Thiamine improves in vitro propagation of sweetpotato [Ipomoea batatas (L.) Lam.] – confirmed with a wide range of genotypes

In vitro propagation of Plant Genetic Resources is a basic step for routine genebank and biotechnology research activities. Accelerating growth and rooting of in vitro plants contributes to an improvement in process efficiency and plant quality. In the present study the effect of supplemental thiamine and explant size on biometric variables, ion content in plant sap, chlorophyll content in leaves and moisture content in plants were assessed in a replicated trial on a group of seven in vitro sweetpotato accessions and validated in a set of other 45 accessions. It was shown that adding 0.1 mg L −1 of thiamine to modified Murashige and Skoog culture medium significantly increased plant height, root length, and number of nodes of in vitro sweetpotato shoot culture plants. No significant differences were observed for N03−, K+, Na+ and Ca++—ion content in plant sap, nor in leaf area, chlorophyll, or moisture content between plants grown with or without thiamine. Uninodal stem segments showed on thiamine-free medium a significantly lower root and plant growth, and reduced number of nodes, than plants grown from uni- and binodal segments on thiamine-supplemented medium. A subsequent experiment tested all the parameters above in a non-replicated screen with a set of 45 diverse sweetpotato accessions. With this diverse set of germplasm, the average plant and root length increased by 41 and 51%, respectively on thiamine-supplemented culture medium compared to the control treatment, confirming that supplemental thiamine is generally beneficial to sweetpotato in vitro shoot culture. Supplementing culture medium with thiamine significantly improved plant and root growth of in vitro sweetpotato plants. The results were confirmed with a diverse set of 45 sweetpotato clones.


Introduction
With a total production of 139 × 10 6 tons on 9.65 × 10 6 hectares, and an average yield of 11.6 tons/hectare, sweetpotato, Ipomoea batatas (L.) Lam., is one of the world's major staple food crops (FAOSTAT, 2022). Sweetpotato is widely consumed in Asian and African countries, and to a lesser extent in the Americas and Europe. Its roots are prepared boiled, steamed, baked, grilled, or fried, and are also used for starch, flour, noodles, and alcohol production. Sweetpotato is a good substitute for potato, apples, and squash, and it finds application in many sweet dishes, as well as in candied and baby food (Thottappilly, 2009).
In vitro propagation of plant genetic resources enables fast multiplication of plant material under sterile conditions which maintains pathogen-free material over time (e.g., free of virus, bacteria, and fungi). In vitro propagation frequently is the initial step for other activities such as distribution of in vitro material, cryopreservation, in vitro conservation, phytosanitary cleaning, DNA/RNA extraction, genetic transformation, assessment of gene expression and secondary metabolites, multiplication in bioreactors, etc. The first publications on in vitro propagation of sweetpotato clones on modified Murashige Skoog medium (MS) date from the late sixties (Elliot, 1969;Murashige and Skoog, 1962). Meristems of four sweetpotato accessions were cultivated on different variations of MS medium, and it was shown that pyridoxine, glycine, and nicotinic acid were not essential for survival nor development of the plants. All three compounds form part of the standard MS medium vitamin supplement. According to Elliot (1969), gibberellic acid (GA 3 ) and coconut water suppressed shoot and root growth while naphthalene acetic acid (NAA) stimulated root and plant growth. Several years later, a genotype-specific response was shown to the type and concentration of growth regulators on the development and growth of plants coming from axillary meristems of sweetpotato clones grown on MS medium supplemented with different combinations of indole acetic acid (IAA), indole butyric acid (IBA) and zeatin (Alconero et al., 1975). Later, investigations continued to assess the effect of plant growth regulators on in vitro growth and development of sweetpotato however these reports generally only included one to four sweetpotato genotypes. In many of the studies callus formation and/or other genotype-specific responses to plant growth regulators were observed (Fadaladeen et al., 2022;Beyene et al., 2020;Alula et al., 2018;Sefasi et al., 2013;Dugassa and Feyissa, 2011;Jarret et al., 1991a;Jarret et al., 1984;Litz and Conover, 1978). It was further reported that supplementing culture media with auxins and cytokinin could induce somaclonal variation in the plants, particularly during indirect organogenesis (i.e., callus formation) [Leva et al., 2012;Bairu et al., 2011Bairu et al., , 2006. These results are problematic because the goal of most in vitro propagation is to maintain true-to-type propagation of genotypes. An assessment of a set of carbohydrates (sucrose, sorbitol, mannitol) and environmental factors (temperature, photoperiod) on growth and development of sweetpotato accessions also confirmed a strong interaction between genotypes and the assessed variable (i.e., genotype-specific response) [Jarret and Gawel 1991b]. In a more recent study, the positive effect of higher temperatures (27 ± 2 °C) on plant growth and leaf number was confirmed with a set of 30 sweetpotato accessions (principally Brazilian landraces) under in vitro conditions [Vettorazzi et al., 2017]. A study on two Kenyan sweetpotato varieties assessed the possibility to replace nutrients of MS medium by locally available fertilizers (Ogero et al., 2011).
Three decades ago, CIP scientists published an auxin-and kinetin-free culture medium for the in vitro propagation of sweetpotato clones that could be applied to a wide range of genotypes, without callus formation during plant regeneration (Lizárraga et al., 1992). The culture media contained standard MS salts (without vitamins), supplemented with gibberellic acid, ascorbic acid, calcium nitrate, L-Arginine, calcium pantothenate and putrescine-HCl. Although this culture medium avoided callus formation and the associated potential somaclonal variation, suboptimum root and plant growth was still a limiting factor in many accessions.
Thiamine (vitamin B1) is an essential enzymatic coenzyme that modulates plant metabolism, although its full role is still not completely understood (Fitzpatrick and Chapman, 2020). Thiamine can be found in many plant organs such as leaves, flowers, fruits, seeds, roots, tubers, and bulbs, and is a cofactor for enzymes involved in the synthesis of amino acids as well as the Krebs and Calvin cycles (Asensi-Fabado and Munné-Bosch, 2010;Subki et al., 2018). Thiamine was a component of the original MS culture media developed for tabaco by Murashige and Skoog and has since been used in the culture media of many plant species. The specific positive effect of thiamine on rooting and/or in vitro plant growth has been shown in multiple species including, wild banana, damask rose, peach × almond hybrid, and soybean (Razaji et al., 2021;Ssekiwoko et al., 2014;Sepahvand et al., 2011;Gamborg et al., 1968). Nevertheless, its effect on growth and development of in vitro sweetpotato shoot cultures has not been assessed.
Optimum explant size for in vitro propagation varies between plant species. In olive (cv. Dolce Agovia) bi-, tri-, and tetra-nodal stem segments have shown significantly better root formation and growth compared to uni-nodal explants and plants coming from tetra-nodal segments were superior to bi-or tri-nodal explants (Haq et al., 2009). In Excoecaria agallocha L., a mangrove species, higher mean shoot length was observed in plants coming from bi-nodal than uni-nodal explants, independent of the hormone type and concentration (Rao et al., 1998).
In the present study the effect of thiamine, autoclaved vs filter-sterilized GA 3 and explant type (uni-and binodal stem segments) on growth, rooting, ion uptake (NO 3 − , K + , Na + , Ca ++ ), chlorophyll, and moisture content of sweetpotato in vitro plants was assessed with a wide range of 52 diverse genotypes. To our knowledge, this is the first study that assessed the effect of thiamine on sweetpotato in vitro plants and in a significant number of diverse accessions.

Propagation of plant material
Per accession, one 18 × 150 mm glass tube containing two in vitro plants was requested from CIP's sweetpotato in vitro medium-term storage, which is stored at 20 ± 1 °C, light intensity of 40 µmol/m2/s, with a photoperiod of 16 h of light (Fluorescent lamp COOL DAYLIGHT, 36 W). In vitro plants of the sweetpotato collection were maintained on solid MS conservation medium and sub-cultured every 6-12 months, depending on genotype and physiological 1 3 conditions of the plants. The conservation medium has a similar composition as the propagation medium described below, but without gibberellic acid and thiamine, and supplemented with 7 g/L of agar instead of phytagel (same pH). After transfer from medium-term storage, in vitro plants were multiplied every 5-7 weeks in 25 × 150 mm test tubes on modified full-strength Murashige Skoog (MS) propagation medium and incubated at 24 ± 1 °C, light intensity of 100 ± 20 µmol.m −2 . s −1 with a 16 h photoperiod. The MS propagation medium was supplemented with 30 g L −1 of sucrose, 10 mg L −1 of gibberellic acid (GA3), 200 mg L −1 of ascorbic acid, 100 mg L −1 calcium nitrate, 2 mg L −1 of calcium pantothenate, 100 mg L −1 of L-Arginine, 20 mg L −1 of putrescine-HCl, and 3.0 g L −1 of Phytagel™ with or without 0.1 mg L −1 , thiamine. The pH of all media was adjusted to 5.70 ± 0.02 prior to autoclaving for 20 min (at 121 °C, 15 psi). Uninodal and binodal stem segments were compared as explant types. The routine sweetpotato culture medium used at CIP was used as one control (routine control), with binodal explants, no thiamine, and GA 3 added prior to autoclaving. A second control used binodal explants with filter sterilized GA 3 added after autoclaving (filter sterilized GA 3 control), without thiamine. All treatments (uninodal versus binodal explants and medium ± thiamine) used filter-sterilized GA 3 which was added post autoclaving after medium had cooled to 35-40 °C.
All five comparisons (routine control, filter sterilized GA 3 control [binodal explant minus thiamine], binodal explant + thiamine, uninodal explant minus thiamine and uninodal explant + thiamine) were tested in a thrice replicated experiment with seven genotypes (CIP 421066, CIP 420952, CIP 420964, CIP 400989, CIP 420530, CIP 440144 and CIP 420701). For each treatment and replicate of each accession, seven to eight test tubes with two explants per tube were propagated. Subsequent to this replicated trial, a set of 45 diverse sweetpotato accessions was screened in a separate non-replicated experiment (single repetition with six tubes per treatment and accession) in order to confirm results carried forward to a larger set of genotypes ( Fig. 1, Table 1).

Evaluation
Biometric data (plant height, root length, root and node number, stem diameter, and leaf area), ion content (in plant sap), photosynthetic activity and moisture content were measured 47-51 days after subculture on six plants from each replicate (three test tubes). The biometric variables plant length (in cm), stem width at base, mid and upper position (in mm), number of nodes, number of roots, root length (in cm) and leaf area were determined photographically, using the ImageJ software (Version 1.51e). Per treatment, six plants were removed from test tubes, and leaves and roots were cut from the stem. Plant parts were separated by organ type and laid out onto small pieces of transparent contact paper (Layconsa, Lima, Peru), that were then stuck onto normal paper sheets. Samples were photographed together with accession and treatment identifiers, and a ruler for calibration of the image processing software. Sample processing was performed quickly to minimize loss of moisture during handling.
The moisture content (in %) of the plant parts used for the biometric measurements was determined using a moisture analyzer (Ohaus, MB120, NJ, US). Plant parts were dried at 105 °C for 15-20 min, the equipment automatically performs the measurement of fresh and dry weights (drying cycle without ramp). After each process cycle, the equipment was auto calibrated.
Plant sap was extracted from a separate set of eight to ten in vitro plants per replicate (four to five test tubes) using a handheld plant sap press. Sap liquid was collected in a sterile petri dish and 200 µl was removed using a micropipetter and placed on the flat sensor of the pocket meters (Horiba, Laq-uaSeries, CA, US) for the measurement of Ca ++ , K + , Na + and NO 3 − -ion concentration (in ppm). After each measurement of eight samples, the pocket meters were recalibrated with its corresponding standard solutions. Photosynthetic activity (in ppm) was determined in middle-positioned leaves with a chlorophyll meter (Spectrum Technologies, SPAD 502, IL, US) [ Fig. 2].

Data analysis
The replicated experiment with seven accessions was set up using a completely randomized design (CRD) and replicated three times with a sample size of three test tubes with two plants per tube for the biometric measurements (total: six plants). For the confirmation of results with a wider set of 45 different accessions a single repetition of 6 plants per treatment was performed. Distribution of data and homogeneity of variance were analyzed with the Anderson-Darling and Leven test, respectively. Data with normal distribution (p > 0.05) was analyzed by an Analysis of Variance ANOVA) and the variables showing significant differences for ANOVA were compared with the Tukey test (p < 0.05). For the number of roots, plant and root length variables, data was transformed with log(x), ln(x) or Johnsons functions before ANOVA analysis. Statistical analysis, data and graphical presentation were performed with MINITAB 17.1.0 and Microsoft Excel 365 (Version 2102).

Results
Sweetpotato in vitro plants grown on culture medium supplemented with thiamine showed a significantly higher average plant height (uninodal explant [uni]  Effect of thiamine and type of explant on physiological and morphological appearance and growth of 7-week-old in vitro sweetpotato accession (CIP 400906), grown on MS culture media. MS media were supplemented with thiamine (+ Thiame) or thiamine-free medium (-Thiamine). Uninodal and binodal stem segments were used as explants for propagation. For the routine control gibberellic acid (10 mg. L −1 ) [GA 3 ] was added prior autoclaving while for the other treatments GA 3 was added as filter sterilized solution after autoclaving. The routine control was grown on thiamine-free medium using binodal stem segments as explants  had autoclaved GA 3 . Moreover, plants showed significantly better root growth on thiamine-rich culture medium (uni: 9.12 cm, bi: 8.97 cm), in comparison to the routine control (4.38 cm) and the filter sterilized GA 3 control (uni: 4.62, bi: 4.82 cm). Finally, with the addition of thiamine, no significant differences were observed for plant or root growth between the use of binodal or uninodal explants (Fig. 3).
A significantly higher number of nodes were formed, when either uninodal or binodal explants were propagated on culture medium supplemented with thiamine (11.1 and 10.8 nodes respectively) relative to the routine control treatment (9.0 nodes). This increase in nodes translates into a ~ 20% higher multiplication rate for subsequent subculture cycles. Regarding the average number of roots, the use of binodal explants in combination with filter-sterilized GA 3 (added after autoclaving), showed a significantly higher number of roots (4.5-4.7 roots), compared to the plants coming from single node explants or grown on culture medium with filter sterilized GA 3 and thiamine (3.4 roots), however there was no significant difference in root number compared with the routine control (autoclaved GA 3 ). Although a significant difference in stem diameter was observed between binodal explants without thiamine and uninodal explants with thiamine, the difference was minor (0.01 cm) and there were no other significant differences in either stem diameter or average leaf area for the treatments (Fig. 4).
No significant differences were observed between genotypes and treatment in the concentration of NO 3 − , K + , Na + and Ca ++ -ions in the plant sap for the five treatments with p values from 0.19 to 0.49. Nevertheless, genotypespecific significant differences for ion concentrations were observed. CIP 420530 had a significantly higher NO 3 − -ion concentration in its sap (5997 ppm), compared to four of the other genotypes (CIP 400989, CIP 420701, CIP 421066, and CIP 440144 (4813-4880 ppm)). For K + content, CIP 440144 had a significantly higher K + -ion concentration in its sap (5397 ppm In general, only a weak linear correlation between biometric variables and ion content in sap was observed, independent of the presence of thiamine in the culture media. Positive and negative linear correlation coefficients ranged from 0.02 to 0.53 and − 0.02 to − 0.76, respectively. The highest negative correlation was observed between stem width and Na + concentration, i.e. sweetpotato in vitro plants with high Na + concentration in plant sap had a thinner stem in the basal and apical stem sections. Interestingly, a higher Na + content in plants grown on thiamine-supplemented medium had a weak positive effect on plant growth (R = 0.53), while this was not the case for thiamine-free medium (R = 0.17) [ Table 2].
Significant differences were observed for chlorophyll content, both on the accession (p = 0.01) and treatment level (p = 0.027), but not for the interaction of both (p = 0.779). Plants cultured on the routine control medium had a significantly higher average chlorophyll content (32.7 ppm) than those coming from uninodal segments grown on culture media with GA 3 and thiamine (28.8 ppm), while there were no significant differences in chlorophyll content observed for the other treatments. On the accession level, CIP 400989 had a significantly higher chlorophyll content (33.8 ppm) than CIP 420530 (29.4 ppm) and CIP 440144 (28.1 ppm). The accessions with the highest plant height (CIP 420701), and longest root length (CIP 420530), also had lower chlorophyll content (29.4-29.5 ppm) [ Table 3].
As no significant differences in moisture content (%) were observed between the five treatments, the genotypic values of moisture content (%) across all treatments is presented. CIP 400989 had a significantly higher moisture content (92.0%) than CIP 420952, CIP 421066, and CIP 440144 (90.7-91.0%). Accessions with the best root growth (CIP 420530 and CIP 400989) also had the highest moisture content (91.7-92.0%) [Fig. 6].
These same treatments were screened with a set of 45 diverse sweetpotato accessions (Table 1), to confirm the biometric results of the replicated experiment over a broad group of genotypes. Adding thiamine to the culture medium and the use of binodal stem segments, resulted in the highest values for 6 of the 8 biometric variables. Compared to the routine control treatment, the average plant height and root length increased by 41% and 52%, respectively. Also, the number of nodes formed with the thiamine treatments was higher (9.0-9.2) compared to the thiamine-free treatments (6.9-7.8). Stems were also thicker in the basal, mid, and top positions of the stem when thiamine was added to the culture media. Plants coming from uninodal segments, showed the highest values for NO3 − , K + and Na + concentrations (culture medium without thiamine) and Ca ++ (culture medium with thiamine). Plants grown on thiamine-supplemented culture medium showed higher moisture levels (90.7-90.9%), than plants grown on thiamine-free medium (89.7-89.9%). Plants grown on the routine control medium, showed the highest chlorophyll content (31.6 ppm). Although uninodal explants on thiamine supplemented medium generally had lower biometric values than the binodal segments, root growth was extraordinarily good (10.1 vs 9.6 cm) [ Table 4].
It is important to note that 36-39 of the screened 45 accessions (= 80-86%), showed better plant and root growth on culture medium supplemented with thiamine. The effect of thiamine-supplemented medium on in vitro sweetpotato plants was most pronounced in the stem diameter of the plants (particularly the basal position of the stem). 42 of 45 accessions had a thicker stem at its base, when they were grown on culture medium supplemented with thiamine. Although 33 of 45 accessions had higher chlorophyll content on thiamine-free culture medium, no correlation with biometric growth and quality parameters were observed. In 87% of the assessed accessions moisture content of the plants was higher when they were grown on thiamine-supplemented medium. Interestingly, 33-34 of 45 accessions showed a higher K + and Na + content in its plant sap on thiamine-free medium (Table 5).

Discussion
From a technical, practical, and economical standpoint it is difficult and impractical to develop optimized protocols (the best individual combination of plant hormones, nutrients concentrations, and environment conditions) for individual vegetatively propagated accessions in large plant genetic resources collections such as those in genebanks. However, it is important to find factors that show significant differences for the treatments but also show low interaction of treatment x genotype and therefore have broad application in such collections. In vitro plants were grown from binodal (bi) or uninodal (uni) stem segments on modified MS culture medium supplemented with gibberellic acid (10 mg L −1 ) [GA 3 ] and thiamine (0.1 mg L −1 ) [Thia] after 47-51 days in culture. GA 3 was added filter sterilized after autoclaving for all treatments, except the routine control where binodal explants were placed on medium containing GA 3 (10 mg L −1 ) which was added prior to autoclaving. Data was transformed using the ln(x) and Johnson functions prior to data analysis however graphs show the original values. Different lower-case letters indicate significant differences for the Tukey test for the means (p < 0.05). In contrast to other crops (olive, potato, Excoecaria sp.), in the present study sweetpotato in vitro plants coming from uninodal stem segments did show similar growth and developmental patterns than those coming from binodal stem segments when the culture medium was supplemented with thiamine. Using uninodal stem segments can potentially double the number of plants obtained, compared to binodal stem segments. This can be of particular importance when a high number of plants must be propagated within a limited period of time.
Compared to the report of Vettorazzi et al. (2017) in an evaluation of 30 Brazilian sweetpotato landraces, growth of sweetpotato clones in the present study was faster. Although Vettorazzi et al. (2017) incubated the in vitro clones at 27 ± 2 °C, i.e., at a temperature of about 4 °C higher than in the present study, the average plant height was close to after 47-51 days in culture. GA 3 was added as filter sterilized stock solution after autoclaving for all treatments, except the routine control where binodal explants were placed on medium containing GA 3 (10 mg L −1 ) which was added prior to autoclaving. Different lowercase letters indicate significant differences for the Tukey test for the means (p < 0.05). 3.0 cm after 60 days, which is lower than the values obtained in the present study for the 45 screened accessions (4.2 cm), however it was similar to the routine control treatment (3.0 cm). As the Vettorazzi et al. (2017) study confirmed a significant increase in plant growth at higher culture temperatures, it is probable that increasing the temperature from 24 to 27-28 °C could further accelerate growth and development of the sweetpotato clones used in the present study. The positive effect of specific concentrations of phytohormones on growth and rooting of in vitro sweetpotato clones, was confirmed in diverse studies. Nevertheless, these works assessed a limited number of genotypes (one to four), frequently varieties of local importance. Beyene et al. (2020) reported the highest shoot length (7.8 cm) for variety 'Kulfo' when MS medium was supplmented with 1.0 mg L −1 of benzylaminopurine (BAP), and the highest node number (8.2) at a concentration of 2.0 mg L −1 of BAP. Compared to the control treatment (0 mg L −1 BAP), Beyene et al. (2020) observed an increase in shoot growth of 53%, which is slightly lower than the 72% increase observed in the present study (seven genotypes; 3 repetitions; Fig. 3) but higher than the relative increase in shoot growth (42%) of our screening experiment (45 genotypes; 1 repetition; Table 3). In contrast, Fadaladeen et al. (2022) observed the best growth for variety 'Mabrokat Al-Shimal' after four weeks (3.55 cm) and the highest node number (8.77) on BAP-free medium, compared to BAP concentrations of 0.5, 1.0, 1.5, and 2.0 mg L −1 (plant lengths of 1.88 to 3.50 cm; node numbers of 3.66 to 6.88). Nevertheless, for a second variety ('Mangawy'), Fadaladeen et al. (2022) obtained a significantly higher shoot length (2.88 cm) and node number (5.22) with 2.0 mg L −1 BAP. These results clearly show a strong genotype-specific response of in vitro sweetpotato clones to BAP. When working with a specific or small number of genotypes, the use of phytohormones can be a good alternative, nevertheless Table 3 Chlorophyll content (ppm) of seven in vitro sweetpotato accessions classified by treatment (thiamine and explant type) and genotype The chlorophyll content (ppm) was expressed as the average value of five leaf measurements per plant. Plants were grown on modified MS culture medium supplemented with gibberellic acid (10 mg L −1 ) [GA 3 ] and thiamine (0.1 mg L −1 ) [Thia]. As explants, binodal and uninodal stem segments were assessed. GA 3 was added as filter sterilized stock solution after autoclaving in all treatments, except for the routine control treatment, where binodal explants were cultured on medium where GA 3 was added before autoclaving. Different lowercase letters indicate significant differences for the Tukey test for the means (p < 0.05). SE: Standard Error    in a genebank conserving thousands of diverse landraces and varieties methodologies with less genotype-specific responses have to be used (e.g. use of thiamine). Using stem width as a biometric variable at three stem positions contributed to an assessment of plant quality, as excessive plant growth with thin stems is not necessarily desirable. It is noteworthy that stem diameters were more robust at the base, mid and top positions when thiamine was added to the culture medium (compared to the routine control treatment). However, considering that plants grown on thiamine-supplemented culture media, also had higher moisture content and root growth, the increase of stem thickness may be a direct result of improved water absorption. It is also interesting to note that hyper-hydration (vitrified plants) was not observed in any of the treatments.
In the present study, chlorophyll content did not appear to be a good indicator for plant quality and growth. Although plants from the routine control treatment showed the highest chlorophyll content in its leaves, virtually all other parameters or metrics were higher in the thiamine treatments. The stimulation of root formation may be a compensation mechanism for plants against limited root growth and water absorption.
It would be interesting to perform basic studies on a potential relationship between thiamine and K + and Na + content in sweetpotato in vitro plants, as 33-34 of 45 screened accessions had lower ion-concentrations on thiamine-supplemented medium. These two cations play an important role in cell membranes (K-Na pump), which in turn is energy-driven through the Krebs cycle with the involvement of coenzymes such as thiamine.
Finally, caution is recommended in generalizing results obtained with a limited number of accessions on the whole species or subspecies, without screening the observed effect with a genetically diverse set of clones.
As an output of the present study, an improved protocol for in vitro propagation of sweetpotato clones was developed and validated with a total number of 52 sweetpotato genotypes. Supplementing modified MS culture medium with thiamine (0.1 mg L −1 ) and using binodal segments as explants for in vitro propagation of sweetpotato clones, resulted in a significant improvement of plant growth and rooting. Table 4 Average values of biometric variables from 45 diverse in vitro sweetpotato accessions as a factor of their response to thiamine and explant type Table 5 Classification of biometric variables of 45 diverse in vitro sweetpotato accessions as a factor of their response to supplemental thiamine (0.1 mg L −1 ) The accessions were classified for plant height, root length, root and node number, stem diameter (basal, mid, and top diameters), leaf area, ion concentration, moisture content and chlorophyll content, in response to thiamine-supplemented (+Thia) versus thiamine-free culture medium (−Thia)