The Mutated Acetolactate Synthase Gene from Rice as a Non-Antibiotic Selection Marker for Transformation of Bamboo Cells

Ogita, Shinjiro; Kikuchi, Nanaka; Nomura, Taiji; Kato, Yasuo

doi:10.4236/ajps.2012.33044

Cited by 5 publications

(4 citation statements)

References 17 publications

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“…The system is applicable to investigations of carbohydrate metabolism involved in growth and cell wall development of bamboo cells [ 12 ]. We also established transformation protocols using particle bombardment in suspension cell culture systems of Phyllostachys bamboo [ 13 , 14 ]. We have now standardized a novel xylogenic suspension culture model in order to unveil the sequential biological steps in lignification of living bamboo cells.…”

Section: Introductionmentioning

confidence: 99%

A novel xylogenic suspension culture model for exploring lignification in Phyllostachys bamboo

et al. 2012

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BackgroundSome prominent cultured plant cell lines, such as the BY-2 cell line of tobacco (Nicotiana tabacum cv. ‘Bright Yellow 2’) and the T87 cell line of Arabidopsis (Arabidopsis thaliana L. Heynh., ecotype Columbia) are used as model plant cells. These suspension cell culture systems are highly applicable for investigating various aspects of plant cell biology. However, no such prominent cultured cell lines exist in bamboo species.ResultsWe standardized a novel xylogenic suspension culture model in order to unveil the process of lignification in living bamboo cells. Initial signs of lignin deposition were able to be observed by a positive phloroglucinol-HCl reaction at day 3 to 5 under lignification conditions (LG), i.e., modified half-strength Murashige and Skoog medium (m1/2MS) containing 10 μM 6-benzyladenine (BA) and 3% sucrose. Two types of xylogenic differentiation, both fiber-like elements (FLEs) with cell wall thickening and tracheary elements (TEs) with formation of perforations in the cell wall, were observed under these conditions. The suspension cells rapidly formed secondary cell wall components that were highly lignified, making up approximately 25% of the cells on a dry weight basis within 2 weeks. Detailed features involved in cell growth, differentiation and death during lignification were characterized by laser scanning microscopic imaging. Changes in transcript levels of xylogenesis-related genes were assessed by RT-PCR, which showed that the transcription of key genes like PAL1, C4H, CCoAOMT, and CCR was induced at day 4 under LG conditions. Furthermore, interunit linkage of lignins was compared between mature bamboo culms and xylogenic suspension cells by heteronuclear single quantum coherence (HSQC) NMR spectroscopy. The presence of the most common interunit linkages, including β-aryl ether (β-O-4), phenylcoumaran (β-5) and resinol (β-β) structures was identified in the bamboo cultured cell lignin (BCCL) by HSQC NMR. In addition to these common features of lignin, several differences in lignin substructures were also found between the BCCL and the bamboo milled wood lignin (BMWL).ConclusionsOur xylogenic suspension culture model could be used for detailed characterization of physiological and molecular biological events in living bamboo cells.

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Section: Introductionmentioning

confidence: 99%

A novel xylogenic suspension culture model for exploring lignification in Phyllostachys bamboo

et al. 2012

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“…Cells in these cultures proliferate in a highly synchronous manner and show uniform morphological features (Ogita 2005;Ogita et al 2012c). We also established highly efficient particle bombardment transformation protocols for suspension-cultured cells of P. nigra and P. bambusoides (Ogita et al 2011(Ogita et al , 2012a. To study the sequential biological events in xylogenesis in living bamboo cells, we developed a culture system that promotes a high degree of lignification in P. nigra suspension cells (Ogita et al 2012b).…”

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Occurrence of hydroxycinnamoylputrescines in xylogenic bamboo suspension cells

Nomura

Shiozawa

Ogita

et al. 2013

Plant Biotechnology

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To explore the chemical basis of xylogenesis in bamboo cells, we compared secondary metabolite compositions between xylogenic and non-xylogenic suspension cell cultures of bamboo (Phyllostachys nigra), which we developed previously. Two compounds, one major and one minor, showed large-scale increases in the cells cultured under two lignification (xylogenic) conditions, compared with cells cultured under proliferation (non-xylogenic) conditions. Based on spectroscopic analyses, the major compound was identified as feruloylputrescine (FP) and the minor compound was identified as p-coumaroylputrescine (pCP). We examined the accumulation profiles of these hydroxycinnamic acid amides during a 16-day culture period. When cells were kept in proliferation conditions for 16 days, the contents of FP and pCP peaked at 2 days (0.32 and 0.25 nmol mg −1 fresh weight, respectively) and decreased to trace levels thereafter. In contrast, the FP content increased throughout the 16-day culture period, reaching maximum levels of 4.3 and 6.8 nmol mg −1 fresh weight in the two xylogenic conditions. The pCP content was lower than that of FP under both xylogenic conditions. The pattern of FP accumulation resembled that of lignin accumulation, as monitored by phloroglucinol-HCl staining. It is likely that FP plays a role in xylogenesis in suspension-cultured bamboo cells.

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“…The enzymatic activity method is a colorimetric test, for rapid diagnosis, often used to detect resistance to imidazolinones in plants, usually weeds, or as part of functional tests in transgenic plant analysis (Sato et al 2009;Ogita et al 2012;Xavier et al 2013). In this test, cyclopropanedicarboxylic acid (CPCA) is used as an inhibitor of ketoacid reductoisomerase (KARI), an enzyme that catalyzes a key reaction of AHAS during the biosynthesis of branched-chain amino acids.…”

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confidence: 99%

Colorimetric and Chlorophyll Fluorescence Assays for Fast Detection and Selection of Transgenic Events of Cotton, Cowpea, Soybean and Common Bean Expressing the Atahas Gene

Citadin

Santos

Andrade

et al. 2022

Plant Breed. Biotech.

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The detection of the presence and expression of transgenes in genetically modified plants is a key step in the process of selecting promising lines. We adapted two methods developed for detecting tolerance to the herbicide imazapyr for selection of transgenic lines expressing the mutated acetohydroxyacid synthase enzyme (AHAS) from Arabidopsis thaliana (Atahas gene). This was achieved using transgenic events from cotton, cowpea, soybean and common bean, which have previously been transformed to express the mutated Atahas gene. In the first method, a colorimetric assay was developed that detects acetoin, an intermediate in the biosynthetic pathway of branched chain amino acids, which is accumulated in the presence of cyclopropanedicarboxylic acid (CPCA), an inhibitor of ketoacid reductoisomerase (KARI). In the presence of the herbicide, it was possible to distinguish non-transgenic from transgenic plants. Qualitative analysis of acetoin formed during the AHAS inhibition allowed to indirectly determine the Atahas transgene expression. The second method measured the kinetics of chlorophyll fluorescence emission. Leaf discs pre-treated with imazapyr for 24 hours were evaluated using the modulated fluorimeter for maximum quantum efficiency of Photosystem II (PSII) (Fv/Fm) and relative electron transport rate (ETR). Results showed that almost all species analyzed presented a marked decrease in Fv/Fm after treatment with imazapyr. In addition, the ETR was significantly reduced in transgenic plants treated with the herbicide. Collectively, our results showed that it is possible to identify transgenic plants expressing Atahas gene and infer their levels of tolerance to imazapyr at a very early stage after transformation.

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The Mutated Acetolactate Synthase Gene from Rice as a Non-Antibiotic Selection Marker for Transformation of Bamboo Cells

Cited by 5 publications

References 17 publications

A novel xylogenic suspension culture model for exploring lignification in Phyllostachys bamboo

A novel xylogenic suspension culture model for exploring lignification in Phyllostachys bamboo

Occurrence of hydroxycinnamoylputrescines in xylogenic bamboo suspension cells

Colorimetric and Chlorophyll Fluorescence Assays for Fast Detection and Selection of Transgenic Events of Cotton, Cowpea, Soybean and Common Bean Expressing the Atahas Gene

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