Effect of Potassium and Sucrose Concentrations on the Production of Potato Microtubers Through Tissue Culture. S. F. El-Sharabasy *, El-Sharnouby, M. E. ** and Zahran, A. A. ** * Central Lab., of Date Palm Res. and Development, Agricultural Research Center. ** Natural Products Depart., National Cent. Rad. Res. and Tech., Nasr City,Cairo, Egypt. ABSTRACT This investigation was carried out on potato ( Solanum tuberosum) cultivar Alpha where shoot tips were cultured on solid MS medium supplemented with 3% sucrose and 1 mg/ l of BA for four weeks. Multinodal (three nodes) explants were excised from proliferated shoots and were subcultured on MS media with different strengths (25, 50 and 75 % strength of MS medium) enriched with potassium nitrate (at concentrations of 475, 950, 1425 and 1900 mg/ l), potassium di-hydrogen-ortho-phosphate (at concentrations of 42.5, 85.0, 127.5 and 170 mg/ l), and sucrose (at concentrations 25, 50, 75 and 100 g/ l) to produce microtubers under long day (LD) and dark conditions on growth- regulator- free mediums. The results indicated that maximum number of microtubers was produced on MS medium with 25% strength supplemented with 25 g/ l sucrose under LD conditions, whereas under dark conditions, best results were obtained from cultures on 75% strength MS mediums enriched with 950 and 1425 mg/ l KNO 3. It was also found that regardless of MS medium strength, sucrose additives resulted in the production of microtubers with the heaviest weights under LD conditions, while under dark conditions, all potassium supplements produced relatively heavy microtubers. Finally, MS medium with 25 % strength supplemented with 170 / l KH 2 PO 4 produced the largest microtubers under LD conditions, while 75 % MS medium with supplemented with 127.5 g/ l KH 2 PO 4 under dark conditions produced the largest microtubers. Key Words: Potato/ Potassium/ Sucrose/ Tissue Culture. INTRODUCTION Potato ( Solanum tuberosum) is the fourth most important food crop after wheat, rice and maize, therefore, it is the most important dicotyledonous and tuber crop (1). It produces protein and calories per unit area per unit of time and per unit of water more than any other major plant (2). Seed tubers are the most common source of plant material in potato reproduction, but because of low multiplication rate in the field (3) and being often prone to attacks by pathogens such as fungi, bacteria and viruses resulting in poor yield and quality, tissue culture has become very popular in producing healthy seed potatoes (4). Microtuber production is one of the strategies under this perspective because of their small size and weight, which gives them tremendous advantages in terms of disease free, storage, transportation and mechanization (5), in addition to the possibility of being stored for extended periods of time (6). It is worth mentioning that microtubers size plays a major role in the successful utilization of in vitro microtubers at the commercial scale (7). In this regard, microtubers were defined as small, in vitro produced tubers varying in size and weight from 3-12 mm and 0.02 1.0 g, respectively (8). Tuberization in potato represents a morphogenetic transition of stolon growth to tuber formation, which happens to be under complex environmental and endogenous regulation (9). In this regard, it suggested that high sucrose levels are required for maintaining the tuberization signal and in another investigation, it was stated that in vitro potato plantlets, 6 8 cm in height could be derived from meristems of potato tubers cultured on half strength MS medium (10). Later, -561-
low net photosynthetic rate and dry matter content in seedlings growing under white light has been reported (11). Variety Alpha, which was used in this study, has large upright vines that produce long tubers with smooth skin and yellow flesh. It is a sturdy plant which happens to be very resistant to poor growing conditions and is utilized as fresh market potatoes. MATERIALS AND METHODS This investigation was carried out in Tissue Culture Laboratory, Agricultural Development System (ADS), California Project, MOALR Egypt, during 2009. Mother tubers of potato cultivar Alpha were selected and washed with water, followed by 5 min dip in 95% alcohol, then packed in craft paper bags which were kept in the dark for one month at 21 o C constant temperature in the growth chamber. Etiolated shoots were used as explant source after being surface sterilized by washing for half an hour in tap water before in vitro culture. Shoot tip explants 0.5 cm long obtained from etiolated shoots were surface sterilized by 1.5 min dip in 70% ethyl alcohol and 20 min dip in 10% chlorax solution plus two drops of Tween- 20 as surfactant followed by 5 washings with double distilled autoclaved water (12). Solid MS (Murashige and Skoog) medium (13) supplemented with 8 g/ l agar and 3 g sucrose and ph adjusted to 5.8 was autoclaved at 1.05 kg/ cm 2 and 121 C for 20 min and left to cool down. The medium was supplemented with BA at a concentration of 1 mg/ l and was ready for explants to be cultured in the jars and kept in a growth room for one month under a temperature of 27 o C and a photoperiod of 16 h for micropropagation. Afterwards, nodal explants (3 nodes) were excised and cultured on MS mediums with different strengths; i.e. 25, 50 and 75 % MS strength, enriched with sucrose (25, 50, 75 and 100 g/ l), KNO 3 (475, 950, 1425 and 1900 mg/ l) and KH 2 PO 4 ( 42.5, 85.0, 127.5 and 170 mg/ l). The concentrations of KNO 3 and KH 2 PO 4 investigated in this trial were 1 4, 1 2, 3 4 and whole concentrations of KNO 3 and KH 2 PO 4 originally present in basal MS medium. Jars were placed in the growth room at a temperature of 22 ± 2 C under 16 h photoperiod with light intensity of 3000 lux or under the same temperature under complete darkness for 6 weeks. Numbers of microtubers/ explant, microtuber weight and microtuber diameter were recorded for all explants and the means were studied. The experiment was laid out in a factorial arrangement (3 medium strengths x 12 additive) in a completely randomized design with four replications/ treatment, each with five observations. All the data were analyzed by analysis of variance (ANOVA), which is the procedure used for testing the differenc es among means of two or more treatments, and the differences between means were detected using least significant difference (L.S.D.) P< 0.05 according to (14). RESULT AND DISCUSSIONS Effect of medium strength, sucrose, KNO 3 and KH 2 PO 4 on the number of potato microtubers/ explant produced in vitro under long day (LD) and dark conditions. As shown in Tabel 1, both media strength and supplements and their interaction significantly affected the number of microtubers/ explant produced under both photoperiods under investigation. It was noticed that inconsistent trends were followed under LD and dark conditions as a result of incubation on media with different strengths. Regardless of supplements and under LD conditions, 75 % MS medium recoded a significantly low value compared to 25 and 50 % MS media, between which an insignificant difference was recorded. On the other hand, under dark conditions, values recorded increased as medium strength increased. As for the effect of different supplements regardless of medium strength, on the number of microtubers/ explant, no steady trend was noticed under LD and dark conditions. Sucrose with a concentration of 25000 and KNO 3 with a concentration of 950 mg/ l led to production of significantly the highest numbers of microtubers/ explant under LD conditions. Contrarily, under dark conditions, 25000 mg/ l sucrose recorded significantly a lower value compared to 75000 mg/ l sucrose and all KNO 3 and KH 2 PO 4 concentrations investigated, between which, insignificant differences were recorded. Meanwhile, -562-
KNO 3 concentrations of 950 and 1425 mg/ l produced more microtubers compared to 475 and 1900 mg/ l KNO 3. Such results oppose the findings of (15) and (16) who reported more numbers of microtubers per flask at lower potassium levels. It is also worth mentioning that 475 and 1900 mg/ l KNO 3 recorded higher values of microtuber number compared to other concentrations and other supplements under investigation. MS medium with 25% strength supplemented with 25 g/ l sucrose and cultured under LD conditions led to production of a significantly high number of microtubers (8.25) compared to other combinations in this trial. Such promoted microtuber induction under low sucrose concentration is in contrast with the findings of (15) who reported that microtuber induction was directly related to high sucrose concentration. It is also worth mentioning that under LD conditions, 950 mg/ l KNO 3 and 127.5 mg/ l KH 2 PO 4 supplements to 25% MS medium led to satisfactory microtuber induction. Moreover, 950 mg/ l KNO 3 proved to be beneficial when added to 50 % MS medium and value recorded (6.00) showed insignificant differences with those recorded for 475 mg/ l KNO 3 and 25000 mg/ l sucrose. Table 1: Effect of medium strength, sucrose, KNO 3 and KH 2 PO 4 on the number of potato microtubers/ explant produced in vitro under long day (LD) and dark conditions. Medium strength (A) Treatments (B) Concentration (mg/ 1) Long day (LD) Dark conditions 25% MS 50% MS 75% MS Mean 25% MS 50% MS 75% MS Mean Sucrose KNO 3 KH 2 PO 4 25000 8.25 a 5.00 bc 2.75 efgh 5.33 a 2.00 bc 1.00 c 2.00 bc 1.67 b 50000 3.25 defg 4.00 cde 2.25 fghi 3.17 bc 2.00 bc 3.00 abc 3.00 abc 2.67 ab 75000 3.00 defg 4.25 cde 1.25 hij 2.83 bc 3.00 abc 3.00 abc 4.00 ab 3.33 a 100000 1.00 ij 3.25 defg 0.25 j 1.50 d 2.00 bc 3.00 abc 3.00 abc 2.67 ab 475 1.75 ghij 5.25 bc 1.25 hij 2.75 bc 3.00 abc 3.50 abc 4.00 ab 3.50 a 950 4.50 bcd 6.00 b 3.25 defg 4.58 a 3.25 abc 4.00 ab 5.00 a 4.08 a 1425 3.25 defg 3.25 defg 2.00 ghi 2.83 bc 3.25 abc 4.00 ab 5.00 a 4.08 a 1900 3.00 defg 3.00 defg 1.75 ghij 2.58 c 2.50 abc 3.50 abc 4.50 ab 3.50 a 42.5 2.00 ghi 1.25 hij 1.00 ij 1.42 d 3.00 abc 3.50 abc 3.75 ab 3.42 a 85.0 2.25 fghi 2.75 efgh 2.75 efgh 2.58 c 3.25 abc 3.75 ab 3.25 abc 3.42 a 127.5 4.00 cde 3.00 defg 3.75 cdef 3.58 b 3.25 abc 3.50 abc 4.25 ab 3.67 a 170.0 3.25 defg 1.25 hij 2.75 efgh 2.42 c 3.00 abc 3.25 abc 4.50 ab 3.58 a Mean 3.29 a 3.52 a 2.08 b 2.79 b 3.25 ab 3.85 a Under dark conditions, and for each/ all sucrose, KNO 3 and KH 2 PO 4 concentrations investigated, insignificant differences were recorded among different medium strengths. On the other hand, all supplements added to 25 % MS medium recorded insignificant differences inbetween. Moreover, other than values recorded for 25000 mg/ l sucrose, insignificant differences were noticed between values recorded for different supplements added to 50 % and 75 % MS media. It was also noticed that under dark conditions, and for each additive, different concentrations and different medium strengths resulted in values showing insignificant differences in- between. As for the sucrose concentrations investigated, 75000 mg/l proved to be the best for the production of the highest number of microtubers with the heaviest weights ( Table 2) and biggest sizes (Table 3). Although increasing the sucrose concentration from 25000 to 50000 mg/ l led to nonsignificant increases in the number of microtubers produced under LD and dark conditions, it led to significant increases in microtuber weight under LD conditions (Table 2) and microtuber size under dark conditions (Table 3). Further increment in sucrose concentration resulted in a further insignificant increment in number under dark conditions, and a significant decrease in microtuber number under LD conditions. Such an effect of sucrose on the number of -563-
microtubers produced is in accordance with the findings of (17) who reported that a significantly high number of microtubers were induced using 6% sucrose concentration in MS medium, while a further increase in sucrose reduced the number of microtubers. Moreover, comparing the grand mean for the LD (2.9 microtuber/ explant) and dark (3.30)conditions reveal that dark conditions was favorable for the production of high numbers of microtubers. Such result is in contrast with what (11) who reported regarding light being of a positive influence on the number of microtubers produced compared to dark conditions. Effect of medium strength, sucrose, KNO 3 and KH 2 PO 4 on the weight (mg/ microtuber) of potato microtubers produced in vitro under long day (LD) and dark conditions. Similar to the first character studied, weight of Alpha microtubers produced in vitro was found to be significantly affected by medium strength and supplements and their interaction together, under both LD and dark conditions. Under LD conditions, and regardless of supplements, MS medium with 50 % strength recorded the highest value followed by 25 % MS and 75 % MS, respectively, where differences where statistically significant. Contrarily, 75 % MS recorded a significantly high value under dark conditions, followed by 25% and 50 % MS, between which an insignificant difference was recorded. As for the effect of supplements regardless of medium strength under LD conditions, 50000 mg/ l sucrose recorded the highest value. Meanwhile, 1425 mg/ l and 127.5 mg/ l were the best concentrations investigated of KNO 3 and KH 2 PO 4, respectively. On the other hand, under dark conditions, all KNO 3 and KH 2 PO 4 concentrations investigated resulted in significantly high records. Sucrose with a concentration of 75000 mg/ l also resulted in a significantly high record which showed nonsignificant differences with records of all KNO 3 and KH 2 PO 4 concentrations. It was also noticed that the least and highest sucrose concentrations investigated recoded values which were significantly lower than all other supplements. In contrast with sucrose additives, dark conditions resulted in the production of heavy microtubers on mediums supplemented with KNO 3, KH 2 PO 4 compared to those produced under LD. Finally, it was noticed that darkness was the condition that led to the best results when media were supplemented with KH 2 PO 4, while LD recorded the best values when sucrose was incorporated. Results presented in Table 2 under LD conditions reveal that for sucrose and KNO 3, the lower the medium concentration, the higher supplement concentration required to enhance microcorm weight. MS media with 25, 50 and 75 % strength recorded best results when supplemented with 75, 50 and 25 g/ l sucrose, respectively, and 1425, 950 and 475 mg/ l KNO 3, respectively. No such trend was noticed for KH 2 PO 4 where best value was recorded when 127.5 mg/ l was added to 25% MS, 85.0 mg/ l was added to 50 % MS and 170 mg/ l was added to 75 % MS. It was also noticed that the highest weight recorded for microcorms on 25 % MS resulted from 75 g/ l sucrose, the highest weight recorded on 50 % MS resulted from 50 g/ l sucrose and the highest weight recorded on 75 % MS resulted from 25 g/ l sucrose. Under dark conditions, 75000 mg/ l sucrose supplements resulted in a higher microcorm weight on 25 and 75 % MS compared to other sucrose concentrations. Meanwhile, 50000 mg/ l sucrose proved to be the best sucrose concentration when added to 50 % MS medium in regards of microcorm weight. For all concentrations of KNO 3 and KH 2 PO 4, except for 85.0 mg/ l KH 2 PO 4, recorded weights increased with the increase in medium strength. It was also noticed that for each sucrose, KNO 3 and KH 2 PO 4 concentration except for 100000 mg/ l sucrose, differences recorded between weights on 25 and 50 % MS were insignificant. Meanwhile the differences recorded between 50 and 75 % MS where significant only when media where supplemented with 75000 mg/ l sucrose and 170.0 mg/ l KH 2 PO 4. as for the differences recorded between 25 % and 50 % MS, statistical significance was noticed only when 1425 and 1900 mg/ l KNO 3 and 127.5 and 170.0 mg/ l KH 2 PO 4 where supplemented to the medea. -564-
Table 2: Effect of medium strength, sucrose, KNO 3 and KH 2 PO 4 on the weight (mg/ microtuber) of potato microtubers produced in vitro under long day (LD) and dark conditions. Medium strength (A) Treatments (B) Concentration (mg/ 1) Long day (LD) Dark conditions 25% MS 50% MS 75% MS Mean 25% MS 50% MS 75% MS Mean Sucrose KNO 3 KH 2 PO 4 25000 190 hijkl 440 bc 450 bc 360 b 130 j 150 ij 230 fghij 170 c 50000 180 ijklm 860 a 270 efg 437 a 260 efghij 240 fghij 210 ghij 237 bc 75000 390 cd 480 b 160 jklmn 343 b 460 abcdef 180 hij 460 abcdef 367 ab 100000 260 efgh 310 ef 160 jklmn 243 c 200 hij 150 ij 180 hij 177 c 475 170 ijklmn 80 op 160 jklmn 137 fg 360 cdefghij 370 cdefghi 440 bcdefg 390 a 950 190 hijkl 170 ijklmn 60 p 140 fg 400 bcdefgh 450 abcdef 560 abcd 470 a 1425 330 de 80 op 70 p 160 def 350 defghij 390 bcdefgh 620 ab 453 a 1900 80 op 70 p 150 klmno 100 g 270 efghij 350 defghij 530 abcd 383 a 42.5 100 nop 200 ghijk 160 jklmn 153 ef 280 efghij 460 abcdef 480 abcde 407 a 85.0 120 lmnop 210 ghijk 170 ijklmn 167 def 370 cdefghi 590 abc 390 bcdefgh 450 a 127.5 240 fghi 170 ijklmn 180 ijklm 197 d 370 cdefghi 440 bcdefg 620 ab 477 a 170.0 230 ghij 110 mnop 220 ghijk 187 de 330 defghij 440 bcdefg 680 a 483 a Mean 207 b 265 a 184 c 315 b 351 b 450 a Finally, it is worth mentioning that under LD conditions, supplementing half strength MS medium with the concentrations of sucrose investigated in this trial was found to be useful in the production of microtubers with acceptable weight (> 250 mg). Moreover, the positive effects recorded for sucrose under LD on tuberisation are in accordance with the general agreement in the literature that sucrose is required in the medium for potato micropropagation (18, 19). As for the photoperiod and its pronounced effect on microtubers production recorded in this investigation, it is in agreement with what Seabrook (2005) stated about light being the most important stimulus that affects the growth, morphogenesis and tuberisation of potato. In this regard, it was stated that the effect of light has a typical phytochrome response and it can affect morphogenesis through photoperiod, light intensity and spectral wavelength (3). Effect of medium strength, sucrose, KNO 3 and KH 2 PO 4 on the average size (mm) of potato microtubers produced in vitro under long day (LD) and dark conditions. Results presented in Table 3 reveal that statistical significant was detected between different medium strengths, different supplements and the interaction. Regardless of supplements, under both photoperiods investigated, a significant difference was detected between 25% and 50 % MS and a nonsignificant difference was detected between microcorm sizes recorded for 50 % and 75 % MS. Moreover, it was noticed that records decreased as medium strength increased under LD conditions, while increases in medium strength under dark conditions were correlated with size increases. As for the effect of different supplements on microcorm size (mm) regardless of medium strength, it was found that 50000 mg/ l sucrose recorded the highest value under both photoperiods under investigation. It was also noticed that under dark conditions all other sucrose and KH 2 PO 4 concentrations and 950 mg/ l KNO 3 recorded values that were nonsignificantly lower than that recorded for 50000 mg/ l sucrose. On the other hand, culturing the explants on media containing KNO 3 resulted in reduced microtubers size values compared to KH 2 PO 4 and sucrose supplements. Finally, it was noticed that the only treatment which led to the production of microcorms with a diameter less than 5 mm under both photoperiods investigated was 1900 mg/ l KNO 3. In addition to that treatment, 475 mg/ l KNO 3 and 42.5 KH 2 PO 4 also led to the production of microtubers with inferior size under LD conditions. -565-
Table 3: Effect of medium strength, sucrose, KNO 3 and KH 2 PO 4 on the average size (mm) of potato microtubers produced in vitro under long day (LD) and dark conditions. Medium strength (A) Treatments (B) Concentration (mg/ 1) Long day (LD) Dark conditions 25% MS 50% MS 75% MS Mean 25% MS 50% MS 75% MS Mean Sucrose KNO 3 KH 2 PO 4 25000 5.59 fghijk 5.87 efghij 6.36 cdefgh 5.94 bc 5.00 def 6.00 bcde 6.00 bcde 5.67 abcd 50000 7.91 ab 7.10 abcde 6.88 bcdef 7.30 a 6.00 bcde 7.13 abc 7.13 abc 6.75 a 75000 6.15 defghi 7.58 abc 4.66 jklmn 6.13 b 6.50 abcd 7.13 abc 6.60 abcd 6.74 a 100000 6.10 defghi 6.63 bcdefg 2.66 p 5.13 de 6.00 bcde 6.12 abcde 6.00 bcde 6.04 abc 475 4.57 jklmn 5.40 ghijk 4.30 klmno 4.76 ef 5.00 def 5.20 cdef 5.00 def 5.07 cd 950 6.50 cdefg 7.33 abcd 5.08 hijklmn 6.30 b 6.00 bcde 6.00 bcde 6.00 bcde 6.00 abc 1425 6.60 bcdefg 5.07 hijklm 5.60 fghijk 5.76 bcd 4.50 ef 5.00 def 7.00 abc 5.50 bcd 1900 4.90 ijklmn 3.80 mnop 3.07 op 3.92 g 4.00 f 4.50 ef 6.00 bcde 4.83 d 42.5 3.58 nop 4.17 lmno 5.00 ijklm 4.25 fg 4.00 f 6.50 abcd 6.50 abcd 5.67 abcd 85.0 3.97 mnop 5.57 fghijk 6.25 cdefghi 5.26 cde 5.00 def 7.50 ab 6.00 bcde 6.17 abc 127.5 6.76 bcdef 4.90 ijjklmn 7.13 abcde 6.26 b 5.00 def 6.00 bcde 8.00 a 6.33 ab 170.0 8.32 a 3.87 mnop 6.10 defghi 6.10 b 4.50 ef 5.50 cdef 7.50 ab 5.83 abcd Mean 5.61 ab 5.26 b 5.13 b 6.05 a 6.48 a From results presented in tables 1, 2 and 3, it can be noticed that dark conditions were favorable for microtuberisation on 75% MS mediums supplemented with KNO 3 and KH 2 PO 4 in regards of microtuber number, weight and size compared to LD conditions. Moreover, under LD conditions, supplementing 75 % MS media with different concentrations of KNO 3 and KH 2 PO 4 led to the production of inferior potato seeds in regards of weight. Such result is in contrast with what (15) concluded regarding significant reductions in microtuber weight as a result of dark conditions when applied immediately after LD. However, it was also stated that darkness without a preceding- shortday treatment promoted premature senescence of microplants and therefore reduced the bulking potential of microtubers. Meanwhile, 1425 mg/ l KNO 3 and 127.5 mg/ l KH 2 PO 4 supplements to three quarters strength MS medium under dark conditions produced more microtubers with satisfactory weights and sizes compared to other concentrations investigated. It was noticed that increasing KH 2 PO 4 concentration from 85.0 to 127.5 mg/ l in 25 % MS medium under dark conditions led to no differences what so ever in the values recorded for the three parameters under investigation. Moreover, values recorded for these concentrations were generally better than those recorded for 42.5 and 170 mg/ l KH 2 PO 4. It was also noticed that supplementing 50 % MS medium under LD conditions with sucrose (all concentrations investigated) is useful in the production of microtubers with acceptable weight (> 250 mg) and size (> 5 mm in diameter). Similar findings were recorded under dark conditions for KNO 3 when added to 50% and 75 % MS media and for KH 2 PO 4 when added to 75 % MS medium. Finally, it is worth mentioning that whenever the production of large numbers of microtubers was coupled with reduced weight and size, especially on reduced medium concentrations, this might be attributed to the competition microtubers exert on available nutrients in the midea. REFERENCES (1) R. L. Jones, Agric. Rev. pl. physiol., 24, 571 (1994). (2) A. Badoni and J. S. Chauhan, Stem cell, 1, 7 (2010). (3) J. Dobranszki, K. Magyr-Tabori and I. Hudak, Fruit, Vegetable and Cereal Science and Biotechnology, 2 (Special Issue 1), 82 (2008). -566-
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