Harry E. Sommer

Differentiation of Plantlets in Longleaf Pine (Pinus palustris Mill.) Tissue Cultured In vitro

Embryos of longleaf pine (Pinus palustris Mill.) were induced to form numerous adventitious buds along their cotyledons when placed in culture on chemically defined nutrient media. The buds when excised and transferred to other media form roots and vigorous plantlets. Plantlets were obtained from numerous embryos using three different seed sources. Histological observations describing these patterns of differentiation are presented.

High-frequency induction of adventitious shoots from hypocotyl segments of Liquidambar styraciflua L. by thidiazuron

The effects of thidiazuron (TDZ) on adventitious bud and shoot formation from hypocotyl segments of sweetgum (Liquidambar styracifiua) were tested alone and in combination with 2,4-dichlorophenoxyacetic acid (2,4-D). The combination of 1 mg/1 TDZ with 0.01 mg/l 2,4-D resulted in the highest frequency of bud production. Lower concentrations of TDZ stimulated shoot production, generating the most shoots at 0.1 mg/1 TDZ with 0.01 mg/1 of 2,4-D. Inhibition of shoot elongation by TDZ was overcome by transferring shoot cultures to a shoot proliferation medium lacking TDZ or containing naphthaleneacetic acid and benzyladenine in addition to TDZ. Shoot production in liquid culture was significantly greater than that in solid culture. Comparisons of in vitro and ex vitro rooting of the adventitious shoots demonstrated that ex vitro rooting produced plants with faster growth rates and more extensive root systems.

Maturation and conversion ofLiriodendron tulipifera somatic embryos

Embryogenic suspension cultures of the hardwood forest tree yellow-poplar (Liriodendron tulipifera) have the potential to produce millions of plantlets. However, low conversion frequencies limit the realization of this potential. Using 4 embryogenic yellow-poplar lines, we first tested the ability of somatic embryos, selected for their similarity to mature zygotic embryos, to convert to plantlets, then tested physical and chemical treatments for their effects on promoting maturation of somatic embryos and subsequent plantlet production. Embryos selected based on resemblance to mature zygotic embryos and transferred to a hormone-free basal medium without casein hydrolysate (CH) produced plantlets at a frequency of 63%. Populations of synchronized somatic embryos were obtained by repeated fractionation of liquid medium-cultured proembryogenic masses (PEMs) on stainless steel sieves. These fractionated embryos failed to mature properly when cultured in liquid basal medium, however. Development of embryos cultured in basal medium supplemented with 5×10−7 M abscisic acid (ABA) was slowed and embryos appeared to mature properly, with separated cotyledons and little precocious germination. However, ABA-treated embryos only rarely converted to plantlets, possibly due to residual effects of the ABA. PEMs fractionated on sieves, transferred to filter paper and placed on solidified basal medium gave a 60–70% synchronous population of mature embryos 10–12 days following plating. Mature embryos transferred to basal medium without CH converted at a frequency of 72%. The percentage of all embryos differentiating from PEMs on filter paper that formed plantlets was 32%.

Relation of the developmental stage of zygotic embryos of yellow-poplar to their somatic embryogenic potential.

The goal of the study was to characterize the optimal developmental stage of zygotic embryo expiants of the hardwood forest tree species yellow-poplar (Liriodendron tulipifera L.) for the initiation of embryogenic cultures, using morphological measurements and polypeptide profiles of the embryos. Developing zygotic embryos from seeds of six full-sib families, collected every two weeks from 4 weeks postpollination until seed maturity (18 weeks postpollination) were divided into 2 subsamples for each collection date. One group was used to initiate tissue cultures. Embryos in the other group were measured (total length, cotyledon length and hypocotyl thickness) and soluble polypeptide profiles of the embryos were analyzed by SDS-polyacrylamide gel electrophoresis. Potential of an expiant to produce an embryogenic culture peaked during the eighth week following pollination, with an average of 28% of the expiants producing proembryogenic masses, and declined to near zero for mature zygotic embryos. The maximum embryogenic potential corresponded to the globular stage of embryo developmet. Soluble protein profiles of zygotic embryos from 5 sampling dates indicated that decline in embryogenic potential appeared to parallel an increase in the level of a polypeptide of approximately 55 kDa, possibly a storage protein.

Transformation of sweetgum via microprojectile bombardment of nodule cultures

SummaryA sweetgum (Liquidambar styraciflua) nodule culture system was developed and integrated with genetic transformation by microprojectile bombardment. Nodule cultures were established from seedling hypocotyls and proliferated in liquid medium containing 0.1 mg (0.45 µM) thidiazuron (TDZ) per 1 and 0.01 mg (0.045 µM) 2,4-dichlorophenoxyacetic acid (2,4-D) per 1. Shoots differentiated from the nodules in liquid media containing (per 1) 1 mg (4.4 µM) benzyladenine (BA), 0.5 mg (2.2 µM) BA, and 0.01 mg (0.054 µM) naphthaleneacetic acid (NAA), or 0.5 mg BA, 0.01 mg NAA, and 0.05 mg (0.23 µM) TDZ under the light. Differentiating shoots required 4 wk of dark treatment for further development on semisolid medium containing 1 mg BA per 1. Elongated shoots were harvested and the basal ends were soaked in a solution containing 10 mg (49.2 µM) indole-3-butyric acid (IBA) per 1 before being planted in potting mix for ex vitro rooting. Roots formed and leaves expanded in 2 wk. Sweetgum nodules were stably transformed by microprojectile bombardment with a 7.4-kb plasmid, pTRA 140, harboring CaMV 35S-HPH and CaMV 35S-GUS. Evidence that nodules growing in the presence of hygromycin B were stably transformed was provided by polymerase chain reaction analysis and β-glucuronidase activity. Sweetgum shoots differentiated in liquid medium in the presence of hygromycin B. Shoots transferred to solid medium lacking hygromycin B elongated and displayed β-glucuronidase activity in their expanding leaves and stems. Southern analysis confirmed the presence of the GUS gene in nodules and shoots. Transgenic shoots initiated roots and showed leaf expansion 2 wk after being planted in potting mix.

In vitro culture of Liriodendron tulipifera

Liriodendron tulipifera L. (yellow-poplar, tulip-poplar, tuliptree), a native of eastern North American, is one of two extant species of Liriodendron (Magnoliaceae). The other, L. chinense (Hemsl.) Sarg. (Chinese tuliptree), is native to central mainland China. The natural range of yellow-poplar is throughout the eastern United States and into southern Ontario, Canada. Although it grows on a wide variety of sites, it is most abundant and reaches its largest size in the lower Ohio river valley and in the mountains of North Carolina, Tennessee, Kentucky and West Virginia [12]. Yellow-poplar is an early stage successional species on most sites, but its rapid growth rate and the large size of mature trees (up to 60 m), make it a dominant canopy tree. Yellow-poplar possesses many desirable traits which make it one of the most important hardwood species in the United States. Besides rapid growth, it is also known for its straight form, self-pruning ability and wood of exceptional working quality [49]. Large amounts of the wood are used for furniture (mostly hidden parts), plywood, corestock, millwork, siding and other light construction lumber. It is also used for pulping and for products such as chipboard and flakeboard [36]. Bees are a primary pollinator of yellow-poplar, producing a popular honey. Finally, with its symmetrical leaves and yellow, green and orange flowers, yellow-poplar is widely planted both in the United States and abroad as an ornamental species. A number of horticultural cultivars have been described for the species [39].

The influence of intermittent soil drying on growth, cell number, and final cell length in the pith of mature internodes in Helianthus annuus L. and Liquidambar styraciflua L.

The sensitivity of cell division in developing internodes to plant water deficits has not been previously documented. In this study two diverse taxa, Helianthus annuus L. and Liquidambar styraciflua L. were chosen because cell divisions in the pith and cortex continue to occur acropetally throughout the period of internode elongation. Potted plants were given 6-d cycles of soil drying between waterings to observe the effects of moderate, intermittent water deficits on final cell pattern in developing internodes. Under this regime, internode and leaf growth were inhibited although leaf and shoot turgidity were restored daily by nocturnal rehydration. The percent inhibition of final internode lengths was similar in both taxa, increasing from 23–58% in contemporaneously developing internodes. Of this inhibition, 9–48% in H. annuus compared with 97–100% in L. styraciflua was attributable to decreases in cell number in mature internodes. While cell divisions were severely inhibited in both taxa, differences in sensitivity appear related to differences in patterns of histogenesis associated with pronounced inherent differences in final cell lengths. Final cell lengths in H. annuus exceed those of L. styraciflua by 7–8-fold and can play a more dominant role in final internode lengths than total cell number. Conversely, in L. styraciflua total cell number, rather than final cell length, accounts for most of the variation in final internode length. These studies demonstrate species differences in sensitivity of cell division in developing internodes to intermittent water deficits.

Tissue Culture of Liquidambar

The genus Liquidambar belongs to the Hamamelidaceae, a dicotyledonous family comprised of 23 genera of deciduous or evergreen trees and shrubs. Liquidambar is in the subfamily Liquidambaroideae (5, 7). The genus has 3 species (1) [twenty extinct species are known according to Peattie (16)]: L. formosana Hance, Formosan gum native to China and Taiwan; L. orientalis Mill., oriental sweetgum native to Turkey; and L. styraciflua L., American sweetgum or red gum, of the eastern United States with disjunct populations in Mexico and Central America (26). Interspecific hybrids of the three species have been produced (17). The species are monoecious, deciduous trees with simple, alternate, palmately 3–7 lobed leaves, and deciduous stipules. Flowers are apetalous in globular heads. Staminate flowers are in terminal racemes or panicles; pistillate flowers are pedunculate with numerous ovules. Fruits are coherent capsules, dehiscent at apex; seed is winged (5, 10, 12,).