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Correlation and Path Coefficient Analysis of Yield and Yield Components of Quality Protein Maize (Zea mays L.) Hybrids at Jimma, Western Ethiopia

Maize is one of the most important staple food crops in many parts of Ethiopia. However, it is not used extensively due to its poor nutritional quality and low productivity. It lacks two essential amino acids, namely, lysine and tryptophan. Knowledge of the interrelationships of grain yield and its various causal (contributory) components is very helpful to improve the efficiency of breeding programs using appropriate selection indices. )is article reports the findings of a study conducted to determine the nature of relationships of grain yield and its contributing components and to identify those components with significant effects on yield with the intention of using them as selection criteria using path coefficient analysis (PCA). )erefore, PCA has shown that yield per hectare had a significant and positive phenotypic correlation with plant height, ear height, number of kernels per row, and 100-grain weight. Moreover, PCA had a significant and positive genotypic correlation with days to 50% tasseling, plant height, ear height, and 100-grain weight.)e highest direct positive effect on yield per hectare was exhibited by ear height.)e findings of this study showed that most genotypes are early maturing and are suitable for areas with short rainy seasons and prone to drought.

Introduction
Zea mays L. (Poaceae) is an important annual food crop of the world. It is the source of primary staple food as well as protein and calorie for millions of people in the world. Maize accounts for about 15 to 56% of the total daily calories in diets of people in several developing countries in Africa and Latin America, where animal protein is scarce and expensive [1]. It is produced for food among low-income families in Ethiopia and served in different dishes.
ough several hundred million people depend on maize, its common (normal) variety lacks two essential amino acids, namely, lysine and tryptophan, which are required in the biosynthesis of proteins [2]. erefore, the discovery of the recessive allele of the opaque-2 maize gene was a significant breakthrough in the alleviation of global protein deficiency. e high level of lysine and tryptophan amino acids in the maize endosperm protein is due to the presence of the recessive allele of the opaque-2 gene in the genome of mutant maize [3]. is has created tremendous interest and enthusiasm in the scientific community for its potential in developing maize with superior protein quality. However, the gene was found to be closely associated with several undesirable traits. e opaque-2 maize kernels were dull and chalky, had 15 to 20% less grain weight, and were more susceptible to several diseases and insects [4], which led to the loss of interest among scientists to work on it. After several trials and systematic studies, breeders succeeded in finding modifier genes that produce the desirable hard endosperm phenotype in materials containing the recessive opaque-2 mutation. ese agronomically acceptable and nutritionally enhanced materials later came to be known as quality protein maize (QPM) [2,[5][6][7]. QPM contains nearly twice as much usable protein as other maize varieties grown in the tropics and yields 10% more grain than traditional maize varieties [2]. e principal goal of maize breeding programs is to develop new inbred and hybrid varieties that outperform the existing varieties with respect to many traits. In this pursuit, special attention is given to grain yield as the most important agronomic characteristic. Grain yield is a complex quantitative trait affected by a number of factors.
us, the knowledge of interrelationships between grain yield and its contributing components improves the efficiency of breeding programs through the use of appropriate selection indices [8,9]. Path coefficient analysis has been widely used in crop breeding programs to determine the nature of relationships between grain yield and its contributing components and to identify the components with significant effects on yield to be used as selection criteria. Path analysis shows the direct and indirect effects of cause variables on effect variables [10][11][12]. According to this method, the correlation coefficient between two traits is separated into the components that measure the direct and indirect effects.
is article reports the findings of a study that aimed at looking into the phenotypic and genotypic correlations between grain yield and other morphological traits and evaluating the direct and indirect effects of morphological traits on grain yield.

Description of the Study Area.
e study was conducted between July and October 2009 at the Eladalle research station of the Jimma University College of Agriculture and Veterinary Medicine (alt.: 1722 m; lat.: 7°33″0′N; and long.: 36°57″0′E). e mean maximum and minimum annual temperatures of the Eladalle research station are 26.8°C and 11.4°C, respectively. Likewise, the mean maximum and the minimum annual relative humidity (RH) of the station are 91.4% and 39.92%, respectively. It also has a mean annual rainfall of 951.5 mm. Its soil is reddish-brown clay soil with pH ranging from 5.07 to 6.0 [13].

Experimental Materials.
e quality protein maize (QPM) hybrids used in this study were acquired from CIMMYT. e hybrids include 43 three-way hybrids and two checks (one commercial and another local). Details of materials are shown in Table 1.

Experimental Designs and Procedure.
e materials were sown in the alpha lattice (5 × 9) with 5 plots per block with two replications in a 5 meters single row plot with the spacing of 0.75 meters between rows and 0.30 meters between plants. It may be argued that the number of replications is small. However, the efficiency of alpha-lattice design increases the precision of the experiment. All agronomic practices including land preparation, weeding, and fertilization were applied to all plots as per the standard practices for maize.

Data Sources and Analyses.
Data for days to 50% tasseling, plant count, and grain yield of the hybrids were collected based on the whole plot. Likewise, data for plant height, ear height, number of kernel-rows per ear, and number of kernels per row were taken based on five randomly selected plants. Finally, 100-kernel weight was taken from composite seeds of all the plants from the plots after removing the plants at the ends of the rows. eir mean performances are given in Table 2. Phenotypic and genotypic correlations and path coefficient analysis were analyzed using GENRES Version 7.01. Phenotypic correlation (the observable correlation between two variables that include genotypic and environmental components) and genotypic correlation were computed using GENRES using the method described in Singh and Chaudhry [14] as where r p and r g are phenotypic and genotypic correlation coefficients, respectively; p cov x·y and gcov x · y are phenotypic and genotypic covariances between variables x and y, respectively; δ 2 px and δ 2 gx are phenotypic and genotypic variances, respectively, for variable x; and δ 2 py and δ 2 gy are phenotypic and genotypic variances, respectively, for variable y.

Path Coefficient Analysis.
Path coefficient analysis was computed as suggested by Dewey and Lu [15] by using genotypic correlation coefficients as where r ij denotes the mutual association between the independent character i (yield-related trait) and dependent character j (grain yield) as measured by the genotypic correlation coefficients; P ij refers to the components of direct effects of the independent character i on the dependent character j as measured by the path coefficients; and r ik P kj refers to the summation of components of indirect effects of a given independent character i on a given dependent character j via all other independent characters k. e contribution of the remaining unknown characters is measured as the residual as given by

Phenotypic and Genotypic Correlations.
Phenotypic and genotypic correlations among the traits considered in the study are presented in Table 3. In the following, the implications of the data generated by the analyses are provided. Table 3 below the diagonal line shows that grain yield per hectare gave a positive phenotypic correlation with all traits except days to 50% tasseling and plant count. Grain yield has the highest statistically significant correlations with plant height (rp � 0.697; p ≤ 0.01) followed by number of kernels per row (rp � 0.626; p ≤ 0.01), ear height (rp � 0.440; p ≤ 0.05), and 100-grain weight (rp � 0.436; p ≤ 0.05). Some researchers reported similar observations, e.g., [11,[16][17][18][19][20][21][22]. e researchers observed that plant height, ear aspect, ear height, ear length, grains per row, and 100-grain weight or grain yield were positively and significantly inter-correlated implying that hybrids with these traits possess high yield potential. e highest positive phenotypic correlation was observed between plant height and ear height (rp � 0.788; p ≤ 0.01). Similar observation was reported by several researchers [19][20][21][22][23]. ese observations indicate that improvements in each of the traits would lead to overall improvements of the genotypes. Such correlations help in making reasonable decisions in selecting traits controlled by multiple genes. Grain yield, as a quantitative trait, is polygenically controlled [24]. ese findings imply that effective yield improvement depends on simultaneous improvements in all yield components. In fact, selection efforts based on grain yield alone are often less effective and efficient [9]. Selections need to be made based on various traits of the crop at hand.

Genotypic Correlation.
Observation of genotypic correlation coefficients shows that all traits examined in our study have a positive correlation with yield per hectare except plant count and number of kernel-rows per ear ( Table 3). Traits that showed high genotypic correlations with grain yield per hectare are plant height (rg � 0.873; p ≤ 0.01), ear height (rg � 0.698; p ≤ 0.01), days to 50% tasseling (rg � 0.585; p ≤ 0.01), and 100-grain weight (rg � 0.506; p ≤ 0.01). Similar findings were reported elsewhere [9,24,25]. On the contrary, significant and negative genotypic correlation was observed between yield per hectare and number of kernel-rows per ear (rg � 0.744; p ≤ 0.01). is finding is contrary to the findings of some researchers [26,27]. e fact that the higher number of kernel-rows per ear does not correlate with yield may imply that the kernels are smaller and lighter. It is helpful to note that the number of rows per ear and the number of grains per row of the local check were not studied.
In general, genotypic correlations among traits affecting grain yield explain true association as they exclude the environmental influences. It can be suggested that improvements in grain yield of maize can be accomplished through selections based on these correlations. Hence, knowledge of associations between yield and its component traits as well as among the component traits themselves can promote the efficiency of selection in maize breeding programs. In fact, it is well established that correlation studies between yield and yield components are pre-requisite in planning effective breeding programs. e same is true with maize breeders [27]. Quantitative traits like grain yield express themselves in close association with many other traits. Change in the expression of one trait is usually associated with changes in the expression of many other traits. erefore, the correlations obtained in the present study are useful in the selection of traits having direct and significant correlation in improving grain yield.

Path Coefficient Analyses.
Results of path coefficient analysis of all other traits to grain yield per hectare are given in Table 4 and Figure 1.
e results of path coefficient analysis revealed that all the characters studied except plant height, plant count, and number of kernel-rows per ear had positive direct effects on grain yield. e highest direct positive effect on yield per hectare was exhibited by ear height (0.6514). is implies that higher ear height leads to increased grain yield; the genotypic correlation between ear height and grain yield (rg � 0.698; p ≤ 0.01) is predominately attributed to the direct effect (rg � 0.651; p ≤ 0.01) of ear height on the grain yield per hectare (Figure 1). Many research findings were in line with this finding [12,28,29]. Similarly, it is also in agreement with the findings of Asrarur-Rehman et al. [23] and Bello et al. [24]. ese researchers reported positive and significant direct effects of ear length and thousand-kernel weight on grain yield. However, the finding of the present study contradicts with the findings of Rafiq et al. [25].
Days to 50% tasseling has yielded the next highest and direct effect on grain yield (0.245). It is stated above that the genotypic correlation between the traits is positive and statistically significant (rg � 0.585; p ≤ 0.01). e correlation explains the true relationship between the two traits; thus,   selection based on days to 50% tasseling would be effective.
is is in contrast with the findings of Arsode et al. [28] who reported significant negative association of days to 50% tasseling with grain yield per plant and plant height, ear length, ear girth, number of kernel-rows per ear, and number of kernels per row.
Plant count showed negative direct effect on grain yield (−0.041) like its genotypic correlation with grain yield (−0.242), implying that there is true association of the two traits. However, plant count yielded high positive indirect effect on grain yield through ear height (0.570). Likewise, number of kernel-rows per ear and number of kernels per row showed higher positive indirect effect on grain yield through plant height.
ese results are in line with the reports of different authors [10][11][12]30]. e negative and significant genotypic correlation between number of kernel-rows per ear and grain yield (rg � −0.744; p ≤ 0.01) explains the true relationship of the traits. Plant height resulted in high and negative direct effect on grain yield (−0.424). Other researchers have reported similar findings [25,32]. However, we observed highest and significant genotypic correlation between plant height and grain yield (rg � 0.873; p ≤ 0.01).
is may be due to the indirect effect of plant height on ear height. us, we recommend that selection based on plant height is made cautiously. We also observed that 100-grain weight had positive and significant direct effect on the grain yield as indicated in Table 4 (0.338; rg � 0.506; p ≤ 0.01). Other studies reported similar results [33,34]. e correlations and intercorrelations show that the seven causal traits (i.e., the causal variables) explain much of the variability in grain yield. In fact, a residual effect of 0.204 (Figure 1) implies that the causal traits explained about 79.6% of the variability in the grain yield, leaving 20.4% of the variability unexplained.

Concluding Remarks
Genotypic correlation coefficients showed that all the traits considered in our study have positive correlation with yield per hectare except plant count and number of kernel-rows per ear. Plant height, ear height, days to 50% tasseling, and 100-grain weight showed high genotypic correlations with grain yield per hectare. Genotypic correlations among traits affecting grain yield explain the true association as they exclude any environmental influences. Hence, it can be concluded that plant height, ear height, days to 50% tasseling, and 100-grain weight are the best traits for selection to improve grain yield per hectare of the maize genotypes tested in our study.

Data Availability
Data used in preparing this manuscript is available from the corresponding author on reasonable request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.


extfieldsofstudy: Medicine Biology

provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2020.jsonl.gz.gz

text:
A simple method to establish an efficient medium suitable for potato regeneration

Potato ( Solanum tuberosum ) is one of the important crop plants, and many potato cultivars consist of a tetraploid genome with high heterozygosity. The techniques of transformation and genome editing require plant regeneration. However, no efficient regeneration method has been established except for some specific cultivars, such as ‘Sayaka’. In general, it is necessary to determine the adequate concentrations of auxin and cytokinin for plant regeneration. We established an efficient method using a 24-well microplate that easily enabled determination of the concentrations of these plant growth regulators suitable for shoot regeneration. Using this method, the optimal concentrations of these factors were analyzed for two representative potato cultivars, ‘Sayaka’ and ‘Konafubuki’. This analysis revealed there was a large difference in the optimal concentrations between them. Based on this result, a specialized medium for the efficient regeneration of ‘Konafubuki’ cultivars was proposed. This assay method was also applied for determination of hygromycin sensitivity of these potato cultivars, and it was observed that ‘Konafubuki’ was rather sensitive to hygromycin. These findings suggested that the selection of a ‘Konafubuki’ transformant could be achieved using a medium containing a lower amount of hygromycin than that used for ‘Sayaka’. 30-day culture, we analyzed alteration of potato tissue morphology, and evaluated shoot regeneration.

Introduction
Potato is one of the important crop plants containing nutrition-rich tubers, and is widely used for potato chips, French fries and other processed products. In addition, potato starch is utilized for additive materials in paper, adhesive agents, and some food products (Kraak 1993). There are hundreds of potato varieties worldwide. Among them, the major species cultivated is Solanum tuberosum, which has a tetraploid genome and commonly shows a vegetative reproduction. Obtaining a new potato cultivar by the traditional crossbreeding method is difficult, and many potato cultivars have high heterozygosity (Muthoni et al. 2015).
Transformation techniques have been developed for potato, enabling the creation of genetically modified potato plants, in which various genetic materials have been introduced with a stable integration into the genome (Barrell et al. 2013). This process also enabled the beginning of a new era for new breeding techniques, such as genome editing of potato plants. Till now, targeted mutagenesis has been achieved using TALEN and CRISPR/Cas9 (Kusano et al. 2018;Sawai et al. 2014;Tuncel et al. 2019). Skoog and Miller (1957) reported that an adequate amount of two plant growth regulators, auxin and cytokinin, strongly affect the promotion of differentiation and dedifferentiation in plant explants. In Arabidopsis, an auxin-rich medium promotes callus formation, and roots and shoots are efficiently regenerated from the callus cultured in a high-auxin or high-cytokinin medium (Valvekens et al. 1988). However, no efficient potato transformation procedure has been established except for some cultivars, such as 'Sayaka' and 'Sassy' (Yamamizo et al. 2006;Yasumoto et al. 2019).
It has been shown that there are great differences in the efficiency of regeneration from cultured cells depending on the potato cultivars. This largely influences the efficiency of obtaining regenerated potato (Ishige et al. 1991). Currently, potato breeding technologies including new breeding techniques can be applied to only a few potato cultivars. In this study, to obtain potato plants possessing a desired trait, we established a general method that may allow the introduction of an appropriate gene with efficient regeneration techniques. Here, we describe a novel method for the determination of optimal concentrations of plant growth regulators for regeneration. We also demonstrate the effect of

Plant material and tissue culture condition
Potato cultivars, 'Sayaka' , 'Konafubuki' , 'Sakurafubuki' and 'Norin-1' , were used in this study. They were kindly provided by Hokkaido Agricultural Research Center, National Agriculture and Food Research Organization (NARO), Japan. In vitrogrown potato plantlets were grown in long-day conditions with 16 h light and 8 h dark at 23°C in a growth chamber. For plant subculture, we used the medium containing MS basal salt mixture (Fuji Film Wako, Tokyo, Japan) (Murashige and Skoog 1962), 3% sucrose, and 0.3% Gelrite (Fuji Film Wako), with pH adjusted to 6.0. For the plant regeneration, 3C5ZR medium (pH 5.8), which contained MS basal salt mixture, 3% sucrose, 0.01% myo-inositol and 0.3% Gelrite, with 1.5 mg l −1 thiamine-HCl, 0.75 mg l −1 nicotinic acid, 0.75 mg l −1 pyridoxine-HCl, and 0.6 mg l −1 aspartic acid, was used as a basal medium. Plant growth regulators and antibiotics were supplemented to this as required.

Assay for shoot regeneration efficiency
Stems of in vitro-grown potato plantlets were cut into approximately 5-8 mm segments using microscissors (MB-50-15, Natsume Seisakusho, Tokyo, Japan). Two pieces of stem segments were placed onto the 2 ml 3C5ZR medium containing a series of concentrations of 3-Indoleacetic Acid (IAA) (Fuji Film Wako) and trans-zeatin riboside (tZR) (Fuji Film Wako), which was set in a well of a 24-well culture plate (nontreated microplate with lid). Potato tissues were grown at 23°C in longday condition with 16 h light and 8 h dark in a growth chamber. After 30-day culture, regenerated shoots were detected. When shoot regeneration was observed in one of the two pieces of potato stem segments in a well, it was noted as the occurrence of regeneration.

Regeneration of four potato cultivars on the improved medium
Stem segments of potato cultivars, 'Sayaka' , 'Konafubuki' , 'Sakurafubuki' , and 'Norin-1' were placed on Medium-I and Medium-II, which were placed in Petri dishes, and they were cultured for 15 days. They were transplanted onto the same media to replenish the components in the medium and cultured for 15 more days. Medium-I consisted of 3C5ZR supplemented with 0.53 mg l −1 IAA and 1.75 mg l −1 tZR, which was conventionally used for potato transformation (Kusano et al. 2016). Medium-II contained 3C5ZR medium supplemented with 0.27 mg l −1 IAA and 2.63 mg l −1 tZR. After 30-day culture, the number of potato tissues showing shoot regeneration was counted. This experiment was performed more than four times each. The regeneration ratio was calculated for each experiment. The statistical significance was determined using a proportion test between regeneration frequencies in the experiments on Medium-I and Medium-II using R software (https://www.r-project.org).

Hygromycin sensitivity test for 'Sayaka' and 'Konafubuki'
Five pieces of stem segments of 'Sayaka' and 'Konafubuki' were placed in each well of a 6-well plate (untreated microplate with lid) containing 8 ml 3C5ZR medium with a series of concentrations of Hygromycin B (Fuji Film Wako) ( Figure  5A). For the cell culture of 'Sayaka' , the medium containing 0.53 mg l −1 and 1.75 mg l −1 of IAA and tZR, respectively, was used. For the 'Konafubuki' , the medium containing 0.27 mg l −1 and 1.75 mg l −1 of IAA and tZR, respectively, was used. After a 30-day culture, we analyzed alteration of potato tissue morphology, and evaluated shoot regeneration.

Determination of ranges of auxin and cytokinin concentrations for shoot regeneration
Regeneration conditions of potato plants vary depending on the cultivar. To establish a general procedure for potato regeneration, it is necessary to use a culture medium containing the optimal concentrations of plant growth regulators. 'Sayaka' is known as a potato cultivar that is efficiently regenerated. Using this cultivar, we examined the effect of various concentrations of auxin and cytokinin in the medium, and found that 'Sayaka' was regenerated on the medium containing concentrations of 0 to 1.06 mg l −1 of auxin (as IAA) and concentrations of 0.44 to 14.0 mg l −1 of cytokinin (as tZR). Based on these findings, we developed an assay system to determine the optimal concentration of each plant growth regulator.
Sections of potato stems of cultivars 'Sayaka' and 'Konafubuki' were placed onto the medium containing various concentrations of plant growth regulators, cultured for 30 days, and regenerated shoots from the cultured cells were observed. To save space and amount of culture medium, we used 24-well culture plates, each of whose wells contained different concentrations of IAA and tZR (Figure 1).
Shoot regeneration was observed in some cultured cells for both 'Sayaka' and 'Konafubuki' . The callus was formed along the cut surface, stem segments were expanded in most wells, and shoot was generated from a part of cultured cells in 30 days after ( Figure  1D, E). Regeneration events were observed in many wells with 'Sayaka' cells, while 'Konafubuki' showed low regeneration frequency (Figure 2). These results indicated that regeneration efficiency was greatly different between these cultivars.
The regeneration efficiency was investigated for 'Sayaka' and 'Konafubuki' using the abovementioned system. Two pieces of the stem segments of these Copyright © 2020 The Japanese Society for Plant Cell and Molecular Biology cultivars were cultured on media containing various concentrations of IAA and tZR, and shoot formation was evaluated. When shoot regeneration was observed in one of the two cultured stem segments in a well, we considered that the regeneration event had occurred on the corresponding culture medium. The number of regeneration events was compared among the different culture media. In the case of 'Sayaka' , it was found that the optimal plant growth regulator concentration for regeneration was broadly distributed within 0 to 1.06 mg l −1 of IAA, and 0.88 to 7.0 mg l −1 of tZR. In contrast, in the case of 'Konafubuki' , this was estimated to lie in the narrow range of 0.13 to 0.8 mg l −1 of IAA, and 1.75 to 7.0 mg l −1 of tZR (Figure 3).

Regeneration ratio for the two media with different plant growth regulator concentrations with four potato cultivars
Based on the results of the estimation for optimal plant growth regulator concentrations, we attempted to improve the culture medium for potato regeneration. In addition to the conventionally used medium, Medium-I, which contained 0.53 mg l −1 IAA and 1.75 mg l −1 tZR (Kusano et al. 2016), we created another medium, Medium-II containing 0.27 mg l −1 IAA and 2.63 mg l −1 tZR. Using these media, regeneration efficiency was determined for the four representative potato cultivars, 'Sayaka' , 'Konafubuki' , 'Sakurafubuki' , and 'Norin-1' .
As shown in Figure 4, the regeneration frequency was evidently different between the stem segments cultured on Medium-I and Medium-II; however, 'Sayaka' showed a similar efficiency of regeneration in both media. The ratio of occurrence of regeneration in both Medium-I and Medium-II was approximately 70% for the 'Sayaka' cultured cells. In contrast, 'Konafubuki' and 'Sakurafubuki' showed higher values of regeneration efficiency when they were cultured on Medium-II (66.3% and 15.5%, respectively) than on Medium-I (46.7% and 5.2%, respectively). For 'Norin-1' , the regeneration efficiency was rather low in both media (3.3%) ( Table  1). These results suggest that Medium-II containing the optimal concentrations of plant growth regulators is highly effective for increasing the efficiency of potato regeneration in case of 'Konafubuki' .

Assay for hygromycin sensibility of potato cultivars
Using this assay system, the hygromycin sensitivity of 'Sayaka' and 'Konafubuki' was analyzed. As shown in Figure 5, 'Konafubuki' showed higher sensitivity to hygromycin than 'Sayaka'. 'Konafubuki' failed to regenerate on the medium containing 1 mg l −1 hygromycin, whereas 'Sayaka' cells increased normally on the medium containing 3 mg l −1 hygromycin. 'Sayaka' survived on the medium containing 10 mg l −1 hygromycin, and sometimes regeneration of the shoot was observed. This suggests that the hygromycin sensibility was rather different depending on the cultivar.

Discussion
For creating a genetically modified plant as well as a genome-edited plant, a technique for plant regeneration is required (Loyola-Vargas and Vázquez-Flota 2006). In the case of potato, it is difficult to obtain a regenerated plant because the conditions of regeneration are greatly different depending on the cultivar. In this study, we developed a new assay system to determine the optimal plant growth regulator concentrations for regeneration from cultured potato cells. Using this assay system, we determined the optimal concentrations of two plant growth regulators, auxin and cytokinin, which are important for the regeneration of potato plants. This assay indicated that the optimal concentrations of these plant growth regulators were different depending on the potato cultivar (Figures 2 and 3).
'Sayaka' is commonly used as a cultivar that has a high regeneration ability (Teo et al. 2017;Yamamizo et al. 2006). However, there has been no report on the regeneration of 'Konafubuki' , which produces a large amount of starch, and has potential as a raw material for industrial starch. If the starch quality is improved by genome editing, new applications of potato starch can be developed and would contribute to increasing its industrial value, resulting in a large ripple (economy) effect. Our assay system indicated that 'Konafubuki' was also showed regeneration but the optimal concentration ranges of plant growth regulators for 'Konafubuki' were narrow as compared with those of 'Sayaka' (Figure 3). Using the improved medium, Medium-II, regenerated 'Konafubuki' plants were obtained with high efficiency. However, Medium-II did not lead to a large improvement in the regeneration frequency for 'Sakurafubuki' and 'Norin-1' , although the frequency was slightly higher for 'Sakurafubuki' (Table 1). These cultivars could be regenerated on the medium containing the optimal concentrations of plant growth regulators other than Medium-I and Medium-II. In this experiment, we selected three potato cultivars whose genetic background was different from that of 'Sayaka' . They might have the genes involved in regeneration with a large divergence, and lead to differences in the regeneration frequency.
Hygromycin is commonly used as a selection marker for transgenic potato plants. However, the concentration  . The occurrence of regeneration was determined using the assay method as shown in the previous figures. When the regenerated shoots were observed on one of the two tissues, it was counted as 'regenerated' . This assay was performed four times each, and numbers of 'regenerated' in total are indicated as the frequency of regeneration. In each box, frequency is shown as the total number as 'regenerated' events, with color gradation.
Copyright © 2020 The Japanese Society for Plant Cell and Molecular Biology of hygromycin is critical because cellular growth inhibition often occurs in presence of excess hygromycin. We examined the hygromycin sensitivity of 'Sayaka' and 'Konafubuki' . The findings indicated 'Konafubuki' was rather sensitive to hygromycin. In contrast, 'Sayaka' grew normally on the medium containing 3 mg l −1 hygromycin and survived on the medium containing 10 mg l −1 ( Figure 5). Usually, we used the medium containing 5 mg l −1 hygromycin for selection of potato transformants of 'Sayaka' (Kusano et al. 2016). This result suggests that our selection medium contains the amount of hygromycin that is effective in reducing the growth of potato cells without killing them. These findings indicate that the selection of a 'Konafubuki' transformant can be achieved using a medium containing a lower amount of hygromycin than that used for 'Sayaka' . In addition, wild type 'Sayaka' survived in the 5 mg l −1 hygromycin condition, and a small number (1 in 5) of plants showed regeneration. We consider these plants were the plants   The sections of potato stems were placed in dishes with Medium-I and Medium-II. Plants were cultured for 30 days, and regenerated shoots were counted. The values indicate the total number of sections of potato stems that regenerated shoots in the stems examined. Values in parentheses show the regeneration ratio. Asterisks indicate the statistical significance between regeneration frequencies in the experiments on Medium-I and Medium-II, which was determined using a proportion test (p<0.1). escaping without hygromycin resistance.
Our method may lead to the establishment of culture conditions for various potato cultivars. The favorable culture condition can be easily obtained for each cultivar and applied for genome editing, which would greatly contribute to crop development by improving an appropriate phenotype at a pinpoint.


text:
Effects of different sample processing protocols on dermatophyte culture yield: A comparative study

Introduction: Dermatophytes fungi infect keratinized tissues such as hair, nails and skin. Different methods for sample collection and transportation of dermatophytes for culture have been described. The standard methods for dermatophytes sample collection, transportation and lab processing, results in low positive culture dermatophyte yield. In this study, we have compared the standard method (Protocol A) with modified sample processing protocol (Protocol B), developed by our department. This modification was based on the core concept of collection, inoculation, starting incubation and transportation of dermatophyte sample, at patient’s point of care. Materials and Methods: A total of 200 clinical isolates of dermatophytes, were subjected to two different sample processing protocols. Standard method was Protocol A and laboratory developed method was labelled as Protocol B. Protocol B, included a small (7ml) transparent plastic tube with special culture media. These tubes were inoculated with sample at patient’s point of care and same tube were utilized as transportation media. Results: Samples processed as per modified protocol (Protocol B), demonstrated positive dermatophyte culture yield in 160 out of 200 samples, whereas figures were 80 out of 200 for samples processed as per standard protocol (Protocol A). Difference in positive dermatophyte culture yields, were statistically significant. The contamination rates of dermatophyte culture by saprophytic fungus and bacteria, was measured, 6% for Protocol A and 1% for Protocol B. Conclusion: Our study demonstrated that a simple, economical modification in the procedure for dermatophyte sample processing by the laboratory, has led to a significant increase the positive dermatophyte culture yield with reduced secondary contamination rates.

Introduction
Dermatophytes are the unique group of fungi that can invade the keratinized tissues. Keratinized tissue infected by dermatophytes includes skin, hair, and nails. 1 Dermatophytes exist in two phases during their life cycle, as most other fungus species do. These states are the anamorph state (imperfect or asexual phase) and the teleomorph state (perfect or sexual phase). State isolated in the laboratory is anamoph state. As teleomorphs state is difficult to detect and identify, it has yet not been described for many species of dermatophyte. Fungus species in Dermatophytes belongs to the genera Trichophyton, Microsporum and Epidermophyton. 2 Based on their natural habitats, Dermatophytes are broadly classified as geophilic, zoophilic and anthropophilic species. Among geophilic species, M. gypseum, a soil-inhabiting saprophytes can occasionally be pathogenic to humans. Animals are infected by zoophilic species and humans are accidental hosts. These group of dermatophytes has acquired the ability, from degrading keratinous debris in the soil to invading keratinized tissue of the animal host. Zoophilic species includes T. equinum, T. verrucosum, T. mentagrophytes and M. canis. Anthropophilic species like T. rubrum, have evolved from zoophilic fungi to infect human host. 3 Dermatophyte infections can involve different areas of the body with varied clinical manifestations. These clinical disease are named using word tinea followed, a Latin term for the specific body part such as Tinea corporis (non-hairy skin), Tinea capitis (scalp), Tinea cruris (groin), Tinea pedis (foot) and Tinea barbae (bearded areas). 4 Tinea pedis is characterized by infection of the interdigital web spaces and feet. These lesion presents as vesicles or pustules lesions. Common etiologic agents are E. floccosum, T. mentagrophytes and T. rubrum. 5 . Tinea capitis is dermatophyte species involving hair shaft either in ectothrix, or endothrix manner. Mainly M. canis and M. audouinii are known for ectothrix infection, whereas endothrix type involvement is by T. tonsurans. 5 Tinea corporis is caused by T. rubrum, T. mentagrophytes and M. canis. Tinea corporis presents as annular lesions with raised borders. 5 Tinea unguium is characterized by the nail involvement presenting as thickened, discolored nails with subungual debris. Common etiological agents are T. rubrum, T. mentagrophytes and E. floccosum. Superficial onychomycosis is mainly caused by T. mentagrophytes. 6 Due to warm humid condition, dermatophyte infection is a common clinical manifestation. It has high prevalence worldwide as the most common fungal pathogens infecting humans. 7 Majority of these infection can be treated with topical application of antifungal agents, however some of these infections especially onychomycoses requires long term treatment due to poor responsive to standard treatment. 8 Mycological examination for species level identification of dermatophytes, is required preferably in fresh cases and mandatory in unresponsive superficial mycosis cases. 9 This is also important in view of recent reports of Azole resistance in the dermatophytes isolates. 10 As per the standard procedure, superficial mycosis sample materials are transported in dry, hard black paper which is folded in a herbarium packet and transferred to the laboratory. 11 At the Mycology laboratory dermatophyte species level identification is performed by microscopic examination and culture characteristic. 11 For dermatophyte culture special media like Sabouraud dextrose agar (SDA), Sabouraud Dextrose Agar containing chloramphenicol and cycloheximide and Dermatophyte test medium may be used. 12 However, by these standard methods positive dermatophyte culture yield is low. 13 This low culture yield is further complicated by frequent secondary contamination by saprophytic fungus or bacteria during transportation. 14 In our department, we have prepared new protocol, with concept of collection, inoculation and incubation of sample at the point of care. In this study, we have compared the effects on dermatophyte culture yield based on two different protocols-Standard protocol (Protocol A) and point of care protocol (Protocol B).

Materials and Methods
In this study, 200 consecutive, non repeat samples from clinically diagnosed dermatophyte patients reporting to a tertiary care hospital were included. Each patient samples were processed as per two different protocols. These protocols were designated as Protocol A and Protocol B. Standard operating procedures of these protocols were issued, which are as follows:-Protocol A-Standard Protocol Collection of Sample: All clinically diagnosed lesion of dermatomycosis was cleaned with 70% alcohol before sample collection. For the skin lesions, sterile blunt scalpel was used and scales from lesion margins were collected. Infected basal portion of hairs were plucked with sterile forceps. For patients presenting with onychomycosis, nail clipping or scrapings were collected with a scalpel blade. All the above samples were placed in a dry black paper. Transport of Sample: Sample material was transported to mycology laboratory in the dry, hard black paper, which was folded in a herbarium packet. The samples were divided into two portions: one for microscopic examination and one for culture.

Laboratory Processing
Microscopy Examination: Samples were received in black paper for direct microscopic examination. Solution for wet mount preparation was prepared in two different strengths and kept in different clean bottles. These preparation were of 10% potassium hydroxide (KOH) was mixed equal quantity of 40% Dimethyle sulphoxide (DMSO) and 40% KOH with equal quantity 40% DMSO. Skin and hair specimen were processed, using 10% KOH mixture and for nail samples, 40% mixture was used. Before microscopic examination, 3-4 drops of the above mixture was placed on a clean glass slide. The representative clinical sample of dermatophyte were placed into the KOH+DMSO drops on the slide. A cover slip was placed carefully to avoid any air bubble formation. Potassium hydroxide (KOH) in presence of DMSO softens keratin present in tissue. After 10 minutes, each slides were examined both under low power (10X) and high power (40X) magnification for the presence of fungal elements including arthroconidia. Dermatophyte Culture: All clinical samples were streaked on the Sabouraud Dextrose Agar (SDA) slants with cycloheximide (50 mg/l) and chloramphenicol (500 mg/l). These culture slopes were placed in incubator and temperature of 30°C was maintained. These samples were incubated for the duration of 4 weeks. Identification of dermatophyte with positive growth was done utilizing both macroscopically and microscopically features. Macroscopic features included dermatophyte colony characteristics, whereas microscopically features included study of type of dermatophytes conidia, including its size, surface characteristics and septations.

Protocol B-Point of Care Protocol
Description of the Modified Culture and Transport Media: As per this protocol the core concept was to collect, inoculate and start incubation at the point of care, so a modified culture and transport media was prepared ( Fig. 1 and 2). The modified culture and transport media included a small, soft, transparent, plastic tube of 7 ml capacity with tightly fitting screw cap. These special tubes were filled with 3ml slant of Sabouraud Dextrose Agar with chloramphenicol (50mg/l) and cycloheximide (500mg/l). As these tubes had tightly fitting screw cap loss of oxygen and water vapor was minimal. Cycloheximide was added to inhibit saprophytic fungi and chloramphenicol was added to inhibit bacteria growth. Collection of Sample: As per protocol A Inoculation and Transport of Sample: Sample were immediately inoculation on to the medium and screw cap placed. As dermatophytes grow at room temperature, these tube were kept upright during transportation. Laboratory Processing: Sample received in special culture and transport media were not opened. They were placed in incubator directly for incubation at 30 0 C. The special culture tube slopes were examined regularly, for any evidence of fungal growth. Cultures were incubated for 1month before discarding them as negative. Identification of fungal growth was performed as per standard methods, which includes colony morphology; pigment production and microscopic examination. Null hypothesis stating that no significant difference between two protocol was rejected (P <0.05). Contaminations of cultures lope by saprophytic fungus and bacteria was noted in 12 tubes for samples processed by the protocol A. So the contamination rate for protocol A was 6%, whereas it was 1% for protocol B.

Discussion
Due to humid environment, dermatophytosis is most common superficial fungal infection in tropical and subtropical countries, like India. 15 Diagnostic Laboratory with facility for rapid and accurate identification of the dermatophytes are essential for definitive treatment and planning prevention measures. In our study, 200 consecutive, non-repeat clinically samples were collected and processed by two different protocols-protocol A nd B. These samples included clinical cases of T. corporis (67%), T. pedis (15%), T. cruris (13%), T. capitis (3%) and Tinea unguium (2%). This finding is in concordance with other studies conducted in India. 16 In our study, 21-30 years (76/200) age group patient were the most common, followed by patients from age group of 31-40 years (64/200). Similar findings have been reported by other Indian authors. 17 Diagnostic laboratories receives clinical samples from patients with dermatophytois, which generally includes skin, nails, and hair. These samples requires careful collection and transportations. Typically, a black paper placed in envelope is used for transportation. Many authors have described, different techniques for the sample collection such as scraping, cotton swabs, cellophane or vinyl strip for skin, 18 hair clipping or brush technique for hair 19 and clipping and microdrill for nail. 20,21 However, the patient samples are contaminated with saprophytic fungi and bacteria 22 at the time of collection and gets secondary contamination from environment during transportation and handling of sample at the diagnostic laboratory. 23 This high risk of secondary contamination, from the environment, during transportation and sample processing warrants use of special culture medium in the laboratory. Various studies have shown high contamination rates and low fungal culture yield, despite using special mediums like Sabouraud dextrose agar supplemented with chloramphenicol and cycloheximide. 14,24 Similar findings of high contamination rate (6%) and low dermatophyte culture yield (40%) was observed in our study, for the samples processed as per standard procedures (Protocol A).
In this study, new protocol (Protocol B) was designed with core concept of sample collection, inoculation, transportation and starting the incubation in the special fungal culture medium for dermatophytes, right at the point of care. This modification of the procedure, resulted in statistically significant increase in positive dermatophyte culture yield (80%). Our study also demonstrated the reduction in the secondary contamination rate of the dermatophyte fungal culture to 1% compared to 6% for the standard procedure. This observable reduction in contamination rate may be multifactorial, among which important ones are reduction in the primary and secondary contamination of the samples by saprophytic fungus and bacteria. Primary contamination was minimized, as sample were collected after cleaning infected area with 70% alcohol. Secondary contamination was reduced as sample were placed inside the screw cap tubes with culture media at the point of care. Further, secondary contamination was avoided due to screw capped dermatophyte culture tubes and by avoiding sample handling at the laboratory, until the growth of pathogenic dermatophyte.
In resource constrain laboratory, this modification may be beneficial as media dispensed per tube was merely 4 ml against 8ml to 15ml medium required for the standard procedure.
Our modification has resulted in higher rate of isolation of dermatophytes in our laboratory, which has translated into better patient management, improved surveillance, effective planning of preventive measures and further research, as dermatophyte antifungal susceptibility pattern of local population.

Conclusion
Our study shows that the simple economical modification in the procedure for dermatophyte sample processing standards, has significantly increased the positive culture yield with reduced secondary contamination rates. This modification of dermatophyte sample processing protocol has potential for positive ramification on patients health, over a long duration, as it has basis of scientific and evidence based management of patients presenting with dermatophyte infection.

Conflict of Interest:
None.


text:
Stem Lettuce and Its Metabolites: Does the Variety Make Any Difference?

The objective of the present study was to characterize chemical composition of hitherto unexamined aerial parts of Lactuca sativa var. angustana cv. Grüner Stern. In contrast to leafy and head varieties of the lettuces, asparagus lettuce grown in Europe is much less studied. Fractionation of a methanolic extract from leaves of L. sativa cv. Grüner Stern, supported with HPLC/DAD and 1H NMR analysis, led to the isolation and/or identification of numerous terpenoid and phenolic compounds, including five apocarotenoids—(-)-loliolide, (+)-dehydrovomifoliol, blumenol A, (6S,9S)-vomifoliol, and corchoionoside C; three sesquiterpene lactones; two lignans—((+)-syringaresinol and its 4-O-β-glucoside); five caffeic acid derivatives; and three flavonoids. Some of the compounds, to the best of our knowledge, have never been isolated from L. sativa before. Moreover, monolignols, phenolic acids and a tryptophan-derived alkaloid were found in the analyzed plant material. Stems, leaves and shoot tips of the asparagus lettuce were examined to assess their phenolics and sesquiterpene lactone content as well as DPPH scavenging activity. Another stem lettuce—L. sativa var. angustana cv. Karola, two cultivars of leafy lettuces and one species of wild lettuce—L. serriola, were also examined as a reference material using HPLC/DAD. The results have been discussed regarding our previous studies and the literature data available.

Introduction
Lettuce (Lactuca sativa L.), one of the most popular leafy vegetables, is present in the market in a wide variety of cultivars, which differ from one another in their taste, color, texture, pathogen resistance, and value as a functional food. This diversity is connected with an array of specialized metabolites produced by the plants. Rapid development of hyphenated analytical techniques brought about an increase in number of metabolomic studies devoted to crop plants, including popular vegetables like lettuce [1][2][3][4][5][6][7][8]. The studies have been chiefly focused on polyphenols, especially flavonoids and hydroxycinnamates, which are believed to carry some health benefits [9]. Another group of widely investigated specialized metabolites produced by the plant are terpenoids, including carotenoids, pentacyclic triterpenes, and sesquiterpene lactones. The last ones are responsible for the bitter taste of lettuce as well as, to some extent, for inhibition of insect feeding [10][11][12][13]. Pharmacological studies proved that lactucin-type guaianolides isolated from lettuce demonstrated anti-inflammatory and antinociceptive activity [14,15]. Metabolomic studies on the cultivated lettuce plants revealed also the occurrence of lignans [2][3][4]6]. The group of specialized plant metabolites seems to be of importance as an estrogenic component of human diet. However, their content in some lettuce cultivars is probably too low to exert any significant effect on the consumer's health [16].

General Experimental Procedures
Optical rotation was determined on a PolAAr31 polarimeter (Optical Activity Ltd., Ramsey, UK). NMR spectra were recorded either in CDCl 3 or in CD 3 OD on a Bruker AVANCE III HD 400 (resonance frequency-400. 17 MHz for 1 H) (Bruker Corp., Billerica, MA, USA). Analytical RP-HPLC separations were performed either at 25 • C, on a Zorbax Eclipse XDB-C18 column 4.6 × 150 mm (Agilent Technologies, Santa Clara, CA, USA) or at 40 • C on a on a Kinetex XB-C18 column (4.6 × 250 mm, 5 µm; Phenomenex, CA, USA) using an Agilent 1200 Series HPLC system (Agilent Technologies) equipped with a Rheodyne manual sample injector, quaternary pump, degasser, column oven, and a diode array detector. Semipreparative RP-HPLC was performed on a Vertex Plus column

Plant Material
Aerial parts of L. sativa L. var. angustana cv. Grüner Stern were collected three times. First, in July 2014, the raw material for isolation work was harvested (leaves, stalks, and shoot tops from the flowering plants; voucher No 03/2014). Next, in June and July 2020, the plant material for HPLC/DAD analyses (leaves from 8 and 15 weeks old plants; voucher No 02/2020) was collected concomitantly with the leaves of three other L. sativa cultivars and the leaves of L. serriola. Seeds of L. sativa L. var. angustana cv. Grüner Stern were obtained from the Botanical Garden of the Bonn University (Germany). Seeds of L. sativa var. angustana cv. Karola, L. sativa cv. Great Lakes, and L. sativa var. crispa cv. Amerikanischer Brauner were purchased from the commercial growers. Seeds of L. serriola L., collected from the wild, were delivered by the Botanical Gardens in Münster (Westfäliche Wilhelms-Universität, Münster, Germany; voucher No 05/2020) and Nantes (Ville de Nantes, France; voucher No 04/2020). All plants were grown in the Garden of Medicinal Plants, Maj Institute of Pharmacology, Polish Academy of Sciences, Kraków, Poland, where the voucher specimens were deposited. Data on cultivation conditions (type of soil, average annual temperature, annual rainfall, and agrotechnical procedures applied) are described elsewhere [23]. The collected plant material was dried at room temperature under shade. The dried plant material (378 g) was powdered and exhaustively extracted with 70% MeOH (4 × 1.5 L) at room temperature with shaking. The combined extracts were concentrated in vacuo providing c. 500 mL of an aqueous suspension. The suspension was successively extracted with n-hexane, CHCl 3 , EtOAc, and n-BuOH. The obtained organic extracts were evaporated under the reduced pressure to yield 3.62, 1.60, 1.76, and 8.56 g of the dry residue, respectively.
The n-BuOH part of the methanolic extract was initially separated by CC on polyamide to give fractions P1-P65 (100 mL each). The separated fractions were monitored by analytical RP-HPLC/DAD, and the relevant ones were combined. The fractions that contained commonly known L. sativa metabolites, easily detectable by HPLC/DAD (protocatechuic acid, caffeic acid, 5-CQA, luteolin-7-O-β-glucopyranoside, and isoquercitrin), as major constituents, were not further separated.

Assessment of the Reducing Capacity of the Plant Material
The reducing capacity of the plant material, referred to as "total phenolic content" (TPC), was estimated using Folin-Ciocalteu colorimetric method, as described earlier [24]. Measurements were done using 20 mg of the dry plant material per sample. Leaves of 8-and 15-week-old plants of asparagus lettuce (cv. Karola and cv. Grüner Stern) and L. serriola (two accessions) were collected and dried separately for each individual plant. Results (means of three samples, each prepared from one plant) were expressed as gallic acid equivalents (mg GA g −1 DW).

DPPH Radical Scavenging Assay
Portions of dried and pulverized leaves, stalks, and shoot tops of L. sativa var. angustana cv. Grüner Stern (100 mg each) were extracted twice with 12.5 mL of 50% MeOH at room temperature. The solutions from two subsequent extractions were pooled together and evaporated in vacuo. The obtained residues were dissolved in 1 mL of 70% MeOH each, left to stand overnight, at 4 • C, centrifuged (11,340× g, 5 min), and the supernatant was diluted 10 times to obtain concentration corresponding to 10 mg of the dry plant material per 1 mL of the sample. DPPH was dissolved in methanol to obtain the stable free radical solution (100 µM). Solution (4 mM) of Trolox (reference compound) was prepared by dissolving of 100 mg of the compound in 100 mL of methanol. To a spectrophotometric cuvette (1 cm pathlength) containing 480 µL of the methanolic DPPH solution, 20 µL of the diluted plant extract (final concentration 10 mg DW mL −1 ) was added. A decrease in absorbance at λ = 517 nm was measured by UV/VIS CE 2021 spectrophotometer (Cecil, UK) after 0.5, 1, 2, 3, 4, 5, 10, 15, 20, and 30 min.

Sesquiterpene Lactone Analysis
Methanol extracts from the dry and pulverized plant tissues (200 mg) were subjected to RP-HPLC/DAD analysis, as it was described before [25]. Lactucin-like guaianolides could be easily detected in the extracts due to their distinctive chromophore (λ max -258 nm).

Quantification of Major Caffeic Acid Derivatives
The dry and pulverized plant tissue (50 mg) was extracted twice with 10 mL of 70% MeOH at room temperature for 3 h on a rotary shaker (100 r.p.m.). The extracts were combined and evaporated to dryness under reduced pressure to give a residue that was redissolved in 1 mL of 70% MeOH and centrifuged (11,340× g, 5 min) prior to HPLC analysis. Analytical RP-HPLC separations of the samples were performed as it was described earlier [26]. Quantification was carried out using an external standard method. The calibration curves were constructed using four concentration levels (0.001, 0.01, 0.1, and 1.0 mg mL −1 ) of 5-CQA, caffeic acid, CTA, and DCTA. Peak areas, measured at 325 nm, were referred to the corresponding calibration curve.
stituent of celtuce stalks [17], and it has been the only report on its occurrence in cultivated lettuce plants.
In order to roughly assess radical-quenching activities of extracts from different parts of the asparagus lettuce, DPPH radical scavenging measurements were done. The experiments revealed substantial differences in activity of the examined extracts. The extract from leaves (10 mg DW mL −1 ) scavenged 72.7% ± 5.8% of the DPPH radical, whereas the extracts from stalks caused quenching of only 7.4% of the available free radical, after 30 min reaction (Figure 2). In order to roughly assess radical-quenching activities of extracts from different parts of the asparagus lettuce, DPPH radical scavenging measurements were done. The experiments revealed substantial differences in activity of the examined extracts. The extract from leaves (10 mg DW mL −1 ) scavenged 72.7% ± 5.8% of the DPPH radical, whereas the extracts from stalks caused quenching of only 7.4% of the available free radical, after 30 min reaction (Figure 2). Except for cichorioside B, identified as one of the components of the complex mixture of compounds from the butanolic fraction, we did not find any lactucin-like guaianolide during the fractionation of the extract from leaves of asparagus lettuce. Roots of the plant, investigated earlier [20], yielded mainly glucosides of 6 and 9α-hydroxyzaluzanin C accompanied by minor amounts of lactucin-like guaianolides, of which cichorioside B was the most abundant one. As 11β,13-dihydrolactucopicrin, another lactucin-like sesquiterpene lactone, was isolated from the celtuce stalks [17], its presence in the material under study was checked by HPLC/DAD method.
To assess the content of 11β,13-dihydrolactucopicrin/lactucopicrin (one of the most characteristic and easily detectable pairs of sesquiterpene lactones from L. sativa) in the analyzed plant material, a series of extracts was prepared from different organs of the asparagus lettuce (Figure 3a), leaves of L. serriola, and leaves of various cultivars of L. sativa (Figure 3b). The aerial parts of L. sativa cv. Grüner Stern, collected in July, contained c. 0.003% DW of 11β,13-dihydrolactucopicrin/lactucopicrin. The content was two times smaller than that found in roots of the plant. In general, L. serriola leaves, and leaves of the analyzed L. sativa cultivars, did not accumulate detectable amounts of the compounds until the eighth week of growth. The exceptions were two individual plants of L. serriola (Nantes) and one plant of the Amerikanischer Brauner cultivar (c. 0.005-0.009% Except for cichorioside B, identified as one of the components of the complex mixture of compounds from the butanolic fraction, we did not find any lactucin-like guaianolide during the fractionation of the extract from leaves of asparagus lettuce. Roots of the plant, investigated earlier [20], yielded mainly glucosides of 6 and 9α-hydroxyzaluzanin C accompanied by minor amounts of lactucin-like guaianolides, of which cichorioside B was the most abundant one. As 11β,13-dihydrolactucopicrin, another lactucin-like sesquiterpene lactone, was isolated from the celtuce stalks [17], its presence in the material under study was checked by HPLC/DAD method. To assess the content of 11β,13-dihydrolactucopicrin/lactucopicrin (one of the most characteristic and easily detectable pairs of sesquiterpene lactones from L. sativa) in the analyzed plant material, a series of extracts was prepared from different organs of the asparagus lettuce (Figure 3a), leaves of L. serriola, and leaves of various cultivars of L. sativa (Figure 3b). The aerial parts of L. sativa cv. Grüner Stern, collected in July, contained c. 0.003% DW of 11β,13-dihydrolactucopicrin/lactucopicrin. The content was two times smaller than that found in roots of the plant. In general, L. serriola leaves, and leaves of the analyzed L. sativa cultivars, did not accumulate detectable amounts of the compounds until the eighth week of growth. The exceptions were two individual plants of L. serriola (Nantes) and one plant of the Amerikanischer Brauner cultivar (c. 0.005-0.009% DW). After 15 weeks of growth, 11β,13-dihydrolactucopicrin/lactucopicrin was not detected in leaves of asparagus lettuce (cv. Grüner Stern and cv. Karola), but was present in leaves of both L. serriola accessions (0.087-0.123% DW) and leafy cultivars of L. sativa (0.008-0.014% DW).  To investigate the diversity in polyphenolic profile among the analyzed plant species and cultivars, a series of hydroalcoholic extracts from leaf samples was prepared and chromatographically analyzed. Six major hydroxycinnamate signals (Figure 4) could be observed in the analyzed samples, i.e., CTA (tR = 5.5 min), 5-CQA (tR = 6.7 min), caffeic acid (tR = 7.4 min), DCTA (tR = 13.0 min), and two unidentified caffeates (tR = 8.9 min and tR = 15.1 min). The compounds were quantified according to the previously described procedure. The results are summarized in Table 2.  To investigate the diversity in polyphenolic profile among the analyzed plant species and cultivars, a series of hydroalcoholic extracts from leaf samples was prepared and chromatographically analyzed. Six major hydroxycinnamate signals (Figure 4) could be observed in the analyzed samples, i.e., CTA (t R = 5.5 min), 5-CQA (t R = 6.7 min), caffeic acid (t R = 7.4 min), DCTA (t R = 13.0 min), and two unidentified caffeates (t R = 8.9 min and t R = 15.1 min). The compounds were quantified according to the previously described procedure. The results are summarized in Table 2.  To investigate the diversity in polyphenolic profile among the analyzed plant species and cultivars, a series of hydroalcoholic extracts from leaf samples was prepared and chromatographically analyzed. Six major hydroxycinnamate signals (Figure 4) could be observed in the analyzed samples, i.e., CTA (tR = 5.5 min), 5-CQA (tR = 6.7 min), caffeic acid (tR = 7.4 min), DCTA (tR = 13.0 min), and two unidentified caffeates (tR = 8.9 min and tR = 15.1 min). The compounds were quantified according to the previously described procedure. The results are summarized in Table 2.   Looking at the obtained chromatograms, distinctly higher content of CTA in leaves of L. serriola in comparison with that in L. sativa cultivars could be noticed in the 8 weeks old plants. Statistical analysis (one-way ANOVA, p < 0.05 followed by Tukey's HSD test) of the quantitative results obtained for caffeic acid derivatives (CTA, 5-CQA, caffeic acid, DCTA) confirmed significant differences in CTA content between L. serriola and cultivated lettuces (irrespectively of the cultivar). Seven weeks later, however, no differences in the major caffeate contents could be found (data not shown).

Discussion
Numerous factors regulate production and accumulation of secondary metabolites in lettuce including light, temperature, availability of nutrients, and presence of pathogens and pests. Accumulation of particular compounds in the plant organs could be also affected by the stage of a plant life cycle [20][21][22][23][24]. Moreover, colonizing microorganisms, e.g., mycorrhizal fungi, can affect secondary metabolism of the host plant [25]. The multiplicity of factors that affect the plant metabolome makes direct comparison of results obtained in different experiments difficult, though some general conclusions can be drawn on the basis of the available data.
The apocarotenoids 1-4 and 14 have not been found previously in cultivated lettuces. Comprehensive metabolomic fingerprints obtained by different HPLC/DAD/MS methods [2,[5][6][7][8] did not reveal the presence of these compounds. Their absence from the investigated plant material, however, is very unlikely. The compounds originated from degradation of carotenoids and are widely distributed within the plant kingdom. Loliolide and β-damascenone are known metabolites of L. serriola, a wild predecessor of the cultivated lettuces [44,45]. Its plausible that the minute amounts of apocarotenoids were difficult to detect in a very complex matrix submitted to metabolomic analysis. Their isolation from the asparagus lettuce may suggest higher contents of the metabolites in the plant material under study in comparison with other lettuce cultivars. It is worth to note that lettuce is frequently used as a model plant to investigate allelopathic activity of apocarotenoids [46][47][48]. The compounds demonstrate diverse biological effects of ecological and pharmacological significance. (+)-Dehydrovomifoliol (1) inhibited germination of L. sativa cv. Roman and Allium cepa L. seeds and inhibited development of L. sativa cv. Napoli V.F. plants, but stimulated shoot and root elongation in Hordeum vulgare L. [46,47]. It moderately inhibited LPS-induced NO production in mouse RAW264.7 cells, after 24 h, by Griess reagent-based assay [49] and expressed cytotoxic effect against some cancer cell lines in vitro [50]. (-)-Loliolide (2), formerly identified as a potent ant repellent [51], recently has been postulated to be an endogenous inducer of herbivore resistance in plants [52]. The compound inhibited cellular senescence in human dermal fibroblasts [53], exerted antimelanogenic and oxidative stress-protective effects in mouse melanoma and human ker-atinocyte cells [54], inhibited HCV virus entry in vitro [55], and, similarly to 1, LPS-induced NO generation in RAW264.7 cells [56]. Blumenol A (3) and its isomer (6S,9S)-vomifoliol (4) demonstrated anti-inflammatory activity in different types of in vitro assays [57][58][59]. Recently, neuroprotective function of vomifoliol in amyloid-beta 1-42 -treated neuroblastoma cells has been also studied [60]. Blumenol A moderately inhibited elongation of shoots and roots of L. sativa cv. Napoli V.F. and growth of some monocots [47,48]. Compound 5 (corchoionoside C, a glucoside of 4) is one of the four stereoisomers of roseoside. Corchoionoside C inhibited histamine release, induced by antigen-antibody reaction, from rat peritoneal exudate cells [61]. Roseoside of unspecified stereostructure inhibited production of hypertension-related molecules by the rat myocardial cells stimulated with angiotensin II [62] and delayed carcinogenesis induced by peroxynitrite and TPA treatment in mice [63]. (+)-Roseoside caused vasorelaxation of the precontracted aortic rings from Sprague-Dawley rats (in endothelium-dependent manner) [64] and demonstrated insulinotropic activity [65].
Phenolic compounds are the most extensively studied lettuce metabolites. Their dietary intake is promoted as they are generally considered beneficial to human health [9]. According to van Treuren et al. [7], wild relatives of L. sativa, as well as primitive forms of domesticated lettuce, contain more polyphenols than modern cultivars. The main groups of the lettuce polyphenols are flavonoids (including anthocyanins from red varieties) and hydroxycinnamic acids derivatives and lignans. Although flavonoids and hydroxycinnamates are major constituents of lettuce plants (see DCTA content in leaves, Table 2), lignans are usually present in minute amounts. Up to 16 µg of lignans in 100 g of fresh lettuce leaves was reported by Milder et al. [16]. Isolation yield of lignans from the leaves of asparagus lettuce (c. 1.4 mg per 100 g dry weight) suggested relatively high content of the compounds in the investigated plant material. Metabolomic studies disclosed lignan accumulation patterns in some cultivated lettuces with syringaresinol and its glycosides as major representatives of this subclass of plant phenolics [2][3][4]. The asparagus lettuce investigated in the present study seems to follow the same scheme. In addition, major flavonoids detected in leaves of L. sativa cv. Grüner Stern corresponded to those found in leafy cultivars of the species. Significant qualitative and quantitative differences in the contents of the major hydroxycinnamates were not observed as well. The reducing capacity (TPC) of extracts from L. serriola and the two examined cultivars of the asparagus lettuce, Karola and Grüner Stern (Table 1), was comparable and falls within the range of TPC values estimated for different leaf cultivars of L. sativa [66]. The DPPH scavenging activity of extracts from different parts of L. sativa cv. Grüner Stern (Figure 2) suggests that the leaves of the plant are the richest in antioxidant compounds.
Sesquiterpene lactones are the most distinctive terpenoid constituents of lettuces. They are responsible for the bitter taste and, to some extent, for the insect resistance of the plants [10][11][12][13]. The compounds are usually present in very small amounts in the plant material intended for consumption (due to their bitterness) and their contents markedly increase at the bolting stage [67]. In the extract from leaves of the asparagus lettuce only 9αhydroxy-11β,13-dihydrozaluzanin C (6) and 9α-hydroxy-4β,11β,13,15-tetrahydrozaluzanin C (7) were present in quantities that allow successful isolation of the lactones. The former compound was a novel constituent of the cultivated lettuce. Cichorioside B (dihydrolactucin glucoside) was identified as a component of a complex fraction from the butanolic part of the extract. Lactucopicrin and/or its dihydroderivative could not be detected in leaves of the asparagus lettuce collected in the initial phase of cultivation (until the 15th week). The compound was accumulated by the plants that enter the flowering stage albeit in small quantities.

Conclusions
Our findings concerning secondary metabolites from leaves of the old cultivar of stem lettuce (cv. Grüner Stern) broaden the knowledge on the chemistry of the garden lettuce-one of the most popular leafy vegetables. Among the newly identified constituents of the plant, apocarotenoids may have been of importance, taking into consideration both their ecological role and their pharmacological activity. It is worth to note that the apocarotenoids (compounds 1-4 and 14) and 9α-hydroxyzaluzanin C derivatives (9α-hydroxy-11β,13dihydrozaluzanin C and 9α-hydroxy-4β,11β,13,15-tetrahydrozaluzanin C) have not been found before in any of the commercial cultivars of the lettuce. The investigated plant material, in terms of polyphenolic content and antioxidative activity, was similar to modern leafy cultivars of L. sativa.


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Multivariate Analysis for the Classification of Chocolate According to its Percentage of Cocoa by Using Terahertz Time-Domain Spectroscopy (THz-TDS)

: Terahertz time-domain spectroscopy is a useful technique for determining some physical characteristics of materials, and is based on selective frequency absorption of a broad-spectrum electromagnetic pulse. In order to investigate the potential of this technology to classify cocoa percentages in chocolates, the terahertz spectra (0.5–10 THz) of five chocolate samples (50%, 60%, 70%, 80% and 90% of cocoa) were examined. The acquired data matrices were analyzed with the MATLAB 2019b application, from which the dielectric function was obtained along with the absorbance curves, and were classified by using 24 mathematical classification models, achieving differentiations of around 93% obtained by the Gaussian SVM algorithm model with a kernel scale of 0.35 and a one-against-one multiclass method. It was concluded that the combined processing and classification of images obtained from the terahertz time-domain spectroscopy and the use of machine learning algorithms can be used to successfully classify chocolates with different percentages of cocoa.

Introduction
The different spectroscopy techniques used in organic products have always explored ranges within the spectra-visible, ultraviolet and infrared-assessing how light-sensitive photoreceptors control many crucial biological processes [1]. The boom in studies at this frequency is due to access to instruments that are available, but some spectra of the intermediate band or terahertz region (THz) are not totally studied and defined yet [2], showing great potential for uses in products of biological origin.
The so-called non-contact and non-destructive methods, such as NIR spectroscopy [3] and multispectral/hyperspectral imaging [4], imaging in the visible range [5] and Raman spectroscopy [6] have been widely used in the food sector as they are sensitive to intra-molecular vibration [7] and are generating applications in the field of food analysis. Likewise, new applications have been identified, such as the classification by origin of various products.
Many of the methodologies that use spectroscopy, such as those mentioned above, are now being used for determining contaminants in food. However, as noted, terahertz spectroscopy (THz) has not been extensively explored in this field yet, because the ranges of this type of spectroscopy are relatively unexplored within the electromagnetic spectrum ranging from 0.1 to 10 THz, which lies between the mid-infrared and microwave ranges [8].
The composition of cocoa beans is directly influenced by genetic variability, geographical origin and processing. Therefore, chemical and biochemical characteristics and their relationship to external parameters are key characteristics for quality control and technological aspects [9]. Currently, there are studies using near-infrared spectroscopy (NIRS) in the cocoa and chocolate industry [10], showing that it can detect differences, but that there are still points for improvement, such as exploring other spectra. Terahertz spectroscopy could generate new and novel information regarding frequency ranges that are not sensitive to thermal radiation [11].
Looking for the applicability of this technology in the chocolate industry, the objective is to determine the level of differentiation of chocolate bars based on their percentage of cocoa in their composition by using THz spectroscopy and multivariate analysis.

Raw Material
The cocoa genotype (Theobroma cacao L.) that was used is called "Marañon Native" and comes from the area of Cajamarca, Peru. It was used to make chocolate bars with 50%, 60%, 70%, 80% and 90% of cocoa in their composition. For this process, 10 samples were used for each percentage used. The bars used had dimensions of 10 × 10 cm with 0.5 mm of depth. This gave us an image for each sample. In total, 50 images were taken, with 2048 wavelengths, which gave us an average of 600 Mb per square centimeter analyzed.

Equipment for the THz Spectroscopy
The TeraPulse 4000 (Teraview Ltd., Cambridge, UK) was used for this research, which is equipped to measure the spectrum in the range of terahertz with a bandwidth of 60 GHz up to 10 THz and a scanning range of 1200 ps. Figure 1 shows the scheme of this equipment, where the generation of an ultra-short laser pulse through a semiconductorphotoconductor emitter with laser opening is highlighted.  Figure 2 shows the sequence of steps followed to classify the calculated absorbance of THz pulses obtained for each chocolate sample, in order to observe if separation by the percentage of cocoa contained in the samples was possible. To define which classifier had the best performance, 7 models were used, which are listed below: decision trees, linear discriminant analysis (LDA), quadratic discriminant analysis (QDA), support vector machines (SVM), nearest neighbor classifiers, ensemble classifiers and naive Bayes classifiers. Each of these algorithms was combined with its level of interpretability and flexibility, obtaining 24 models that were used in this research. The selection of discriminat-ing variables was carried out using a feature selection technique based on the staggered decorrelation of variables. Cross-validation was used to randomly divide the original dataset of THz spectra into a training set and test set, measuring the mean cross-validation error as a performance indicator. For the other parameters, a heuristic procedure was used to select the scale value based on the kernel function to calculate the best classifier. The best model was determined based on its accuracy. Finally, once the best model was determined, the characteristics were transformed with a PCA to reduce its dimensionality.

Terahertz Imaging Analysis
The chocolate samples contained different percentages of cocoa in their composition and, in order to place them in the THz spectrometry equipment, they were arranged in blocks of 10 × 10 cm. The characteristic pulse of THz is shown in Figure 3a. This, after data processing, could be transformed into indicators that were more suitable for analysis, such as the absorbance shown in Figure 3b. Pre-treatment ranged from noise reduction to the application of fast Fourier transform for conversion into absorbance data [12]. As shown in Figure 3b, the measurement range used was 0.1 to 8 THz in order to observe the behavior of each sample in a broad spectrum. However, after pre-treatment, it was observed that not all the spectra generated adequate information for a classification process as that intended for chocolate samples; thus the range with greater differentiation was used, which was 0.1 to 2.0 THz.
In this shorter range, the absorbance spectra obtained could be better differentiated. In the range of 1.6 THz it could be observed that there was crossing of information. This may have been due to the fact that air and water vapor were not properly purged and can generate this type of noise; but despite this, it could be observed that it was possible to differentiate the samples appropriately, based on the cocoa content within their composition.

Multivariate Analysis
Within the THz classification procedure, two types of models were used to achieve this objective, which were divided into 24 algorithms used, of both LDA and SVM type. To differentiate them, the accuracy indicator (%) and machine learning techniques executed in MATLAB were used, all of them with 15 interactions in cross validation. The five algorithms that reached the highest level of accuracy were taken, which are shown in Table 1. After testing the 24 models, and according to the accuracy, it was obtained that SVM reached the highest accuracy, so that an extra step was made to improve the model with an optimization function. In this case, the selected function was of the Bayesian type with 30 interactions, which achieved an optimized accuracy level of 93%. This SVM model has already been reported as very efficient for food classification [13].
After identifying the best classification function for chocolates based on their cocoa content, it was sought to reduce their dimensionality and test their classification level, for which a PCA was applied, showing that the first component can explain 63.8% and the second component explained 36.2%. In addition, to show the classification level, a confusion matrix was performed as shown in Figure 4a, achieving appropriate classification levels. Additionally, a regression was made, since the exact percentages of cocoa in the chocolates were known. It was tested with the same SVM function and a RMSE of 0.171751 was achieved, which is a very promising value for future work in the prediction of cocoa in chocolate samples. This regression can be seen in Figure 4b.  Terahertz spectroscopy has shown great potential for use in the field of chocolate production. A study by Catapano [14] examined work on quality control of chocolate bars contaminated with foreign objects, where THz technology showed a great ability to detect and discriminate different types of materials based on their composition. Methods for carrying out these quality processes, especially those based on composition, have always used techniques such as mass spectroscopy [15], so the importance of evaluating novel techniques such as time-domain spectroscopy becomes necessary, especially for the food industry, by assessing the importance of non-destructive inspection, meeting the needs of modern and rapid techniques for international trade in food.
Finally, it can be said that spectroscopy in the terahertz range can have a penetration level in the product, which gives an advantage over other spectroscopy technologies, besides the possibility of analyzing the level of vibration and behavior of food macroand micronutrients in a little-studied range [16]. Although with this type of spectroscopy many topics are still under discussion, such as water vapor interference, applications in organic and biological systems and the high costs of generating this type of spectrum, as demonstrated, represent an important and emerging point for food control in the future.

Conclusions
The overall results show that terahertz time-domain spectroscopy together with classification modeling can successfully identify the composition of chocolate bars based on their cacao percentage. The ability of this technique to characterize the molecular structure of many biological substances makes it an attractive analytical process tool for better monitoring in food quality control. However, while terahertz time-domain spectroscopy is demonstrating efficiency in classification methods, as in chocolate, there are still many parameters to take into account in the use of this type of technology.

Funding:
The authors acknowledge the financial support of the Project Concytec-World Bank "Development of Predictive Models of Food Quality Based on THz Imaging Technology", through its executing unit Fondecyt, contract number 006-2018-FONDECYT/BM-Mejoramiento de la infraestructura para la investigación (equipamiento).


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Antibacterial Activity of Marula [Sclerocarya Birrea] and Brominated Marula Seed Oil

Over the last decade, the use of hand sanitizers has increased substantially. Recently, with this outbreak of COVID 19 virus, all natural hand sanitizers have taken the spotlight for their simplicity, powerful cleaning abilities, natural moisturizing formulas and the ability to never leave the skin feeling dry. Marula [Sclerocarya birrea] seed oil was soxhlet extracted with n-hexane and then brominated. The oil quality parameters of marula oil (MO) and brominated Marula oil (BMO) were found to be; for MO: oil content (58.5 ± 0.435 %), moisture content (4.3 ± 0.158 %), saponification value (180 ± 0.7150 mgKOH/g ), peroxide value (10.52 ± 0.006 meq/kg), acid value (3.48 ±0.002) meq/kg ), FFA (1.73 ± 0.000 % as oleic),iodine value (61.02 ± 0.477 gI2/100g ). For brominated marula oil (BMO) ; saponification value ( 168.7 ± 0.930 mgKOH/g ), iodine value ( 8.6 ± 0.490 gI2/100g ), peroxide value (10.63 ± 0.505 meq/kg ), acid value ( 3.42 ± 0.000 meq/kg ), FFA (1.61 ± 0.000 % as oleic). Fourier Transform Infrared (FTIR) spectroscopy was used to confirm the bromination process. The following peaks of absorptions were observed for BMO: C–Br at 600 cm -1 , C–H stretch, (2950 to 2850 cm -1 for alkane), 1750cm -1 for carbonyl stretch, 1450 cm -1 for ester, 1680 cm -1 for alkene. The BMO showed a broad spectrum activity against E. coli, Salmonella sp, Staphylococcus aureus and Streptococcus epidermidis microorganism which gave the inhibitions zones of 19.50 mm, 20.50 mm, 19.00 mm, 18.60 mm respectively. DMSO was used as the negative control while streptomycin as the positive control at 400 mg/ml concentration. Based on these findings, BMO may be use as disinfecting oils or hand sanitizers and will discourage its use as food additive.

INTRODUCTION
Brominated Vegetable Oils (BVO) are complex mixtures of plant derived triglycerides that have been reacted to contain atoms of the element bromine bonded to the molecules [12]. Brominated vegetable oil has extensively been used as a food additive in soft drink manufacture, where it has been used as an emulsifying agent. However, in other applications, it has been utilized for its fire retardant properties a practice that have brought to question in its continued use as a food additive [10]. This is arising from reported cases of Bromism [5] and the observed increase in the level of brominated fatty acids that have been noted to be stored away in the body's lipid [10,11]. To support this assertion is the reported effort of BVO on yeast population [7] and thus its possible effect on human body cells, a fact that have been pointed out in studies with experimental animals. Marula oil is extracted from the kernels (nuts) of the marula tree (Sclerocarya birrea), from the Anacardiaceous family. Marula oil is traditionally used in cosmetics, in food as cooking oil and as a meat preservative and to treat leather [4].

II. MATERIALS AND METHODS
The kernel of plant Sclerocarya birrea was obtained within A T B U Bauchi metropolis. The sample was authenticated by the biological science department A.T.B.U Bauchi Nigeria.
The marula oil was soxhlet extracted using n-hexane and was brominated.The oil quality parameters of marula oil (MO) and brominated marula oil (BMO) was determined using a standard method by [1]. Infrared (Ir) spectroscopy was use to confirm the bromination process.

 Bromination process
This involves direct bromine adding with a dropping funnel into the oil sample in a Erlenmeyer flask at a maintained temperature of not more than 50 0 C. The reaction took up to 8 hours under vigorous strirring with magnetic stirrer with a condenser connected under a fume curpboad.

 Antibacterial Evaluation of MO and BMO
The Disc Diffusion Method for antimicrobial susceptibility testing was adopted [6,9] to access the presence of the antibacterial activity of MO and BMO. Bacteria culture of E Coli, Salmonella species, Staphylococcus aureus and Streptococcus epidermidis were used. In this method discs which have been impregnated with MO and BMO was placed on the Muller-Hinton Agar (MHA) plate surface, with each test plate comprising of 6 discs. The plates were incubated at 37 0 C for 18 to 24 hours, after which the plates was examined for inhibition zones diameter was measured using calipers and were recorded. Controls were conducted in line with the above procedure, using streptomycin antibiotic for positive control while dimethylsulphoxide (DMSO) for negative control.

 Extract Preparation
About 0.4ml of the extract (each of MO and BMO) were dissolved and made up to 1ml with DMSO to give a concentration of 400mg/ml each.

III. RESULTS AND DISCUSSION
The moisture content of the Marula oil (4.3%) was moderately high which could be responsible for the hydrolysis of triglycerides to yield high level of free fatty acids and will lead to high refining losses of oil yield (58.5%) as consequences as shown in table 1. Other parameters which include; acid value (AV), perioxide value (PV), saponification value (SV), free fatty acid (FFA), and iodine value (IV) were also presented at  Table 1:-Oil quality parameters of /MO and BMO The results are express in terms of mean ± standard deviation) Iodine value (IV) is the measure of the overall unsaturation in the structural matrix of the oil and it is usually used to characterize oils and fats as nondrying, semi drying and drying. However, the IV of MO and BMO falls within the nondrying class. The reduction of IV in BMO (8.6 gI 2 /100g) from (61.02 gI 2 /100g) for MO (table 1.) indicate that bromination has occurred but not 100% completion.
Evidence that bromination has occurred, is the sharp weak absorption of carbon-bromine bond at 600cm -1 at table 3. And its absence in that of MO at table 2. Presence of some targeted functional groups like the carbonyl stretch 1750cm -1 ,1400cm -1 for C-O ester and 1660 to 1680cm -1 for C=C alkene were observed in both IR spectra of MO and BMO (table 2&3).   BMO showed a broad activity against E coli, salmonella sp, staphylococcus aureus and streptococcus epidermidis microorganism which gave the inhibition zones of 17 mm, 16.5 mm, 15 mm and 16.0 mm respectively. Dimethylsulphoxide (DMSO) was used as the negative control while Streptomycin as the positive control at 400mg/ml sample concentration.

IV. CONCLUSION
This research work was successful since marula oil was extracted and brominated. The oil quality parameters of the marula oil and its brominated derivative was determined. The IR spectroscopy of MO and BMO were conducted to verify the presence of some targeted functional groups and to confirm the bromination process.
Antibacterial activity of the two (MO and BMO) shows activity with microbes that was used.
Focusing on the activity BMO has on some bacteria, a suggestion of it as an antimicrobial agent, can be explored and exploited for it application as sanitary products such as hand sanitizers or as disinfecting oils.