Effect of 5-Aminolevulinic Acid on Development and Salt Tolerance of Potato (Solanum tuberosum L.) Microtubers in vitro

The effects of 5-aminolevulinic acid (ALA), a key precursor in the biosynthesis of porphyrins such as chlorophyll and heme, on development and salt tolerance of microtubers of two potato (Solanum tuberosum L.) cultivars Jingshi-2 and Zihuabai were examined under in vitro conditions. ALA at 0.3–3 mg/l promoted microtuber formation by increasing the average number, diameter, and fresh weight of microtubers especially under 0.5% NaCl stress conditions, but further increase in ALA concentration resulted in a reduction of microtuber yield irrespective of NaCl stress. Under 1.0% NaCl stress conditions, microtuberization was seriously repressed and could not be restored by the addition of ALA. The accumulation of malondialdehyde in the microtubers treated with 30 mg/l ALA increased by 22% compared to the controls (no salinity), while only a 7% increase was observed when the microtubers were exposed to 0.5% NaCl, indicating that ALA functions as a protectant against oxidative damages of membranes. Under 0.5% NaCl stress conditions, the highest activities of peroxidase and polyphenoloxidase were detected in microtubers treated with ALA at 0.3 and 3 mg/l, being by 73% and by 28% greater than those in the untreated controls, respectively. These results demonstrate that ALA at lower concentrations of 0.3–3 mg/l promotes development and growth of potato microtubers in vitro and enhances protective functions against oxidative stresses, but ALA at 30 mg/l and higher concentrations seems to induce oxidative damage probably through formation and accumulation of photooxidative porphyrins.     

The effect of gamma irradiation on potato microtuber production in vitro

The effects of low doses of gamma irradiation and potato (Solanum tuberosum L.) cultivar on the production of microtubers in vitro were investigated. Nodal segments from virus free explants of three potato cultivars (cv.) were placed on tuberization inducing medium and irradiated with 4 doses of gamma radiation (2.5, 5, 10, 15 Gy). Cv. Diamant produced the highest number of microtubers followed by Draga and Spunta. Irradiation of the explants with 2.5 Gy of gamma radiation led to a significant increase in the number of microtubers (38% increase over the control). Average weight of microtubers was not significantly influenced by low doses of gamma irradiation. Draga microtubers were the largest followed by Diamant and Spunta. Microtubers resembled mature tubers in shape (Spunta was oval and Draga and Diamant were spherical). Size of microtubers was crucial for sprouting in vivo. It is suggested that only microtubers larger than 5 mm in diameter (250 mg) be used to produce minitubers in vivo. Since 2.5 Gy is a low irradiation dose, it can be used to enhance tuberization in vitro without fear of genetic changes in the used cultivars. This revised version was published online in June 2006 with corrections to the Cover Date.     

Carboxylic acids affect induction,development and quality of potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.) microtubers grown <Emphasis Type="Italic">in vitro</Emphasis> from single-node explants

The role of three carboxylic acids with increasing alkyl-chain length, viz., formic, acetic and propionic acids in microtuberization was investigated in three potato (Solanum tuberosum L.) genotypes in vitro. Different concentrations of these carboxylic acids (0.0, 1.5, 3.0, 4.5 and 6.0 mM) were supplemented in microtuber induction medium, which was based on MS medium containing 8% sucrose, and their efficacy for induction, development and quality of microtubers was studied using single-node explants under continuous darkness at 20 °C. The carboxylic acids exhibited a strong stolon- and root-inhibiting effect on single-node explants with their increasing concentrations as well as alkyl-chain length (i.e., formic < acetic < propionic acids), and their mode of action was synonymous with antigibberellin substances. However, they did not have any significant inductive effect on microtuberization as compared to that under 8% sucrose medium. Rather they did show a detrimental effect on microtuber development in terms of average microtuber fresh weight with increasing concentrations as well as alkyl-chain length; both acetic and propionic acids at 6.0 mM induced the smallest microtubers in vitro. The carboxylic acids could, however, significantly increase the harvest indices suggesting their possible role in the regulation of source-sink co-ordination during microtuberization from single-node explants. But the most favourable effect of carboxylic acids on microtubers was apparent in terms of dry matter concomitant with higher starch synthesis and enhanced accumulation of reducing and total sugars. Acetic acid was the most effective in increasing the percentage of microtuber dry matter. The higher percentage of dry matter with higher carbohydrate reserves in microtubers induced by the carboxylic acids could be assumed to affect the quality of microtubers for subsequent storage, dormancy release and sprout growth.     

The role of GA,IAA and BAP in the regulation of <Emphasis Type="Italic">in vitro</Emphasis> shoot growth and microtuberization in potato

The effect of auxin, GA and BAP on potato shoot growth and tuberization was investigated under in vitro condition. The shoot length of potato explants increased with the increasing of concentrations (0.5 – 10 mg dm⁻³) of IAA treatment especially with the addition of GA₃ (0.5 mg dm⁻³), but was inhibited by BAP (5 mg dm⁻³). The root number and root fresh weight of potato explants increased with the increasing of IAA levels either in the presence of GA₃ (treatment IAA+GA) or not (IAA alone). However, no root was observed in the treatment IAA+BAP, instead there were brown swollen calli formed around the basal cut surface of the explants. The addition of GA₃ remarkably increased the fresh weight and diameter of calli. Microtubers were formed in the treatments of IAA+BAP and IAA + GA + BAP but not observed in the treatments of IAA alone or IAA + GA. IAA of higher concentrations (2.5 – 10 mg dm⁻³) was helpful to form sessile tubers. With the increasing of IAA levels, the fresh weight and diameter of microtubers increased progressively. At 10 mg/L IAA, the fresh weight and diameter of microtubers in the treatment of IAA + GA + BAP were 409.6 % and 184.4 % of that in the treatment of IAA + BAP respectively, indicating the interaction effect of GA and IAA in potato microtuberization.     

Relative importance of maltose and sucrose supplied during a 2-step potato microtuberization process

A 2-stage in vitro tuberization process comprising first micropropagation via nodal explants and then tuber induction in the resultant in vitro plantlets was studied using 2 cultivars of potato, Iwa and Daeji. In particular, the effects on both plantlet growth and subsequent in vitro tuberization of Murashige and Skoog (1962) basal medium containing either sucrose or maltose, each at 3 % (w/v), used for micropropagation were investigated. Sucrose and maltose were found to be equally effective in supporting development of vigorous plantlets from the nodal explants of both potato cultivars. Upon transfer to a medium with an optimised level of sucrose (i.e. 8 %, w/v) for in vitro tuberization, only the plantlets previously grown in the sucrose-containing medium were capable of forming more microtubers of the larger size category (greater than 0.5 g). The relative importance of sucrose supply at the mircropropagation stage was further confirmed when the resultant plantlets grown in the 3 % sucrose-containing medium were transferred to an in vitro tuberization medium containing either sucrose or maltose, each at 8 % (w/v). In this experiment, maltose and sucrose had indistingushable effects on in vitro tuberization.     

Effects of temperature fluctuation during in vitro phase on in vitro microtuber production in different cultivars of potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.)

An experiment, including potato cultivars Gloria (very early), Marfona (mid-early) and Agria (late), was carried out to assess the effects of different temperatures during two phases of the day on in vitro potato microtuber production. Temperature significantly (P < 0.01) affected the percentage of cuttings that produced microtubers. The highest temperatures in either phase resulted in the lowest percentage of cuttings that produced microtubers. With lower temperature during either phase, we found more microtubers per cutting and larger microtuber sizes. The effects of temperature on individual microtuber weight were not statistically significant. However, increasing the temperature during different thermophases increased both length and weight of sprouts formed on the microtubers. Moreover, the highest temperatures resulted in the lowest levels of tuberization (as shown by bud status) and the largest sprout growth. The temperature amplitude had a significant effect as well: very large temperature amplitudes resulted in poorer tuber formation compared with smaller temperature amplitudes with the same average temperature. All three cultivars showed different responses with regard to the percentage of explants that produced microtubers. After 45 days of incubation, the percentage of explants producing microtubers, the number of microtubers and the length of the sprouts were significantly increased compared with 35 days of incubation. Nonetheless, the status of the microtubers (sprouted or not-sprouted) and the microtuber size did not change beyond 35 days of incubation. Polynomial analysis of temperature effects showed that almost all traits assessed showed a significant linear trend.     

Effect of γ-radiation on development,yield and quality of microtubers <Emphasis Type="Italic">in vitro</Emphasis> in <Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.

Explants obtained from in vitro-propagated plantlets of two potato cultivars, Shepody and Atlantic, were treated with five doses of γ-radiation (0, 2, 4, 6 and 8 Gy) to investigate the stimulating effects of low irradiation on the production and quality of microtubers in vitro. Microtubers of both cultivars treated with γ-radiation initiated 5 d earlier than in the non-irradiated control. The whole period of microtuberization was prolonged by 10 – 15 d with 4, 6 and 8 Gy irradiation treatment for cv. Atlantic. Irradiation of the plantlets (4 Gy) led to a significant increase not only in the microtuber number (116.7 and 34.5 % over the control) but also in the fresh mass (77.6 and 23.2 % in Shepody and Atlantic, respectively). Low dose irradiation (2 – 4 Gy) increased the starch content of microtubers. High doses (6 – 8 Gy) enhanced ascorbic acid and reducing sugar contents. 4 – 6 Gy doses also effectively increased the protein contents of microtubers.     

Gelrite as an alternative to agar for micropropagation and microtuberization of Solanum tuberosum L. cv. Baraka

Summary Phytagel™ allowed the production of longer internodes, faster in vitro tuberization, and larger tubers in Solanum tuberosum L. cv. Baraka as compared to Difco Bacto-agar during both an 8-h photoperiod or in darkness. It also allowed a higher tuberization percentage in the dark. Only a 0.2% (wt/vol) Phytagel allowed optimal micropropagation and microtuberization under the photoperiod regime used. Water availability does not account for the observed differences in growth and tuberization between media containing the above gelling agents. In consequence, Phytagel appears as an advantageous alternative to agar for micropropagation and microtuberization.     

In vitro induction of minitubers in yam (<Emphasis Type="Italic">Dioscorea cayenensis</Emphasis>- <Emphasis Type="Italic">D. rotundata</Emphasis> complex)

Two methods were used to produce yam minitubers from two different yam cultivars (cv. Krengle and cv. Kponan) using in vitro culture techniques. Method 1: Yam microtubers were first initiated in vitro and then transplanted to soil to generate plants from which minitubers were produced. Yam plants were obtained either by directly planting the microtubers to soil, or by inducing the germination of the microtubers using various chemical and physical treatments, before their transfer to soil. Method 2: Yam plantlets were first produced in vitro and then transplanted to soil for further development and tuber production. In both methods, the presence of jasmonic acid (JA) in the culture medium was found to be essential for yam tuberization, as well as for the germination of yam microtubers. In vitro production of yam microtubers was variety dependant. Compared to cv. Krengle, cv. Kponan responded better to microtuberization, and 2.5 μM JA was the optimum concentration resulting in 70 and 90% explants producing microtubers in the MS medium and the Tuberization medium (T-medium), respectively. Germination of the microtubers required treatment of JA at concentrations ranging from 1.0 to 2.5 μM. The overall length of the process to produce minitubers from microtubers took 32 weeks. In contrast, minitubers were obtained within 20 weeks when plantlets were directly transferred to soil. In this case, plantlets were first grown for 8 weeks on medium containing JA (0.1–1.0 μM) and 8% sucrose to initiate plant growth and rooting.     

Jasmonate effects on in vitro tuberization and tuber bulking in two potato cultivars (Solanum tuberosum L.) under different media and photoperiod conditions

Summary Jasmonic acid (JA) effects on in vitro tuberization of potato nodal explants cvs. Sangre and Russet Burbank were tested under liquid and solid media conditions and 0,8, and 16h photoperiod. Explants taken from stock plants grown on 2.5μM JA-supplemented medium tuberized first, particularly in darkness. The most pronounced benefits of the JA pretreatment were recorded under 16h photoperiod, which is known to inhibit tuberization. Cultivar Sangre benefited from the JA preconditioning of stock plants more than Russet Burbank. Russet Burbank required the JA supplement in tuberization media to reach the same degree of stimulation. Overall, microtubers produced either from JA preconditioned stock plants or on the JA-containing tuberization media were more uniform and larger than from other treatments. Eight hours photoperiod was by far the best treatment for the production of high-quality uniform microtubers. JA conditioning of stock plants prior to taking explants for tuberization is being proposed as a treatment enhancing the quality of microtubers.     

Effects of kinetin,paclobutrazol and their interactions on the microtuberization of potato stem segments cultured in vitro in the light

Single-nodal cuttings of Solanum tuberosum (four cultivars) and Solanum chacoense were induced to produce in vitro microtubers on Murashige & Skoog (MS) medium supplemented with 8 g l^–1 sucrose and various concentrations of kinetin and paclobutrazol. The cultures were kept 10 days in darkness and then transferred to a 14 h daylength with 100 µE m^–2 sec^–1 light intensity at 21 °C. Kinetin (2.5 mg l^–1) had no significant influence on tuber formation. However, its addition together with paclobutrazol (0.001 mg l^–1) significantly enhanced tuberization. Paclobutrazol alone stimulated early tuber initiation and inhibited stem growth. Despite some genotype × treatment interactions, all genotypes (from very early to late and wild type) formed the maximum proportion of explants bearing microtubers on the media containing both plant growth regulators.     

Effects of photoperiod,mineral medium strength,inorganic ammonium,sucrose and cytokinin on root,shoot and microtuber development in shoot cultures of Dioscorea alata L. and D. bulbifera L. yams

The effects of photoperiod (8, 12 or 16 h), mineral medium strength (dilutions of a tuberization medium, the T medium), sucrose (0, 2, 4, 8% w/v) and kinetin (0, 2.5μM) on the development of roots, shoots and microtubers in shoot cultures of Dioscorea alata L. and D. bulbifera L. yams were evaluated. All of the factors were found to have substantial effects on microtuber induction in these two species. The effects of high and low inorganic ammonium containing media on microtuberization of yam shoot cultures indicated that ammonium ions inhibited microtuber induction in D. alata but not in D. bulbifera. Microtubers of D. alata were only formed on shoot cultures if these were held under 8-h days. D. bulbifera cultures on the other hand produced microtubers under this photoperiod treatment as well as under 16-h photoperiods provided that kinetin was present in media at 2.5μM. Most microtuberization in D. alata shoot cultures occurred on full-strength T medium supplemented with 2% sucrose, 2.5μM kinetin held under 8-h photoperiod at 25°C, whereas most microtuberization in D. bulbifera shoot cultures occurred on full-strength MS medium supplemented with 4% sucrose, 2.5μM kinetin held under 8-h photoperiods at 25°C. Under these two sets of conditions, yam shoot cultures consistently produced microtubers with individual weights in excess of 100 mg which were large enough to be capable of direct planting and subsequent growth in unsterilized soils.     

In vitro production of microtubers for conservation of potato germplasm: Effect of genotype,abscisic acid,and sucrose

Summary With the objective of using microtubers for conservation of potato germplasm, the main effects of genotype, abscisic acid (ABA), and sucrose level, and of their interactions on biomass production, microtuberization, microtuber dormancy, and dry matter content, were studied. ABA decreased both microtuber production and microtuber dormancy, whereas higher concentrations (60–80 gl⁻¹) of sucrose promoted biomass production, microtuber production as well as microtuber dry matter content. Microtubers stored under diffused light had longer dormancy than those kept continuously in the dark. Interactions among various factors conditioned the main effects for some characters. In vitro performance of the genotypes studied was related to their known performance under in vivo conditions for most of the characters. Microtubers produced on media devoid of ABA and containing high sucrose concentrations and N⁶-benzyladenine (44.38 μM) could be stored for 12 mo. under diffused light at 6±1°C.     

Effect of photoperiod on in vitro tuberization of potato (Solanum tuberosum L.)

Single-node cuttings of potato cultivars Jemseg, Katahdin, Russet Burbank and Superior were cultured on a multiplication medium containing MS salts and no growth regulators. Cultures were exposed to 8 h (SD) and 16 h (LD) photoperiodic regimes. The subsequent plantlets were excised and single node cuttings from each photoperiodic regime were placed under SD or LD on a second medium containing growth regulators which promoted tuberization. Production of microtubers was strongly influenced by genotype and by photoperiodic treatments. Superior produced stunted plantlets and some microtubers under SD conditions in the multiplication medium. The number of microtubers formed by Jemseg was not influenced by photoperiod. However, Katahdin and Russet Burbank formed fewer microtubers under LD-LD conditions compared to LD-SD, SD-SD and SD-LD regimes. Compared with the other regimes, LD-SD photoperiod generally promoted microtuber formation with larger diameters and significantly (p<0.05) greater fresh weight. The intensity of the tuberization stimulus was affected by daylength, and this was characterized by microtubers with secondary tubers, the growth of more than one axillary microtuber, and microtubers subtended by stolons. The maturity group of the potato cultivars and photoperiodic regime in vitro strongly influenced the production of microtubers. These results can be employed to adapt light regimes for multiplication and tuberization to the specific requirements for cultivars from different maturity groups, and thus increase the efficiency of potato multiplication protocols.     

The effect of NaCl on proline accumulation in potato seedlings and calli

The effects of salt stress were studied on the accumulation and metabolism of proline and its correlation with Na⁺ and K⁺ content in shoots and callus tissue of four potato cultivars, viz., Agria, Kennebec (relatively salt tolerant), Diamant and Ajax (relatively salt sensitive). Na⁺ and proline contents increased in all cultivars under salt stress. However, K⁺ and protein contents decreased in response to NaCl treatments. The activities of enzymes involved in proline metabolism, Δ¹-pyrroline-5-carboxylate synthetase (P5CS) and proline dehydrogenase (ProDH) increased and decreased, respectively, in response to elevated NaCl concentrations. The changes of P5CS and ProDH activities in more salt sensitive cultivars (Diamant, Ajax) were more than those in the tolerant ones. Then the stimulation of synthesis in combination with a partially increase of protein proteolysis, a decrease in proline utilization and inhibition of oxidation resulted in high proline contents in seedlings and calli under salt stress. In callus tissue, reduced growth and cell size may be partially responsible for high proline accumulation in response to high NaCl levels. However, although the basic proline contents in the seedlings of more salt tolerant cultivars were higher than the sensitive ones, a clear relationship was not generally observed between accumulation of proline and salt tolerance in potato.     

In vitro microtuberization in potato (Solanum tuberosum L.) cultivars

Mechanism of microtuberization in three elite cultivars kufri badhsha (KB), kufri chandramukhi (KCM) and kufri jawahar (KJ) of potato was studied. Sprouts of all the three cultivars were used to obtain in vitro shoot cultures. MS medium supplemented with chlorocholine chloride was found to be most suitable for all the cultivars. Maximum tuberization was obtained under incubation conditions of continuous darkness at 20 degrees +/- 1 degrees C. The highest number of micro-tubers per plant basis was produced under continuous darkness and KCM recorded the highest yield of micro-tubers and was found significantly superior to KJ and KB.     

Comparison of tissue culture and whole plant responses to salinity in potato

Salt sensitivities of six potato cultivars using six levels of sodium chloride (0.0 to 0.25M) were studied in a greenhouse. Responses of these cultivars were also determined in tissue culture by studying rooting of stem segments, increase in length of cultured roots and inhibition of growth of cell suspension cultures using similar salt concentrations. Responses of cultured stem segments and cell suspensions differed from those expressed by whole plants. A close similarity was observed between the salt stress response of whole plants and of cultured roots. The latter technique may provide a preliminary screening method for assessing salt tolerance in potato genotypes.     

The effect of NaCl on antioxidant enzyme activities in potato seedlings

The effect of NaCl on the growth and activity of antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX) were investigated in the seedlings of four potato cultivars (Agria, Kennebec; relatively salt tolerant, Diamant and Ajax; relatively salt sensitive). The shoot fresh mass of Agria and Kennebec did not changed at 50 mM NaCl, whereas in Diamant and Ajax it decreased to 50 % of that in the controls. In Agria and Kennebec, SOD activity increased at 50 mM NaCl, but no significant changes observed in Diamant and Ajax. At higher NaCl concentration, SOD activity reduced in all cultivars. CAT and POD activities increased in all cultivars under salt stress. Unlike the other cultivars, in Ajax seedlings, APX activity increased in response to NaCl stress. We also observed new POD and SOD isoenzyme activities and changes in isoenzyme compositions under salt stress. These results suggest that salt-tolerant potato cultivars may have a better protection against reactive oxygen species (ROS) by increasing the activity of antioxidant enzymes (especially SOD) under salt stress.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation,microtuberization, and molecular characterization of three potato cultivars

Sprouts of potato tubers were excised from the three potato cultivars Agria, Hermes, and Spunta, sterilized and subjected to shoot formation and propagation on Murashige and Skoog (MS) medium supplemented with 1 mg dm^-3 6-benzylaminopurine (BAP) + 0.5 mg dm^-3 gibberellic acid. Shoots were rooted on MS medium supplemented with 1 mg dm^-3 indole-3-butyric acid. To increase shoot vigour prior tuber formation, shoots were subcultured on MS medium supplemented with 0.56 mg dm^-3 BAP, 0.11 mg dm^-3 2,4-dichlorophenoxyacetic acid, and 0.96 mg dm^-3 naphthaleneacetic acid. Under dark, microtuberization on MS media supplemented with 4 mg dm^-3 of both BAP and kinetin was better than 4 mg dm^-3 BAP alone, where they induced higher number of microtubers per shoot and/or the percentage of shoots that formed microtubers. The highest frequency of microtuber formation was achieved when sucrose at high concentration (8 %) was used as carbon source in culture media. Glucose ranked at the second position whereas fructose reduced the microtuber formation frequency when it was used alone or in combination with glucose. Under the applied culture conditions, cvs. Agria and Hermes showed better micropropagation and microtuberization in comparison to cv. Spunta. In addition, isozyme and RAPD techniques revealed that Agria and Hermes are closer to each other when compared with the third cultivar.     

Effect of water stress mediated through agar on <Emphasis Type="Italic">in vitro</Emphasis> growth of potato

With the objective to develop a practical method of screening potato for drought tolerance, shoot and root growth in plantlets raised in vitro (from nodal cuttings drawn from in vivo as well as in vitro grown plantlets) were studied in three genotypes with known root mass production under field conditions. Different levels of water stress were induced using five concentrations of agar in MS (Murashige and Skoog in Physiol Plant 15:473–497, 1962) medium. Water potential of various media ranged from −0.70 MPa to −0.98 MPa. Water stress in culture adversely affected plantlet growth, and the responses varied with genotype and explant source. Genotype IWA-1 was less affected than Konafubuki and Norin-1. In the experiment with explants from in vivo grown plants, the time to rooting was considerably delayed in Konafubuki and Norin-1 by an increase in agar concentration, but no such effect was observed in IWA-1. In all media, the mean number of roots and root length was greater in IWA-1 than Konafubuki and Norin-1, and the latter two genotypes were at par. At 10 gl⁻¹ agar, IWA-1 had taller plantlets, heavier foliage dry weight, root volume, as well as root dry weight than Konafubuki and Norin-1, whereas the latter two genotypes were at par for all these characteristics. This pattern was similar to the reported pattern of these genotypes for root dry weight under field conditions. However, such similarity in the in vitro and field behavior of the tested genotypes was not observed when nodal cuttings drawn from in vitro plantlets were used as explants. It is concluded that in vitro screening of potato under specific and limited water stress conditions by raising plantlets from nodal cuttings drawn from in vivo grown plants may provide a system for effectively differentiating the genotypes for their expected root mass production under field conditions.