Effect Of Heterologous Expression Of Populus Euphratica PeCPD And PeDWF4Genes On Callus Induction And Growth Of Arabidopisis

Brassinosteroids (BRs) act as a new class of plant hormones and play the important roles in the growth and development of plant. Both CPD and DWF4were found firstly in Arabidopsis, and encoded key enzymes in BR biosynthesis. PeCPD and PeDWF4are two genes that were cloned from Populus euphratica and are homologous with Arabidopsis CPD(AtCPD) and DWF4(AtDWF4) respectively. The present studies on biological function of BRs are limited on individual level of plant, rarely on the level of plant tissue and cell culture. In addition, studies on the biological function of BRs are still largely focused on exogenously applied BRs, the effect of endogenous BR on plant growth and development is rarely reported. In this study, in order to understand the fuctions of endogenous BR in growth and development of plant, the functions of PeCPD and PeDWF4, and the functional differnces between PeCPD and PeDWF4, PeCPD and AtCPD as well as PeDWF4and AtDWF4through observing the response of the three transgenic Arabidopsis lines to exogenous hormones in vitro tissue culture level. Arabidopsis wild-type (WT), Arabidopsis-PeCPD (PeCPD) and Arabidopsis-PeDWF4(PeDWF4) transgenic lines were used as material. The main results were as follows:1. On the MS solid medium containing2.0mg/L2,4-D and1.0mg/L6-BA,WT and PeCPD formed callus at the cotyledon and hypocotyl. However, callus of PeDWF4transgenic line formed firstly at hypocotyls and shoot apical meristem, and subsequently extended to the ends of cotyledons. Callus formation in WT and PeCPD transgenic lines was earlier than that in PeDWF4transgenic line, but growth rate of their callus was slower than PeDWF4. The paraffin section analysis showed that the outside of callus cells in PeCPD transgenic line and PeD WF4transgenic line arranged in tightening, stained deeply by safranine,and cell nucleus was very obvious. Callus cells inside were stained lightly by safranine, cell nucleus was not obvious. Compared with PeCPD and PeDWF4lines, callus cells in surface of WT arranged looser, cell nucleus was smaller, stained lighter by safranine, internal cells near-surface layers of callus were stained deeply.2. In the basic medium with0.5mg/L BL, the Paraffin section analysis showed that the callus cells of WT, PeCPD transgenic line and PeDWF4transgenic line arranged in loose, irregular, and the cell nucleus was not obvious. Compared with callus grown in the basic medium, growth rate of WT, PeCPD and PeDWF4transgenic lines increased, and the callus became greener, the callus cells became longer and wider, and the cell volume increased.3. In comparsion with seedlings on basic medium, the length of hypocotyls of WT, PeCPD and PeDWF4lines grown on basic medium that6-BA was replaced by BL were increased and the size of cotyledons were decreased,and hypocotyls became bent.Moreover, Callus formation time delayed, the growth rate of callus decreased. With the increasing concentration of BL, callus induction rate decreased, callus was easier to brown, brown degree was WT> PeCPD=PeDWF4.4. In the basic medium that2,4-D was replaced by BL, WT, PeCPD and PeDWF4transgenic seedlings could not be induced into callus, hypocotyls became shorter, thicker, leaf area increased. With increasing concentration of BL, the root growth was completely or partly inhibited.5. PeCPD gene expressed only in PeCPD transgenic lines, and the expression level in seedlings was higher than that in callus. Compared with callus grown on the basic medium, the expression level of PeCPD was higher in callus grown on the basic medium added with BL.6. PeDWF4gene expressed only in PeDWF4transgenic lines, and the expression level in callus was higher than that in seedlings,and the expression level had no significant difference in callus grown on the medium that adding with BL or not.7. AtCPD and AtDWF4genes expressed both in seedlings and callus of WT, PeCPD transgenic line and PeDWF4transgenic line. AtCPD expression level had no significant difference in seedlings and callus among three lines. However, expression level of AtDWF4in PeCPD transgenic seedlings was higher than that in WT and PeDWF4transgenic lines seedlings, and expression level of AtDWF4in callus of PeCPD transgenic line and PeDWF4transgenic line was higher than that in callus of WT, but the expression level of AtDWF4had no significant difference in callus grown on the medium that adding BL or not.Those result indicated that:1) endogenous and exogenous BL could promote callus by promoting cell growth, exogenous BL keep callus bright green.2) During callus induction and growth of Arabidopsis, the role of6-BA (cytokines) was not completely replaced by BL, and BL promoted callus browning. In addition, interaction between BL and6-BA did not induce callus in Arabidopsis. Namely, during callus induction and growth, the role of2.4-D (auxin) can’t be replaced by BL.3) During callus induction and growth, the role of PeCPD and PeDWF4was not identity, the roles of PeCPD and AtCPD, PeDWF4and AtDWF4were also not identity.4) there was synergistic effect in transcriptional level between AtDWF4and PeCPD/PeDWF4,and there was no interaction between AtCPD and PeCPD/PeDWF4.