Asexual Propagated Clone Induction Of Red Algae And Preliminary Study On Its Developmental Mechanism

Typical red algae, Chondrus ocellatus, Gracilaria lemaneiformis, G. asiatica andHalymenia sinensis, were choose as candidates for studying the early development ofsporelings, induction of asexual propagated clone from red algal tissue, DNA andRNA analysis correlated with the development and differentiation.The tetraspores and carpospores of C. ocellatus were cultured indoor for theobservation of early development. It showed that tetraspore and carpospore hadsimilar early development fate, there were mainly three stages during the earlydevelopment: early divided stage, discoid crust stage and erect thalli formation stage.There were mainly two valid methods to obtain the red algal asexual propagatedclone from the fragments. One was that fragments can directly regenerate thebranches and formed new plantlets under tissue culture, another was that, fragmentscan be induced into callus or callus-like, which develop into discoid crusts and formjuvenile seedlings.The asexual propagated clones of G. lemaneiformis and G. asiatica were obtainedfrom fragments culture, which directly formed the new plantlets. 6-BA (0.25 mg/L)exhibited obvious effects on fragments' regeneration, and the new plantlets inductionpercentage could reach 84.4%. The results showed that the size of scar affected thepercentage of new plantlets regeneration, and too big scar increased the regenerationrate. High concentration of 6-BA or IAA (4 mg/L) could result in the death offragments. Polarity was observed during the fragments culture of G. lemaneiformis.The asexual propagated clones of H. sinensis were mainly obtained fromfragments, which formed callus-like, and developed into discoid crust and formedjuvenile seedlings. It showed that typical plant hormones (IAA and 6-BA) have noobvious effect on the filaments induction of H. sinensis, but it could be induced intofilaments when cultured in PES media. Filaments of H. sinensis came from healthythalli and discoid crusts. The rates of filaments formation were about 80% and 90%respectively, it grew normally under the conditions at 13-23℃, with optimization at18℃;and 10-15 μE?m?2?s?1. High light intensity (50 μE?m?2?s?1) was deleterious toits normal growth.According to the different obtained methods, there were two developmental waysof red algal asexual propagated clones. One of the developmental ways of asexualpropagated clone was that, the asexual propagated clones obtained by directlyfragments tissue culture, first the differentiated cells showed the totipotency andrestored dividing ability and formed new branches, and the other end of fragmentsembodied the polarity and developed into holdfast, and the whole new plant formatted.The other developmental way was that, the differentiated cells showed the totipotency,dedifferentiated and formed callus or callus-like, then the callus or callus-like adheredto the substratum and redifferentiated and formed discoid crusts, and developed intoerect thalli.Crude proteins analysis was conducted by SDS-PAGE, aimly to screen theproteins correlated with the development of red algal asexual propagated clone. It wasshowed that there were distinctive proteins: 30 KD protein, which was only detectedin the regenerated new branches, and 150 KD and 125 KD protein were detected onlyin the normal thalli and the basal part of fragments of G. lemaneiformis.Five pair of primers for gene clone were designed according to the genesequence correlated with germination and development of meristem and callus. Toobtain the related genes involved in the early development of red algae, PCR andRT-PCR detection and DNA sequence analysis were performed, to preliminary studythe related gene sequences.