Advanced biotechnological approaches and gene manipulation in Populus species

The biotechnology of plant tissue culture has been developed as an applied science for micropropagation, tree improvement, and basic physiology studies. Biotechnological approaches in forest science are advanced as a desirable tool to obtain superior genotypes and to screen for insect resistance. As a basic technique, in vitro cloning is a powerful tool using different explant types such as immature ovules or stem nodes for tree improvement. Typically, plants can be regenerated in vitro by apical or vascular cambial meristems. Genetic transformation is another option to improve the variable lines by genetic modification in host genetic materials with desirable traits. In addition to cloning and gene transfer, in vitro screening methods are applicable for early selection with modified plantlets;In this advanced biotechnology program, several aspects were addressed with Populus species: (1) light and scanning electron microscope study of shoot formation in developing ovules of cottonwood (Populus deltoides), (2) evaluation of shoot proliferation from alternative in vitro nodal cultures of a cottonwood hybrid (Populus deltoides X P. nigra), (3) investigation of morphological in vitro shoot abnormalities in hybrid aspen (Populus alba L. X P. grandidentata Michx.), and (4) early screening of resistance to the cottonwood leaf beetle (Coleoptera: Chrysomelidae) in transgenic poplar (Populus deltoides X P. nigra);Immature ovule cultures can provide early cloning from zygotes that are composed of diverse genetic sources and the explant of immature ovules can be a desirable target for genetic transformation. Therefore, the culture system was developed and shoot formation from immature embryos was investigated via electron and light microscopes. The regeneration frequency was increased substantially by treatment with zeatin;Ordinarily, an intact node is used as explant material in plant tissue culture. However, shoot production was significantly enhanced by using internode and modified stem segment explants in this study. Besides the apical or vascular cambial meristems, shoots could be produced from the phellogen (lenticel) which is an organ for secondary growth in bark. Therefore, shoot formation from lenticel area was investigated through stereo and light microscopy;Even though the development of in vitro systems is important in biotechnological research, abnormal plantlets need to be studied to overcome in vitro problems. The cells and structure of normal and vitrified leaves of hybrid aspen were investigated with scanning electron and light microscopy. Vitrified leaves had more circular, larger stomata; an absence of palisade parenchyma; and large, vacuolated spongy mesophyll cells. Chloroplasts in vitrified leaves were less developed than in normal leaves;Finally, an in vitro system was integrated with entomological knowledge to screen for resistance to the cottonwood leaf beetle. This study was conducted to evaluate the potential of the proteinase inhibitor gene II (pin 2). The in vitro system provided a quick, highly controlled method to screen the transgenic poplar in an in situ condition directly