg always dried fast even at 97% RH. In contrast, an embryo mass of 80 mg generated different kinetics of water loss, from fast drying at 63% RH to slow drying at 97% RH. Drying rates similar to those obtained with 80 mg embryos were also generated by combining 40 mg embryos with 40 mg water. The effects of drying rate and embryo MC on germination rate, root elongation, and plantlet regeneration were examined. A fast drying rate to 4–5% embryo MC, obtained under 63% RH, was detrimental to germination and plantlet development. However slower drying rates, obtained under 79–97% RH and generating 7–19% MC in the embryos, gave developmental responses similar to the control. Synchronization of root emergence was improved only for embryos desiccated to approx. 16% MC under 97% RH. The optimal desiccation protocol using large desiccation chamber at 97% RH and a constant embryo mass of 40 mg embryos plus 40 mg water was applied to five genotypes of black spruce. For all genotypes, desiccated embryos gave plantlet regeneration rates similar to the control undesiccated embryos.

Selection and micropropagation of nodulating and non-nodulating clones of Alnus crispa (Ait.) Pursh

600,000 seedlings of Alnus crispa were inoculated with a 1:1:1 mixture of the Frankia strains ACN1 AG , AGN1 exo AG and MGP10i. After 3 successive inoculations and screenings, one individual, AC-4, was selected as non-nodulating (Nod−) with Frankiae. This selected individual AC-4 (Nod−) and two other clones of A. crispa, AC-2 and AC-5, known for their ability to nodulate (Nod+) with Frankia were in vitro propagated. The different clones of A. crispa in culture required different kinds and concentrations of sugar during the in vitro multiplication and rooting stages. Nodulation tests using 7 Frankia strains indicated that the clone AC-4 (Nod−) was non-nodulating with 6 of the 7 Frankia strains tested. One strain, Frankia ANNI, isolated from one unique nodule produced on the mother-plant AC-4, induced 38% of the AC-4 plantlets to nodulate but with a number of nodules 10 to 20 times less than the clones AC-2 (Nod+) and AC-5 (Nod+). Morphological observations of the roots of AC-4 (Nod−) indicated that this clone had few and abnormally short root hairs.

Callus regeneration from Alnus incana protoplasts isolated from cell suspensions

Abstract Nagata and Takebes (NT) medium, supllementedte with 2.5 μm 2,4-dichlorphenoxyacetic acid (2,4-D), induced development of friable calluses from leaves of axenic shoot cultures of Alnus incana . Fast-growing cell suspensions were established in the same medium without agar. Suspensions gave high yields of viable protoplasts after an overnight incubation in an enzyme mixture consisting of 1% (w/v) Onozuka R-10, 0.5% (w/v) Rhozyme HP-150, 0.03% (w/v) Macerase, CPW salts, and 13% (w/v) mannitol (pH 5.8). Protoplasts cultured on K8p medium underwent cell wall regeneration within 24 h. The optimum protoplast-derived colony formation and growth was obtained on the NT medium supplemented, as was the K8p medium, with glucose as the osmoticum, growth regulators, coconut milk and casein hydrolysate. Compared with other culture techniques, the agarose bead technique of Shillito et al. (Plant Cell Reports, 2 (1983) 244) improved cell division and colony formation frequency. Protoplast-derived macrocalluses grew under the same conditions as those used for leaf calluses.

Effects of sealed and vented gaseous microenvironments on the maturation of somatic embryos of black spruce with a special emphasis on ethylene

Maturation of black spruce somatic embryos in sealed and vented microenvironments was investigated. The sealed microenvironment induced a larger number of well-formed mature embryos and less precocious germination than the vented microenvironment. Maturation rate of somatic embryos was not changed either by injection of ethylene into the culture vessel or by its removal by potassium permanganate traps. Increased as well as decreased ethylene concentrations, by the addition of either 1-aminocyclopropane-1-carboxylic acid (precursor of ethylene) or cobalt chloride (inhibitor of ethylene biosynthesis), resulted in a decreased number of embryos produced. However, inhibition of ethylene action by the addition of silver nitrate to the maturation medium did not affect either ethylene concentration or somatic embryo production. It was concluded that ethylene accumulation during maturation has no effect on somatic embryo production. Neither the microenvironment nor the modification of the ethylene metabolism affected conversion rate of somatic embryos into plantlets growing in soil.