Study Of Protoplast Formation, Regeneration And Fusion And Spore Fusion Of Gracilaria Lemaneiformis (Rhodophyta)

The macroscopic red alga Gracilaria lemaneiformis is an economically important seaweed, particularly in China, where it is a major agarophyte. Agar is widely used in the food, pharmaceutical and bioengineering industries, as well as other fields. Significant research on the basic biology of G. lemaneiformis has included investigations of genetic rules, hybridization experiments, strain selection and population structure. Beginning during the early 1970s, research on the growth characters and quality of agar produced by G. lemaneiformis has also been widely conducted; Large-scale culture of G. lemaneiformis gradually expands from the Northern Seaarea of lower temperature (Bohai Sea, Yellow Sea) to the Southern Seaarea of higher temperature (Donghai Sea and part of the Nanhai Sea). However, vegetative propagation is still the main cultivation approach of G. lemaneiformis currently, so germplasm degradation is the facing problem which may hinder the development of G. lemaneiformis industry. Therefore, using modern biotechnology to breed new lines of G. lemaneiformis with properties of growing fast, good quality and high temperature resistance is still an important project.Plant cell engineering technology is a well-known modern biotechnology approach which provides an effective method in breeding. In this study, three kinds of plant cell engineering technologies have been carried out on G. lemaneiformis. The main methods and conclusions are as follows:1. Protoplast isolation and regeneration technology. G. lemaneiformis was taxonomy lower developed algae, which did not own truly differentiation root, stem and leaves. However, the protonema was simply divided into three parts - holdfasts (the basal segments), middle segments (or stem) and tips. Our preliminary study had confirmed that the biochemical characters maybe different in these three parts, so we carried out an analysis of protoplast formation and regeneration among these three parts. Protoplasts were isolated by enzyme and regenerated by MES liquid medium. The result indicates that holdfasts, middle segments and tips have different optimal enzymolysis time at which the best protoplast yield was also different. Middle segments displayed the shortest enzymolysis time and the highest protoplast yield. However, the protoplasts of tips had the strongest vitality. Finally, only the protoplasts of tips regenerated successfully. This study not only provided an important reference for the success in the protoplast regeneration of G. lemaneiformis, but also provided basic research data for the application of cell engineering technology, such as protoplast fusion technology in breeding of G. lemaneiformis.2. G. lemaneiformis cell fusion technology. First, protoplast fusion was used to obtain somatic cell hybrids of Gracilaria lemaneiformis and Gracilaria vermiculophylla. Second, tetraspore fusion was used to obtain somatic cell hybrids of these two species. Both of these methods were based on the lack of cell wall, that is, no barrier for cell fusion. Based on the same principle of spore fusion, this study also conducted two exploratory experiments: First, the tetraspore (haploid) and carpospore (diploid) were fused to obtain triploid spores; second, the carpospores were self-fused in order to obtain tetraploid spores. The results showed that PEG solution had toxic effects on the cells and the tolerance of protoplast was stronger than the tolerance of spores; finally only tetraspore fusion cultures of G. lemaneiformis and G. vermiculophylla developed into young plant, which of pale green-brown color was similar to G. vermiculophylla. A few young plants were with properties of growing faster and branching more, but they must be determined by chromosome observation experiment to ensure somatic cell hybrids or not. This study not only provided various ways to obtain somatic cell hybrids of G. lemaneiformis and the second specie, but also provided basic research data for the application of cell engineering technology in breeding of G. lemaneiformis.3. G. lemaneiformis chromosome preparation technique. In this study, carbol fuchsin stain- squash method was used to observe and count the chromosomes of G. lemaneiformis. The results showed that G. lemaneiformis cells were of multi- nucleus and the number of nuclei in different cells was different. Within the observation range, there were at least 2, at most more than a dozen nuclei in the G. lemaneiformis cells; The chromosome counting suggested that the chromosome number of G. lemaneiformis haploid probably was 15 or 16. This experiment provided technical support for using chromosome counting method to determine somatic cell hybrids, and provided basic research for the application of cell engineering technology in breeding of G. lemaneiformis.