Yu. I. Dolgikh

The Stimulatory Effect of the Antibiotic Cefotaxime on Plant Regeneration in Maize Tissue Culture

In order to elucidate the effects of the antibiotic cefotaxime on callus growth and morphogenesis, we incubated embryogenic maize calli (Zea mays L.) of A188 and R91 lines and of their F1 hybrid with 50–500 mg/l cefotaxime throughout several subcultures. Cefotaxime did not affect the induction frequency and growth of the embryogenic callus but enhanced its morphogenesis. In both tested lines and a hybrid, the highest increase in the number of regenerated plants was observed at the antibiotic concentration of 150 mg/l. The degree of morphogenesis stimulation and the range of cefotaxime concentrations effective in stimulation of plant regeneration depended on the properties of calli obtained from tested genotypes.

Analysis of Specific RAPD and ISSR Fragments in Maize (Zea mays L.) Somaclones and Development of SCAR Markers on Their Basis

RAPD (Random Amplified Polymorphic DNA) and ISSR (Inter Simple Sequence Repeats) markers were used to analyse the genetic divergence between the regenerated plants derived from callus cultures and the original maize line A188. Analysis of polymorphism by using 38 RAPD- and 10 ISSR-oligonucleotide primers showed that the differences between eight examined somaclones and the original line ranged from 6.5 to 23%. As confirmed using new primers, the regenerants derived from callus cultures grouped into two clusters according to their origin. The regenerants isolated from calluses grown for eight months differed from one another and the original line to a larger extent than the regenerants obtained from two-month callus cultures. In some somaclones, molecular marking of the regenerants revealed specific RAPD and ISSR fragments that were absent in other somaclones or the original maize line. On the basis of six specific fragments (five RAPD and one ISSR), SCAR (Sequence Characterized Amplified Region) markers were developed. Specific polymorphism revealed with random primers was completely confirmed using five SCAR markers. Polymorphism of one SCAR marker differed from that revealed with random primers. Five SCAR fragments were inherited as simple dominant traits. One SCAR fragment displayed codominant inheritance.

Optimization of Agrobacterial ( Agrobacterium tumefaciens ) Transformation of Maize Embryogenic Callus

The method for genetic transformation of maize (Zea mays L.) via embryogenic callus infection with Agrobacterium tumefaciens was developed. Calli were co-cultivated with the overnight culture of A. tumefaciens strain LBA4404 harboring the pBI121 plasmid with the nptII and uidA genes. Thereafter, the sensitivity of calli and regenerated plantlets to kanamycin (Km) was determined. It was shown that kanamycin selection was more efficient at the stage of regenerated plantlets than in callus culture. Both vacuum infiltration at the infection step and preliminary activation of Agrobacterium by acetosyringone or by tobacco leaves exudate increased the frequency of Km-resistant plants. The frequency of Km-resistant plants also varied depending on the morphogenic ability of calli. Polymerase chain reaction confirmed the presence of the nptII gene in the genome of regenerated plants and their progeny. β-Glucuronidase gene expression was observed in roots of T1 plants.

Effect of the ipt gene expression on wheat tolerance to root flooding

The effect of enhanced cytokinin synthesis due to expression of the ipt gene from Agrobacterium tumefaciens on plant tolerance to root flooding was studied. Transgenic wheat (Triticum aestivum L.) plants carrying the ipt gene were more tolerant to flooding than wild-type plants. The effect of transformation was manifested in the higher yield and less growth inhibition during flooding. The measurements of activities of antioxidant enzymes, superoxide dismutase and catalase, as well as MDA content during flooding revealed differences between wild-type and transgenic plants that correlated with their tolerance. These results point to the protective role of cytokinins during wheat root flooding.

The Response of Sugarcane (Saccharum officinarum) Cultured Cells to Anoxia and the Selection of a Tolerant Cell Line

The effect of anoxia on the sugarcane (Saccharum officinarum L.) cultured cells was studied in order to elaborate a technique for in vitro selection of cell lines, which would be tolerant to anaerobic stress. Inhibitory and lethal doses of anaerobic incubation were established from the state of the mitochondrial ultrastructure during the anaerobic incubation of cells either with or without exogenous glucose, as well as from the pattern of the post-anaerobic callus growth. An intact state of the mitochondrial ultrastructure and the viability of some cells in the presence of 3% glucose were shown to be maintained for at least 14 days of anaerobic incubation, while the index of post-anaerobic growth decreased by almost 50% even after 72-hour-long anaerobiosis. In the absence of exogenous glucose, a marked destruction of mitochondria and a twofold decrease in the callus growth index were observed as early as after six-hour-long anaerobic stress. A 48-hour-long incubation under these conditions resulted in the maintenance of the intact ultrastructure only in 7–10% of cells, while a 96-hour-long anaerobiosis brought about the complete degradation of the subcellular structure and cell death. A 48-hour-long anaerobiosis without exogenous glucose was chosen for selecting the anoxia-tolerant cell lines. After three cycles of selection, the anoxia tolerance of the selected cell line exceeded the respective index of the initial callus several-fold. In selected line, about 50% of cells retained viability and could resume growth even after 96-hour-long anaerobic incubation. The experimental results obtained were used to determine the possible causes of the heterogeneity of callus cells as regards their anoxia resistance.

RAPD Analysis of Maize Somaclones

The genetic difference between maize line A188 and A188-derived somaclones was assessed via analysis of randomly amplified polymorphic DNA (RAPD). In total, 17 decanucleotide primers used each allowed amplification of 2–17 fragments ranging 200–2000 bp. The RAPD patterns did not differ between individual plants of line A188, which demonstrated again its high genetic homogeneity. The difference between the initial line and the somaclones was high, ranging 64–74%. On evidence of the genetic divergence, the somaclones formed two clusters. The distribution of somaclones between these clusters was consistent with their origin.

Oligosaccharide Inhibits Ethylene Synthesis and Stimulates Somatic Embryogenesis in a Cotton Cell Culture

The effect of a three-component oligosaccharide fragment of xyloglucan FucGalXyl (XG3) on callus-tissue growth and somatic embryogenesis was investigated in a cotton (Gossypium hirsutumL.) cell suspension culture. The oligosaccharide introduced into an induction medium at 10–8and 10–7M concentrations did not affect the frequency of callus formation from hypocotyl segments; however, it enhanced the monthly increment of callus-tissue weight 1.5- and 3-fold, respectively. Induction and culturing of the callus on an XG3-containing medium adversely affected its morphogenetic potential. Addition of XG3 to the culture medium during the cell suspension preparation stimulated cell division resulting, after 40 days, in a 3.4-fold (at 10–8M XG3) and a 1.7-fold (at 10–7M XG3) increase in the cell number as compared to the control. Exclusion of 2,4-D, kinetin, and oligosaccharide from the culture medium caused, after two weeks, a 3.8-fold increase in the number of embryos in the 10–7M XG3-treated suspension culture as compared to the control. The stimulation of somatic embryogenesis by the oligosaccharide was accompanied by a 12-fold decrease in ethylene emission. The morphogenetic effect of oligosaccharide is suggested to result from its anti-auxin action, which, in particular, inhibited the auxin-dependent ethylene synthesis.

The level of genetic variability of cells in prolonged suspension culture of Arabidopsis thaliana

The level of heterogeneity and genetic variability of cells in a suspension of Arabidopsis thaliana cultured in vitro for more than seven years was studied. The considerable heterogeneity of the suspension in cell size was shown. As revealed by nuclear DNA cytophotometry, the suspension culture was mixoploid and the amount of DNA in the cells varied from 4 to 16 C. However, PCR with 6 RAPD- and 4 ISSR-primers and their intragroup combinations showed the lowest degree of variability of DNA markers. The genetic distances of clones obtained from a suspension culture of the parent plant were only 1.5%. Differences between the clones were identified with only one pair of 31 primer combinations tested, indicating low level of genetic heterogeneity of the suspension. The results showed that variations in the amount of DNA in the suspension culture cells are not accompanied by significant changes in the DNA sequence.

Patterns of ISSR and REMAP DNA markers after cryogenic preservation of spring wheat calli by dehydration method

Inter-Simple Sequence Repeat (ISSR) and retrotransposon-microsatellite amplified polymorphism (REMAP) markers were applied to study the influence of successive steps of dehydration cryopreservation on DNA in recovered calli and regenerated plants of spring wheat (Triticum aestivum L.). The precultivation step had no influence on the genetic stability of plant material. After the dehydration step, a new fragment appeared in the REMAP profiles for one DNA sample of calli of Nv16 line. A fragment of similar length was observed in one DNA sample for calli regenerated after complete procedure of cryopreservation in liquid nitrogen (−196°C). However, in samples of calli cultured in vitro for two and four weeks after any type of treatments, the amplicon spectra exhibited no difference from those of starting materials. The amplicon profiles of plants regenerated from calli after successive steps of cryopreservation were also identical to the profiles of the mother plants.

Freezing of dehydrated calli of spring wheat (Triticum aestivum L.) in liquid nitrogen and their morphogenetic potential.

A cryopreservation method developed earlier was modified for freezing of calli derived from mature embryos of four spring wheat (Triticum aestivum L.) pure lines. The effects of particular stages of cryopreservation protocol on water content, number of calli recovering growth, and rate of morphogenesis were analyzed. Regrowth was observed in 90.5–100% of calli after dehydration and in 93.3–100% after freezing-thawing. Dehydration, but not freeze-thawing significantly decreased the frequency of morphogenetic variation.