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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.

Based on casual qualitative observations, grain sorghum [Sorghum bicolor (L.) Moench] is less tolerant to salt than the noxious weed and potential biomass energy crop, johnsongrass [S. halepense (L.) Pers.]. The objective was to quantitatively compare whole plant growth and physiological responses to salt stress of these two sorghum species. Salt stress was induced by adding incremental levels of NaCl to a vermiculite medium until concentrations of 0.1 and 0.2 M were attained (...)

Grain sorghum [Sorghum bicolor (L.) Moench], is reported to be less tolerant to salt than the noxious weed and potential biomass energy crop, johnsongrass [S. halepense (L.) Pers.]. The objective was to compare callus growth and osmotic responses to salinity of these two sorghum species. Callus was grown for 2 wk in a liquid Murashige and Skoog medium to which NaCl was added to final concentrations of 0.05, 0.1, and 0.15 M (...)

Slash pine (Pinus elliottii Engelm.) is one of the hard yellow pines indigenous to the southeastern United States (Lohrey and Kossuth 1990). It is one of the two southern pines used for naval stores, and is one of the most frequently planted timber species in North America (Lohrey and Kossuth 1990). It is favoured by many forest managers because of its fast growth and excellent utility for pulp, lumber, and poles (Sheffield et al. 1983). It has the smallest native range of the four southern pines and grows naturally from South Carolina south to central Florida and west to Louisiana. It has been established by planting as far north as Tennessee and as far west as eastern Texas, where it now seeds naturally (Lohrey and Kossuth 1990). In the three decades prior to 1980, the P. elliottii ecosystem increased by 22% and peaked in the late 1970s and early 1980s (Sheffield et al. 1983). Today, the rate of planting outside the natural range has slowed compared to the 1950 to 1970 period.

The induction of gene expression in response to environmental or hormonal cues, has been studied mostly at the of transcriptional level. The importance of post-transcriptional events in determining the final protein levels, however, is now becoming clear. As part of a program to investigate gene expression in woody plants growing under water deficit, we have isolated several cDNA clones of a Proteinase Inhibitor-like gene family from the woody desert shrub, Atriplex canescens. Northern analysis shows a broad inducible signal in response to water deficit. Reverse Transcription -PCR (RT-PCR) experiments show that levels of the individual transcripts are altered to different degrees by exposing plants to water deficit or Abscisic acid. Sequence analysis reveals several motifs in the 3′-untranslated region (3′-UTR) of the mRNA which may contribute to RNA stability or translatability. In a parellel program a cDNA for water deficit-repressible gene is shown to have unusual structure. The longest open reading frame begins over halfway through the mRNA and the 5′-untranslated region (5′-UTR) has extensive secondary structure and several upstream open reading frames (uORFs). These 5′-UTR features are involved in post-transcriptional regulation of gene expression in other eukaryotic systems. Elucildating the function of these sequences will assist in understanding the regulation of stress-inducible genes and in the design of plant transformation vectors to optimize the expression of foreign genes.

Slash pine (Pinus elliottii Engelm.) is a hard yellow pine indigenous to southeastern United states. It is a rapid-growing, straight, symmetrical tree attaining a height of 100 ft, with a diameter of 1 m. The wood is considered to be the hardest, strongest, and heaviest of all commercial conifers in the United States. It starts producing seeds at the age of 15–20 years with good production every 2–3 years. It is one of the two southern pines used for naval stores and also one of the most frequently planted timber species in North America (Lohrey & Kossuth, 1990). It is favored by many forest managers because of its fast growth and excellent utility for pulp, lumber, and poles (Sheffield et al., 1983). It has the smallest native range of the four southern pines and grows naturally from south of South Carolina to central Florida and west to Louisiana. However, its range has been extended with northern plantings in Tennessee and western planting in east Texas where it now seeds naturally (Lohrey & Kossuth, 1990). Plantings outside its natural range mostly occurred over three decades spanning from the early 1950s through the latter 1970s. During this time, the slash pine ecosystem increased by 22% (Sheffield et al., 1983).

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 Effects of water stress on protein synthesis were investigated in heterotrophic callus cultures derived from slash pine (Pinus elliottii Engelm.) cotyledon explants. Cultures were transferred to medium containing O-15% mannitol to provide water potentials between -0.4 and - 2.5 MPa. Beginning between 0 and 22 h after transfer to the new medium, cultures were incubated for 2 h with T-methionine. Both the uptake of label and the incorporation of label into protein decreased with decreasing medium water potential. Incorporation, however, was reduced to a greater extent than uptake suggesting that the reduced incorporation was at least partly the result of reduced protein synthesis. Separation of labeled proteins by one-dimensional SDS-polyacrylamide gel electrophoresis and visualization by fluorography revealed the induction of two new protein bands after incubation at - 1.8 MPa for 24 h. Induction of new proteins was not observed at other water potentials or after shorter incubation times. Protein bands from cultures incubated at - 1.8 MPa for 24 h and then transferred for 24 h to mannitol-free medium ( -0.4 MPa) were comparable to those from control cultures maintained on mannitol-free medium throughout.

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.

Silvicultural treatments and the development of site-adapted genotypes can aid in the management of drought-stress in forest species. Both pre- and post-planting silvicultural treatments of the seedling and site have proven effective in enhancing survival and growth under drought-stress conditions. Silvicultural treatments modify the stand and its environment and, consequently tree physiological mechanisms in response to drought; this modifies the water balance in forest stands. Evapotranspiration is directly related to stand leaf area. A balance between water loss and productivity can be accomplished by use of silvicultural regimes which manipulate crop leaf area and eliminate non-crop competing leaf area.

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.