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Transcriptome Analysis of Melocactus glaucescens (Cactaceae) Reveals Metabolic Changes During in vitro Shoot Organogenesis Induction.

ABSTRACT: Melocactus glaucescens is an endangered cactus highly valued for its ornamental properties. In vitro shoot production of this species provides a sustainable alternative to overharvesting from the wild; however, its propagation could be improved if the genetic regulation underlying its developmental processes were known. The present study generated de novo transcriptome data, describing in vitro shoot organogenesis induction in M. glaucescens. Total RNA was extracted from explants before (control) and after shoot organogenesis induction (treated). A total of 14,478 unigenes (average length, 520 bases) were obtained using Illumina HiSeq 3000 (Illumina Inc., San Diego, CA, USA) sequencing and transcriptome assembly. Filtering for differential expression yielded 2,058 unigenes. Pairwise comparison of treated vs. control genes revealed that 1,241 (60.3%) unigenes exhibited no significant change, 226 (11%) were downregulated, and 591 (28.7%) were upregulated. Based on database analysis, more transcription factor families and unigenes appeared to be upregulated in the treated samples than in controls. Expression of WOUND INDUCED DEDIFFERENTIATION 1 (WIND1) and CALMODULIN (CaM) genes, both of which were upregulated in treated samples, was further validated by real-time quantitative PCR (RT-qPCR). Differences in gene expression patterns between control and treated samples indicate substantial changes in the primary and secondary metabolism of M. glaucescens after the induction of shoot organogenesis. These results help to clarify the molecular genetics and functional genomic aspects underlying propagation in the Cactaceae family.

SUBMITTER: Torres-Silva G

PROVIDER: S-EPMC8417902 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundOur aim is to improve knowledge of gene regulatory circuits important to dedifferentiation, redifferentiation, and adventitious meristem organization during in vitro regeneration of plants. Regeneration of transgenic cells remains a major obstacle to research and commercial deployment of most taxa of transgenic plants, and woody species are particularly recalcitrant. The model woody species Populus, due to its genome sequence and amenability to in vitro manipulation, is an excellent species for study in this area. The genes recognized may help to guide the development of new tools for improving the efficiency of plant regeneration and transformation.ResultsWe analyzed gene expression during poplar in vitro dedifferentiation and shoot regeneration using an Affymetrix array representing over 56,000 poplar transcripts. We focused on callus induction and shoot formation, thus we sampled RNAs from tissues: prior to callus induction, 3 days and 15 days after callus induction, and 3 days and 8 days after the start of shoot induction. We used a female hybrid white poplar clone (INRA 717-1 B4, Populus tremula x P. alba) that is used widely as a model transgenic genotype. Approximately 15% of the monitored genes were significantly up-or down-regulated when controlling the false discovery rate (FDR) at 0.01; over 3,000 genes had a 5-fold or greater change in expression. We found a large initial change in expression after the beginning of hormone treatment (at the earliest stage of callus induction), and then a much smaller number of additional differentially expressed genes at subsequent regeneration stages. A total of 588 transcription factors that were distributed in 45 gene families were differentially regulated. Genes that showed strong differential expression included components of auxin and cytokinin signaling, selected cell division genes, and genes related to plastid development and photosynthesis. When compared with data on in vitro callogenesis in Arabidopsis, 25% (1,260) of up-regulated and 22% (748) of down-regulated genes were in common with the genes regulated in poplar during callus induction.ConclusionThe major regulatory events during plant cell organogenesis occur at early stages of dedifferentiation. The regulatory circuits reflect the combinational effects of transcriptional control and hormone signaling, and associated changes in light environment imposed during dedifferentiation.

Project description:A β-lactams that act by inhibiting the bacterial cell wall biosynthesis are one of the most common classes of antibiotics applied to suppress the growth of latent bacterial infection associated with the plant tissue culture, as well as in the Agrobacterium-mediated transformation techniques. Plant sensitivity to antibiotics usually is species-, genotype-, or even tissue-specific and mainly depends on concentrations, growth conditions, and culture system. In the presented article, we estimated a comparative effect of four β-lactam antibiotics (Claforan®, timentin, amoxicillin, and Amoxiclav®) at different concentrations in an agar-solidified Murashige and Skoog (MS) culture medium supplemented with 5 mg L-1 6-benzylaminopurine (6-BA) and 0.1 mg L-1 indole-3-acetic acid (IAA) on in vitro callus induction and shoot organogenesis from hypocotyl and cotyledon explants of two tomato cultivars (Rekordsmen, Moryana). The role of clavulanic acid in combination with amoxicillin (Amoxiclav®) in the shoot organogenesis frequency and number of shoots per explant has been demonstrated. Additionally, the growth inhibition of Agrobacterium tumefaciens AGL0 strain according to agar disk-diffusion assay was studied. As a result, both stimulatory (timentin, amoxicillin, and Amoxiclav®) and inhibitory (Claforan®) effects of β-lactam antibiotics on in vitro morphogenetic responses of tomato were noted. It was found that clavulanic acid, which is part of the commercial antibiotic Amoxiclav®, significantly increased the shoot regeneration frequency from cotyledon and hypocotyl explants of Rekordsmen tomato cultivar. Possible reasons for the stimulating effect of clavulanic acid on the induction of shoot organogenesis are discussed. According to agar disk-diffusion assay, the maximum diameter of growth inhibition zones (43.9 mm) was identified using 200 mg L-1 timentin. The in vitro antibacterial activity of tested β-lactam antibiotics was arranged in the following order: timentin > Claforan® > amoxicillin ≥ Amoxiclav®. Thus, to suppress the growth of internal and latent bacterial infection of tomato plant tissue culture, as well as for transformation of Moryana and Rekordsmen cultivars by A. tumefaciens strain AGL0, we recommend adding of 100-200 mg L-1 timentin or 400-800 mg L-1 Amoxiclav® to the shoot induction medium.

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Transcriptome Analysis of Melocactus glaucescens (Cactaceae) Reveals Metabolic Changes During in vitro Shoot Organogenesis Induction.

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