Sweetpotato Calli Research Articles

LncRNA-mediated ceRNA regulatory network provides new insight into chlorogenic acid synthesis in sweet potato.

Sweet potato (Ipomoea batatas L.) is considered a highly nutritional and economical crop due to its high contents of bioactive substances, such as anthocyanin and chlorogenic acid (CGA), especially in leaves and stems. The roles of noncoding RNAs (ncRNA), including long noncoding RNA (lncRNA) and microRNA (miRNA), in CGA synthesis, are still unknown. In this study, the differentially expressed (DE) mRNAs, miRNAs, and lncRNAs in two leafy vegetable genotypes "FS7-6-14-7" (high CGA content) and "FS7-6" (low CGA content) were identified. The cis-regulation between lncRNA and mRNA was analyzed. Then, the CGA synthesis-related modules MEBlue and MEYellow were identified to detect trans-regulation mRNA-lncRNA pairs. The GO and KEGG annotations suggested that mRNA in these two modules was significantly enriched in the secondary metabolite synthesis biosynthesis category. A competing endogenous RNAs (ceRNA) network, including 8730 miRNA-mRNA and 444 miRNA-lncRNA pairs, was constructed by DEmiRNA target prediction. Then, a CGA synthesis-related ceRNA network was obtained with lncRNA and mRNA from MEBlue and MEYellow. Finally, one relational pair, MSTRG.47662.1/mes-miR398/itb04g00990, was selected for functional validation. Overexpression of lncRNA MSTRG.47662.1 and mRNA itb04g00990 increased CGA content in both tobacco and sweet potato callus, while overexpression of miRNA mes-miR398 decreased CGA content. Meanwhile, regression analysis of the expression patterns demonstrated that MSTRG.47662.1, acting as a ceRNA, promoted itb04g00990 expression by competitively binding mes-miR398 in CGA synthesis in sweet potato. Our results provide insights into how ncRNA-mediated ceRNA regulatory networks likely contribute to CGA synthesis in leafy sweet potato.

Heat Shock Treatment Promoted Callus Formation on Postharvest Sweet Potato by Adjusting Active Oxygen and Phenylpropanoid Metabolism

This study aimed to investigate that rapid high-temperature treatment (RHT) at an appropriate temperature could accelerate callus formation by effectively promoting the necessary metabolic pathways in sweet potato callus. In this study, the callus of sweet potato was treated with heat shock at 50, 65, and 80 °C for 15 min. The callus formation was observed within 1, 3, and 5 days, and the accumulation of intermediates in the metabolism of phenylpropane and reactive oxygen species and changes in enzyme activities were determined. The results showed that appropriate RHT treatment at 65 °C stimulated the metabolism of reactive oxygen species at the injury site of sweet potato on the first day, and maintained a high level of reactive oxygen species production and scavenging within 5 days. The higher level of reactive oxygen species stimulated the phenylalanine ammonia-lyase (PAL), 4-coumarate-CoA ligase and cinnamate-4-hydroxylase activities of the phenylpropane metabolic pathway, and promoted the rapid synthesis of chlorogenic acid, p-coumaric acid, rutin, and caffeic acid at the injury site, which stacked to form callus. By Pearson’s correlation analysis, catalase (CAT), PAL, and chlorogenic acid content were found to be strongly positively correlated with changes in all metabolites and enzymatic activities. Our results indicated that appropriate high-temperature rapid treatment could promote sweet potato callus by inducing reactive oxygen species and phenylpropane metabolism; moreover, CAT, PAL, and chlorogenic acid were key factors in promoting two metabolic pathways in sweet potato callus.

Genome-wide identification of cold-tolerance genes and functional analysis of IbbHLH116 gene in sweet potato

Sweet potato (Ipomoea batatas L.) originated from South America; therefore, it is vulnerable to low temperature. Here, the evolutionary analysis of 22 cold-responsive genes in 35 plant species revealed that the identified MYC-type basic helix-loop-helix (bHLH) transcription factors exhibit diverse structures. We found that the number of bHLH gene family members was significantly lower than that of cold-tolerant species. We further systematically evaluated the gene structure, promoter analysis, synteny analysis, and expression pattern of 28 bHLH gene family members in sweet potato. The basic helix-loop-helix protein 116 (IbbHLH116) has the closest phylogeny to the AtICE1 protein of A. thaliana. However, the IbbHLH116 protein from cold-tolerant variety FS18 showed a 37.90% of sequence homology with AtICE1 protein. Subcellular localization analysis showed that IbbHLH116 is localized in the nucleus. The transcripts of IbbHLH116 were highly accumulated in cold-tolerant genotype FS18, particularly in new leaves and stems, compared to the cold-sensitive genotype NC1 under cold stress. Overexpression of IbbHLH116 in the wild type (Col-0) A. thaliana significantly enhanced cold tolerance in transgenic plants by regulating activities of oxidative protective enzymes, such as peroxidase (POD), superoxide dismutase (SOD), and the contents of malondialdehyde (MDA), proline and soluble proteins. Moreover, overexpression of IbbHLH116 in ice1 mutant A. thaliana fully rescued the cold-sensitive phenotype by promoting the expression of C-repeat binding factors 3 (CBF3). Overexpression of IbbHLH116 in the sweet potato callus also induced the expression of CBF3 under low temperature. These results imply that IbbHLH116 can perform the function of the ICE1 gene in conferring cold tolerance in sweet potato.

Overexpression of the Golden SNP-Carrying Orange Gene Enhances Carotenoid Accumulation and Heat Stress Tolerance in Sweetpotato Plants.

Carotenoids function as photosynthetic accessory pigments, antioxidants, and vitamin A precursors. We recently showed that transgenic sweetpotato calli overexpressing the mutant sweetpotato (Ipomoea batatas [L.] Lam) Orange gene (IbOr-R96H), which carries a single nucleotide polymorphism responsible for Arg to His substitution at amino acid position 96, exhibited dramatically higher carotenoid content and abiotic stress tolerance than calli overexpressing the wild-type IbOr gene (IbOr-WT). In this study, we generated transgenic sweetpotato plants overexpressing IbOr-R96H under the control of the cauliflower mosaic virus (CaMV) 35S promoter via Agrobacterium-mediated transformation. The total carotenoid contents of IbOr-R96H storage roots (light-orange flesh) and IbOr-WT storage roots (light-yellow flesh) were 5.4–19.6 and 3.2-fold higher, respectively, than those of non-transgenic (NT) storage roots (white flesh). The β-carotene content of IbOr-R96H storage roots was up to 186.2-fold higher than that of NT storage roots. In addition, IbOr-R96H plants showed greater tolerance to heat stress (47 °C) than NT and IbOr-WT plants, possibly because of higher DPPH radical scavenging activity and ABA contents. These results indicate that IbOr-R96H is a promising strategy for developing new sweetpotato cultivars with improved carotenoid contents and heat stress tolerance.

A single amino acid change at position 96 (Arg to His) of the sweetpotato Orange protein leads to carotenoid overaccumulation.

IbOr-R96H resulted in carotenoid overaccumulation and enhanced abiotic stress tolerance in transgenic sweetpotato calli. The Orange (Or) protein is involved in the regulation of carotenoid accumulation and tolerance to various environmental stresses. Sweetpotato IbOr, with strong holdase chaperone activity, protects a key enzyme, phytoene synthase (PSY), in the carotenoid biosynthetic pathway and stabilizes a photosynthetic component, oxygen-evolving enhancer protein 2-1 (PsbP), under heat and oxidative stresses in plants. Previous studies of various plant species demonstrated that a single-nucleotide polymorphism (SNP) from Arg to His in Or protein promote a high level of carotenoid accumulation. Here, we showed that the substitution of a single amino acid at position 96 (Arg to His) of wild-type IbOr (referred to as IbOr-R96H) dramatically increases carotenoid accumulation. Sweetpotato calli overexpressing IbOr-WT or IbOr-Ins exhibited 1.8- or 4.3-fold higher carotenoid contents than those of the white-fleshed sweetpotato Yulmi (Ym) calli, and IbOr-R96H overexpression substantially increased carotenoid accumulation by up to 23-fold in sweetpotato calli. In particular, IbOr-R96H transgenic calli contained 88.4-fold higher levels of β-carotene than those in Ym calli. Expression levels of carotenogenesis-related genes were significantly increased in IbOr-R96H transgenic calli. Interestingly, transgenic calli overexpressing IbOr-R96H showed increased tolerance to salt and heat stresses, with similar levels of malondialdehyde to those in calli expressing IbOr-WT or IbOr-Ins. These results suggested that IbOr-R96H is a useful target for the generation of efficient industrial plants, including sweetpotato, to cope with growing food demand and climate change by enabling sustainable agriculture on marginal lands.

Selection and regeneration of purple sweet potato calli against drought stress simulated by polyethylene glycol

Background: Water shortage due to natural and/or technical drought stress, widespread throughout Sumatra, Java, Sulawesi and Kalimantan islands, significantly reduces crop production. The development of varieties tolerant to drought stress is important since it is more effective rather than improving irrigation infrastructure to increase the sweet potato productivity. Methods: Selection and regeneration experiments assessing purple sweet potato callus tolerance of drought stress, simulated by polyethylene glycol (PEG), were conducted to generate new variant plants tolerant of drought stress. Sterile explants (leaf and petiole) generated from previous in vitro culture were inoculated to the Murishage and Skoog (MS) medium containing plant growth regulator combination as treatments to induce calli. The calli were then transferred to half-MS medium containing 0, 5, 10, 15 and 20% PEG as selection agent for drought tolerance. The surviving calli were regenerated in the MS medium containing 0, 0.5, 1 or 1.5 mg l-1 6-benzylaminopurine (BAP). The callus formation, growth and survivability during in vitro culture were measured. Results: Calli were successfully formed in almost all media containing 2,4-Dichlorophenoxyacetic acid (2,4-D ) with the concentration of 1, 2, 3 and 4 mg l-1 and BAP (concentration: 0.5 and 1 mg l-1), but the medium of MS + 2 mg l-1 2,4-D + 0.5 mg l-1 BAP resulted in the highest number of induced calli per treatment (mean=11.36), with the percentage of responsive explants standing at around 96%. The higher the concentration of PEG, the lower the number of surviving calli. At 20% PEG, only 54.42% calli survived. There were five plants successfully regenerated from the survived calli at 20% PEG, using MS medium containing 1.5 mg l-1 BAP. Conclusions: The experiment has successfully produced putative drought-tolerant plants by callus screening using PEG as drought-tolerance-selecting agent in purple sweet potato.

Down-regulation of lycopene ε-cyclase expression in transgenic sweetpotato plants increases the carotenoid content and tolerance to abiotic stress

Carotenoids are required for many biological processes in plants and humans. Lycopene ε-cyclase (LCY-ε) catalyzes the conversion of lycopene into lutein via the α-branch carotenoid biosynthesis pathway. Down-regulation of IbLCY-ε by RNAi increases carotenoid accumulation and salt stress tolerance in transgenic sweetpotato calli. As the role of IbLCY-ε in carotenoid biosynthesis and environmental stress responses in whole plants is poorly understood, transgenic sweetpotato (RLE plants) with reduced expression of IbLCY-ε were developed. RLE plants contained higher levels of total carotenoid and β-carotene, due to an elevated β-carotene/lutein ratio rather than increased de novo biosynthesis. RLE plants showed high reactive oxygen species/radical-scavenging activity. They also exhibited an enhanced tolerance of both salt and drought stress, which was associated with lower membrane permeability and a higher photosynthetic rate, respectively. Elevated carotenoid accumulation in RLE plants mitigated the reductions in leaf photosystem II efficiency and chlorophyll induced by abiotic stress. Expression of the carotenoid cleavage genes 9-cis-epoxycarotenoid dioxygenase, carotenoid cleavage dioxygenase 1 (CCD1) and CCD4 was higher in RLE plants, as was abscisic acid accumulation. IbLCY-ε silencing thus offers an effective approach for developing sweetpotato plants with increased tolerance to abiotic stress that will grow on global marginal lands with no reduction in nutritional value.

Selection and regeneration of purple sweet potato calli against drought stress simulated by polyethylene glycol

Background: Water shortage due to natural and/or technical drought stress, widespread throughout Sumatra, Java, Sulawesi and Kalimantan islands, significantly reduces crop production. The development of varieties tolerant to drought stress is important since it is more effective rather than improving irrigation infrastructure to increase the sweet potato productivity. Methods: Selection and regeneration experiments assessing purple sweet potato callus tolerance of drought stress, simulated by polyethylene glycol (PEG), were conducted to generate new variant plants tolerant of drought stress. Sterile explants (leaf and petiole) generated from previous in vitro culture were inoculated to the Murishage and Skoog (MS) medium containing plant growth regulator combination as treatments to induce calli. The calli were then transferred to half-MS medium containing 0, 5, 10, 15 and 20% PEG as selection agent for drought tolerance. The surviving calli were regenerated in the MS medium containing 0, 0.5, 1 or 1.5 mg l-1 6-benzylaminopurine (BAP). The callus formation, growth and survivability during in vitro culture were measured. Results: Calli were successfully formed in almost all media containing 2,4-Dichlorophenoxyacetic acid (2,4-D ) with the concentration of 1, 2, 3 and 4 mg l-1 and BAP (concentration: 0.5 and 1 mg l-1), but the medium of MS + 2 mg l-1 2,4-D + 0.5 mg l-1 BAP resulted in the highest number of induced calli per treatment (mean=11.36), with the percentage of responsive explants standing at around 96%. The higher the concentration of PEG, the lower the number of surviving calli. At 20% PEG, only 54.42% calli survived. There were five plants successfully regenerated from the survived calli at 20% PEG, using MS medium containing 1.5 mg l-1 BAP. Conclusions: The experiment has successfully produced putative drought-tolerant plants by callus screening using PEG as drought-tolerance-selecting agent in purple sweet potato.

Stress-induced expression of the sweetpotato gene IbLEA14 in poplar confers enhanced tolerance to multiple abiotic stresses

Late embryogenesis abundant (LEA) proteins are small, highly hydrophilic proteins that act as protectors of macromolecules and increase abiotic stress tolerance in plants. We previously reported that overexpressing sweetpotato IbLEA14 under the control of the CaMV 35S promoter increased osmotic and salt stress tolerance in transgenic sweetpotato calli. In this study, we generated transgenic poplar plants (Populus alba × P. glandulosa) expressing IbLEA14 under the control of the oxidative stress-inducible SWPA2 promoter (referred to as SL plants). Among the 15 SL plants obtained, three lines (SL2, SL7, and SL12) were established based on IbLEA14 transcript levels, tolerance to salt stress and Southern blot analysis. The SL plants exhibited less damage in response to methyl viologen-mediated oxidative stress than non-transgenic (NT) plants. SL plants also showed enhanced tolerance to drought, salt, and heat stress, which was associated with higher photosystem II efficiency and lower malondialdehyde levels compared with NT plants. Furthermore, SL plants had higher levels of monolignol biosynthesis-related gene transcripts under drought stress compared with NT plants. Finally, SL plants exhibited increased tolerance to heat stress, which is associated with the high thermostability of IbLEA14 protein. SL plants might be useful for reforestation on global marginal lands, including desertification and reclaimed areas.

Evaluation of the Phosphomannose Isomerase-Based Selection System for Genetic Transformation of Sweetpotato

In response to increased public concern on antibiotic or herbicide resistance genes usage in genetically modified plants, mannose was evaluated as selectable agent for the genetic transformation of sweetpotato. Nontransformed sweetpotato stem explants of cv. KSP36 were cultured on media containing various combinations and concentrations sucrose and mannose ranging from 0 to 30 g/l. Likewise, efficacy of hygromycin and kanamycin as selection agents for transformation of sweetpotato was evaluated on media containing varying concentrations of antibiotics ranging 0 to 100 mg/l. Selection agent effectiveness was determined by detecting the minimal concentration of the selection agent required to fully inhibit sweetpotato calli growth. Hygromycin was the most effective selection agent as it inhibited cell growth at concentrations above 20 mg/l. Kanamycin was moderately effective as it inhibited cell growth at 60 mg/l. Sweetpotato calli were able to grow and even produce embryos even when mannose was the only source of carbohydrates.DOI: http://dx.doi.org/10.3126/ijls.v9i3.12466 International Journal of Life Sciences 9 (3): 2015; 46-53

Overexpression of the sweet potato IbOr gene results in the increased accumulation of carotenoid and confers tolerance to environmental stresses in transgenic potato

In a previous study, we have evidenced that the overexpression of the IbOr gene isolated from sweet potato conferred a tolerance activity against salinity and methyl viologen (MV) treatment in transgenic sweet potato calli along with an enhanced carotenoid content. In this study, to further examine the function of the IbOr gene in heterologous organism, we transformed the IbOr gene into potato under the direction of SWPA2 promoter, a strong inducible promoter upon treatment with various environmental stresses. Consistently with our previous study of sweet potato calli, the level of total carotenoid was elevated up to 2.7-fold (38.1μgg−1DW) compared to the non-transgenic control, Atlantic cultivar. However, the composition of carotenoid was not influenced by the overexpression of the IbOr gene since only pre-existing carotenoids in the non-transgenic control including violaxanthin, lutien and β-carotene were elevated at a similar level of total carotenoids. In general, the transcript levels for most of carotenogenesis-related genes were elevated in transgenic tuber, whereas they remained at similar levels in transgenic leaf tissues compared to those of non-transgenic controls. The increased levels of carotenoid content in the leaf or tuber tissue of transgenic lines were correlated with the enhanced tolerance activity against salt- or MV-mediated oxidative stresses and DPPH radical-scavenging activity. Our preliminary results suggest that further investigation is required for the development of a crop tolerant to salinity and other environmental stresses through the overexpression of the IbOr gene.

Cloning and characterization of an Orange gene that increases carotenoid accumulation and salt stress tolerance in transgenic sweetpotato cultures

The Orange (Or) gene is responsible for the accumulation of carotenoids in plants. We isolated the Or gene (IbOr) from storage roots of orange-fleshed sweetpotato (Ipomoea batatas L. Lam. cv. Sinhwangmi), and analyzed its function in transgenic sweetpotato calli. The IbOr gene has an open reading frame in the 942 bp cDNA, which encodes a 313-amino acid protein containing a cysteine-rich zinc finger domain. IbOr was strongly expressed in storage roots of orange-fleshed sweetpotato cultivars; it also was expressed in leaves, stems, and roots of cultivars with alternatively colored storage roots. IbOr transcription increased in response to abiotic stress, with gene expression reaching maximum at 2 h after treatment. Two different overexpression vectors of IbOr (IbOr-Wt and IbOr-Ins, which contained seven extra amino acids) were transformed into calli of white-fleshed sweetpotato [cv. Yulmi (Ym)] using Agrobacterium. The transgenic calli were easily selected because they developed a fine orange color. The expression levels of the IbOr transgene and genes involved in carotenoid biosynthesis in IbOr-Wt and IbOr-Ins transgenic calli were similar, and both transformants displayed higher expression levels than those in Ym calli. The contents of β-carotene, lutein, and total carotenoids in IbOr-Ins transgenic lines were approximately 10, 6, and 14 times higher than those in Ym calli, respectively. The transgenic IbOr calli exhibited increased antioxidant activity and increased tolerance to salt stress. Our work shows that the IbOr gene may be useful for the biotechnological development of transgenic sweetpotato plants that accumulate increased carotenoid contents on marginal agricultural lands.

Effect of genotype, gelling agent, and auxin on the induction of somatic embryogenesis in sweet potato ( Ipomoea batatas Lam.)

Lateral buds of six cultivars of sweet potato were induced to form embryogenic callus in a culture medium solidified with two types of gelling agents, Agar or Gelrite, and supplemented with various concentrations of auxins, 2,4-D, 2,4,5-T and Picloram. Of the six cultivars screened, only three gave an embryogenic response. Best results with an average of 3.53% embryogenic response were obtained with the medium solidified with Agar, while in Gelrite only 0.45% of lateral buds gave rise to embryogenic callus. The interaction between the genotype and auxins was highly significant; particularly the optimal response was obtained with cv. Zho and 865 yielding 10.7 and 14.7% somatic embryogenesis, respectively, in the medium containing 2,4,5-T or Picloram. The plant conversion was dramatically improved by subculture of the embryogenic callus on the medium with the combination of 1 μM 2,4-D and 1 μM Kinetin or 5 μM ABA alone before transfer of mature embryos onto hormone-free medium. The embryogenic callus of sweet potato and its sustained ability to further regenerate plants have regularly been maintained for several years by frequent subculture in 5 μM 2,4,5-T or the combination of 10 μM 2,4-D and 1 μM BAP or kinetin. The embryo-derived plants seemed apparently genetically stable and similar to the hexaploid parental plants, based on morphological analysis and their ploidy level determined by using flow cytometry. To cite this article: Z. Triqui et al., C. R. Biologies 331 (2008).

A LINE-type Retrotransposon Active in Meristem Stem Cells Causes Heritable Transpositions in the Sweet Potato Genome

We isolated a LINE-type retrotransposon, LIb, which showed high transposition activity in sweet potato callus. A copy transposed in the callus was 6303 bp in length and showed key features of a LINE element. Apparently full-length copies sharing the 5' UTR sequence with the 6303-bp copy increased dramatically in the callus as several original copies in the sweet potato genome. These apparently full-length copies had almost identical sequences to other transposed copies, many of which were truncated at the 5' end upon transposition. These results indicate that active LIb is confined to a single LINE family, and that members containing a long functional 5' UTR are present in limited numbers in the sweet potato genome. This is despite their copy numbers being estimated at over 100. The transcription of LIb was not completely suppressed, even in wild-type plants. Spontaneous transpositions were found among local variant lines of the cultivar Koukei14, from which the callus with high LIb activity was derived. Meristem culture of this cultivar appeared to facilitate transpositions of LIb in a mericlone plant. This is the first experimental demonstration of retrotransposition in a plant species without the imposition of cell differentiation. LIb transpositions appear to occur in single founder cells in the meristem because the LIb insertion was found throughout mericlone plant tissues. Transpositional activities in meristem cells might be essential characteristics of plant retrotransposons that cause heritable changes in host plant genomes and genetic systems.

サツマイモの器官別デンプンの特性に関する研究

To study starch biosynthesis in terms of molecular structures of organ-specific starches within one plant species is interesting. The structural properties of starches from tuberous roots of various sweetpotato varieties and breeding lines, and also starches from leaf and callus of sweetpotato cv. Koganesengan were investigated in this study. Examination of the structure and pasting properties of the 16 kinds of root starches suggested that, statistically, the variation in pasting properties of the starches was more accurately reflected by amylopectin structures than by amylose. During the course of examination on starch structures of the latest breeding lines, we found two lines having root starches with unique characteristics. One had low amylose starch and the other had low temperature gelatinizing starch. The physicochemical properties of these starches were likewise characterized. In leaves where the starch structures and their diurnal-nocturnal changes were examined from leaves harvested at different times of a day. Gel-permeation chromatography of the leaf starches and amylopectins showed that, compared to root starch, the leaf starches had higher amount of materials eluted at the low molecular weight fraction which contained both amylose and a large amount of small amylopectin molecules. It was found that the amylose content of the leaf starches dropped during the daytime whereas the branch aspect of the leaf amylopectins remained relatively constant throughout the whole day. Finally, the structural properties of starch formed in the sweetpotato callus were studied. The starch content increased from nearly zero to the maximum content in the first week of culture, and then decreased afterward. Based on the chain-length distributions of the callus starches at different culture periods, it was demonstrated that the structural change from waxy to normal feature of starch granule occurred during the culture of the callus. Thus, the structural characteristics of starches from tuberous root, leaf and callus of sweetpotato have been revealed.

Developmental change in starch granules in sweetpotato callus

Developmental change in starch granules formed in sweetpotato callus was examined. The starch content increased from nearly 0 to 10.6% of the dried calli over one week. The chain-length distribution of the debranched starches determined by gel-permeation chromatography revealed that no amylose was present in the one-day sample. During development, amylose fraction increased continuously for seven days to 12.3% of total starch. On the other hand, the ratio of two unit-chain fractions from amylopectin was nearly identical, and the short-chain distribution (DP=6–35) was also similar throughout the culture period. Furthermore, all starch granules showed an A-type crystallite. These results suggest that the amylopectin molecules were completed as a granule as early as the first day before the appearance of the amylose fraction. With respect to the structural change of starch granules, the transcript of the GBSSI gene was detected in a constant level during the culture for one month. These results indicate that in spite of the occurrence of the GBSSI transcript at the start of starch accumulation, the structural change from waxy to normal feature of starch granule occurred during the culture of sweetpotato calli.

Cloning and Characterization of a cDNA for Manganese Superoxide Dismutase from Callus of Sweet Potato

A full-length complementary DNA (cDNA) clone encoding a putative manganese superoxide dismutase (Mn-SOD) from the callus of sweet potato was amplified by polymerase chain reaction technique from cDNAs synthesized from callus mRNA. Nucleotide sequence analysis of this cDNA clone revealed that it comprises a complete open reading frame coding for 233 amino acid residues and a 29 amino acid transit peptide at the N terminus. The deduced amino acid sequence showed greater identity (67-75%) with plant mitochondrial Mn-SOD than with Mn-SOD from mammalian mitochondria (53%), yeast (47%), or bacteria (44%). The residues required to coordinate the single tervalent manganese ion and the 11 residues putatively involved in the active center are conserved as they are among all reported Mn-SOD sequences. These suggest that the sweet potato callus cDNA clone encodes a mitochondrial Mn-SOD. In addition, the coding region of Mn-SOD cDNA from sweet potato callus was introduced into an expression vector, pET-28a(+) and transformed into Escherichia coli BL21(DE3). A predominant protein band was detected by Coomassie blue staining of native PAGE, and activity staining confirmed the result of Coomassie blue staining. These indicate that this Mn-SOD cDNA clone can express Mn-SOD enzyme in E. coli.

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.

Starch-accumulating Sweet Potato Callus Tissue Devoid ofβ-Amylase but with Two Starch Phosphorylase Isozymes

By controlling the concentrations of kinetin, auxin, and sucrose in the Murashige–Skoog medium, starch contents in callus culture induced from sweet potato tissues could be manipulated. Activity staining and Western analysis on PAGE plates and activity assays made on starch phosphorylase in the presence and absence of mercuric ions showed that β-amylase is absent in callus cultures regardless of whether their starch content is high or low. This would imply that β-amylase induction in sweet potato calli is not linked to the metabolic control through which the expression of storage function is associated, as proposed by Nakamura et al. [Plant Physiol., 96, 902 (1991)] for sweet potato leaf-petiole cuttings. Analyses of starch phosphorylase in crude extracts suggested the presence of a new starch phosphorylase in tuberous root and callus tissue. This phosphorylase is immunologically different from the tuberous root and leaf enzymes that we studied previously.

Isolation and culture of protoplasts from high anthocyanin-producing callus of sweet potato

Optimum conditions for the isolation and culture of protoplasts from high anthocyanin-producing callus of sweet potato (Ipomoea batatas) were investigated. Growth phase of callus and the ratio of callus-enzyme solution affected the yield of protoplasts. Composition of the medium and protoplast density were examined for protoplast culture.Small colonies were regenerated from the protoplasts. Upon transfer to light a high amount of anthocyanin was accumulated in these colonies.