Institute of Plant Physiology and Biochemistry, Russian Academy of Sciences
Krasnodar Research Institute for Agriculture
Characterization of callusogenesis in A344, A344-CP5, Sg25 inbred lines and their hybrids
--Shmakov, VN, Mashnenkov, AS, Konstantinov, YM
The aim of the present work was to try to reveal some polymorphism if any in A344, A344-CP5, Sg25 inbred lines and their hybrids on the level of in vitro cell culture. It was shown previously that the subline A344-CP5 causes heterosis in the hybrid, including the initial line A344 (Mashnenkov, MNL 56:92-98, 1982). Callus culture for all genotypes studied was initiated from scutellum and seedling shoots. The explants consisted of scutellum and embryo excised from mature kernels of maize under aseptic conditions. Previously, kernels were submerged for about 8 h in 3% H2O2 in the dark. Then kernels were surface sterilized in 0.1% mercuric chloride with 0.1% saponin for 20 min, and subsequently rinsed three times with sterile water. Excised explants were transferred to vessels for plant tissue culture, containing 20 ml of culture medium under aseptic conditions as blocks of 3-4 explants each. The vessels were incubated at 24±1 °C in the dark. The callus induction and maintenance medium was MS basal salt containing 0.5 mg/l each of thiamine HCl, pyridoxine HCl and nicotinic acid, 80 mg/l inositol, 1 g/l each of casein hydrolysate and L-proline, 30 g/l sucrose, 2 mg/l 2,4D, and 0.2 mg/l BA.
A344-CP5 subline and hybrid A344 x 165 had the best characteristics of callusogenesis based on shoots as an explant (Table 2). It was observed that there was no case for callusogenesis on leaf explants (Table 2). It was also rather rare for epicotyls.
The calli growth on scutellum as an explant was relatively weak for all 6 genotypes studied (Table 1). Among all genotypes, only subline A344-CP5 demonstrated sufficient callus formation (Table 1). Sg25 inbred line scutellum couldnt serve as material for calli initiation at all. The longevity of calli cultivation on the medium we used in these experiments was restricted by about 2 weeks. After this period of calli cultivation, their growth was stopped as a result of cell damage.
Table 1. Callusogenesis and seedling development characteristics of genotypes measured after 2 days of cultivation.
|Genotypes||No. of explants||No. of developed seedlings||Length of shoot, mm||Length of root, mm||Initiation of callus, days||Volume of scutellum, mm3|
|A344-CP5||9||9||8 ± 0.5||-||9||233 ± 22.3|
|A344||10||10||6 ± 0.9||-||8||232 ± 34.0|
|A344 × A344-CP5||10||9||11± 1.2||5 ± 0.6||9||163 ± 22.7|
|Sg 25||10||8||7 ± 1.4||3 ± 0.3||8||190 ± 27.0|
|A344 × Sg 25||10||8||12 ± 0.7||3 ± 0.5||8||267 ± 29.2|
|A344 × 165||10||10||12 ± 1.6||2 ± 0.3||9||228 ± 28.7|
Table 2. Shoot explant callusogenesis characteristics of genotypes studied.
|Genotypes||Number of explants||Initiation of leaf callus, days||Initiation of epicotyl callus, days||Initiation of callus from the upper segment of the hypocotyls, days||Initiation of callus from the medium segment of the hypocotyls, days||Initiation of callus from the lower segment of the hypocotyls, days|
|A344 × A344-CP5||12||-||-||6||3–10||6–12|
|A344 × Sg 25||12||-||-||-||3–12||6|
|A344 × 165||12||-||12||6–12||6||6|