Efficient protoplast isolation from ovule-derived embryogenic callus inCitrusvolkameriana

The present study reports on the isolation of viable protoplast from ovule-derived embryogenic calli of Volkameriana (Citrus volkameriana L.), which is a rootstock in high demand for lemon production.Ovules of C. volkameriana isolated at 3 different immature fruit stages, comprising4, 8, and 12 weeks after anthesis (WAA),were cultured on5 different media in order to produce embryogenic callus lines as a source material for protoplast isolation. EME medium (MT basal medium + 0.5 gL–1 malt extract), with the addition of phytohormones [kinetin (KIN), 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BAP)] at different concentrations, were tested for callogenesis. According to 2-way ANOVA, significant effects were determined as a result of the immature fruit stage and type of culture media (P ≤ 0.01) on the callogenesis and embryogenic callus induction frequency. First, callus formation was recorded after 4 WAA on medium comprising EME + 2,4-D (1.0 mg L–1) + BAP (0.5 mg L–1). Callus induction frequency was the highest (90.00%) in the same culture medium when the ovules wereculturedat8 WAA. In addition, culturing the ovules isolated from 12 WAA immature fruits of C. volkameriana resulted in the highest indirect somatic embryogenesis (24%). Embryogenic callus initiation was the highest (25.56%) using EME + KIN (1.0 mg L–1) and ovules cultured at 8 WAA (14%) resulted in the highest embryogenic callus formation. Effects of different enzyme concentrations on the efficiency of protoplast isolation were calculated using the hemocytometer cell counting method. The combination of 2% cellulase and 0.2% pectinase gave the highest numbers of protoplasts, at 12.33 × 105protoplast/mL. Embryogenic callus lines obtained by culturing ovules of C. volkameriana yielded high-quality protoplasts after isolation and could be useful as a protoplast source for further somatic hybridization studies.

Real-time detection of somatic hybrid cells during electrofusion of carrot protoplasts with stably labelled mitochondria

Somatic hybridisation in the carrot, as in other plant species, enables the development of novel plants with unique characteristics. This process can be induced by the application of electric current to isolated protoplasts, but such electrofusion requires an effective hybrid cell identification method. This paper describes the non-toxic fluorescent protein (FP) tagging of protoplasts which allows discrimination of fusion components and identification of hybrids in real-time during electrofusion. One of four FPs: cyan (eCFP), green (sGFP), yellow (eYFP) or the mCherry variant of red FP (RFP), with a fused mitochondrial targeting sequence, was introduced to carrot cell lines of three varieties using Agrobacterium-mediated transformation. After selection, a set of carrot callus lines with either GFP, YFP or RFP-labelled mitochondria that showed stable fluorescence served as protoplast sources. Various combinations of direct current (DC) parameters on protoplast integrity and their ability to form hybrid cells were assessed during electrofusion. The protoplast response and hybrid cell formation depended on DC voltage and pulse time, and varied among protoplast sources. Heterofusants (GFP + RFP or YFP + RFP) were identified by detection of a dual-colour fluorescence. This approach enabled, for the first time, a comprehensive assessment of the carrot protoplast response to the applied electric field conditions as well as identification of the DC parameters suitable for hybrid formation, and an estimation of the electrofusion success rate by performing real-time observations of protoplast fluorescence.

Obtaining barley genotypes in selective in vitro systems with complex resistance to soil stress factors

The aim of the work was to develop selective in vitro systems and obtain on their basis the initial breeding material of spring barley adapted to adverse soil conditions – increased acidity, toxicity of aluminum and heavy metals, drought. In the process of research, optimal patterns for selecting callus lines on selective media using various combinations of stress factors were identified: Al3 + (20–40 mg/l), H+ (4.0–6.0 pH units), Cd2 + (10-20 mg/l), Mn2 + (100–250 mg/l) and osmotic (10–20 % polyethyleneglycol). In the proposed in vitro selective systems, more than a thousand regenerated plants were obtained. Varieties created on the basis of regenerants exceed the standard in yield, have high productive tillering (29.0–67.5 % higher than the standard) and dense spike (4.5–6.6 % higher than the standard). Their advantage is due to resistance to lodging, a high level of survival, germination and environment-forming activity of the root system.

Variability of rice haploids obtained in anther culture in vitro

Morphological variability of haploid plants and doubled haploids of rice obtained on one callus line in anther culture in vitro was studied. The work was carried out on rice plants Oryza sativa L. subspecies japonica Kato varietу Cascade. Regenerant plants of one callus line obtained from one rice anther (four in total) were divided into two or three groups of 20-30 plants, depending on the sample size in order of their differentiation on callus and transplantation on the rooting medium. Two callus lines (15.1 and 18.1) formed half of the haploids, half of the doubled haploids, and two other callus lines (5.1 and 7.2) numerous haploids. On callus lines with numerous haploids (5.1 and 7.2), as the regenant number increases, the size of plants decreases (plant height, number of flowers on the main panicle, number of panicles). On the lines 15.1 and 18.1 between groups of haploids and between groups of doubled haploids statistically significant differences absent. In breeding purposes for the induced doubling of the number of chromosomes in haploid regenerants with antitubulin substances such as colchicine, it is advisable to use plants that form on callus among the first. Between haploids of four callus lines and doubled haploids of two callus lines, statistically significant differences (at p=0.001) were revealed using the Hotelling's T2-criterion, calculated for the whole complex of biometric features. Haploids of different lines differed in three or four of them, doubled haploids on three of the five signs (length of panicle, productive bushiness and plant height). Varieties of interest to breeders may be improved by anther culture in vitro.

In Vitro Organogenesis of a Slipper Orchid, Paphiopedilum ‘Alma Gavaert’

The aim of the present study was to improve the regeneration efficiency of callus lines in a slipper orchid, Paphiopedilum ‘Alma Gavaert’. Three kinds of vegetative tissues, root, stem and leaf segments, were used as explants to induce callogenesis; out of these, only root explants formed callus and was subcultured in the presence of 5 mg/L dicamba and 5 mg/L 2,4-D combined with 1 or 2 mg/L TDZ. The resulting four callus lines, assigned as 5Di1T, 5Di2T, 5D1T and 5D2T, respectively, were used to test the effect of NAA to BA ratios on re-differentiation, wherein the highest number of shoots (approximately 2 shoots/0.1 g callus clump) were obtained in callus line 5D2T at ratios of 0.001 and 0.002. A largely improvement of shoot regeneration efficiency was obtained by continuous selection of callus lines which derived from different explant positions. Eventually, six callus lines, including 5D2T-T6-G5 to 5D2T-T6-G10, were able to produce approximately 10 times of shoots per callus clump when compared with the parental callus line 5D2T.