Evaluation of the capacity for direct regeneration of maize inbreds of the Lancaster selection group
AbstractIn connection with the necessity of bringing elite maize inbreds of the Lancaster germplasm group, which have potential for cultivation in Ukraine, into the system of genetic tranformation, the aim of this investigation is to identify the ability of maize inbreds of this group to regenerate by direct organogenesis and to determine the optimal mineral basis for their nutritional environment using segments of the node area of shoots. As explantats we used sterile 4-day old seedlings of 4 maize inbreds of Lancaster germplasm and model inbred Chi31 exotic germplasm. The seedlings were obtained by germination of sterile seeds in Petri dishes between two layers of moist sterile filter paper at a temperature of 27 ºC in dark conditions. A single 1 cmlong segment was cut from each from each seedling, running from 0.5 cmbefore the node to 0.5 cmafter the node. A cut was made in each segment of the node in order to create a wounded surface. Explantats were planted in a nutrient environment with mineral bases of MS or N6, modified by the addition of 10 mg/l silver nitrate, 100 mg/l casein hydrolyzate, 690 mg/l L-proline, 30 g/l sucrose, 1.0 mg/l 2,4-dychlorphenoksiacetic acid and 0,1 mg/l abscisic acid. Cultivation was carried out at 25–27 ºC in the light. Direct hemogenesis in this environment on the 14th day of cultivation in vitro reached 100% for each line. This meant that all researched lines of Lancaster germplasm and the model line showed a high capacity for direct regeneration through direct hemogenesis, which does not depend on the composition of the mineral content of their nutritional environment. Callus formation was observed in all genotypes on the 14th day of cultivation in vitro and the extent of its formation increased during the following month of cultivation. The callus formation was observed only at the site of the wounded surface. The calluses were transparent. Although green areas appeared in these calluses, they were nonembryogenic and did not lead to regeneration. All explantats were transplanted into fresh nutrient media with simultaneous removal of germinated shoots on the 14th day of cultivation. Regeneration on the 45th day of cultivation took place by direct hemogenesis (formation of shoot or leafy structures) or direct hemoryzhogenesis (formation seedlings which had both shoot and root). Formation of ryzhogenic calli was observed in most inbreds. The frequency of regeneration of shoots and leaf structures was characterized by a tendency to increase indicators in the culture environment with mineral basis N6, and the frequency of regeneration of shoots was significantly marked using a medium with mineral basis MS. However, regeneration on the 45th day took place at a much lower rate than on the 14th day of cultivation, which must be taken into account when a system of genetic transformation systems using nodal segments of seedling shoots is being developed. Thus, using nodal sections of 4-day old seedling shoots of maize inbreds of Lancaster germplasm and model inbred Chi31, we can obtain viable seedlings for use in biological systems of agrobacterial transformation. We can recommend two variants of provoking regeneration by direct organogenesis in vitro for application in genetic transformation. The first variant suggests for all studied genotypes 100% induction on the 14th day of in vitro cultivation of direct organogenesis by hemogenesis and this requires further rooting of obtained shoots before planting into soil. Both variants of the nutrient environments of cultivation proposed us can be used. The second variant involves low frequency of regeneration by direct hemoryzhogenesis in a limited number of genotypes, but excludes the rooting stage. The cultivation environment with the mineral basis MS is recommended for this variant. The choice of either variant for obtaining direct regeneration depends on the particularity and aim of the research.
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