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Embryogenic callus growth of sweetpotato (Ipomoea batatas (L.) Lam.) was selectively enhanced by subculture on basal callus proliferation medium modified to contain 15 m M NH4NO3. Embryogenic callus production was doubled on basal callus proliferation medium modified to contain 60 m M K+, while nonembryogenic callus production was reduced 40%. Additions of up to 40 mM NaCl to basal callus proliferation medium did not affect callus proliferation. The development of embryos from calli subculture to embryo production basal medium was unaffected by the KCl or NaCl treatments of the callus proliferation phase. However, embryo production was increased by subculturing callus from callus proliferation medium containing 20 m M to embryo production medium containing 10 m M Our results demonstrate that changes in mineral nutrition, in addition to growth regulator differences between callus proliferation and embryo production media, are important factors in sweetpotato somatic embryogenesis. Clonal propagation via somatic embryogenesis has been pro- posed by Cantliffe et al. (1988) for synthetic seed production of sweetpotato. Sweetpotato somatic embryo production is ac- complished in two stages (Chee and Cantliffe, 1988a). Embry- ogenic callus is continuously proliferated by subculture on media containing growth regulators; embryos are produced by trans- ferring embryogenic callus to media without growth regulators. Formation of embryogenic callus was enhanced by addition of 10 µM (2,4-dichlorophenoxy)acetic acid (2,4-D) and 1 µ M N- (phenylmethyl)-1 H- purine-6-amine (BA) (Chee and Cantliffe, 1988b). Further improvements in embryogenic callus growth and embryo production could be expected by optimizing in- organic salts in the basal medium. However, information on the mineral nutrition of embryogenic callus is insufficient (Ammir- ato, 1983; Vasil, 1984). Nitrogen is quantitatively the major mineral nutrient taken up during growth of plant tissues (Marschner, 1986). Supplying N in its reduced rather than oxidized form to tissue culture likely would be more efficient for growth, since NO3 reduction is energetically expensive (Clarkson and Hanson, 1980). How- ever, cultured cells were unable to grow on as the sole N source when media did not include organic acids as buffers, or without continuous titration of the medium (Chaleff, 1983; Dou- gall and Verma, 1978; Fukunaga et al., 1978; Gamborg and Shyluk; 1970; Martinet al., 1977). This result is to be expected since uptake is physiological ly acidic (Marschner, 1986). The relative and absolute amounts of also in- fluence embryogenesis in cell cultures (Tazawa and Reinert, 1969). Generally, it is thought that the ionic form of N affects growth and nutrient uptake by altering cation-anion balance, changing organic anion accumulation and ultimately affecting medium and cytosol pH values (Marschner, 1986; Poole, 1978

Embryogenic callus of sweetpotato ( Ipomoea batatas (L.) Lam.) disassociates in liquid medium to form a heterogeneous population of embryogenic and nonembry- ogenic cell aggregates of varying sizes. To improve embryo production, such cell ag- gregate populations were obtained by manually fragmenting calli 5 to 10 mm in size into liquid medium. The resulting suspensions were analyzed and the embryogenic fraction identified. The percentage of embryogenic aggregates and the percentage of aggregates forming embryos decreased with decreasing aggregate size. Thus, 76% of the 710- to 1000-µm-diame ter aggregates but only 14% of the 180- to 250-µm aggre- gates had embryogenic potential. However, only 20% of the 710- to 1000-µm aggregates and only 2% of the 180- to 250-µm aggregates actually formed embryos. Conversely, embryogenic callus and embryo production per milligram of cultured embryogenic callus increased quadratically with decreasing aggregate size. Individual torpedo-stage embryos were produced from cell aggregates 180 to 250 µm in size. Sweetpotato, a major food crop and po- tential biomass crop, is conventionall y prop- agated using stem cuttings. This propagation method is costly since intensive labor and large nurseries and storage facilities are re- quired. The lack of resistance to virus dis- eases further complicates the maintenance and multiplication of virus-free stocks. Mass production of disease-free plant material can be expected using tissue culture. The pro- duction of synthetic seed, through somatic embryogenesis, and direct seeding would re- duce the production cost of this vegetatively propagated crop. The application of somatic embryogenesis to synthetic seed production will require many

Plant formation from somatic embryos in response to BAP, NAA, and sucrose was studied in sweetpotato [Ipomoea batatas (L.) Lam.]. A maximum of 15% embryos at the torpedo stage of development formed plants of agar-solidified basal medium containing 3% sucrose and no growth regulators. The percentage of embryos forming shoots was increased to 53% by 4 μm BAP, but BAP reduced whole plant formation and promoted callusing at the root axis end of embryos. The frequency of plant development was increased to 38% by adding 0.1 μm NAA to the basal medium. Reducing sucrose concentration to 1.6% in basal medium increased the frequency of plant development to 32%. Chemical names used: 6-benzylaminopurine (BAP; α-naphthaleneacetic acid (NAA).

Summary The mechanism by which exogenous auxins inhibit embryo development from embryogenic callus is not known. In sweet potato [ Ipomoea batatas, (L.) Lam.] we investigated the hypothesis that embryo development was subjected to endogenous IAA transport by studying embryogenesis from calli in response to various concentrations of either 2,4-dichlorophenoxyacetic acid (2,4-D), 2,3,5-triiodobenzoic acid (TIBA), 7-aza-indole (AZI) or p-chlorophenoxyisobutyric acid (PCIB). Additions of AZI and PCIB at 1 to 5 μM did not affect morphogenesis. Additions of TIBA and 2,4-D at 5 μM inhibited embryo formation and promoted embryogenic callus growth. Embryo development was restricted to increasingly earlier stages, i.e. from the torpedo stage toward the pro-embryo stage, as TIBA was gradually increased from 0 to 6μM, or as 2,4-D was gradually increased from 0 to 5μ/tM. TIBA specifically inhibits polar IAA transport, thus, our results suggest that embryo development may coincide with endogenous IAA transport and that, inhibition of embryogenesis from embryogenic calli by exogenously supplied auxins may be a consequence of the disruption by exogenous auxins of endogenous IAA efflux from embryogenic loci.

Patterns of somatic embryo development from callus were elucidated inIpomoea batatas Poir. (sweet potato). Embryos at three stages of development were able to grow into plants. They included embryos arrested at the late torpedo and cotyledonary stages, and an arrested torpedo embryo that resulted from precocious hypocotyl expansion. Early torpedo-stage embryos rooted on reculture but did not form shoots. A diversity of other embryo stages were also produced which upon reculture formed only adventitious embryos and roots. Morphologic variants with similar growth potential were noted among embryos at similar stages of development. Plasticity in somatic embryo developmental patterns led to different forms that were mature enough to produce plants.