Water Spectral Patterns Reveals Similarities and Differences in Rice Germination and Induced Degenerated Callus Development

In vivo monitoring of rice (Oryza sativa L.) seed germination and seedling growth under general conditions in closed Petri dishes containing agar base medium at room temperature (temperature = 24.5 ± 1 °C, relative humidity = 76 ± 7% (average ± standard deviation)), and induced degenerated callus formation with plant growth regulator, were performed using short-wavelength near-infrared spectroscopy and aquaphotomics over A period of 26 days. The results of spectral analysis suggest changes in water absorbances due to the production of common metabolites, as well as increases in biomass and the sizes of the samples. Quantitative models built to predict the day of the development provided better accuracy for rice seedlings growth compared to callus formation. Eight common water bands were identified as presenting prominent changes in the absorbance pattern. The water matrix of only rice seedlings showed three developmental stages: firstly expressing a predominantly weakly hydrogen-bonded state, then a more strongly hydrogen-bonded state, and then, again, a weakly hydrogen-bonded state at the end. In rice callus induction and proliferation, no similar change in water absorbance pattern was observed. The presented findings indicate the potential of aquaphotomics for the in vivo detection of degeneration in cell development.

Optimize 2,4-D concentration and callus induction time enhance callus proliferation and plant regeneration of three rice genotypes

Carsono N, Juwendah E, Liberty, Sari S, Damayanti F, Rachmadi M. 2021. Optimize 2,4-D concentration and callus induction time enhance callus proliferation and plant regeneration of three rice genotypes. Biodiversitas 22: 2555-2560. The development of callus in the course of transgenic rice avoids the somaclonal variants. To obtain a high number of normal phenotypes and a low number of somaclonal variants requires an appropriate 2,4-D concentration. In this study, we obtained the best callus induction time and a high number of green plant regeneration for three responsive rice genotypes on different 2,4-D concentrations in NB5 medium. The mature seeds of rice embryos were used as explants. A completely randomized factorial design was applied with four levels of 2,4-D concentrations (0, 1, 3, and 5 ppm), two levels of induction time (one and two weeks), and three rice genotypes (cv. Fatmawati, Nipponbare, and Kitaake). The study revealed that there was no interaction effect among genotype, 2,4-D concentration, and callus induction time. Three rice genotypes performed best in callus proliferation and regeneration. One-week callus induction time showed higher callus growth capacity (CGC) as compared to two-week callus induction time. Shoot regeneration capacity (SRC) was independently affected by genotype as well as by callus induction time. The interaction effect between 2,4-D concentration and callus induction time was observed on plant regeneration capacity (PRC). Without the addition of 2,4-D and 1 ppm of 2,4-D, the green plant regeneration capacity (GRC) was comparatively higher. Addition of 2,4-D showed a significant effect, especially at the plant regeneration stage. We found that one-week callus induction was the best treatment for callus proliferation and plant regeneration. We recommend the use of one-week callus induction and 1 ppm of 2,4-D for rice callus proliferation (sub-culture) and subsequent plant regeneration.

Overexpression of Rice Metacaspase, OsMC4, Increases Endoplasmic Reticulum Stress Tolerance in Transgenic Rice Calli

Endoplasmic reticulum (ER) is an important organelle responsible as protein synthesis regulator in plant. High salinity can also lead to the activation of ER stress, caused by the accumulation of misfolded protein. This could lead to a stress response mechanism, unfolded protein response (UPR). Failure of UPR to reverse the effect of protein misfolding will activate Programmed Cell Death (PCD). Metacaspase genes regulate programmed cell death (PCD) in plants. The present study was focused on comprehensive gene analyses of the expression patterns of type II rice metacaspase (OsMC) genes in response to the endoplasmic reticulum (ER) and salinity stress in rice leaf and OsMC4 in callus. A strong evidence of unfolded protein response (UPR) during tolerance to both ER and salinity stress was found in the present study. Overexpression of OsMC4 in rice callus as a fusion protein with TagRFP and controlled by the CaMV35 promoter caused major changes in the expression of the stress ER-marker genes, protein disulfide isomerase (PDI) and Binding immunoglobulin Protein (BiP), and OsMC4 in overexpressing calli. These expression analyses of the OsMC family provide valuable information for further functional studies on the biological roles of OsMCs in PCD related to ER and salinity stress responses.

Transcriptome comparison between pluripotent and non-pluripotent calli derived from mature rice seeds

In vitro plant regeneration involves a two-step practice of callus formation and de novo organogenesis. During callus formation, cellular competence for tissue regeneration is acquired, but it is elusive what molecular processes and genetic factors are involved in establishing cellular pluripotency. To explore the mechanisms underlying pluripotency acquisition during callus formation in monocot plants, we performed a transcriptomic analysis on the pluripotent and non-pluripotent rice calli using RNA-seq. We obtained a dataset of differentially expressed genes (DEGs), which accounts for molecular processes underpinning pluripotency acquisition and maintenance. Core regulators establishing root stem cell niche were implicated in pluripotency acquisition in rice callus, as observed in Arabidopsis. In addition, KEGG analysis showed that photosynthetic process and sugar and amino acid metabolism were substantially suppressed in pluripotent calli, whereas lipid and antioxidant metabolism were overrepresented in up-regulated DEGs. We also constructed a putative coexpression network related to cellular pluripotency in rice and proposed potential candidates conferring pluripotency in rice callus. Overall, our transcriptome-based analysis can be a powerful resource for the elucidation of the molecular mechanisms establishing cellular pluripotency in rice callus.

In vitro response and effect of gamma irradiation on four local indica rice varieties

In vitro response of four local Indica rice cultivars viz. Sadamota, Kachamota, Moulata and Dudhkalam was evaluated. The aim of this study is to develop an efficient protocol for callus induction, plant regeneration and to observe the effect of gamma radiation on plant regeneration for creating possible genetic variability. In Different concentration of 2,4-D and growth regulators were supplemented with MS medium (Murashige and Skoog’s) to observe their callus induction frequency using mature embryo as explant. Among the cutivars, the highest primary callus (92.55%) as well as embryogenic callus induction (56.26%) was showed in sadamota at 3.0 mgl-1 2,4-D and 10 mgl-1 kinetin under dark condition. Twenty one days old embryogenic calli were exposed to 0, 2, 4 and 6 Gy of gamma rays and transferred to regeneration medium. Both callus growth and regeneration capacity were found to be decreased with increasing level of exposure to gamma rays. The doses of 4 Gy of gamma radiation were found to be the 50% inhibition dose for callus growth and plant regeneration in sadamota and kachamota, repectively whereas the 50% inhibition dose for moulata and dudhkalam at 2 Gy. This results indicate that sentivity of gamma radiation on rice callus depends on genotype of a genus.