Intervarietal and interspecific chimera formation by in vitro graft-culture method in Brassica

Abstract

An efficient chimera formation method by tissue culture combined with grafting was studied in Brassica. Cabbage cultivars "Ruby-ball" and "YR-ranpou" (Brassica oleracea) were used for intervarietal chimera formation. Seven-day-old seedlings leaving one of two cotyledons were approach-grafted and cultured in vitro (AGSC method). Chimeric shoots were obtained by the subculture of directly growing chimeric leaves (DGCL) from grafted part and cross-cut section of the united part after the graft culture. These were rooted and grown to complete chimera. An approach-grafted culture method was also available for interspecific chimera formation between "Komatsuna" (B. campestris) and "Ruby-ball" (B. oleracea), resulting in 20% formation per culture. Interspecific chimeras were identified as complex and peripheral-sectorial type by microscopic observation and the electrophoretic analysis of acid phosphatase isozyme pattern. The AGSC method was more effective than the usual Winkler's graft method and a simple mixed culture of heterogeneous cells or tissues.

Artificially-Synthesized Intergeneric Chimera between Brassica oleracea and Raphanus sativus by In Vitro Grafting.

“…One chimeral shoot from one graft out of 20 grafts was grown by in vitro approach -graft according to our established method (Noguchi et al,1992;Oguni et al,1996). Transplantation and propa- gation were performed from the chimeral tissues, resulting in 77 chimeras as the first generation of intergeneric chimeras.…”

“…Type definition of 77 chimeras was done by morphological characteristics (Fig.1), anthocyanin pigmentation pattern (LII and/or L III layer), leaf tissue structure by making the transversal sections (Fig.2), surface layer (LI) structure by sump method (Fig.3), and amplified DNA pattern by PCR analysis using atp6 gene primer (radish specific marker is 1489bp fragment and cabbage marker is 925bp fragment in Fig.4). These chimeral structures were classified (Table1) according to three layers of origin of the apical meristem (Schmidt, 1924) as in the case of interspecific chimeras (Noguchi et al,1992). Results showed that more than half of the chimeras (43/77= 55.8%) were revertant to radish (LI-LII-L1II =MMM from the surface layer to the inner one), four (5.2%) were revertant to red cabbage, 14 (18.2%) were periclinal MMR type, only one (1.3%) was peripheral chimera MRR type, and the other 15 (19.5%) were more or less mericlinal chimeras of the mixed type with MMM, MMR and/or MRR.…”

“…To synthesize chimera we applied the in vitro approach-grafting method to young seedling just after the emergence (Noguchi et al,1992). Twenty grafts were done and the resulting chimeral apical part was subcultured on the modified B5 medium (Gamborg et al,1968) supplemented with 3.75gl-1 CaCl2.2H20 and lppm 6-benzylaminopurine (BA).…”

“…For the purpose in vivo grafting and in vitro graft culture methods (Noguchi et al,1992) were developed and four kinds of interspecific chimeras were artificially synthesized in Brassica crops. Morphogenic, physiologic and genetic interactions between different cells and tissues have been studied using these chimeras.…”

Comparative studies on chloroplast DNA in Ogura-type CMS lines derived from a cybrid (Brassica oleracea L. var. capitata) and a chimera (Brassica rapa L. var. peruviridis) and on a distinction of chloroplast DNA by PCR in Brassicaceae

“…) was extracted from 'Ruby Ball', 'Komatsuna', 'Shogoin', 'MS-Gensuke', 'PDC' line and 'CDC' line (progeny of the 'CDC' line used previously, Noguchi et al 1992;Noguchi and Hirata 1994;Hirata et al 1999 and, by the cetyltrimethylammoniumbromide (CTAB)-based method (Aldrich and Cullis 1993), and PCR was performed with the two primer pairs (primers A and B, and C and D) to find that variation region (Table 2, Figure 1). These primers were designed based on the sequence alignments between clone 24 and the corresponding region of Arabidopsis thaliana (AP000423).…”