Heritability of regeneration in tissue cultures of sweet potato (Ipomoea batatas L.)

Summary A population of open-pollinated progeny from 12 parents, and the 12 parents, was surveyed for in vitro growth and regeneration characteristics. Four different tissue culture procedures involving different media and the use of different explants to initiate the cultures were used. Petiole explants from young leaves were used as explants for initiation of callus cultures. These were evaluated for callus growth rate, friability, and callus color and texture, before transferring to each of three different regeneration media for evaluation of morphogenetic potential. Small shoot tips also were used to initiate callus cultures, which were evaluated for the same growth characteristics and transferred to growth-regulator free regeneration media. Regeneration occurred through root or shoot regeneration or through embryogenesis. Tissue culture treatment effects, as well as genotypic effects, were highly significant in determining: the types of callus produced, callus growth rates, color and texture on the two types of media used for the second and third subcultures. The family x treatment interaction was generally not statistically significant, affecting only callus color. Estimates of narrow sense heritability for callus growth rate in both the second and third subcultures were high enough (0.35 and 0.63, respectively) for the evaluation of parental lines for selection procedures. These characteristics were also the only early culture callus traits that were consistently correlated with later morphogenesis of the cultures. They were negatively correlated with root or shoot regeneration. The occurence of somatic embryogenesis was not correlated with early callus growth characteristics. Genetic and treatment effects were highly significant in the evaluation of morphogenetic potential, through root or shoot regeneration, or through embryogenesis. Regeneration of all types was of low frequency for all procedures, expressed in 11% of the cultures of the total population.Paper No. 9906 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7601, USA. From a thesis submitted by the senior in partial fulfillment of the requirements for the Ph.D. degree     

Isozyme modifications and plant regeneration through somatic embryogenesis in sweet potato (Ipomoea batatas (L.) Lam.)

Summary The potential of somatic embryogenesis was evaluated for 10 cultivars of sweet potato through extensive embryogenic response and isozyme analysis. Embryogenic callus was induced by incubating lateral buds on Murashige and Skoog medium containing 10 M 2,4-dichlorophenoxyacetic acid for 6–8 weeks. The frequency of embryogenic response was low, and varied with genotypes, ranging from 0 to 17%. Embryo to plantlet formation could be enhanced by the use of the combination of 2,4-dichlorophenoxyacetic acid with kinetin, both used at 0.01 M. Embryogenic callus with its potential of plantlet formation has constantly been maintained for over two years. However, after several subcultures, 0.5 to 12% of embryogenic callus reverted irreversibly into friable fast-growing non-embryogenic callus whose ability to regenerate shoots was then definitively lost. The isozymes of esterase, peroxidase, glutamate oxaloacetate transaminase and acid phosphatase investigated in this study were found appropriate to distinguish compact embryogenic from friable non-embryogenic callus in sweet potato. In fact, the callus reversion was associated with a loss of bands or a decline in isozyme activity. On the contrary, very small changes in isozyme activity or no specific changes at all were observed during the differentiation of embryogenic callus into globular embryos.Abbreviations Acp acid phosphatase - BAP 6-benzylaminopurine - cv cultivar - df degree of freedom - 2,4-D 2,4-dichlorophenoxyacetic acid - Est esterase - Got glutamate oxaloacetate transaminase - IAA indole-3-acetic acid - MS Murashige and Skoog (1962) medium - Prx peroxidase - Tris tris(hydroxymethyl)aminomethane     

Interpretation of randomly amplified polymorphic DNA marker data for fingerprinting sweet potato (Ipomoea batatas L.) genotypes

In this paper we present a method for the generation of randomly amplified polymorphic DNA (RAPD) markers for sweet potato. These were applied to produce genetic fingerprints of six clonal cultivars and to estimate genetic distances between these cultivars. The level of polymorphism within the species was extremely high. From the 36-decamer random primers used, 170 fragments were amplified, of which 132 (77.6%) were polymorphic. Ten primers resulted in no detected amplification. Of the remaining 26 primers for which amplification was achieved, only one did not reveal polymorphism. Six primers used alone enabled the discrimination of all six genotypes. Pattern analysis, which employed both a classification and ordination method, enabled the grouping of cultivars and the identification of primers which gave greatest discrimination among the cultivars.     

Touch-induced gene (IbTCH1) from sweet potato [Ipomoea batatas (L.) Lam.]: molecular cloning and functional analysis

The cDNA of the touch-induced genes (TCH) of the sweet potato [Ipomoea batatas (L.) Lam.] has been cloned and analyzed. IbTCH1, which exists as at least two-copy genes in the genome of the sweet potato, encodes for 148-amino acid polypeptides, and harbors four conversed Ca²⁺-binding motif EF-hands. IbTCH1 was shown to be expressed in the flower, leaf, thick pigmented root, and particularly in the white fibrous root, but expressed only weakly in the petiole. IbTCH1 is upregulated upon exposure to environmental stresses, dehydration, and jasmonic acid. Furthermore, IbTCH1 is developmentally regulated in the leaf and root. These results strongly indicate that the gene performs functions in both plant development and in defense/stress-signaling pathways.     

A comparison of shoot-forming and non-shoot-forming somatic embryos of sweet potato [Ipomoea batatas (L.) Lam.] using computer vision and histological analyses

Diagnostic structural features for competence to form shoots were tested among sweet potato embryos by combining morphological image capture (using a computer vision system) with anatomical analyses (using light microscopy). Five major morphological variants (`perfect', `near perfect', `limited/no meristematic activity', `disrupted internal anatomy', and `proliferating') were identified among torpedo- and cotyledonary-stage embryos. Among these, only the first two were found to be competent for conversion into plantlets. Lack of organized shoot development in somatic embryos of sweet potato was associated with the following abnormalities: lack of an organized apical meristem, sparcity of dividing cells in the apical region, flattened apical meristem, and multiple meristemoids and/or diffuse meristematic activity throughout the embryo. Diagnostic separation of most shoot-forming and non-shoot-forming torpedo and cotyledonary embryo variants was achieved. Received: 27 January 1997 / Revision received: 28 January 1998 / Accepted: 12 February 1998     

Plant regeneration from stem and petiole protoplasts of sweet potato (Ipomoea batatas) and its wild relative,I. lacunosa

A protoplast-to-plant regeneration system has been established for sweet potato (Ipomoea batatas (L.) Lam.) and its wild relative, I. lacunosa L. Viable protoplasts, isolated from preplasmolyzed stems and petioles of in vitro-grown plants, were cultured on liquid MS (Murashige & Skoog 1962) medium that supported cell division and colony formation. Embryogenic calli of sweet potato were induced on agar-solidified MS medium supplemented with 3% (w/v) sucrose, 50 mg l^-1 casamino acids, 0.2–0.5 mg l^-1 2,4-d, 1.0 mg l^-1 kinetin and 1.0 mg l^-1 ABA. On average, 3 plants were regenerated from a single sweet potato callus subcultured on semi-solid MS medium containing 3% (w/v) sucrose, 800 mg l^-1 glutamine, 2.0 mg l^-1 BA or 1.0 mg l^-1 kinetin and 1.0 mg l^-1 GA₃. Embryogenic calli of I. lacunosa L. were initiated on semi-solid MS medium containing 0.2–0.5 mg l^-1 IAA and 1.0–2.0 mg l^-1 BA. An average of 5 plants was regenerated from a single sweet potato callus subcultured on semi-solid MS medium containing 0.5 or 1.0 mg l^-1 GA₃.Abbreviations ABA abscisic acid - BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole acetic acid - MES 2-(N-morpholino)-ethane sulfonic acid - NAA -naphthaleneacetic acid     

Computer vision analysis of somatic embryos of sweet potato [Ipomoea batatas (L.) Lam.] for assessing their ability to convert to plants

Apical and axial shoot tips of sweet potato were cultured to produce somatic embryos that mature and develop into plants in basal nutrient medium. However, the lack of high regeneration efficiency is an impediment to the use of somatic embryos to produce synthetic seeds. Conversion experiments with mature embryos over a 20-day period revealed that 80–90% of the embryos formed roots but only 40–50% formed shoots. Using computer vision and canonical or Fisher discriminant function (CDA) analysis along with conversion results, it was possible to correctly classify competent embryos 40–50% of the time based on size features, 50–60% of the time based on shape features, and 55–60% of the time based on color features. Non-competent embryos were correctly classified 65–75%, 55–60%, and 70–75% of the time based on size, shape, and color, respectively. These results can be used effectively to identify and select competent embryos for improved regeneration efficiency. Received: 2 January 1997 / Revision received: 21 January 1998 / Accepted: 12 February 1998     

Effects of medium composition and culture duration on in vitro morphogenesis of sweet potato

In vitro morphogenesis of sweet potato (Ipomoea batatas) shoot explants after cultures in callus initiation medium (CIM) with two sucrose contents and plant regeneration medium (PRM) with three growth regulator combinations for different durations was studied. After 4 weeks, explants on 5 % sucrose CIM had significantly more shoots but similar or lower root fresh mass and callus fresh mass than those on 3 % sucrose CIM subsequent to transfer for 6 weeks on all three PRM. Cultures transferred to growth regulator-free PRM after 4 and 12 weeks on 5 % sucrose CIM formed plants through organogenesis and embryogenesis, respectively. Embryogenic cultures from 4 weeks on CIM + 10 weeks on callus proliferation medium when transferred to PRM without growth regulator for 4 and 8 weeks produced multiple embryos in the prior and both embryos and shoot buds in the later.     

Abscisic acid-induced growth inhibition of sweet potato (Ipomoea batatas L.) in vitro

The inhibitory effects of abscisic acid (ABA) on in vitro growth and development of axillary buds from nodal segments of sweet potato (Ipomoea batatas L.) was investigated. ABA at concentrations of 0.01, 0.1, 1.0 or 10.0 mg 1^-1 inhibited axillary bud and root development and subsequent plantlet growth. ABA at 10 mg 1^-1 completely inhibited axillary shoot development but did not affect the viability of cv. Jewel explants over a culture period of 365 days. Transfer of nodal segments cultured for 90, 180 or 365 days from basal medium containing 10 mg 1^-1 ABA to growth regulator-free media resulted in rapid and normal plantlet development. Gibberellic acid at 0.1, 1.0 or 10.0 mg 1^-1 in the presence of ABA at 0.1, 1.0 or 10.0 mg 1^-1 did not counteract the ABA-induced growth inhibition. Although ABA totally inhibited the growth of 6 sweet potato plant introductions at a concentration of 10.0 mg 1^-1, the efficacy of ABA as a suppressant of shoot growth varied with genotype.Abbreviations ABA abscisic acid - GA gibberellic acid - cDNA complementary DNA - PI plant introduction - SE standard error     

Characterization of an oviposition stimulant from the surface of sweet potato Ipomoea batatas storage roots for the sweet potato weevil, Cylas formicarius elegantulus

An improved laboratory bioassay was used to characterize an oviposition stimulant from the surface of sweet potato Ipomoea batatas (L.) Lam. storage roots for the sweetpotato weevil, Cylas formicarius elegantulus (Summers). Filter paper discs impregnated with a methylene chloride surface extract of sweet potato storage roots induced significantly (p<0.05) higher oviposition on root cores than those treated with solvent only. Significantly higher oviposition was also observed in the nonpolar fractions, especially one that contains a tentatively identified triterpenoid present in susceptible cultivars.

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Caractérisation d'une substance extraite de la surface des racines tubéreuses d'Ipomoea batatas induisant la ponte de Cylas formicarius elegantulus

Résumé La caractérisation des substances extraites de la surface des racines tubéreuses d'I. batatas Lam et stimulant la ponte de C. formicarious elegantulus Summers, a été effectuée à partir d'une technique améliorée. Différents substrats ont été essayés: extraits au chlorure de méthylène ou péridermes intacts de cultivars résistants ou sensibles, racines tubéreuses après élimination du périderme, morceaux de Solanum tuberosum ou de papier filtre présentés au milieu de plaques à 24 plots pour culture de tissus. Le meilleur substrat s'est révélé être de petits disques de papier filtre (diamètre 0,4 cm), fixés au milieu d'un morceau de racine avec encore un peu de périderme sur lequel le coléoptère pouvait pondre. Cette méthode a été utilisée dans les tests ultérieurs pour caractériser la nature du stimulant.Des disques de papier filtre inhibés d'extraits au chlorure de méthylène du contenu des structures superficielles de racines tubéreuses de cultivars sensibles induisent une ponte significativement plus importante (p<0,05) sur les morceaux de racines par comparaison avec ceux traités au chlorure de méthylène pur. Une ponte significativement plus importante a aussi été obtenue avec des fractions non-polaires, particulièrement celles qui contiennent un triterpènoïde de cultivars dont l'identification a été tentée.

     

Use of Ipomoea trifida germ plasm for sweet potato improvement. 3. Development of 4x interspecific hybrids between Ipomoea batatas (L.) Lam. (2n=6x=90) and I. trifida (H.B.K) G. Don. (2n=2x=30) as storage-root initiators for wild species

Summary More than 28,000 pollinations were carried out between 5 Ipomoea batatas and 41 diploid I. trifida accessions of diverse origins to obtain 4x interspecific hybrids. From the resultant 730 seeds, 248 plants were finally obtained. Ploidy level determination of the progeny showed unexpected results: 52 individuals were hexaploid, 5 were pentaploid, 190 were tetraploid, as expected, and one was not determined. The existence of 5x and 6x progenies from 6x x 2x crosses not only confirmed the presence of 2n gametes but also their successful function in gene flow between ploidy levels and polyploidization within this genus. The progeny and their cultivated parents were planted in an observation field. The cultivated parents produced 0.49 kg/plant or less. Most 4x progenies did not produce storage roots or had very poor yields; nonetheless, and despite their cultivated parents' poor yields, 8 genotypes yielded between 0.81 and 1.50 kg/plant.A new scheme, using the 4x interspecific hybrids, is proposed for evaluating 2x and 4x wild accessions of the section Batatas to which the sweet potato belongs. Other possible uses of the 4x hybrids in breeding and genetics of the sweet potato are also discussed.     

Abscisic acid and osmotic induction of synchronous somatic embryo development of sweet potato

Summary Somatic embryos of sweet potato have potential as synthetic seeds. The effects of abscisic acid (ABA) (0,0,0.1, 1.0, 10.0 and 50.0 μM) were examined to improve synchrony and proliferation of somatic embryos. Transferring embryos compared to those cultures transferred at day 0. The development of embryos in suspension culture supplemented with ABA was poor. However, when calli proliferation cultures were in gelled medium and pulsed with 0.1 μM ABA for 14 d, the number of somatic embryos increased. Proembryonic masses cultured in mannitol-containing medium (Y=−1.5 MPa) increased embryo development and synchrony of embryo development. Thus, in this work ABA and mannitol have been shown to improve both the total number and the synchrony of sweet potato somatic embryos.     

Carotenoids with a 5,6-dihydro-5,6-dihydroxy-beta-end group, from yellow sweet potato "Benimasari", Ipomoea batatas Lam

A series of carotenoids with a 5,6-dihydro-5,6-dihydroxy-beta-end group, named ipomoeaxanthins A (1), B (2), C1 (3) and C2 (4) were isolated from the flesh of yellow sweet potato "Benimasari", Ipomoea batatas Lam. Their structures were determined to be (5R,6S,3'R)-5,6-dihydro-beta,beta-carotene-5,6,3'-triol (1), (5R,6S,5'R,6'S)-5,6,5',6'-tetrahydro-beta,beta-carotene-5,6,5'6'-tetrol (2), (5R,6S,5'R,8'R)-5',8'-epoxy-5,6,5',8'-tetrahydro-beta,beta-carotene-5,6-diol (3), and (5R,6S,5'R,8'S)-5',8'-epoxy-5,6,5',8'-tetrahydro-beta,beta-carotene-5,6-diol (4) by UV-Vis, NMR, MS and CD data.     

Mesophyll protoplast culture of sweet potato (Ipomoea batatas L.)

Mesophyll protoplasts of sweet potato (Ipomoea batatas L.) were readilyisolated by soaking chopped leaf tissue in distilled water for 16 h prior to enzymatic digestion. Isolated mesophyll protoplasts began to divide three days after start of culture in liquid modified N₆ medium and and formed colonies after 30 days of culture. The colonies transferred to solid medium grew rapidly and differentiated into calli. Some of the calli transplanted onto regeneration medium produced roots.     

Cryopreservation of shoot tips from in vitro plants of sweet potato [Ipomoea batatas (L.) Lam.] by vitrification

 Routine cryopreservation of shoot tips from sweet potato [Ipomoea batatas (L.) Lam] has been hampered by their survival variability after cryogenic exposure. We examined the effects of light conditions on stock plants, sucrose preculture and cryoprotectant loading on survival after vitrification using PVS2 solution. The survival of vitrified sweet potato shoot tips cooled to approximately –208  °C was increased by preculturing with 0.3 M sucrose for 24 h at 22  °C. Survival was also enhanced by excising shoot tips immediately after the 8-h dark photoperiod. The best survival after cryogenic exposure was obtained using 2 M glycerol +0.4 M sucrose for 1 h at 22  °C followed by dehydration with PVS2 for 16 min at 22  °C. Rapid cooling was used and achieved by the immersion of foil strips into partially solidified nitrogen. Successfully vitrified and warmed shoot tips directly developed shoots on a medium containing 1 μM NAA, 0.5 μM BA and 0.1 μM kinetin with only minimum callus formation. Shoot formation occurred in all surviving shoot tips. This procedure shows promise for cryopreserving sweet potato shoot tips. Received: 2 March 1999 / Revision received: 21 September 1999 / Accepted: 29 September 1999     

Transgenic sweet potato plants obtained byAgrobacterium tumefaciens-mediated transformation

Stable expression of foreign genes was achieved in sweet potato (Ipomoea batatas (L.) Lam) plants using anAgrobacterium tumefaciens mediated system. Embryogenic calluses produced from apical meristems of cultivar White Star were multiplied and cocultivated withA. tumefaciens strain EHA101 harboring a binary vector containing the -glucuronidase (GUS) and neomycin phosphotransferase (NPT II) genes. The calluses were transferred to selective regeneration medium and kanamycin resistant embryos were recovered which developed into morphologically normal plants. Histochemical and fluorimetric GUS assays of plants developed from the kanamycin resistant embryos were positive. Amplified DNA fragments were produced in polymerase chain reactions using GUS-specific primers and DNA from these plants. Transformation was confirmed by Southern analysis of the GUS gene. With the developed method, transgenic sweet potato plants were obtained within 7 weeks. This method will allow genetic improvement of this crop by the introduction of agronomically important genes.Florida Agricultural Experiment Station Journal Series N-02231. This research was partially supported by CNP_q/RHAE (Brazil).     

Cryopreservation of sweet potato (Ipomoea batatas [L.] Lam.) shoot tips by vitrification

Summary Vitrification is a technically simple method for cryopreserving plant germplasm, requiring only the application of suitable cryoprotectants and rapid cooling rates. Sweetpotato (Ipomoea batatas [L.] Lam.) shoot tips obtained from in vitro plants survived liquid nitrogen (–196°C) exposure following a vitrification-inducing pretreatment. Shoot tips were treated in a stepwise manner with a vitrification solution containing 30% glycerol, 15% ethylene glycol and 15% dimethylsulfoxide in growth medium. Incubation of shoot tips for 1 to 2 h in low concentrations of the vitrification solution enhanced survival. Most surviving shoot tips developed callus, and a variable percentage subsequently formed shoots. Survival was not achieved using two-step cooling procedures. The percentage of shoot tips surviving vitrification and those subsequently forming a shoot varied widely among replications.Abbreviations BA N⁶-benzyladenine - IBA indole-3-butyric acid - EG ethylene glycol - DMSO dimethylsulfoxide - MS Murashige and Skoog (1962) minerals and vitamins - LN liquid nitrogen - PI plant introduction