In VitroCell. Dev. Biol.--Plant 33:269-274, October-December 1997 © i997 Societyfor In VitroBiology 1071-2690/97 $05.00+0.00 GENETIC CONTROL OF IN VITRO DIFFERENTIATION PROCESSES IN RADISH L. A. LUTOVA, I. S. BUZOVKINA, O. A. SMIRNO\\~. O. N. TIKHODEYE\: S. O. SHISHKOV~, .~.~DI. M. TRIFONOVA Department of Genetics, St. Petersburg State Lniversit); St. Petersburg, 1990.34 Russia (Received 16 November 1995; accepted 10 Ma) 1997: editor D. ~. Altman) SUMMARY Genetic control of differentiation processes in radish was studied in vitro on the level of morphogenic capacities of explants. We have shown that when cultured on hormone-free MS medium (Murashige and Skoog, 1962), isolated radish cotyledons can produce callus and/or roots. At the same time, excised seedling apices placed on MS medium supplied with exogenous cytokinin can form multiple shoots or crop-root-like structures. In our model, the ability of explants to undergo the above morphogenic events in cuhure under certain in vitro conditions was examined as a genetic marker. As forms tested, highly inbred radish lines maintained by tight inbreeding for 28-34 generations were used. ~,~ have shown that ability of excised cotyledons to produce callus is controlled by a single gene, while their root-producing capacity is under di-genic control with some additional influence of the cytoplasm. Analysis of inheritance of seedling apex capacity to produce crop-root-like structures in response to exogenous eytokinin led us to propose the interaction of three genes in control of this trait. Key words: in vitro differentiation; explant sensitivity; cytokinin; inbred lines; phytohormone variation. INTRODUCTION Although differentiation processes in higher plants are the subject of special interest in many labs, the precise mechanisms of plant morphogenesis are still poorly understood. It is widely accepted that phytohormones, especially auxins and cytokinins, play the key roles in regulation of plant differentiation (Reid, 1990; Hobble et al., 1994). Therefore, studying genetic control of plant differentiation appears to be tightly related to identification and characterization of genes establishing plant phytohormonal status and response. Several approaches are traditionally used in this area: • genetic transformation of plant cells with agrobacterial oncogenes affecting plant auxin/cytokinin system (Klee and Romano, 1994); • use of "reverse genetical" methods to clone plant genes encoding for auxin/cytokinin system receptors (Palme et al., 1992) or enzymes (Bartling et al., 1994); • analysis of plant forms possessing some deviations in their in vivo morphogenesis (Featherstone et al., 1990) or in vitro phytohormonal response (Muller et al., 1985; Estelle and Sommerville, 1987; Suter et al., 1988; Wilson et al., 1990). In the model developed in our lab, plant differentiation properties are examined on the level of in vitro morphogenic capacities of ex- plants (e.g., their ability to produce shoots, roots, or callus in the absence or presence of exogenous phytohormones) (Fadeyeva et al., 1974, 1979). By use of this model, six genes controlling in vitro differentiation processes in radish (Raphanus sativus L.) have been identified. rally cross-pollinating species, this long period of inbreeding has led to ho- mozygosity of various recessive mutations persisting in the initial cultivars at the heterozygous state. At the present time, our collection includes about 30 inbred lines possessing different morphological and physiological devia- tions such as dwarfism/giantism, agravitropic growth, reduced apical domi- nance, intensive crop-root cracking, formation of spontaneous genetic tumors, etc. The expected high-level homozygosity of the inbred lines was confirmed by genetic analysis of their isozymic spectra (Fadeyeva et al., 1975). To obtain aseptic seedlings, radish seeds were sterilized in 30% H202 (or 10-15 min and germinated on MS medium (Murashige and Skoog, 1962) at 24-26 ° C and 14 h light (250 tamol/m2/s)--10 h darkness photopeviod. On Day 10 after germination, the seedling apices and 2A of each cotyledon were excised and placed on MS medium with 2 mg/1 (8.87 pM) N6-benzyladenine (BA) and hormone-free MS medium containing 0.1 mg/1 thiamine, 0.1 mg/1 pyridoxine, and 0.05 rag/1 nicotinamide, respectively. Morphogenic capacity of explants was examined on Day 30 of culturing. In case of cotyledons, their ability to produce roots and/or callus was determined. The excised apices were studied for their ability to form crop-root-like structures (CRS: huge callus appearing at the basal part of the apex and pigmented like the intact crop-roots of the same inbred line). It is well known that in vitro differentiation traits often manifest in a frequent manner when not all explants of the same genotype display the certain morphogenic response (see Fadeyeva et al., 1974; Kopertekh and Butenko, 1995). Therefore, the in vitro phenotype of each line was estimated by the value of the trait penetrance (frequency of the trait manifestation). If both contrasting phenotypes have frequent manifes- tation, the observed F2 ratios will not exactly correspond to those at the genotypie level. This discrepancy meant that some F 2 segregants, which ge- notypes were associated with a dominant trait, displayed recessive phenotype and vice versa. Therefore, the observed F 2 ratios were corrected by use of formulae (Buzovkina et al., 1993): nd°m Paom MATERIALS AND METHODS In the present work, we used highly inbred lines of radish (Raphanu~ sativuz L.) derived from single plants of Saksa and Virovsky Bely cultivars by selfing for 28-34 generations (Narbut, 1966). In radish, which is a natu- wherein N = volume of F2 sample; ndo,, = actual number of F 2 segregants, which genotypes were associated with the dominant trait; fj.°, = observed number of Fz segregants possessing the dominant pbenotype; P,~om= pene- trance of the dominant trait; n~, = actual number of F 2 segregants, which 269