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Clonal propagation by tissue culture of Afghan pine (Pinuseldarica Medw.) could be useful for improving Christmas tree production and afforestation of semiarid lands in North America. The objectives were (i) to develop organogenesis procedures for clonal propagation and (ii) to evaluate plantlet performance in the field. Shoot production from cotyledon explants was accomplished on solid Shenk and Hildebrandt medium amended with kinetin (11 μM) and benzyladenine (11 μM). Over 100 shoots per clone were obtained with this method. Spontaneous rooting was prevalent in some of the induced shoots. Shoots without root primordia were induced to form roots with naphthalene acetic acid (5 μM), indole butyric acid (10 μM), and benzyladenine (0.22 μM) in 1/2 strength Shenk and Hildebrandt medium. The rate of plantlet elongation in the field was similar to that of seedlings; however, survival of plantlets was lower than that of seedlings. The lower survival of plantlets was directly related to their smaller size at the time of planting. Organogenesis appears to be a very suitable method for mass micropropagation of this environmentally and economically useful tree.

Summary The physiological responses of osmotic adjustment and turgor maintenance may be critical for seedling survival under drought stress. The objectives were to determine: 1) if osmotic adjustment in shoots and changes in shoot tissue elasticity could be induced by cyclical gradual development of water deficits in seedlings, 2) if osmotic adjustment occurred in roots, and 3) to characterize important solutes contributing to osmotic adjustment. Cyclical drought stress treatments were applied to 5- to 6-month-old Pinus taeda L. seedlings from two half-sib families in a controlled-environment chamber by withholding irrigation until predawn xylem potentials reached desired levels. Predawn xylem water potentials did not fall below -0.6MPa in the irrigated, nonstressed seedlings, while in the stressed treatment, seedlings were irrigated to saturation only when predawn xylem potentials reached -1.5 to - 2.0 MPa. Pressure-volume curve analysis and solute analyses after five or more stress cycles confirmed significant osmotic adjustment. Osmotic adjustment through proline and monosaccharide (fructose, glucose) accumulation in fascicles was observed. Shoot tissue elasticity was reduced by drought stress. Osmotic adjustment was also demonstrated in both suberized and nonsuberized root portions of 16-month-old seedlings after only one drought cycle. No differences between the two families in cell wall elasticity capacities were observed. Pressure-volume curve analyses showed no significant differences in osmotic adjustment between the two families.

Clonal propagation of Virginia Pine (Pinus virginiana Mill.) was achieved by organogenesis on cotyledon explants. The influence of several cytokinins and abscisic acid on adventitious shoot production from cotyledon explants was investigated. Benzyladenine was more effective in shoot induction than three other cytokinins tested. Benzyladenine (22.2 μM) in combination with naphthaleneacetic acid(0.05 μM) in a Gresshoff and Doy (1972) medium was found to increase shoot bud production. Abscisic acid (7.6 μM) in combination with benzyladenine and naphthaleneacetic acid enhanced shoot formation by an additional 65%. Root initiation was achieved with 0.5 strength Gresshoff and Doy media amended with naphthaleneacetic acid (1.3 μM), indole-3-butyric acid (1.2 μM) and benzyladenine (0.4 μM). Over 2400 plantlets from 2 families survived and were transferred to a greenhouse in preparation for field planting. After ten months, the maximum number of surviving plantlets/seed explant from these two sources was 57 for family ALPV-38 and 41 for family ALPV-78, respectively.

Summary The relationship between mannitol-induced water stress and protein synthesis was investigated in hypocotyl slices of loblolly pine (Pinus taeda L.). Mannitol-.aduced water stress inhibited the incorporation of L-[%]methionine into protein. As the water potential decreased, incorporation of label into protein decreased in both the soluble- and membrane-protein fractions. There were no significant differences in response to water stress among seed sources from four different geographical regions.

Embryogenic callus development was initiated in cultured immature embryo explants of four different genotypes of slash pine (Pinus elliottii) on several culture media supplemented with auxin and cytokinin. Callus induction, either from the suspensor cells at the base of immature embryos or from the cotyledons of immature embryos, started after 1–2 weeks. As the callus started to proliferate, three types of calli were distinguished; white mucilaginous, loose globular, and light green-loose. Histochemical staining, with acetocarmine, indicated that only white mucilaginous calli were embryogenic. Non-embryogenic loose globular and light green-loose calli continued to retain their nature even after several subcultures. The origin of somatic proembryos in Pinus elliottii was from single cells of the suspensor cell callus. The developmental stage of the zygotic immature embryos, genotype and culture medium are critical factors for the production of embryogenic callus. Somatic proembryos are being cultured for further development.

Water status of Pinus taeda L. callus supported on Murashige and Skoog (MS) liquid medium was characterized over an 8 week period using thermocouple psychrometry. Medium with 30 gl−1 sucrose was used to produce a high water potential (Ψw) of −0.4 MPa (H), and the same medium was used to create a moderate Ψw of −0.7 MPa (M) by the addition of 10% polyethylene glycol (PEG, w/v, MW=8000). Calli were produced from cotyledon explants on H medium for 2 weeks and then transferred to either M or H medium. Callus absorption of PEG accounted for 40% of the callus dry weight and less than 7% of the callus fresh weight. Callus dry weight (without the PEG fraction) on M medium was 40% of that observed on H medium. Fresh weight on M medium was only 15% of that observed on H medium. The Ψw of both H and M media remained constant throughout the culture period. On H medium, callus Ψw and osmotic potential (Ψs) both increased 0.05 MPa/week with the callus Ψw approaching that of the external medium. On M medium, callus Ψw and Ψs both decreased more than 0.1 MPa/week with the callus Ψw decreasing greatly below that of the external medium. The latter was attributed to a rapidly produced osmotic shock induced upon callus transfer and/or PEG which caused less callus hydration and resulted in reduced growth. Callus turgor potential (Ψp) was estimated to be +0.02 to +0.09 MPa and turgor was maintained as callus Ψw increased or decreased. After 8 weeks, cell volumes from callus on M medium were 50 to 60% less than on H medium, suggesting that reduced cell volumes were related to turgor maintenance.

Embryogenic callus developed in 55% of the mature embryo explants of Norway spruce (Picea abies L.) growing on a LP medium minus the amino acids and sugars (except sucrose). This is the highest reported yield of embryogenic callus from mature embryos of P. abies that has ever been reported. Callus induction from either the middle or the end of the hypocotyl of the embryos began after 2-3 weeks. Three types of calli were recovered: (a) globular, (b) light green-compact, (c) white mucilaginous. Only the white mucilaginous calli were embryogenic. The globular and light green-compact calli never become embryogenic, even after several subcultures. The development of somatic embryos was accomplished on half-strength macro-elements of NSIII medium containing 1 μM α-naphthaleneacetic acid, 1 μM abscisic acid, and 3% sucrose. The addition of 10(-7) M buthionine sulfoximine to the medium increased the development of somatic embryos by three fold. These results suggest that there is a great potential for increasing the frequency and development of somatic embryos in P. abies. Careful selection of the genotype and modification of the culture medium is required.

Osmotic adjustment is a physiological process by which trees accumulate solutes in response to drought stress. It benefits the plant by maintaining turgor pressure and hence continued but reduced growth. Osmotic adjustment operates by the accumulation of soluble carbohydrates and amino acids within stressed tissues. An indirect consequence of osmotic adjustment is that it raises the nutritive value of affected tissues as a food source for herbivores. Osmotic adjustment without other metabolic changes therefore increases the vulnerability of stressed trees to pest attack.

Plant regeneration from protoplasts facilitates gene transfer, somatic cell fusion, and mutation. Genetic variability in plants regenerated from tissue culture is termed “somaclonal variation” (Larkin and Scowcroft, 1981), and we propose that variation observed in protoplast derived plants be referred to as “proto-variation”. Our aims were 1) to regenerate plants from Brassica napus L. protoplasts; and 2) to select desirable proto-variants (p-variants) with herbicide resistance and high seed quantity.