Number Of Microtubers Research Articles

In Vitro Screening of Different Potato Genotypes for Salinity Tolerance

In vitro screening of several potato genotypes (29 cultivars and lines) to salinity stress was conducted. Singlenode explants were evaluated on the basis of their growth, microtuberization and biochemical analysis under differentconcentrations of salt (NaCl) stress. In most cases, increasing NaCl in the tissue culture medium resulted in reduction inplantlet growth, rooting and microtuberization potential in varying degrees, depending on the cultivar. Some genotypessuch as Oceania, 97f-267 and Picasso produced higher number of microtuber under 150 mM NaCl than control.Generally, 97f-267, Oceania and Universa genotypes were ranked among salt tolerant, while Nicola, Safran, Diamantwere ranked as moderate and Elodi, Triomph, Marabel, Bolista and 99-981 were ranked as salt sensitive based onmorphological characters. Photosynthetic pigments decreased under salt stress, however, Universa and Safranmaintained higher chlorophyll content under salt conditions (100 mM NaCl) comparing with control (0.0 mM NaCl)plantlets. Also, Universa and Safran accumulated more proline and free amino acids under stress treatment comparingwith control, than other tested genotypes. The activities of antioxidant enzymes were different among potato genotypes.In this respect, Universa and Safran had higher catalase (CAT) activity under salt stress (100 mM NaCl) than controltreatment (0.0 mM NaCl), however, superoxide dismutase (SOD) activity was not good indicator for salinity tolerancein the tested potato genotypes.

Effect of growth regulator on in vitro potato growth var. Arnova and Borin

This study was focused to test the effect of BA concentrations 0, 0.5, 1, and 2 mg/l on multiplication of two potato varieties, namely Arnova and Borin. In vitro microtubers production was tested by adding 4 mg/l of BA or kinetin with 80 gm sucrose/l. Results of multiplication indicated that there were significant differences between genotypes in leaves number. Studied of BA concentration and interaction between cultivars and BA revealed that there were significant effect in plant height, branch length/plant, leaves number/plant and cut number/plant. In case of microtubers induction, the result showed that there were no significant effect of varieties and plant growth on number and diameter of microtubers.

English

The effect of exogenous phytohormones and sucrose on micropropagation and microtuberization from nodal cuttings of Manihot esculenta was studied. Direct and indirect organogeneses were established from these explants. When nodal cuttings were cultured in the presence of 0.01 to 0.1 mg.L-1 of BAP or NAA there was induction of the budding of axillary buds, with the highest percentages of nodal cuttings with axillaries buds budded of 98 and 73% with 0.07 mg.L-1 BAP and 0.05 mg.L-1 NAA, respectively. When sub cultured in the presence of 0.05 to 0.1 mg.L-1 NAA, the nodal cuttings with axillaries buds budded produced yellowish and friable callus with the highest percentage of callogenesis of 59.8% with 0.08 mg.L-1 NAA. The highest fresh weight of callus of 1334.7 mg was obtained with 0.07 mg.L-1 of NAA. When sub cultured in the presence of 0.05 to 0.1 mg.L-1 BAP, the nodal cuttings and callus induced shoots with the highest percentages of 72.3% and 92.3% for nodal cutting and callus respectively with 0.08 mg.L-1. Also, the highest numbers of shoots per nodal cutting 17.9 and per callus 28.1 were obtained with 0.08 mg.L-1. Multiple shoots, when isolated from nodal cuttings or callus and sub cultured in the presence of different ratio of BAP/NAA, differentiated into plantlets. With 0.07/0.05 mg.L-1 and 0.08/0.05 mg l-1 ratio, all shoots (100%) gave rise to plantlets with the highest growth parameters (14.8 ± 3.1 to 15.3 ± 1.7 cm of high, 12.3 to 12.7 leaves and 7.8 to 8.3 roots). These plantlets were used to induce microtubers on the basal medium supplemented with 0.1 to 0.6 mg.L-1 of NAA or Kin or NAA/Kin ratio and 10 to 60 g.L-1 sucrose. The ratio NAA/Kin of 0.4/0.4 mg.L-1 combined with 20 to 30 g.L-1 sucrose was more effective on the microtuberization than NAA and Kin used separately. In fact, it gave rise to 57.8 to 61.3% of plantlets which produce microtubers with the highest number of microtuber per plantlet (4 to 5), the highest diameter (130.4 to 131.8 mm) and the highest fresh weight of microtubers (403.3 to 408.1 mg). When the plantlets were acclimatized in different substrates, 100 % survived in the mixture red soil/black soil at equal volume (V/V) These results of this work show that BAP, NAA, and Kin used separately or in ratio can be used for organogenesis and micropropagation in Manihot esculenta var TMS 96/0023 in vitro.   Key word: NAA, Kin, BAP, sucrose, organogenesis, microtuberization, cassava.

Researches Concerning Improving Methods of In Vitro Microtubers Production

Five genotypes of potato (Solanum tuberosum L.) were induced to form microtubers under two in vitro culture conditions (continuos darkness and light). Cultures maintained under continuos darkness (with a temperatue of 170C) had a higher yield with a greater number of microtuber (1.394/plantlet) than those maintained under long days (16 h of light/day) combined with temperature of 200C. In the last case the microtubers were higher, with with the highest average weight of 0.602 g. Three phytohormones it was used: coumarine, benzylaminopurine and salicylic acid. Regarding the average number of microtubers obtained when it was used coumarine and salicylic acid, the highest average number of microtubers was recorded (1,135 microtubers/plantlet). The study had been performed also on two fractions of the of sugar quantity (80 and 140 g/l), but they had less influence compared to the varieties.

Microtuber Induction of Two Potato (Solanum tuberosum L.) Varieties

Two potato varieties namely, ‘Hunde’ and ‘Ararsa’ were tested for their microtuber induction under five levels of sucrose (40, 60, 80, 100 and 120 gl-1) in completely randomized design with 2×5 factorial combinations. The objective was to determine optimum concentration of sucrose for microtuber induction. In both varieties, among the five concentrations of sucrose, MS medium supplemented with 60 gl-1 sucrose exhibited a better response than the other concentrations in mean values of microtuber number, diameter, and weight and was found optimum. Accordingly, this medium produced, after 42.57 ± 0.58 days of culture, an average value of (1.97 ± 0.02) microtuber number, (3.60 ± 0.04 mm) microtuber diameter, and (0.08 ± 0.002 g) weight of microtuber in the variety Ararsa. On the other hand, it gave, after 35.67 ± 0.58 days of culture, mean value of (2.90 ± 0.031) microtuber number, (2.95 ± 0.01 mm) microtuber diameter, and (0.06 ± 0.001 g) weight of microtuber in the variety Hunde.

In Vitro Microtuberization of Potato (Solanum Tuberosum L.) Cultivar through Sucrose and Growth Regulator

Microtubers which are produced in tissue culture have benefits for disease free potato production. As growth regulators and sucrose concentration affect the initiation and growth of microtuber, therefore, an experiment was conducted to discover the suitable growth supplement to produce disease free potato microtuber. Four potato cultivars viz. Granula, Daimond, Sarpoaxona and Lal Pakri1 with three different treatments viz. T1 = 8% Sucrose, T2 = 6% Sucrose + 4 mg/L Kinetin, T3 = 8% Sucrose + 4 mg/L Kinetin + 1 mg/L BAP were used for micro tuberization as supplement of Murashige and Skoog (MS) medium. Among tested cultivars, Diamond performed better compare to other three potato cultivars with treatment combination T3. Maximum number of culture showing microtuber (56.0), explant formed microtuber (93.3%), number of microtuber (2.7), average diameter (4.42 mm) and average weight (97.61 mg) were found from Diamond cultivar in 8% Sucrose + 4 mg/L Kinetin + 1 mg/L BAP.

Involvement of jasmonic acid and light periods on potato microtuber growth response

The present study was conducted to determine the effects of Jasmonic acid (JA) and light periods on performance of microtuber growth response in five potato cultivars (Diamant, Atlanta, All Blue, Shepody and Shilbilaty). Microtubers were produced from in vitro grown plantlets treated with or without 5µM JA on micropropagation and microtuberization media under 0 and 8 h light periods. Microtuberization was established in MS liquid media having 8% sucrose along with the above treatments. Among the investigated cultivars, the highest number of microtubers (12.97 microtubers per vessel) was obtained in All Blue. On the other hand, Diamant microtubers gave maximum fresh weight (106.7 mg) and Shilbilaty yielded the highest percentage (57.72%) and number of larger microtubers (8.25 larger microtubers out of total 12.78 microtubers per vessel) followed by Diamant and All Blue. Atlanta showed lower values at all investigated microtuber growth traits. Microtuber growth was better in 5µM JA supplemented microtuberization media with 8 h photoperiodic incubation than those were in micropropagation media with the same treatment. The 0 h period was found significantly inferior than 8 h at all growth traits.

Potato leafroll virus (PLRV) and Potato virus Y (PVY) influence vegetative growth, physiological metabolism, and microtuber production of in vitro-grown shoots of potato (Solanum tuberosum L.)

Effects of Potato leafroll virus (PLRV) and Potato virus Y (PVY) on vegetative growth, physiological metabolism and microtuber production were investigated using in vitro shoot cultures. The results showed that parameters of shoot growth including bud break percentage, shoot length, and node number and length were markedly reduced in the diseased shoots. These negative effects were much more pronounced in shoots co-infected with PLRV and PVY than in those singly infected with either PLRV or PVY. The inhibitive effects on root developments measured by root number and length were observed only in shoots co-infected with PLRV and PVY. Significantly lower contents of chl-a, chl-b and total chl were found in virus infected shoots than in healthy ones. There were striking differences in contents of total soluble protein observed between healthy shoots and PLVR and PVY co-infected ones. The content of total soluble sugar was highest in shoots co-infected with PLRV and PVY, and lowest in healthy shoots. Furthermore, there were no significant differences found in the level of endogenous indole-acetic acid among healthy shoots verses virus infected shoots. However, the level of zeatin-ribosome was much higher in healthy shoots than in virus infected ones. Yet, both healthy and single PLRV infected shoots produced similar levels of gibberillic acid 3, which were much higher than those of PVY single-infected shoots and PLRV and PVY co-infected shoots. Also, there were no significant differences in the number of microtubers among healthy shoots, PLVR single or PVY single infected shoots, but shoots co-infected with PLRV and PVY produced the lowest number of microtubers. Overall, the healthy shoots produced the largest size of microtubers and the highest percentage of microtubers ≥5 mm in diameter.

The Effects of Sucrose on in vitro Tuberization of Potato Cultivars

Two potato varieties namely, ‘Hunde’ and ‘Ararsa’ were tested for in vitro Tuberization response under five levels of sucrose (40, 60, 80, 100 and 120 gram liter-1) in completely randomized design with 2×5 factorial combinations. The objective was to determine optimum concentration of sucrose for in vitro Tuberization. In both varieties, among the five concentrations of sucrose, Murashige and Skoog (MS) medium supplemented with 60 gram liter-1 sucrose exhibited a better response than the other concentrations in mean values of microtuber number, diameter, and weight and was found optimum. Accordingly, this medium gave an average value of (1.97 ± 0.02) microtuber number, (3.60 ± 0.04 mm) microtuber diameter, and (0.08 ± 0.002 gram) weight of microtuberin variety ‘Ararsa’ after 42.57 ± 0.58 days of culture. On the other hand, it gave mean value of (2.90 ± 0.031) microtuber number, (2.95 ± 0.01mm) microtuber diameter, and (0.06 ± 0.001 gram) weight of microtuber in variety ‘Hunde’ after 35.67 ± 0.58 days of culture.

Optimization of immersion frequency and medium substitution on microtuberization of Chlorophytum borivilianum in RITA system on production of saponins

In this study, different immersion times were compared for optimization of Chlorophytum borivilianum microtuber production in a RITA (Récipient à Immersion Temporaire Automatique) system. Significantly higher mean number and length of microtubers and growth index were obtained (16, 52mm and 16, respectively) with 15min immersion in microtuberization medium (MM) for every 60min rest period, and could eliminate the occurrence of hyperhydricity. The substitution of MM with liquid hormone-free MS (MSO) medium on week 6 of culture differed significantly in terms of mean number and length of microtubers and growth index compared to earlier substitution at week 3 with MSO but did not differ significantly in comparison to cultures maintained throughout the 9 weeks in MM. Comparison of total saponin of TIS-derived microtubers with tubers of field grown mother plant showed a 1.3-fold increase of saponin (≈21% diosgenin) in the microtubers. The above results hold a promising application of TIS for large scale production of microtubers and accumulated saponin in C. borivilianum.

Microtuberization of Potato (Solanum tuberosum L.) as Influenced by Supplementary Nutrients, Plant Growth Regulators, and In Vitro Culture Conditions

Five experiments with in vitro-grown plantlets of potato cvs ‘Superior,’ ‘Early Valley,’ ‘Golden Valley,’ and ‘Winter Valley’ were carried out to investigate in a stepwise approach the potential for enhancing microtuber production of (1) adding supplementary nutrients (SN) including KNO3, NH4H2PO4, Ca(NO3)2.4H2O, and MgSO4 in full- or half-strength Murashige and Skoog (MS) medium, (2) adding a pretested combination of plant growth regulators (PGRs), i.e., 10 mg l−1 6-benzyl aminopurine plus 200 mg l−1 succinic acid 2,2-dimethylhydrazide, (3) varying the timing of the supplemental applications, and (4) varying the replenishment of the medium. Plantlets of ‘Superior’ for microtuberization were grown from two-node explants for 75 days under environmental conditions consisting of 22 ± 2 °C and a 16-h photoperiod on a solid basal medium consisting of half-strength MS (1/2 MS) medium containing 6 % sucrose and a moderate concentration of supplementary nutrients, SN-2 (KNO3, 1.22 g l−1; NH4H2PO4, 1.41 g l−1; Ca(NO3)24H2O, 0.23 g l−1; and MgSO4, 0.74 g l−1). Thereafter, plantlets were grown for 60 days under dark conditions. Liquid media containing different combinations of MS, SN, and PGR levels plus 8 % sucrose were added as a supplement and were evaluated for their ability to increase tuberization compared to the control composition of the supplement (MS with 8 % sucrose). In the first experiment, addition of 1/2 MS liquid medium containing 8 % sucrose and SN-2 before dark incubation induced the highest total number of microtubers (on average 1.4 per plantlet), a significantly higher weight and size than the control, and the highest percentage (50 %) of large-sized (>500 mg) microtubers. In the second experiment, addition of 1/2 MS liquid medium containing 8 % sucrose, SN-2, and PGRs produced the highest number (on average 1.5 per plantlet) and total yield (on average 971 mg per plantlet) of microtubers, and significantly the highest percentage (66 %) of large-sized (>500 mg) microtubers. The size of the microtubers was higher than for the treatment without growth regulators. The optimum time for addition of supplemental medium containing 8 % sucrose, SN-2, and PGRs was shown in experiment 3 to be latest 1 week before dark incubation. This timing resulted in both the highest microtuber number (on average 1.6 per plantlet) and yield (on average 1208 mg per plantlet), and the highest percentage (64 %) of large-sized microtubers. Finally, it was found that a second supplemental treatment consisting of 50 % replacement of the liquid medium (by volume) 2 weeks after transfer to dark conditions gave rise to the highest number (on average 1.9 per plantlet), yield (on average 1346 mg per plant), and also significantly the highest percentage (25 %) of large-sized microtubers (>1000 mg) as compared to replacement of a greater or smaller proportion of the medium. Four potato cultivars, ‘Superior,’ ‘Early Valley,’ ‘Golden Valley,’ and ‘Winter Valley’ produced acceptable numbers and yield of microtubers of relatively large-sized tubers using a process that included supplemental liquid medium with 1/2 MS, 8 % sucrose, moderate concentration (SN-2) of SNs and PGRs, followed by a 50 % replacement of the same medium.

둥근마(Dioscorea opposita Thunb.) 기내 소괴경 생산을 위한 배양환경과 배지의 교체효과

둥근마 액체배양에서 소괴경의 생산을 위하여 배양온도와 일장의 전환과 배양 배지의 교체 효과를 조사하였다. 둥근마 액체배양에서 식물체의 증식을 위한 일장시간은 6주의 배양기간 동안 <TEX>$25^{\circ}C$</TEX>에서 16시간보다는 24시간이 효과적이었다. 기내소괴경의 생산을 위하여 배양기간을 12주로 연장하여 배양하였으며, 배지교체의 효과를 검토하기 위하여, 배양 중기인 배양 후 6주째에 새로운 배지로 교체하였더니, 배지를 교체하지 않은 것보다 소괴경의 형성수가 평균 5개씩 더 많았고, 소괴경무게도 70% 이상 증가하였다. 배양환경의 전환이 소괴경 형성에 미치는 효과를 알아보기 위하여, 12주의 배양기간 중에 식물체 생장이 활발한 초기 6주간은 <TEX>$25^{\circ}C$</TEX>에서 24시간의 일장환경에서 배양하고, 이후 6주간의 배양온도와 일장시간을 전환하였다. 배양온도는 <TEX>$25^{\circ}C$</TEX>가 <TEX>$15^{\circ}C$</TEX>보다 효과적이었고, <TEX>$25^{\circ}C$</TEX>에서 일장시간은 24시간이 암처리 또는 8시간 일장처리보다 효과가 있었다. 12주간의 배양기간중에 배지 교체의 적절한 시기는 배양 후 2, 4, 6, 8, 10주째 또는 배지 무교체 처리구중에서, 생체중, 마디수, 소괴경수에서는 2주 또는 6주째에 교체하는 것이 좋았고, 소괴경중은 8주째 교체 처리구에서 가장 무거웠으나 소괴경의 수와 무게를 모두 고려하였을 때, 초기 배양 후 6주째에 배지 교체하는 것이 가장 효율적이었다. 12주간의 배양기간 중 배양 후 6주째에 교체할 배지의 성분은 sucrose 30, 60 <TEX>$g{\cdot}L^{-1}$</TEX> 또는 sucrose 60 g + BA 0.1 <TEX>$mg{\cdot}L^{-1}$</TEX> 함유 배지 중에서 sucrose 60 <TEX>$g{\cdot}L^{-1}$</TEX>이 함유된 배지로 교체하였을 때, 소괴경의 총무게가 가장 무거워 소괴경의 비대에 가장 효과적이었다. 따라서, 둥근마의 액체배양시 <TEX>$25{\pm}1^{\circ}C$</TEX>, 24시간 광조건으로, 마디접종 후 6주째에 sucrose 60 <TEX>$g{\cdot}L^{-1}$</TEX>를 함유한 배지로 교체하여 6주간 추가 배양하는 방법이 기내 식물체와 소괴경의 생장과 형성에 가장 효과적이었다. To figure out optimum culturing condition for <TEX>$in$</TEX> <TEX>$vitro$</TEX> yam (<TEX>$Dioscorea$</TEX> <TEX>$opposita$</TEX> Thunb.) production, various media components and environmental controls were evaluated, effective temperature, light condition and timing of the liquid media replacement for multiplication of yam in liquid culture were determined in this study. There was no visible difference detected for <TEX>$15^{\circ}C$</TEX> and <TEX>$25^{\circ}C$</TEX> temperature conditions. At <TEX>$25^{\circ}C$</TEX>, continuous light condition was more effective compared to 16 hour light/ 8 hour dark condition. Effect of media replacement was tested, and approximately 5 more microtubers obtained and 70% of increase in weight was detected when media replacement was performed. Timing for media replacement was tested at 2, 4, 6, 8, and 10 weeks after initial culture for 12 weeks. Considering both number and weight of microtubers, replacement of media 6 weeks after initial culture was most effective. In terms of component of media, significant increase in weight of microtubers was observed in MS media containing sucrose <TEX>$60g{\cdot}L^{-1}$</TEX>. In summary, the most effective condition for <TEX>$in$</TEX> <TEX>$vitro$</TEX> propagation of chinese yam is replacing medium 6 weeks after initial inoculation with MS medium containing sucrose <TEX>$60g{\cdot}L^{-1}$</TEX> in continuous light condition.

The Effect of Plant Growth Regulator and Active Charcoal on the Development of Microtubers of Potatoes

With the detoxicated seedling of a potato cultivation breed named “Mire” as the material, the effect of auxins CCC, 6-BA, and active carbon to microtubers of potato (Solanum tubersum L.) was investigated under the in-vitro circumstances. The result indicated the exogenous auxins improved the production and quality of microtubers of potatoes. The effect of induction can be described as CCC > CCC + 6-BA > 6-BA > CK, the number of microtubers in per flask is 8.17 > 7.67 > 7.29 > 5.46, and the number of large potatoes in per flask is 6.33 > 5.17 > 3.17 > 1. In addition, by adding 0.5‰ of active charcoal, the growth period was shortened from 25.0 days to 9.33 days on average, and the amount of larger potatoes increased 8.54%. These results benefited the growth of microtubers of potato.

Application of artificial seeds in rapid multiplication of Pseudostellaria heterophylla

In this study, a method which produces artificial seeds of Pseudostellaria heterophylla was presented. The micro-tubers induced from virus-free seedlings were used as explants, and the coating material was sodium alginate. The results show that the appropriate medium for P. heterophylla micro-tubers formation was the MS medium supplemented with 2.0 mg l -1 NAA, 500 mg l -1 CCC and 50 g l -1 sucrose; and the average number of micro-tuber reached 7.3 in each seedling under these conditions. Moreover, the optimum medium for the highest germination rate (83.8%) was 1/4 MS (without sugar) + 1.0 mg L -1 6BA + 0.1 mg L -1 NAA, and the artificial seeds were more perishable in sucrose-rich environments. It also indicated that the artificial seeds were still active with a seedling rate of 80% after they were preserved at 4°С for 90 days, which could meet the requirement of winter-produced seeds and plants in spring.

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.

Induksi Umbi Mikro Tanaman Daun Dewa (Gynura pseudochina (Lour.) DC) Secara In Vitro dengan Perlakuan Sukrosa dan Daminozide

The effects of sucrose and daminozide on in-vitro microtuber formation were evaluated for producing microtubers to supply year round microtubers and to facilitate sterilized explants exchange regionally and internationally. Uninodal stem segment explants were cultured on agar solidifi ed Murashige and Skoog medium supplemented with 3% sucrose, 0.5 mg L-16-benzylaminopurine (BAP), 0.1 mg L-1 α-naphthaleneacetic acid (NAA) for bud multiplication for 3 months. Three nodals stem segment which already formed three perfect leaves were cultured on agar solidifi ed MS medium, 3% sucrose and 1 mg L-1 NAA for two weeks to induce root formation (until ± minimum 60% of the explants rooted). The next step was the induction of three nodal stem segments for microtuber formation on agar solidifi ed MS medium supplemented with 5 mg L-1 BAP and two levels of sucrose, four concentrations of daminozide for 10 weeks. Sucrose at 6% resulted in the signifi cantly highest number of microtuber. The daminozide 41.29 mg L-1 stimulated tuberization at base of the stems and reduce number of microtuber formation in stolon. Keywords: daminozide, Gynura pseudochina (L.) DC, in vitro, microtuber, sucrose

Effects of temperature fluctuation during in vitro phase on in vitro microtuber production in different cultivars of potato (Solanum tuberosum 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.

Functional analysis of a class I patatin gene SK24-1 in microtuber formation of transgenic potatoes

Expression of a class I patatin cDNA clone, SK24-1, in Escherichia coli revealed that the cDNA clone possessed lipid acyl hydrolase (LAH) activity. Transformed potato plants were obtained via Agrobacterium-mediated transformation using the chimeric constructs containing the sense and antisense cDNA under the control cauliflower mosaic virus 35S (CaMV 35S) promoter. In some sense transformed plants, both sense patatin RNA and LAH activity were increased and further resulted in a significant increase of percentage of plantlets that formed microtubers and numbers of microtubers per plantlet in vitro. All antisense plants displayed a reduction in LAH activity. Both sense and antisense RNA could be detected in antisense plants, but transcripts of antisense RNA resulted in a reduction of endogenous sense RNA. Moreover, expression of antisense cDNA in some antisense transformed plants led to a significant decrease in the number of microtubers formed. These results suggest that SK24-1 was involved in regulating microtuber formation. Key words: Patatin, potato, Escherichia coli, sense RNA, antisense RNA

Evaluating asymbiotic seed culture methods and establishing Disa (Orchidaceae) germinability in vitro: relationships, requirements and first-time reports

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.

Effect of γ-radiation on development, yield and quality of microtubers in vitro in Solanum tuberosum 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.