Characteristics Of Growth And Development And Adventitious Ogan Origin In Chimera Of Brassica Juncea And Brassica Oleracea

Tuber mustard (Brassica juncea ) and red cabbage (B. oleracea) are importantvegetable crops of the Brassica in the Cruciferae. The plant chimeras obtained by invitro grafting between tuber mustard and red cabbage were used as the materials forthis study. The characteristics of vegetative and reproductive stages at the aspects ofbotany, cytology, biochemistry and molecular biology, and the photosyntheticcharacteristics of the chimeras were studied for clarifying the interactions betweengenetically different apical cell layers.The regeneration of adventitious organs indifferent explants of the chimeras was investigated in quest of the origin and ontogeny.The interactions in the chimeras can result in changes of phenotypes and improvemany characters, which can be useful for plant breeding. In addition, these studieswill provide academic supports for applying chimeras to plant improvement and putforward some novel ideas and viewpoints at the aspect of plant development. Themain results as follows:1 The characterization of chimera was observed at the level of morphology,cytology, molecular biology and so on. The results were that the chimeras not onlycombined the morphological characters from both donor plants, tuber mustard and redcabbage, but also they undergo some changes in phenotypes. The stomata density ofthe leaf epidermis in the chimeras surpassed the tuber mustard and red cabbage, andthe morphology of chloroplast and starch grains were altered in the chimeras. Inaddition, there were novel bands in the soluble proteins of the chimeras bySDS-PAGE analysis. However, no novel band sepsific for chimeras was detected inRAPD and PCR analysis of the chimeras compared with their donor plants. Thesestudies suggested that there were interactions at the level of morphology, cytology andbiochemistry in the interspecific chimeras of tuber mustard and red cabbage. Theoptimal medium and method for the chimera vegetative proliferation was selected.The chimeras could be conserved in half strength MS medium and propagated in half strength MS medium with 0.1 mg/L BA in which periclinal chimeras would notchange to sectorial and mericlinal chimeras by culturing the nodes with axillary buds.2 In this study, the photosynthesis, chlorophyll fluorescence and chlorophyllcontent, Rubisco activities and the large and small subunits of Rubisco were assayedin the periclinal chimera TCC (Lâ… -Lâ…¡-Lâ…¢=T-C-C, Lâ… -the outmost layer of shootapical meristem; Lâ…¡-the middle layer; Lâ…¢-the innermost layer T=tuber mustard, C=red cabbage) synthesized by grafting in vitro between tuber mustard (Brasscia juncea)and red cabbage (B. oleracea). The net photosynthesis rate of TCC chimera was18.09μmol CO2·m^-2·s^-1, much higher by 24.8% than that of its donor plant redcabbage, and the stomatal conductance and intercellular CO2 concentration of TCCchimera markedly higher than that of both donor plants. The quantum efficiency ofphotosystemâ…¡(φPSâ…¡) and photochemical quenching coefficient (qP) were almostsame in TCC and red cabbage, but distinctly lower than that of tuber mustard. Thetotal content of chlorophyll a and chlorophyll b in TCC chimera was close with that oftuber mustard, but remarkably higher by 97% than that of red cabbage. The initial andtotal activities of Rubisco of TCC chimera were 1.76 and 3.75μmolCO2·g^-1·min-¹,intermediate between tuber mustard and red cabbage, while the expression of rbcLand rbcS of Rubisco in TCC chimera exceeded both donor plants. The resultssuggested that the enhancement of stomatal conductance, chlorophyll content,Rubisco activities and transcription of rbcL and rbcS genes may contribute to higherPN of TCC chimera than the donor red cabbage, and heterogenous epidermis (Lâ… ) inTCC chimera exerted large effects on these physiological characteristics determinedby inner tissues (Lâ…¡and Lâ…¢).3 The characteristics of the TCC chimera reproductive organs and crossing weredetermined. It was noted that TCC chimeras required strict vernalization condition toflower, which was same with red cabbage, while tuber mustard was not so strict as redcabbage and TCC chimeras. Moreover, the flowering habit and morphologycharacteristics of inflorescence in TCC chimeras were more similar with red cabbage.However, the length and width of flower buds and petals, the size of pistils, stamensand pollens were intermediate between those of red cabbage and tuber mustard. In addition, TCC chimeras were artificially pollinated with the pollens of TCC chimera,red cabbage and tuber mustard. The capsule setting frequency of TCC chimera wasclose with both donor plants, but there was no seed in the capsule while the seedsettings of red cabbage and tuber mustard in every capsule were 5.3 and 14.6 for selfpollination, and 0.2 and 7.6 for cross pollination with TCC pollens. It suggested thatthe reproductive characteristics of TCC chimeras were changed by the effects ofinteractions between genetically different apical cell layer Lâ… and Lâ…¡, Lâ…¢.4 Adventitious shoots were induced from nodal segments and leaf discs of TCC(Lâ… -Lâ…¡-Lâ…¢, Lâ… -the outmost layer of shoot apical meristem; Lâ…¡-the middle layer;Lâ…¢-the innermost layer. T=Tuber mustard, C=Red cabbage) chimeras. The origin ofshoots was analyzed by histology and molecular biology. As a result, the frequency ofadventitious shoot induction rose with the increase of BA in MS medium in the areaof nodes. However, there was no different induction frequency of adventitious shootsfrom nodal segment bases in the media with different BA concentrations. Mostadventitious shoots (clustered shoots) arose from node area were TTT (Tubermustard- Tuber mustard- Tuber mustard) but only 4 shoots were chimeras, whichindicated that more shoots originated from Lâ… than from Lâ…¡and Lâ…¢. All shoots fromnodal segment bases were CCC (Red cabbage-Red cabbage- Red cabbage), indicatingthe shoots originated from Lâ…¡or Lâ…¡and Lâ…¢. There were significant differences ofthe regeneration rate in the margin of leaf discs among the three combinations of BAand NAA. Most adventitious shoots from the margin of leaf discs were CCC but 2 of70 were chimeras, which indicated that more shoots originated from Lâ…¡or Lâ…¡andLâ…¢than from Lâ… . All chimeras obtained by regeneration in types were different fromthe original of explants in the present study. The origin of adventitious shoots variedwith the sites of origin on the plants, and could be multicellular and multhistogenic.5 Adventitious roots were induced from stems and leaves of chimera TCC(Lâ… -Lâ…¡-Lâ…¢= TCC, T = Tuber mustard, C = Red Cabbage) synthesized by in vitrografting between tuber mustard and red cabbage previously. The induction frequencyof adventitious roots from TCC stems and leaf discs were 86% and 94%, markedlyhigher than its parents, 38% and 25% from TTT (tuber mustard), 47% and 73% from CCC (red cabbage), and the number and fresh weight of adventitious roots from TCCshoots, 13.11 and 0.274 respectively, was also significantly high when compared to itsparents. This investigation demonstrated that the replacement of LI in plants with adifferent genotype might improve the adventitious root regeneration ability because ofa probable positive co-operation between LI and the two inner apical cell layers.Subsequently, the origin of these adventitious roots was examined by morphology,PCR (Polymerase Chain Reaction) and histology, and it was found that adventitiousroots were originated from the Lâ…¢.These studies showed that the growth and development of the interspecificchimeras was affected by the interactions between the different genotypic apical celllayers. Many quantitative traits were intermediate between two donor plants. Theregeneration ability of adventitious roots and the photosynthetic characteristics ofchimeras were remarkably improved by the interactions of genetically different tissuecell layers, compared to the genotype which they derived from. The floralcharacteristics of TCC chimeras were also changed by the interactions. In conclusion,the interactions between the different genotypic apical cell layers and their derivatesresult in many characters and phenotype variation which will contribute to plantbreeding in the future.