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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).

Potassium starch polyacrylamide, potassium acrylate, a copolymer of potassium acrylate and acrylamide, and hydroxyethylcellulose carrier gels were tested to find a fluid drilling material suited for synthetic seeding of sweet potato (Ipomoea batatas (L.) Lam.) somatic embryos. Somatic embryo developmental stage and size, and maturation (incubation) time were also evaluated to improve plantlet formation. All embryos suspended in the fluidized hydroxyethylcellulose gel were viable after six days and 7% developed into plantlets after two weeks. Up to 97% of the somatic embryos suspended in acrylate and/or acrylamide gels died within six days. Root development was at least 10% and plantlet development at least 30% greater when embryos were subcultured on basal medium for 16 instead of 25 days prior to placement and suspension in hydroxyethylcellulose gel. Up to 25% more plantlets were obtained from embryos at the elongated torpedo stage than those at the cotyledonary or torpedo stages of development. When suspended in hydroxyethylcellulose gel embryo length had no effect on the percentage of plantlets obtained.

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.

Embryogenic suspension cultures of Ipomoea batatas Poir. contain heterogeneous populations of discrete cellular units. In order to optimize embryo production, a study was conducted to identify the embryogenic fraction of such cultures. Suspension cultures were fractionated with sieves of 1000, 710, 500, 355, 250, 180, 125, 90 and 63μm mesh openings and the composition of each fraction was determined. Cellular units larger than 355 μm were primarily calli and made up 75% of the total mass of cultures in the stationary phase of growth. These calli were composed of embryogenic and non-embryogenic subunits, and 98% of the embryogenic subunits measured 355–1000 μm. Calli and embryogenic calli subunits produced clusters of embryos at various stages of development upon transfer to liquid or solidified media without 2,4-D. The 125–355 μm fraction of suspension cultures was composed of cell aggregates of which 20% were embryogenic. The embryogenic cell aggregates produced single globular embryos upon transfer to liquid media containing 0 or 1 μM 2,4-D. The 63–125 μm fraction of suspension cultures contained only 2% of embryogenic cell aggregates. It can be inferred from our results that the embryogenic fraction of cultures was essentially represented in calli, and that proliferation of the embryogenic fraction occurred through the separation of embryogenic cell aggregates from larger calli when cultures approached their stationary growth phase.

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.

High numbers of embryos are difficult to obtain in liquid cultures of sweet potato (Ipomoea batatas (L.) Lam.) because discrete cell aggregates, produced through calli fragmentation, do not support embryo growth. In an effort to demonstrate that embryo development is possible from discrete cell aggregates, we compared embryo formation from cell aggregates 250–355 μm in diameter cultured either in suspension in liquid medium, on agar solidified medium, or immobilized on alginate beads floated in liquid medium. Embryos were initiated but remained arrested in their globular stage on cell aggregates cultured in suspension. Embryos developed to the torpedo stage from cell aggregates cultured on solidified medium and from cell aggregates anchored on alginate beads. Thus, embryos continued to develop beyond the globular stage when a structural polarity, which led probably to the establishment of a physiological polarity, was created. The production of sweet potato embryos in liquid culture can be improved by using alginate beads or culture conditions and protocols leading to the release during calli fragmentation of polarized individual cell aggregates.

Embryogenic callus cultures of Ipomoea batatas Poir. produce fast growing non-embryogenic material which soon dominates the cultures. Our objective was to selectively enhance the proliferation of the embryogenic fraction. For this, the effect of BAP and 2,4-D concentrations on growth of embryogenic and non-embryogenic callus were studied and consequently, nutrient media for the production and indefinite maintenance of embryogenic callus without embryo formation were defined. Selective proliferation of embryogenic callus was obtained on solid media with 10 μM 2,4-D and 1 μM BAP and in liquid media with 5 μM 2,4-D. Selective proliferation of non-embryogenic callus occurred in liquid medium with 1 μM 2,4-D. In embryogenic liquid culture, embryos were produced with 0–2 μM 2,4-D. Increasing 2,4-D concentration from 0 to 2 μM in these cultures restricted embryo development.