Histological and physiological changes of potato starch derived from seed and TPS (True Potato Seed) grown tubers under different cold storage duration

Starch granules in potato tubers exist with varying sizes and size dis­ tribution in nature. In this study, both the tubers of seed potato (‘Lady Rosetta’) and true potato seed (TPS) (‘BARI TPS­1’) varieties were stored at 5°C for 0 to 4 months, and the changes in the starch break down were analyzed physiologically and histologically to investigate how cold storage affects the starch break down. Although the starch content of both varieties reduced dur­ ing cold storage, the reduction of starch content in ‘BARI TPS­1’ was higher than that of ‘Lady Rosetta’. However, both volume and ovality (length:width) of starch granule did not change significantly throughout the storage period irre­ spective of variety, suggesting a non­uniform breakdown of starch granules. Scanning Electron Microscope (SEM) images of starch granule showed non­uni­ formed deformation and enlarged cavity or hole along the storage period, which indicated that starch breakdown occurred at a specific part of starch granule rather than peripherally and penetration would be deeper in ‘BARI TPS­ 1’ than that of ‘Lady Rosetta’. However, there was no significant change in granule size distribution in spite of rapid degradation of amylopectin percent­ age in ‘BARI TPS­1’ than that of ‘Lady Rosetta’, suggesting more susceptibility of ‘BARI TPS­1’ to starch degrading enzyme and higher enzymatic action would cause deeper penetration in ‘BARI TPS­1’ than that of ‘Lady Rosetta’.


Introduction
Potato (Solanum tuberosum L.) crop is usually cultivated by plant ing seed tubers which are genetically identical clones. On the other hand, True Potato Seed (TPS) is the actual botanical potato seed pro duced by the potato plant. Potato production from seed tuber derived from TPS (seedling tuber) is emerging as a promising alternative of using seed tuber due to the advantages of less disease transmission, physiological maturity until planting season, cheap storage cost, and many choices of varieties (Pangaribuan, 1994). As potato tuber comes to maturity, starchrich perimedullary region forms the major portion of the tuber (Gupta and Kaur, 2000), and potato granules are synthesized and stored as roughly spherical shapes in amyloplasts (Naeem et al., 1997;Fajardo et al., 2013) As the tuber matures, new starch granules are also synthe sized in newly produced amyloplasts. There is usually a large granule size distribution within individual tubers in terms of percentages of small, medium, and large granules (Singh et al., 2016). The size of small and large granules ranged from 0.6 to 6 μm and from 10 to 100 μm in potato starch, respectively (Wang et al., 2018), consisting of smoothsurfaced, oval and irregular shape (Singh et al., 2003). Singh et al. (2008) reported small starch granules of 1 to 10 μm, medi um granules of 11 to 30 μm and larger granules of >30 μm in diameter in four different New Zealand potato cultivars.
Cold storage of potato tuber results in disintegra tion and disappearance of the amyloplast mem branes around the starch granules, which bring in contact with the degradative enzymes such as ᾳand β amylases and their substrates (Badenhuizen, 1965;O'donoghue et al., 1995). Thus, prolonged storage of potato tuber at low temperatures can result in starch degradation and conversion of starch into reducing sugar (Zhang et al., 2014). When potato tubers (Solanum tuberosum) are stored at temperatures below 910°C, the accumulation of sucrose and reducing sugars glucose and fructose occurred because of 'lowtemperature sweetening' (LTS) (Pinhero et al., 2007). The rate of starch degradation and sugar accumulation depends largely on cultivar and storage temperatures (Kazami et al., 2000). Starch content was found to be decreased about 2 times after storage at 02°C for 8 weeks in both seed potato and TPS potato tuber by (Karim et al., 2008). A decrease of starch content was also reported after 60105 days of storage at 4°C in several Indian potato varieties (Yamdeu et al., 2015). On the other hand, Biemelt et al. (2000) reported that the starch content did not alter throughout the storage period. The starch degradative enzyme not only affects the starch content in cold storage also starch granule. The enzy matic susceptibility of starch granules has been stud ied by various authors (Franco et al., 1988;Srichuwong et al., 2005;Adejumo et al., 2013). Differences in the enzymatic attack or susceptibilities of starches depend on many factors such as starch source, granule size, extension of association between starch components, rate of amylose and amylopectin, crystalline structure, particle size, sur face porosity, type of enzyme (Hoover and Zhou, 2003;Kong et al., 2003;Li et al., 2004;Tester et al., 2006). A shift to lower granule size distribution has been reported in raw starches of different potato varieties after invitro enzymatic hydrolysis (Kimura and Robyt, 1995). Cold Induced Sweetening (CIS) sus ceptible cultivars can cause smaller starch granules when stored for 4 or 12 weeks at 4°C rather than CIS resistant cultivars, which does not change until 24 weeks (Barichello et al., 1990). Based on the previous observations, it was hypothesized that when potato tubers are stored at low temperature conditions, starch granule content and size change in relation to starch degradation. Starch granules may change ran domly or according to size because several studies showed that the starch granule size is an important factor for influencing the digestibility of raw starch by amylase (Noda et al., 2008). Ezekiel et al. (2010) observed that the number of small granules decreased, and the number of large granules increased in potato after cold storage. Granule size and surface area affect the hydrolysis rate of starch by amylase, large granules with higher diameter have smaller surface area than small granules with lower diameter and therefore larger granules digested more slowly (Tester et al., 2006;Kasemwong et al., 2008;Noda et al., 2008). Because of the susceptibility to hydrolytic enzyme attack, deformation like exter nal corrosion, pits or endoerosion occurred on the starch granule (Planchot et al., 1995).
Even though starch granules change in relation to starch degradation, it is unclear whether and how these happen in case of 'seed tuber' and TPS tuber at the same storage condition. So, the aim of this exper iment was to study whether the starch degradation occurs similarly or differently in seed potato and TPS tuber; and how the starch degradation affects the starch granule deformation morphologically in both tubers.

Plant material
In October 2015, each of 50 potato tubers of 'BARI TPS1' (TPS) and 'Lady Rosetta' (seed potato) were obtained from Bangladesh. Volume, size, and weight of all the tubers were measured and were stored at 5°C for 0 to 4 months in a refrigerator at the laboratory of the Department of Agronomy, Faculty of Agriculture, ShereBangla Agricultural University, Bangladesh. The 'Lady Rosetta' variety is one of the widely cultivated commercial seed potato in Bangladesh, which is also used as processed pota to. On the other hand, 'BARI TPS1' is one of the well cultivated 'True Potato Seed' variety, which is culti vated from true seed in first year and from the tuber lets from second year. Both of the tuber was collect ed and cultivated in the experimental plot of Shere Bangla Agricultural University with integrated crop management. Ten tubers per each variety were taken out monthly from the refrigerator, the skin was peeled off and the tubers were grated using a grater of about 45 mm diameter. Half of the grated sample was then blended and washed with desalted water, and filtered through filter paper. This step was repeated 3 times and then filtrated and dried in sun light under shaded condition for few days when the environmental temperature was around 34°C. This unheated samples were prepared for starch granule observation using SEM (Scanning Electron Microscope). The other half of the grated sample was added to 80% MeOH (100 mL) and heated at 80°C for 30 minutes. The supernatant was decanted, and the residue was extracted by 80% MeOH (100 mL). This step was repeated for 3 times in total and then washed by pure acetone. Then, it was heated and dried on a hot plate at 50 to 70°C only in the daytime and stored at the ambient temperature at night. It took 3 days to dry completely and was weighed repetitively up to constant dry weight. The completely dry sample was cooled to the ambient temperature, stored in a freezer at 20°C. This 80% MeOH (100 mL) extracted samples were used for sugar and starch analysis. The chemical analysis was done at the laboratory of the Department of Bioproduction, Faculty of Agriculture, Yamagata University, Japan.
Soluble sugar content A 0.2 g of dry powder from each cultivar was taken into a test tube, then 9 mL of 80% MeOH was added, and it was heated at 80⁰C for 30 min. The extract was centrifuged at 3000 rpm using a cen trifuge (KS500, KUBOTA, and Tokyo) for 10 min, and the supernatant was decanted. This extraction proce dure was repeated for 3 times, and the combined supernatant was made up to 50 mL volume with 80% MeOH. Reducing sugar content in 0.5 mL of the solu tion was measured by Somogyi Nelson method (Nelson, 1944), and the 0.5 mL of the solution was added by 2 units of invertase (pH 4.5, Kanto, Kagaku, Tokyo) and hydrolyzed at 50°C for 30 min. Sucrose content in the solution was also measured using the same technique as reducing sugar. Copper reagent and nelson reagent were used to prepare a standard solution of glucose. Absorbance was measured at 660 nm, and a standard curve was prepared to calcu late reducing sugar. Nonreducing sugar was mea sured from hydrolytic degradation of sucrose, and absorbance was measured at 660 nm.
Abs obtained from the analysis of reducing sugar was denoted by ABS 1 … (1) Abs obtained from the analysis of nonreducing sugar was denoted by ABS 2 … (2) Concentration of nonreducing sugar was calculat ed from = {Abs (2) Abs (1)} × 0.95 … (3) Concentration of total soluble sugar was calculat ed from = (1) + (3) These steps were repeated for 5 times for each cultivars and for each storage sample.

Starch content
The insoluble solid from the 80% MeOH extract was added with 1.5 mL of distilled water and heated at 100°C for 1 hr. Starch in the pellet was hydrolyzed using amyloglucosidase (Yakult) at 55°C for 3 hrs then neutralized by 0.1% NaOH solution. Starch content was also measured in 0.5 ml of the solution using the same technique of sugar analysis and Glucose Oxidase method. Glucose standard curve was pre pared to measure starch content. Absorbance was measured at 660 nm for Somogyi Nelson Method and at 500 nm for Glucose Oxidase method and was repeated for 5 times for each cultivars and for each storage sample.
Abs obtained from the analysis of starch was denoted by Abs … (4) Starch content was calculated from = Abs 4 × 0.9.

Histological analysis of starch granule
Starch powders were scattered on an adhesive carbon tape and were fixed with 2% osmium tetrox ide (OsO 4 ) and successively washed with 50 mM cacodylate buffer and ultrapure water. The dried samples were coated by Pt using each sample slides by Pt ion coater (JFC1200, JEOL, Tokyo), and then observed under SEM Scanning Electron Microscope (SEM, TM3000, Hitachi, Tokyo). The length and width of the starch granules were measured by using Motic Image Plus 2.0 software from SEM images. The length and width ratio of starch granule was mea sured as the ratio of length:width.
Starch granule was regarded as an ellipsoid, and the volume of starch granule was measured of each axis of the ellipse. For histogram analysis (volume and length/width ratio) 10 SEM image of x 500 mag nification and 200 µm across from each treatment were chosen and 5 granules from each image were measured randomly (total 500 starch granules).

Amylose percentage determination
Amylose content was measured using an assay kit (Megazyme, Amylose/Amylopectin Assay Kit, Ireland) according to the procedure outlined by the manufac turer. Percentage of amylose was directly calculated following the specific Megazyme equation based on the measured absorbance values, no additional stan dard curve or equation was generated for this study. Amylopectin content was calculated by 100% differ ence of the amylose content (Aristizábal et al., 2007). This step was repeated for 5 times for each cultivar and for each consecutive storage sample.

Statistical analysis
Data were subjected to analysis of variance and the difference between cultivars was compared with ttest using SPSS software.

Soluble sugar content
In 'BARI TPS1' tubers, reducing sugar content reached the highest value (57.80 mg/g DW) after 1 month of storage then decreased rapidly thereafter. Reducing sugar of 'Lady Rosetta' showed the same tendency but the highest value after 1 month of stor age (25.86 mg/g dry weight) was less than half of that of 'BARI TPS1' and decreased gradually there after (Fig. 1). Therefore, reducing sugar content of 'BARI TPS1' was significantly higher than that of 'Lady Rosetta' during 1 to 3 months of storage. Although, sucrose content also showed the highest value after 1 month of storage, the value decreased rapidly thereafter. The decline of 'BARI TPS1' was slower than that of 'Lady Rosetta', resulting in a sig nificant difference between the two varieties during 2 to 3 months of storage (Fig. 1). Changes in total sugar content were similar to that of sucrose content and significantly higher content was observed also in 'BARI TPS1' during 2 to 3 months of storage (Fig. 1).

Starch content
Although starch content of both potato varieties decreased continuously throughout the storage peri od, starch content of 'BARI TPS1' decreased more rapidly than that of 'Lady Rosetta' variety, resulting in a significant difference from 2 to 4 months of storage (Fig. 1). It is noticeable from figure 1 that decreasing level of starch content was on par with the increasing reducing sugar content, where 'BARI TPS1' showed rapid starch degradation with higher reducing sugar content after 2 to 4 months of storage than that of 'Lady Rosetta'. Similarly increase in sucrose level of 'BARI TPS1' after 1 month of storage paralleled with the decreased level of starch content, resulting signif icant differences between two varieties.

Starch granule sizes
Starch granule length, width, length:width ratio and volume did not change apparently throughout the storage period. Though 'Lady Rosetta' had a ten dency to have slightly higher values in length, width and volume, there was no significant difference between the both 'BARI TPS1' and 'Lady Rosetta' (Fig. 2).
Histogram was shown for starch granule volume and ovality (length:width) within the range of (0.5 to 81.5) x 10 3 µm 3 and (1.05 to 2.22) respectively ( Fig. 3  and 4). The highest frequency was observed at (7.6 13.5) x 10 3 µm 3 and (1.19 and 1.31) for volume and ovality, respectively and there was no considerable differences between the two cultivars. Moreover, no apparent change also occurred during storage period.

Starch granule morphology
Both 'BARI TPS 1' and 'Lady Rosetta' tubers showed normal and smooth starch granule surface at 0month storage (Fig. 5). Starch granules of both tubers changed after storage condition. Pit and hole like structures were observed after 1 month storage in both varieties. After 2 nd , 3 rd and 4 th month storages prominent depression or cavity was observed in both potato cultivars. However, both potato tubers showed a similar pattern of deformation.

Amylopectin percentage
Although the percentage of amylopectin in both potato varieties decreased continuously throughout the storage period, Amylopectin of 'BARI TPS1' decreased more rapidly than that of 'Lady Rosetta' variety, resulting in a significant difference from 2 and 4 months of storage (Fig. 6). Although 'Lady Rosetta' variety had slightly higher percentage than TPS after 3 months of storage, there was no signifi cant difference between them.

Discussion and Conclusions
In potato tuber, starch is converted to sugar dur ing cold storage (Malone et al., 2006). The starch content of potato tuber decreased markedly during prolonged storage at 48°C through the process of conversion of starch into sugars (Nourian et al., 2003;Smith et al., 2005). Ohad et al. (1971) also reported reduction of starch content by 26% after 17 days of storage. Mature dormant potato tuber produced sug ars by degradation of a small fraction of starch (Isherwood, 1973). Thus starch content decreased because of the hydrolysis of starch by starch degrad ing enzymes (Nielsen et al., 1997), suggesting why reducing sugar content increased as starch content decreased during cold storage (Fig. 1). Storage at 0 5°C increases sugar accumulation in potato tuber (Wismer et al., 1995;Blenkinsop et al., 2003), which coincides with these experiments where both vari eties showed higher reducing sugar content after storage with a significant difference between 'BARI TPS1' and Lady Rosetta (Fig. 1). This increased level of reducing sugar and decreased level of starch con tent is related with the increased activity of hydrolysing enzyme (Sowokinos, 2001), which might explain the higher starch degradation in 'BARI TPS1' (Fig. 1).
During lowtemperature storage, the enhance ment of sucrose and hexoses (glucose and fructose) levels is known as "cold sweetening' or 'low tempera ture sweetening', which is an important metabolic process in the roots of many species as well as potato tuber (Wismer et al., 1995;Espen et al., 1999;Galindo et al., 2004;Galindo et al., 2007). The soluble sugar content of potato tuber increases at low tem perature such as 4°C, when stored for 6 to 12 weeks because of starch decomposition and then inclines to decrease (Cochrane et al., 1991;Chen et al., 2012). Total sugar and sucrose content both showed a simi lar changing pattern after storage with significantly higher content in 'BARI TPS1' during 2 and 3 months (Fig. 1). Cold storage condition triggers the tuber starch to breakdown into sucrose through various hydrolytic enzymes which further hydrolyzed into reducing sugars (glucose and fructose) (Sowokinos, 2001). The amount of soluble sugar accumulated in  tuber also depends on different cultivars (Zommick et al., 2014).), which could explain the higher reducing sugar in 'BARI TPS1' after storage than Lady Rosetta (Santos et al., 2020). However, there was a sudden increase in starch content between 3 rd to 4 th months of storage in 'Lady Rosetta' variety ( Fig. 1), suggesting recondition of starch or increase of respiration losses during stor age. The difference in respiration rates depend on cultivar, growing conditions, experimental condi tions, and physiological status of the tubers (Fennir et al., 2003). Comparatively lower starch degradation after 3 to 4 months of storage might have con tributed to lower presence of reducing sugar in 'Lady Rosetta' (Fig. 1). This can also be explained that likely the rise of starch content between 3 and 4 months of storage, amylopectin percentage also showed similar kind of tendency in 'Lady Rosetta' variety ( Fig. 6). An increase in amylopectin percentage from 3 to 4 months of storage suggested lower enzymatic degra dation (Fig. 6) (Hofvander et al., 2004). It seemed amaylase enzyme activity reduced during 3 to 4 months of storage because of sprouting of seed pota to varieties or it can be explained that the amaylase enzyme activity was not enough to degrade the starch in seed potato (Lewis et al., 1994). As the experimental condition was same for both of the tubers, hence these may explain faster physiological aging in seed potato variety than TPS variety. This may also suggest that experimental storage tempera ture might have affected the respiration losses; and the sugar had been used for tuber germination in case of seed tuber (Olsen et al., 2003).
The TPS variety showed higher starch degradation rate than the seed potato (Fig. 1), and sharp decrease in amylopectin percentage after storage (Fig. 6), moreover there was higher percentage of amy lopectin degradation in 'BARI TPS1' than 'Lady Rosetta' starting after 1 month of storage and contin ued as the storage progressed (Fig. 6), but there was no apparent changes in starch granule volume and ovality (legth:width ratio) (Fig. 2). This result suggest ed a nonuniform degradation pattern of starch gran ules in ''BARI TPS1'and 'Lady Rosetta' varieties.
Histograms were also carried out to investigate, if the starch granule deformation primarily started in smaller sized granules, or larger sized granules (Figs.  3 and 4). Singh et al. (2008) reported a shift of gran ule size range to smaller granule sizes in isolated starches of New Zealand Taewa (Maori potato) when they were stored for three and six months at 4°C temperature. This experiment also reported erosion and pitting on the surface of stored potato starch. In case of banana, surface erosion by enzymatic degra dation resulted in smaller granules with elongated shape and exocorrosion process caused pits on the surface of the starch granule with high frequency (PeroniOkita et al., 2013). This shift of granule size distribution has been related to rapid digestion of starch granule and composition variation between small and large granules (Salunkhe et al., 1989). However, there were no considerable changes of starch granule sizes in both potato cultivars in our study. This suggested that, in spite of having signifi cant change in starch degradation along storage in both variety, it had hardly influenced the size of starch granule (Fig. 2, 3 and 4).
Moreover, it was found that even after 4 months of storage periods, there was no significant change in starch granule sizes in both cultivars, in spite of 'Lady Rosetta' having slightly higher tendency in the value of length, width and volume (Fig. 2). Fajardo et al. (2013) also reported unchanged granule size after storage, which was attributed to the nearly unde tectable change in volume of starch granule. Russet Burbank potato tubers also showed similar granular size distribution after storage at 3.9°C (Johnston et al., 1968).
SEM observation showed that starch granules from both harvested tubers presented smooth gran ular surface under SEM without any storage treat ment or at 0month storage (Fig. 5). Although there is argument about the appearance of natural potato starch granular surface, several studies showed smooth granular surface of natural potato starch under microscopic observation. Cottrell et al. (1993) reported smooth granular surface of potato starch of Record and Brodick potato cultivar at harvest. (Sarikaya et al., 2000) also reported smooth granular surface of potato starch before any enzymatic or freezing treatment. As the SEM observation in this experiment showed hole or pit formation on granular surface of both varieties after storage (Fig. 5) (Sarikaya et al., 2000;Noda et al., 2005), it is suggest ed that the long term storage at low temperature of this experiment lowered the starch content which led to susceptibility to hydrolytic enzyme attack that changes the properties and composition of starch granule (Barichello et al. 1990). The cold storage temperature might have given damage to amyloplast membrane by starch hydrolysis (Ohad et al., 1971), which enhanced membrane permeability through starch hydrolysis enzymes, and resulted the forma tion of cracked region or surface hole on the granule (Sujka and Jamroz, 2010).
However, no apparent change was observed in the ratio of granules and histogram analysis of both potato starch granule, but the SEM observation clear ly showed deformation of starch granule forming hole or pit like structures gradually started from 1 month of storage and continued to 4 months of stor age (Fig. 5). On the other hand, gradual starch degra dation and decreased amylopectin percentage indi cated activation of enzymatic degradation in both cultivars after storage ( Fig. 1 and 6). Therefore, it is suggested that starch granule erosion may occur at specific surface region of the granule causing no change in the granular size. Starch hydrolyging enzyme like aamylase might have attacked at partic ular points on the granule surface, forming tunnels into the granule, thus hydrolyzed the granule from the inside (Lindeboom et. al., 2004). Similar finding was reported by PeroniOkita et al. (2013) where starch granules of green banana maintained the rounded shape after low temperature but presented pits on the granule surface which was enlarged by the corrosion process, suggesting partial degradation of starch granules.
Thus enzyme molecules can affect the starch granule in different patterns either by forming pin holes, medium sized hole, sponge like erosion or selected point at the surface leading to a single hole (Sujka and Jamroz, 2010). Enzyme can gain access to innermost region of the starch granule by faster digestion than at the periphery region of the granule, which can form shallow hole like structure (Duffus, 1984). This may explain how the both starch granules showed no changes in their sizes despite of granular deformation.
However, 'BARI TPS1' showed a higher starch degradation percentage throughout the storage peri od than the seed tuber potato. This suggested higher susceptibility of starch degrading enzyme in TPS tuber than that of seed potato tuber. Variation in starch granule morphology and their crystalline orga nization may explain the susceptibility to enzymatic degradation (Gallant et al., 1992). Amylase plays the major role in invivo breakdown of starch (Manners, 1985). Amylose and amylopectin ratio may explain the degradation pattern of starch granules in both TPS and seed potato varieties. Both the potato tuber showed decreased percentage of amylopectin along the storage period, whereas 'BARI TPS1' resulted lower amylopectin percentage than the seed potato (Fig. 6). Bach et al. (2013) described that as amy lopectin degrades more rapidly than amylose, it caus es activation of more starch degrading enzymes. Therefore, 'BARI TPS1' might have allowed deeper penetration of starch degrading enzymes than the seed tuber in spite of no apparent changes in granule size (Fig. 5). As the extent of pit or cavity of starch granules were not determined in this experiment, it can be suggested that probably a different inner mol ecular organization of TPS starch granule may be allowed differentiated enzyme attack, which could be responsible for slightly smaller starch granule than 'Lady Rosetta' during Storage (Fig. 6) with no signifi cant difference. This also suggests that starches vary in their resistance to enzymatic susceptibility (Srichuwong et al., 2005).
There are different opinions about the presence of pores or holes on the surface of potato starch granules. Although several observations concluded that some factors could cause an increase in the number and size of pores on starch granules surface (Fannon et al., 1992). Moreover, the expansion of granule degradation increased with the increased enzyme concentration (Mu et al., 2015). During enzy matic hydrolysis, some regions of granules are more susceptible to enzyme attack because of less orga nized amorphous rings, whereas the crystalline lamella provides higher resistance to enzymatic ero sion (Oates, 1997). This kind of enzymatic hydrolysis was characterized by forming a hole by creating channel through the less resistant granule core (Jung et al., 2017), which may explain deeper enzyme pen etration in TPS tuber. Action pattern of both endo and exoamylase at a lower temperature may explain overall amylolytic activity in TPS and seed potato tubers (Shin et al., 2002;Nabubuya et al., 2012).

Conclusions
In this experiment, the starch content of TPS tuber degraded more rapidly and produced higher reducing sugar content than the seed potato variety after storage. This result indicated that, TPS tuber may not be acceptable for low temperature storage compare to seed potato variety. There were no sig nificant changes in granule size and volume, indicat ed that granule did not change as a ratio in both tubers. However, both potato granules deformed by forming holes or cavities at innermost surface region after storage, suggesting the starch granule degraded partially rather than concentrically. As both the tubers showed a similar kind of degradation pattern of starch granule in spite of higher starch degrada tion rate in TPS tuber, similarly the rapid degradation rate of amylopectin in TPS tuber explained higher amylase activities in TPS than the seed tuber. This result suggested possibilities of deeper penetration in TPS starch granule than the seed tuber starch granules. Further studies on the susceptibility of starch degrading enzyme on TPS and seed potato can be done to foresee the starch degradation pattern in TPS and seed potato tuber.