Role of Endogenous Abscisic Acid in Potato Microtuber Dormancy

Potato (Solanum tuberosum L. cv Russet Burbank) microtubers generated in vitro from single-node explants contained substantial amounts (approximately 250 pmol/g fresh weight) of free abscisic acid (ABA) and were completely dormant for a minimum of 12 weeks. Microtubers that developed in the presence of 10 [mu]M fluridone (FLD) contained considerably reduced amounts (approximately 5-25 pmol/g fresh weight) of free ABA and exhibited a precocious loss of dormancy. Inclusion of exogenous racemic ABA in the FLD-containing medium suppressed the premature sprouting of these microtubers in a dose-dependent manner. At a concentration of 50 [mu]M, exogenous ABA restored internal ABA levels to control values and completely inhibited FLD-induced precocious sprouting. Exogenous jasmonic acid was ineffective in suppressing FLD-induced sprouting. Application of FLD to preformed, fully dormant microtubers also resulted in a reduction in internal ABA content and precocious sprouting. These results indicate that endogenous ABA is essential for the induction and maintenance of potato microtuber dormancy.     

Synchronization of tuber formation in potato grown in vitro by cell division synchronization in axillary meristems of stem explants

Optimization of in vitro tuberization (formation and growth of stolons and microtubers) by synchronization of cell divisions in axillary meristems of initial stem explants induced by low nonfreezing temperatures was studied in potato (Solanum tuberosum L., cv. Lugovskoi) plants. The proportion of simultaneously dividing cells in axillary meristems of stem explants subjected to 2-h cold treatment at 4°C was in 2.6 times greater than in control material (without chilling). The analysis of growth of stolons and microtubers produced from the explants exposed to cold showed that synchronization of cell divisions in the meristems of initial explants resulted in synchronization of stolon and microtuber formation and production of microtubers of identical physiological age.     

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.     

The use of growth retardants to improve microtuber formation by potato (Solanum tuberosum)

The growth retardant chlormequat stimulated microtuber formation by a recalcitrant cultivar of potato (Solanum tuberosum), but reduced microtuber fresh weight in a cultivar that tuberised readily in its absence. Inhibition of microtuber growth by high concentrations of chlormequat was confirmed using a different in vitro system where all cultivars tuberised in the absence of growth retardants.Alternative growth retardants were tested. Daminozide also had a detrimental effect on microtuber fresh weight, but ancymidol and paclobutrazol did not inhibit microtuber growth at the concentrations required for stimulation of tuberisation by recalcitrant cultivars. In addition, 10^-5 M ancymidol and paclobutrazol inhibited premature sprouting of microtubers in vitro.Abbreviations BA benzyladenine - DMSO dimethyl sulphoxide - FW fresh weight - PAR photosynthetically active radiation - SE standard error     

Novel bioreactor technology for mass propagation of potato microtubers

Potato (Solanum tuberosum L. ssp. tuberosum) microtubers were produced in vitro with Liquid Lab™ Rocker system. A thin-layer liquid culture was applied together with a regular pitch in autoclavable simple plastic vessels. All cultures were carried out at room temperature without contamination problems. Each cultivar tested (Asterix, Timo, Van Gogh, Velox) formed microtubers in the Liquid Lab system. The mean number of microtubers per vessel (50 explants) varied between 30 (cv. Asterix in 8 weeks tuber induction) and 75 microtubers (cv. Velox in 11 weeks tuber induction). Majority (63%) of the microtubers was sufficient by size and weight (above 200 mg) for further storage at dormancy (4°C). The cv. Velox yielded the highest number of microtubers with cultivation capacity. As a result of prolonged microtuber induction of 2–3 weeks, more microtubers with competence for cultivation were obtained per cultivar, except for cv. Van Gogh. Still, the mean weight of Van Gogh microtubers was significantly higher after prolonged microtuber induction (0.67 g) than after short induction (0.51 g). In conclusion, Liquid Lab™ Rocker system is a novel, efficient and rapid system for mass propagation of potato.     

Chemical inhibition of potato ABA-8'-hydroxylase activity alters in vitro and in vivo ABA metabolism and endogenous ABA levels but does not affect potato microtuber dormancy duration

The effects of azole-type P450 inhibitors and two metabolism-resistant abscisic acid (ABA) analogues on in vitro ABA-8'-hydroxylase activity, in planta ABA metabolism, endogenous ABA content, and tuber meristem dormancy duration were examined in potato (Solanum tuberosum L. cv. Russet Burbank). When functionally expressed in yeast, three potato CYP707A genes were demonstrated to encode enzymatically active ABA-8'-hydroxylases with micromolar affinities for (+)-ABA. The in vitro activity of the three enzymes was inhibited by the P450 azole-type inhibitors ancymidol, paclobutrazol, diniconazole, and tetcyclasis, and by the 8'-acetylene- and 8'-methylene-ABA analogues, with diniconazole and tetcyclasis being the most potent inhibitors. The in planta metabolism of [(3)H](±)-ABA to phaseic acid and dihydrophaseic acid in tuber meristems was inhibited by diniconazole, tetcyclasis, and to a lesser extent by 8'-acetylene- and 8'-methylene-ABA. Continuous exposure of in vitro generated microtubers to diniconazole resulted in a 2-fold increase in endogenous ABA content and a decline in dihydrophaseic acid content after 9 weeks of development. Similar treatment with 8'-acetylene-ABA had no effects on the endogenous contents of ABA or phaseic acid but reduced the content of dihydrophaseic acid. Tuber meristem dormancy progression was determined ex vitro in control, diniconazole-, and 8'-acetylene-ABA-treated microtubers following harvest. Continuous exposure to diniconazole during microtuber development had no effects on subsequent sprouting at any time point. Continuous exposure to 8'-acetylene-ABA significantly increased the rate of microtuber sprouting. The results indicate that, although a decrease in ABA content is a hallmark of tuber dormancy progression, the decline in ABA levels is not a prerequisite for dormancy exit and the onset of tuber sprouting.     

Effects of storage conditions on sprouting of microtubers of yam (Dioscorea cayenensis–D. rotundata complex)

The control of field tuber dormancy in the yam (Dioscorea cayenensis–D. rotundata complex) is poorly understood. Although studies have examined single environmental factors and chemical treatments that might prolong tuber dormancy and storage, only a few were focused on further tuber sprouting. The present study concerns microtubers obtained by in vitro culture. When microtubers were harvested (after 9 months of culture) and directly transferred on a new medium without hormones, the tubers rapidly sprouted in in vitro conditions. No dormancy was observed in this case. Harvested microtubers were also stored dry in jars in sterile conditions during 2 to 18 weeks before in vitro sprouting. In this case, microtubers stored during 18 weeks sprouted more rapidly than those stored 8 weeks. A constant “dormancy-like period” (storage duration + sprouting delay) was observed, between 20 and 28 weeks respectively for the more rapid and the slower microtubers. The size of the tubers used for the storage had great influence on further sprouting. The larger they were, the better they sprouted. Light during storage had no effect on the sprouting delay while a temperature of 25 °C permit a quicker sprouting than 18 °C. The medium used to obtain microtubers could also have an effect on sprouting rate.     

Effects of reducing sugar concentration on in vitro tuber formation and sprouting in yam (<Emphasis Type="Italic">Dioscorea cayenensis–D. rotundata</Emphasis> complex)

The effects of reducing sucrose level on tuber formation (% of cultures with microtubers), development (length and fresh weight of microtubers) and sprouting in yam Dioscorea cayenensis–D. rotundata complex in vitro were investigated. Only 29% of the explants showed tuber formation after 3 weeks in the presence of 1% sucrose in contrast to 100% with 3%. After 120 days of culture, the length and the weight of the tubers obtained in the presence of 1% sucrose were less than with 3% sucrose. Addition of sorbitol to keep osmolarity at the same level did not restore normal rate of tuber formation. Similar results were obtained with the use of reduced fructose or glucose level. Microtuber sprouting was also affected by sucrose level incorporated into the tuberisation medium. Tubers obtained on reduced sucrose level sprouted later and the increase of osmolarity with sorbitol did not restore normal sprouting. The bigger tubers obtained on high sucrose media could contain more carbohydrate reserves that could partially explain a higher sprouting rate. These results can be used for optimising in vitro conditions for mass production of microtubers in yam and especially in Dioscorea cayenensis–D. rotundata complex, a very important species in West Africa. They specially showed the importance of tuberisation conditions on precocity of tuberisation, on tuber length and weight and on their further sprouting.     

Silver nitrate-induced in vitro shoot multiplication and precocious flowering in <Emphasis Type="Italic">Catharanthus roseus</Emphasis> (L.) G. Don,a rich source of terpenoid indole alkaloids

Catharanthus roseus (L.) G. Don is an economically and medicinally important plant since its leaves and flowers contain terpenoid indole alkaloids. The present study, for the first time, encompasses the influence of silver nitrate (AgNO₃), in consort with cytokinins like N ⁶-benzyladenine (BA) and 6-furfurylaminopurine (kinetin), to regenerate multiple shoots from nodal segments explants and to induce high-frequency precocious flowering of C. roseus under in vitro condition. Synergistic effect of equal concentrations of BA and kinetin was enhanced following the amalgamation of AgNO₃. As high as 98% explants responded to multiple shoot initiation and proliferation in Murashige and Skoog medium supplemented with 3 µM BA, 3 µM kinetin and 0.1 µM AgNO₃. As many as 7 shoots were developed per explant following 12 days of inoculation. Continuous culture in the same medium for 21 days induced precocious flowering from 75% shoots, wherein a maximum of ~?6 (5.67?±?0.88) flowers was observed per in vitro shoot. On the other hand, in the combinations of BA and kinetin excluding AgNO₃, a maximum of 6.67% explants responded and initiated merely 3.33 shoots per explant. Nevertheless, no induction of flower was observed in the media devoid of AgNO₃. Our results on the induction and proliferation of multiple shoots with simultaneous flowering would help the global pharmaceutical industry to produce in vitro shoots and flowers in bulk, as an alternative source of alkaloids.     

Sucrose utilization during potato microtuber growth in bioreactors

 Potato microtubers are used as pathogen-tested in vitro stocks for certified seed potato production. Microtubers grown in a rotating bioreactor grew at a faster rate when the medium was replaced frequently. Although the total microtuber number was not affected, the number of microtubers over 1 g quadrupled when 75% of the medium was replaced every 2 weeks when compared with no medium refreshment. Significantly slower microtuber growth rates resulted when a lower sugar concentration (40 g 1⁻¹ instead of 80 g 1⁻¹) was used or when a mixture of glucose and fructose replaced sucrose. Although high sucrose levels are necessary for optimal microtuber production, the sucrose supplied was rapidly hydrolyzed into glucose and fructose, making the long-term maintenance of desirable sucrose levels difficult. These results indicate that successful strategies to reduce sucrose hydrolysis without inhibiting microtuber growth will improve the efficiency of sucrose utilization in potato microtuber bioreactors. Received: 1 December 1998 / Revision received: 6 May 1999 · Accepted: 19 May 1999     

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 Induction and Development of Potato Microtubers In Vitroon Media Free of Growth Regulating Substances

Reliable microtuber production has been obtained by culturingnodal explants of potato on Murashige and Skoog medium withoutthe addition of growth regulating substances. The use of sucroseat 8 %, as compared with 4 or 12%, advanced the initiation oftuberization and gave more and larger microtubers. A periodof 1 month under 16 h days, followed by transfer to 8 h photoperiodsgave most rapid microtuber development. A reduction in the totalnitrogen supply or an increase in the ratio of ammonium to nitratereduced the size and number of microtubers Potato, Solanum tubersum L;, microtubers, in vitro induction and development, sucrose, photoperiod, nitrogen nutrition     

Efficient plant regeneration from cotyledon explants of bottle gourd (<Emphasis Type="Italic">Lagenaria siceraria</Emphasis> Standl.)

Using cotyledon explants excised from seedlings germinated in vitro, an efficient plant regeneration system via organogenesis was established for bottle gourd (Lagenaria siceraria Standl.). Maximum shoot regeneration was obtained when the proximal parts of cotyledons from 4-day-old seedlings were cultured on MS medium with 3 mg/l BA and 0.5 mg/l AgNO₃ under a 16-h photoperiod. After 3–4 weeks of culture, 21.9–80.7% of explants from the five cultivars regenerated shoots. Adventitious shoots were successfully rooted on a half-strength MS medium with 0.1 mg/l IAA for 2–3 weeks. Flow cytometric analysis revealed that most of the regenerated plants derived from culture on medium with AgNO₃ were diploid.     

Ethylene inhibitors promote in vitro regeneration of cowpea (Vigna unguiculata L.)

Summary Ethylene is a plant growth regulator that is known to influence in vitro morphogenesis. This study investigated the effects of three ethylene inhibitors, silver nitrate (AgNO₃), 2,5-norbornadiene, and cobalt chloride (CoCl₂), on the regeneration of cowpea from cotyledon explants. Significant increases in the percentage of regeneration occurred as a result of adding either 50 μM AgNO₃ or 100 μM 2,5-norbornadiene. The number of shoots produced per explant was enhanced by adding 25 μM CoCl₂ or 100 μM norbornadiene. Maximum shoot elongation was obtained with 25 μM of either CoCl₂ or norbornadiene. The effect of the duration of exposure to AgNO₃ was also determined. The greatest percent regeneration was obtained with the addition of 60 μM AgNO₃ either to both the initiation and regeneration stages, or to only the regeneration stage. The promotive effects on organogenesis in response to ethylene inhibitors suggests an important role for ethylene in the process of in vitro morphogenesis of cowpea and may contribute to its normally low regeneration frequency.     

Role of polyamines on de novo shoot morphogenesis from cotyledons of Brassica campestris ssp. pekinensis (Lour) Olsson in vitro

Summary The promotive effect of ethylene inhibitors (Els), i.e. AgNO₃ and aminoethoxyvinylglycine (AVG) on de novo shoot regeneration from cultured cotyledonary explants of Brassica campestris ssp. pekinensis cv. Shantung in relation to polyamines (PAs) was investigated. The endogenous levels of free putrescine and spermidine in the explant decreased sharply after 1–3 days of culture, whereas endogenous spermine increased, irrespective of the absence or presence of Els. AgNO₃ at 30 M did not affect endogenous PAs during two weeks of culture. In contrast, explants grown on medium containing 5 M AVG produced higher levels of free putrescine and spermine which increased rapidly after three days and reached a peak at 10 days. An exogenous application of 5 mM putrescine also resulted in a similar surge of endogenous free spermine of the explant. More strikingly, shoot regeneration from explants grown in the presence of 1–20 mM putrescine, 0.1–2.5 mM spermidine, or 0.1–1 mM spermine was enhanced after three weeks of culture. However, exogenous PAs generally did not affect ethylene production, and endogenous levels of 1-aminocyclopropane-1-carboxylate (ACC) synthase activity and ACC of the explant. This study shows the PA requirement for shoot regeneration from cotyledons of B. campestris ssp. pekinensis in vitro, and also indicates that the promotive effect of PAs on regeneration may not be due to an inhibition of ethylene biosynthesis.Abbreviations PAs polyamines - AVG aminoethoxyvinylglycine - SAM S-adenosylmethionine - ACC 1-aminocyclopropane-1-carboxylate - Els ethylene inhibitors     

Synergistic effect of ethylene inhibitors and putrescine on shoot regeneration from hypocotyl explants of Chinese radish (Raphanus sativus L. var. longipinnatus Bailey) in vitro

Summary The role of ethylene and putrescine on shoot regeneration from hypocotyl explants of Chinese radish (Raphanus sativus L. var. longipinnatus Bailey cv. Red Coat) was investigated. Explants were recalcitrant in culture, but exogenous application of ethylene inhibitor [20–30 M aminoethoxyvinylglycine (AVG) or AgNO₃] enhanced shoot regeneration of explants grown on medium supplemented with 2 mg/l N⁶-benzyladenine and 1 mg/l 1-naphthaleneacetic acid. The best regeneration occurred in the medium containing AgNO₃ in combination with AVG. Culture medium solidified with agarose in the presence of AgNO₃ but not AVG was also beneficial to shoot regeneration. Exogenous putrescine, 2-chloroethylphosphonic acid and 1-aminocyclopropane-1-carboxylate had no effect on shoot regeneration. However, regeneration was greatly promoted by 10–25 mM putrescine in combination with 30 M AgNO₃ or AVG. Explants with high regenerability grown in the presence of AgNO₃ or in combination with putrescine emanated high levels of ethylene throughout the 21-d culture period. By contrast, AVG or putrescine alone resulted in a decrease in ethylene production. For rooting of shoot cuttings, IAA and IBA at 1–5 mg/l were more effective than NAA.Abbreviations ACC 1-aminocyclopropane-1-carboxylate - AVG aminoethoxyvinylglycine - BA N⁶-benzyladenine - CEPA 2-chloroethylphosphonic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) medium - NAA 1-naphthaleneacetic acid - PAs polyamines - SAM S-adenosyl-L-methionine     

Role of Ethylene on de Novo Shoot Regeneration from Cotyledonary Explants of Brassica campestris ssp. pekinensis (Lour) Olsson in Vitro

The promotive effect of AgNO₃ and aminoethoxyvinylglycine (AVG) on in vitro shoot regeneration from cotyledons of Brassica campestris ssp. pekinensis in relation to endogenous 1-amino-cyclopropane-1-carboxylic acid (ACC) synthase, ACC, and ethylene production was investigated. AgNO₃ enhanced ACC synthase activity and ACC accumulation, which reached a maximum after 3 to 7 days of culture. ACC accumulation was concomitant with increased emanation of ethylene which peaked after 14 days. In contrast, AVG was inhibitory to endogenous ACC synthase activity and reduced ACC and ethylene production. The promotive effect of AVG on shoot regeneration was reversed by 2-chloroethylphosphonic acid at 50 micromolar or higher concentrations, whereas explants grown on AgNO₃ medium were less affected by 2-chloroethylphosphonic acid. The distinctive effect of AgNO₃ and AVG on endogenous ACC synthase, ACC, and ethylene production and its possible mechanisms are discussed.     

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.     

The influence of ethylene and ethylene modulators on shoot organogenesis in tomato

The influence of ethylene and ethylene modulators on the in vitro organogenesis of tomato was studied using a highly regenerating accession of the wild tomato Solanum pennellii and an F1 plant resulting from a cross between Solanum pennellii and Solanum lycopersicum cv. Anl27, which is known to have a low regeneration frequency. Four ethylene-modulating compounds, each at four levels, were used, namely: cobalt chloride (CoCl₂), which inhibits the production of ethylene; AgNO₃ (SN), which inhibits ethylene action; and Ethephon and the precursor 1-aminocyclopropane-1-carboxylic acid (ACC), which both promote ethylene synthesis. Leaf explants of each genotype were incubated on shoot induction medium supplemented with each of these compounds at 0, 10 or 15 days following bud induction. The results obtained in our assays indicate that ethylene has a significant influence on tomato organogenesis. Concentrations of ethylene lower than the optimum (according to genotype) at the beginning of the culture may decrease the percentage of explants with buds (B), produce a delay in their appearance, or indeed inhibit bud formation. This was observed in S. pennellii and the F1 explants cultured on media with SN (5.8–58.0 μM) as well as in the F1 explants cultured on medium with 21.0 μM CoCl₂. The percentage of explants with shoots (R) and the mean number of shoots per explant with shoots (PR) also diminished in media that contained SN. Shoots isolated from these explants were less developed compared to those isolated from control explants. On the other hand, ethylene supplementation may contribute to enhancing shoot development. The number of isolable shoots from S. pennellii explants doubled in media with ACC (9.8–98.0 μM). Shoots isolated from explants treated with ethylene releasing compounds showed a higher number of nodes when ACC and Ethephon were added at 10 days (in F1 explants) or at 15 days (in S. pennellii) after the beginning of culture. Thus, the importance of studying not only the concentration but also the timing of the application of regulators when developing regeneration protocols has been made manifest. An excess of ethylene supplementation may produce an inhibitory effect, as was observed when using Ethephon (17.2–69.0 μM). These results show the involvement of ethylene in tomato organogenesis and lead us to believe that ethylene supplementation may contribute to enhancing regeneration and shoot development in tomato.     

Accumulation of salicylic acid-elicited alkaloid compounds in in vitro cultured Pinellia ternata microtubers and expression profiling of genes associated with benzoic acid-derived alkaloid biosynthesis

The alkaloid compounds found in Pinellia ternata tubers have major bioactive components, and thus, these plant products are one of the most widely used ingredients in traditional Chinese medicines (TCMs). Under field agricultural growth conditions, however, it usually takes 2 years for tuber formation and growth. In vitro induced microtubers provide an alternative approach for the commercial production of P. ternata tubers for use in the TCM industry. The elicitation effect of supplementation with salicylic acid (SA) on the accumulation of alkaloid compounds in tubers and the related molecular regulation mechanism for biosynthesis are not well understood. In this study, we address this knowledge-gap through the development of an efficient induction system of in vitro cultured microtubers subsequently used to study the mechanism for elicitation of alkaloid compound accumulation by SA. Efficient microtuber induction was achieved by inserting petioles inversely into solid Murashige & Skoog medium (MS) followed by subculturing the morphologically expanded lower portion of the culture petioles in suspension culture, without the additional application of plant growth regulators. The in vitro microtuber induction rate achieved was 100% within 25 days of culture. When treated with 50–150 μM of SA, in vitro cultured microtubers showed higher accumulation of alkaloid compounds over the negative control. The highest accumulation detected showed an increase of 2.5-, 2.1-, 2.8-, and 3.1-fold in the concentration of total alkaloid compounds, guanosine, inosine and ephedrine, respectively, in the presence of 100 μM SA, 15 days after induction. qRT-PCR analysis of candidate genes for key enzymes in alkaloid biosynthesis indicated that CNL, CHY and BALDH are most probably responsible for the accumulation of benzoic acid and other alkaloid derivatives in the in vitro cultured microtubers following SA elicitation. This study developed an efficient in vitro microtuber induction system, and used this to determine that SA-promoted accumulation of alkaloids is associated with genes in the benzoic acid and alkaloid derivative biosynthesis pathway in P. ternata.