Callus regeneration and polyploidy induction of Allium cepa L var. Bima Brebes using oryzalin

Polyploidy induction could increase shallot bulb-size to raise consumer preference and local shallot productivity. The research aimed to obtain an effective method of polyploidy induction on callus of onion (Allium cepa) var. Bima Brebes. The experiment was consisted of two experimental steps, which were callus induction of onion and polyploid induction of the callus. A 1×1 cm callus was treated by two drops of oryzalin with concentrations 0, 25, 50, 75, 100, and 120 μM. The ploidy level was identified based on morphological trait, stomatal analysis and DNA content using a flow cytometry. The results showed callus diameter, number of green spots, and number of shoots were decreased with increasing oryzalin concentration. The planlet leaves regenerated from oryzalin treated callus were darker than that of control. The flow cytometry analysis showed that planlets with 75 μM oryzalin was tetraploid, had longer and wider stomata than that of the control.

Validating Field Regeneration Capacity for Selected Accessions of Gossypium hirsutum Using Callus Induction and Regeneration Capacity

Gossypium hirsutm undergoes a rapid clonal propagation to regenerate a mature plant through tissue culture. In this research, cotton leaf regeneration level for 21 accessions in the field (new leaves) was observed after the first harvest, and a comparison between field regeneration level and callus induction with its regeneration capacity (new shoots and roots) for the same 21 accessions was carried out. During the flowering stage of Gossypium hirsutum, biochemical (Proline), physiological (chlorophyll and carotenoid content) analysis was carried out. Phenotypic observations (plant height, leaf area, fresh leaf weight, dry leaf weight, flower and boll number) were also carried out on 21 accessions for each accession. Callus induction and regeneration capacity of roots and shoots for hypocotyl, cotyledons and shoot tip tissues was used to validate field regeneration capacity through analysis of variance. ZS061, LuMian378, JiMian863, and ZS065 have highest drought tolerance while ZhongMianSuo24, LiaoYangDuoMaoMian, and BeiZheGongSheMian have the lowest tolerance to drought stress. Accessions with both field and callus regeneration capacity were identified.

Development of an efficient in vitro callus proliferation protocol for edible wild rhubarb (Rheum ribes L.)

Rheum ribes L. is a perennial wild species. Young shoots and flower bunches are freshly consumed, and root and rhizomes are generally used for medicinal purposes. The aim of the present study was to improve the callus proliferation protocol for R. ribes L. under in vitro conditions. For callus induction, hypocotyl explants taken from 14-day old plantlets germinated in Murashige and Skoog (MS) media were cultured in MS media with 9 plant growth regulator (PGR) combinations containing 6-benzylaminopurine (BAP) (2, 3, and 4 mg/L) + naphthylacetic acid (NAA) (0.1, 0.5, and 1 mg/L). Then, for callus proliferation, 4 PGR combinations containing NAA (0.2 mg/L) + thidiazuron (TDZ) (0.5, 1, 2, and 3 mg) were used in the first set of experiments, and 36 PGR combinations containing BAP (1, 2, 3, and 4 mg/L) + indole-3-butyric acid (IBA) (0.2, 0.5, and 1 mg/L), BAP (1, 2, 3, and 4 mg/L) + NAA (0.2, 0.5, and 1 mg/L), and TDZ (1, 2, 3, and 4 mg/L) + NAA (0.2, 0.5, and 1 mg/L) were used in the second set of experiments. At the end of the second set of experiments, the greatest callus regeneration ratios were obtained due to the combinations including BAP and IBA as well as the low-dose TDZ- (especially 1 mg/L) and NAA- (0.2, 0.5, 1 mg/L) combinations. Regarding callus fresh weights, TDZ + NAA combinations were found to be more successful. The greatest callus fresh weight (12.7 ±0.4 g) was obtained from MS medium supplemented with 2 mg/L TDZ and 0.2 mg/L NAA.

Stabilization of soft wheat selection material which is the result of crossing with wild relatives

Aim. Testing the haploproduction ability of 30 hybrids of winter soft wheat. Methods. In vitro culture of isolated anthers of wheat. The percentage of callus and regeneration of green plants for each genotype calculated as a percentage of the planted anthers. Results. The differences in the frequency of callus induction and the ability to regenerate plants in the process of androgenesiss in vitro of winter soft wheat were detected. The microspores of 17 of 30 hybrids formed callus by in vitro anther culture were shown. The intensity of one process was different: more than half of the genotypes (18 pcs.) were characterized by a low percentage of callus (from 0.10 to 1.0%), 6 genotypes - medium (from 1.0 to 3.0%), and three - high (4.36%; 15.11% and 15.81%, respectively). Conclusions. Genotype-specific of microspores morphogenetic reactions of soft winter wheat in the process of androgenesis in vitro were revealed Samples P26 and P27 showed the highest level of callus formation. The 10 green regenerating plants were obtained. Keywords: hybrids, soft winter wheat, anther culture in vitro, callus, regeneration.

Genotypic variation in the response to embryogenic callus induction and regeneration in Saccharum spontaneum

Embryogenic callus induction and regeneration are useful in many aspects of plant biotechnology, especially in the functional characterization of economically important genes. However, in sugarcane, callus induction and regeneration vary across genotypes. Saccharum spontaneum is an important wild germplasm that confers disease resistance and stress tolerance to modern sugarcane cultivars, and its genome has been completely sequenced. The aim of this study was to investigate the effect of genetic variations on embryogenic callus induction and regeneration in S. spontaneum and to screen genotypes having high tissue culture susceptibility. The study was performed using nine genotypes of S. spontaneum and the following five parameters were assessed to determine the response of genotypes to embryogenic callus induction and regeneration: callus induction, embryogenic callus ratio, embryogenic callus induction, embryonic callus regeneration and regeneration capacity. All the genotypes varied significantly (P < 0.01) in all the parameters, except for embryonic callus regeneration, which was high (>80%) for all the genotypes. High broad-sense heritability (86.1–96.8%) indicated that genetic differences are the major source of genotypic variations. Callus induction was found to be strongly positively correlated with embryogenic callus induction (r = 0.890, P < 0.01) and regeneration capacity (r = 0.881, P < 0.01). Among the nine tested genotypes, VN2 was found to be the most responsive to tissue culture and could therefore be used to characterize functional genes in S. spontaneum. We also suggested an approach with potential applications in facilitating the rapid identification of sugarcane genotypes susceptible to tissue culture.

Adventitious shoot regeneration from leaf and stem explants of Salix matsudana Koidz

Currently, the leaf and stem as explants for rapid propagation in vitro remained unknown in Salix matsudana. Multiple shoots were regenerated from leaf and stem (stem apex and stem with axil). The optimum medium for callus regeneration and shoots induction from leaf was on WPM medium containing 6.0 mg/l zeatin (Z) and 0.75 mg/l NAA. The optimum medium of shoot induction from stem apex was on MS medium supplemented with 8.0 mg/l Z and 1.0 mg/l NAA, and from stem with axil was on MS with 8.0 mg/l Z and 0.5 mg/l NAA. Rooting of regenerated shoots was obtained on the same medium supplemented with 1.0 mg/l activated charcoal.

Effects of Hormones and Epigenetic Regulation on the Callus and Adventitious Bud Induction of Fraxinus mandshurica Rupr.

Fraxinus mandshurica Rupr. (hereafter “F. mandshurica”) is known as one of northeast China′s important, valuable hardwood timber species. However, tissue culture and micropropagation of the species are difficult and have low efficiency, limiting asexual propagation. In this manuscript, stem explants were utilized to establish an effective regeneration system through adventitious bud organogenesis. The factors influencing callus regeneration in vitro were determined, and callus regeneration technology was established. The mechanism of adventitious bud formation was analyzed. Thidiazuron (TDZ) played a crucial role in the formation of adventitious buds. Elevated concentrations of TDZ were beneficial to callus induction and low concentrations of 6-benzyladenine (BA) led to loose state callus formation. The order of callus induction rates for different explants was stem cotyledon (100%) > segment (98.54%) > hypocotyl (92.56%) > root (50.71%). The effects of exogenous addition of 6-BA and TDZ on the endogenous hormone content of plants during the regeneration of adventitious buds were also assessed, as well as the expression characteristics of genes related to the regeneration pathway. The comprehensive analysis results showed that the suitable medium for callus induction and adventitious bud differentiation was c12 medium (MSB5 + 30 g/L sucrose + 7 g/L Agar + 5 mg/L 6-BA + 8 mg/L TDZ + 2 mg/L glycine + 0.1 mg/L IBA + 5% coconut water). The induction rates of callus and adventitious buds were 99.15% and 33.33%. The addition of 2.4 mg/L of the DNA demethylation reagent 5-azacytidine (5-aza) and 0.15 mg/L of the histone deacetylase inhibitor trichostatin A (TSA) increased the rates of adventitious bud induction by 17.78% over the control. This further laid the foundation for large-scale cultivation of excellent varieties and genetic transformation techniques.