Induction of somatic embryogenesis by salt stress in carrot.

Abstract

h is well known that embryogenic cells can be induced by exogenously applied auxin and that their development into somatic embryos are inhibited by the same hormone.1) A number of researchers have investigated morphological and biochemical aspects of somatic embryo development from embryogenic cells, 2-8) but only a few reports deal with an inductive process of embryogenic cells from somatic cells.. The reason for this is mainly due to diverse actions of auxin on plant tissues, such as the promotionn of adventitious root differentiation, callus formation and cell enlargement. To conduct biochemical and physiological studies on induction of embryogenesis from a somatic cell, it is necessary to develop aa new tissue culture system which does not require any auxin treatment of explants. Recently, we9") demonstrated that carrot somatic embryogenesis could be induced by treatment with high concentration of sucrose, mannitol or heavy metal ions such as Cd2+, Zn2+, Ni2+ and Co2+ in auxin-free medium andd suggested that osmotic and other physiological stress might trigger the somatic embryogenesis. In this report, we describe the effects of salt stress on carrot somatic embryogenesis. Seven-to ten-day-old seedlings of Daucus carota L, cv. US-Harumakigosun grown on vermiculite were surface-sterilized with 10% (v/v) sodium hypochlorite solution (available chlorite concentration of ca. 1%) for 15 min, then rinsed three times with sterilized distilled water. Five mm long segments of cotyledons, hypocotyls, roots and apical tips were cut out from the seedlings. These explants were cultured in plastic Petri-dishes (60 x 15 mm) containing 8 ml of Murashige and Skoog's10) agar (0. 8% Difco Bacto agar) medium with 0. 1 M sucrose (hereafter referred to as MS medium) to which no plant growth regulator was added and NaCI was supplemented at a concentration of 0. 1 M to 0. 4 M. After 1 to 3 weeks in culture, the explants were transferred onto hormone-free MS medium without NaCI. Cultures were carried out under a 16 hr light/8 hr dark illumination (approx. 3, 000 lux) at 25C. Each, treatment consisted of at least twenty replicates and was repeated three times. When apical tips were used as explants, the first and second leaves protruded and elongated during 1-2 week culture on MS medium containing NaCI at a low concentration (0. 1 M), but they did not form somatic embryos before and even after the transfer to hormone-free MS medium without NaCI... On the other hand, apical tips cultured with a high concentration (0. 2-0. 4 M) of NaCI or those cultured for 3 weeks with a low NaCI concentration (0. 1 M), turned to red and formed somatic embryos on the surface of the explants and/or elongated leaves without visible callus formation after the transfer toa hormone-free MS medium without NaCI (Figs. 1, 2). These embryos developed into young plantlets. (Fig. 3). When cotyledon, hypocotyl and root explants were cultured on MS medium with 0. 1 M to 0. 4 M NaCI, their color turned brown or white during the culture and no somatic embryos was observedd before as well as after the transfer to hormone-free MS medium without NaCI. Frequency of somatic embryo formation obtained with apical tip segments has been summarized in.. Fig. 4. Higher frequency with the shorter treatment has been observed at the higher concentration