Antibiotic treatment impaired growth of tobacco in vitro shoots is associated with oxidative injury and reduced microbiome diversity

Plant in vitro cultures initiated from surface-sterilized explants often harbor complex microbial community. Antibiotics are commonly used to decontaminate plant tissue culture or during genetic transformation, however, the effect of antibiotic treatment on the diversity of indigenous microbial population and consequences for performance of the tissue culture are not completely understood. Therefore, the aim of this study was to assess the effect of antibiotic treatment on the growth and stress level of tobacco (Nicotiana tabacum L.) in vitro shoots as well as the composition of plant-associated microbiome. The study revealed that shoot cultivation on medium supplemented with 250 mg L−1 timentin resulted in 29 ± 4% reduced biomass accumulation and 1.2–1.6 fold higher level of oxidative stress injury compared to control. Moreover, the growth properties of shoots were only partially restored after transfer to medium without antibiotic. Shoot microbiome analysis using multi-variable region-based 16S rRNA sequencing revealed abundant microbial community in the control tobacco shoots, including bacteria from 59 families, however, it was largely dominated by Mycobacteriaceae. The antibiotic treatment resulted in a decline of species richness (the number of families was reduced 4.5-fold) and increased domination by the Mycobacteriaceae family. The results imply that the diversity of plant-associated microbiome might represent a significant factor contributing to the efficient propagation of in vitro tissue culture.

Cryopreservation of Hazelnut (Corylus avellana L.) Axillary Buds from In Vitro Shoots Using the Droplet Vitrification Method

Cryopreservation by droplet vitrification was applied to hazelnut (Corylus avellana L.). axillary buds of the Italian cultivated variety Tonda Gentile Romana, which were collected from in vitro growing shoots, immersed in ice cooled PVS2 or PVS3 for 60 or 90 min, then transferred to a droplet of vitrification solution, placed on a strip of aluminium foil, and plunged into liquid nitrogen (LN). Additionally, the effect on the recovery of the mother plant after cryopreservation was evaluated, following a cold pre-treatment at 4 °C for 3 months. The highest regrowth percentage (56.7%) was obtained after applying PVS3 for 60 min, while the application of PVS2 for the same amount of time reduced regrowth to 41.5%. Increasing the exposure to vitrification solutions to 90 min reduced regrowth to 43.3% when PVS3 was applied, and 35.6% if PVS2 was used. The cold pre-treatment on the mother plant did not significantly improve overall regrowth. The cryopreservation process did not decline the rooting ability of the recovered shoots.

Kriopreservasi Tunas in Vitro Pepaya ‘Sukma’ Dengan Perlakuan Prakultur, Loading, Dan Dehidrasi Dengan Pvs2 Dan Modifikasinya

Papaya has high genetic variability because it is an open-pollinated plant and has genotype and phenotypeare that are easily changed due to environment changes. Cryopreservation is a storing method of germplasm in liquid nitrogen (-196 oC) which can maintain the genotype and phenotype of germplasm. The experiment aimed to obtain the best preculture, loading, and dehydration for cryopreservation of papaya ‘Sukma’ in vitro shoots. For preculture, we planted shoots on MS media with 0.3 M and 0.4 M sucrose for 1, 2, and 3 days. In the loading treatment, we immersed shoots in loading solution (liquid MS+1.2M glycerol+0.4M sucrose) for 0, 10, 20, and 30 minutes. For dehydration, we immersed shoots in cryoprotectant (PVS2 and its modification) for 5, 10, and 15 minutes. Then, shoots were immersed in liquid nitrogen. The results showed thatshoots had the best survival rate while they had been precultured on MS medium with 0.3 M sucrose for 3 days. The best loading treatment time was 20–30 minutes. The best dehydration treatment was obtained by modification of PVS2 for 10 minutes. The shoots have not been able to recovery after cryopreservation, so it can be concluded that cryopreservation of in vitro papaya ‘Sukma’ shoots has not been successful.

Effect of Three Types of Ion Beam Irradiation on Gerbera (Gerbera hybrida) In Vitro Shoots with Mutagenesis Efficiency

Gerbera in vitro shoots were irradiated using three types of ion beams with different line energy transfers (LETs) to investigate the effective LET and absorbed doses for mutagenesis. Furthermore, genomic mutation analyses were conducted on the obtained mutants. Survival rate analysis showed a lower lethal dose 50% (LD50) with ion beams with higher LETs. Trait/morphological mutations exhibited changes in the color and shape of petals and male sterility. Irradiation conditions with the highest growth change and trait/morphological mutation rates in each ion were C irradiation at 10 Gy, Ar irradiation at 5 Gy, and Fe irradiation at 5 Gy, with a range of absorbed dose of around LD50 to about 10 Gy lower. The highest trait/morphological mutation rate was 14.1% with Ar irradiation at 5 Gy, which was one of the criteria for ion beam irradiation of gerbera in vitro shoots. Furthermore, the genomic mutation in the flower color, petal shape, and male sterile mutants were confirmed by genotype analysis using Genotyping by Random Amplicon Sequencing-Direct technology. This is the first study to report the efficient production of gerbera mutants that could be analyzed. Our findings may lead to more efficient gerbera mutant production and analysis technology.

Phytotoxicity and Other Adverse Effects on the In Vitro Shoot Cultures Caused by Virus Elimination Treatments: Reasons and Solutions

In general, in vitro virus elimination is based on the culture of isolated meristem, and in addition thermotherapy, chemotherapy, electrotherapy, and cryotherapy can also be applied. During these processes, plantlets suffer several stresses, which can result in low rate of survival, inhibited growth, incomplete development, or abnormal morphology. Even though the in vitro cultures survive the treatment, further development can be inhibited; thus, regeneration capacity of treated in vitro shoots or explants play also an important role in successful virus elimination. Sensitivity of genotypes to treatments is very different, and the rate of destruction largely depends on the physiological condition of plants as well. Exposure time of treatments affects the rate of damage in almost every therapy. Other factors such as temperature, illumination (thermotherapy), type and concentration of applied chemicals (chemo- and cryotherapy), and electric current intensity (electrotherapy) also may have a great impact on the rate of damage. However, there are several ways to decrease the harmful effect of treatments. This review summarizes the harmful effects of virus elimination treatments applied on tissue cultures reported in the literature. The aim of this review is to expound the solutions that can be used to mitigate phytotoxic and other adverse effects in practice.

Problems Encountered during In vitro Culture Establishment in Terminalia arjuna

The main aim of present study was to overcome the problems associated with the in vitro culture initiation in Terminalia arjuna. The micropropagation of tree species is not easy as shrubs and herbs. Many problems encountered from explant collection to in vitro culture establishment. The problems that have been occurred during T. arjuna micropropagation were culture contamination, phenolic exudation, bud growth inhibition, shoots yellowing and leaf fall. All these problems have been solved by applying certain treatments prior to explant collection and inoculation. The mother tree was lopped in November months (six months prior to explant collection) to remove any inhibitory substance and release bud growth. Different sterilizing agents were used to minimize the bacterial and fungal contamination. Some modification in culture media (use of different concentration of NH4NO3 and KNO3 salts and adenine sulphate) was done. Surface sterilization of nodal explants collected from lopped branches with 0.1% HgCl2 for 8 min., treatment with chilled antioxidant solution (Ascorbic acid, Citric acid and PVP) and half strength of NH4NO3 and KNO3 salts of MS medium supported 100% bud break response with proliferation of green and healthy in vitro shoots. Removing these hurdles already in the initial stage of micropropagation is very important and maximize mass in vitro propagation of this medicinally important Arjun tree.