| Plant hormones play important roles in organs regeneration. In order to study further how plant hormones regulating the organogenesis in vitro, we used a series of concentration of three kinds of auxin, named indole-3-acetic-acid (IAA), 1-naphthylacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D), combining with two kinds of cytokinin, named zeatin (ZT) and 6-benzylaminopurine (6-BA), to induce organ regeneration from stem segment of Arabidopsis thaliana L.The results showed that, with appropriate cytokinin concentrations, IAA between 0.1μM and 75.0μM had the abilities to induce organ regeneration, and the suitable concentration rang for redifferentiation was 0.1μM to 20.0μM. In the optimum concentration, leaf or shoot primordium could be regenerated in 12 days, and the high frequency of organ regeneration was obtained. In suitable concentrations of cytokinin, NAA, ranging from 0.1μM to 10.0μM, could induce organ redifferentiation, and the appropriate concentration range for organ regeneration was 0.1μM to 1.0μM. These combinations could induce leaf or shoot primordium egenetation in 14 days and the frequency of organogenesis was high. Under the condition of appropriate cytokinin, 2,4-D ,which induced leaf or shoot primordium regeneration in 15 days, could induced the regeneration of leaf, shoot and inflorescence at concentrations between 0.01μM and 0.1μM. 2,4-D could not induce the root regeneration.Two kinds of cytokinin, zeatin (ZT) and 6-benzylaminopurine (6-BA) were used to examine how cytokinin controling organ regeneration. The resuls showed that , under the appropriate condition of auxin, 6-BA could induce leaf or shoot primordium regeneration in 14 days while ZT did in 12 days; ZT had higher frequencies of leaf and inflorescence regeneration than 6-BA, while both of them were similar in frequencies of root and shoot regeneration.To understand the influence of the ecotype of explant on organ regeneration in vitro, we used stem segment as explant from two different kinds of ecotype seedlings, wassilewskija (WS) and columbia (Col), to induce organogenesis in the condition of gradient concentrations of IAA combinated with ZT. The results exhibited that, explant of Col ecotype regenerated leaf or shoot primordium in 14 days, while explants of WS ecotype did in 12 days; Compared with explant in WS ecotype regenerating root, leaf, shoot and inflorescence, explants in Col ecotype could induce root, leaf and shoot regeneration; The frequencies of organs regeneration in WS ecotype were much higher than those in Col ecotype, for example, the frequencies of leaf and shoot in WS ecotype were up to 89.2% and 75.2%, respectively, while only 20% and 11.9% in Col ecotype, respectively.In order to examine the formation of apical meristem during the organogenesis, we analyzed the expression pattern of WUS and TFL1 by using pWUS::GUS and pTFL1::GUS construct. The results showed that, shoot apical meristem (SAM) identity gene WUS expression was induced at day 6 of culture, and inflorescence meristem identity gene TFL1 was expressed in 15 days after culture in the optimum conditions. The results suggest that the inflorescence reproduction required much more time than shoot regeneration.Our results indicate that the genetic background of explant, the type and concentration of hormone can influence the type and frequency of regenerated organs significantly, and the most suitable hormone combinations were screened out that could induce shoot/leaf, inflorescence and root reproduction with high frequency, respectively, which will be helpful for our understanding the molecular mechanism of organ regeneration in vitro further.
|
PDF Full Text Request