High Temperature Modifies Resistance Performances of Rice Varieties to Brown Planthopper,Nilaparvata lugens(Stl)

To investigate the effect of temperature on the resistance characteristics of rice varieties with different resistance genes to brown planthopper(BPH),Nilaparvata lugens(Stl),the resistances of IR26(Bph1)and IR36(bph2)to BPH population in Hangzhou,China were monitored in greenhouse during September in 2007 and 2008 by using the standard seedling screening techniques(SSST)developed by the International Rice Research Institute(IRRI).Furthermore,the changes in resistance of IR26 and IR36 to BPH,soluble sugar and oxalic acid contents in 25-day-old rice plants of susceptible variety TN1 and resistant varieties IR26 and IR36 were detected at five temperatures(22℃,25℃,28℃,31℃ and 34℃).IR26 completely lost resistance both in greenhouse and at the five tested temperatures.IR36 still had moderate resistance at natural temperature,but its resistance decreased gradually when the temperature increased from 25℃ to 34℃,and fully lost its resistance at 31℃ and 34℃.The highest durable resistance of IR26 and IR36 were recorded at 25℃.The soluble sugar content in plants of the three tested rice varieties increased with temperature increase,and the oxalic acid content increased with the temperature increase at first,maximized at 25℃,and then declined.Two-way ANOVA indicated significant effects of temperature and rice variety on contents of soluble sugar and oxalic acid in rice plants.     

Nuclear and chloroplast diversity and phenotypic distribution of rice (Oryza sativa L.) germplasm from the democratic people’s republic of Korea (DPRK; North Korea)

Background

Rice accounts for 43% of staple food production in the Democratic People’s Republic of Korea (DPRK). The most widely planted rice varieties were developed from a limited number of ancestral lines that were repeatedly used as parents in breeding programs. However, detailed pedigrees are not publicly available and little is known about the genetic, phenotypic, and geographical variation of DPRK varieties.

Results

We evaluated 80 O. sativa accessions from the DPRK, consisting of 67 improved varieties and 13 landraces. Based on nuclear SSR analysis, we divide the varieties into two genetic groups: Group 1 corresponds to the temperate japonica subpopulation and represents 78.75% of the accessions, while Group 2 shares recent ancestry with indica varieties. Interestingly, members of Group 1 are less diverse than Group 2 at the nuclear level, but are more diverse at the chloroplast level. All Group 2 varieties share a single Japonica maternal-haplotype, while Group 1 varieties trace maternal ancestry to both Japonica and Indica. Phenotypically, members of Group 1 have shorter grains than Group 2, and varieties from breeding programs have thicker and wider grains than landraces. Improved varieties in Group 1 also show similar and/or better levels of cold tolerance for most traits, except for spikelet number per panicle. Finally, geographic analysis demonstrates that the majority of genetic variation is located within regions that have the most intensive rice cultivation, including the Western territories near the capital city Pyungyang. This is consistent with the conscious and highly centralized role of human selection in determining local dispersion patterns of rice in the DPRK.

Conclusions

Diversity studies of DPRK rice germplasm revealed two genetic groups. The most widely planted group has a narrow genetic base and would benefit from the introduction of new genetic variation from cold tolerant landraces, wild accessions, and/or cultivated gene pools to enhance yield potential and performance.     

Evaluation of frost resistant tuber-bearing solanum hybrids

Frost resistance of interspecific and varietal potato crosses was evaluated at Bogotá, Colombia using intact plants in a programmed low temperature growth cabinet. Plants were exposed to a 2 hour frost during the dark period and the minimum test temperature was successively lowered 1 C on subsequent nights. The foliage was visually scored for damage 10–12 hours after each frost. While most commercial varieties were killed at—2 to—3 C (28 to 26 F), 101 of 121 clones which have been bred specifically for frost resistance survived to at least—5 C (23 F) and some crosses resisted—9 C (16 F) without observable foliar damage. This suggests the possibility of obtaining cultivated varieties from varietal parents of cultivated species such as G. Andigena, G. Phureja, G. Stenotomum,S. ajanhuiri, and others, which are reasonably frost resistant.     

The effects of ‘Vicia faba L.’ polyphenols on absorption,growth and metabolism in the rat

Two varieties of ‘Vicia faba L.’equina andminor, were studied. Theequina variety had a higher polyphenolic content than theminor variety. Both varieties significantly inhibited rat growth rate and ‘in vivo’ intestinal glucose transport, theequina variety being a more powerful inhibitor than theminor variety. However the raw legume fed animals showed a normal urinary excretion rate of 3-Methylhistidine.     

Glucosinolate Variation in Leaves of Brassica rapa Crops

Total and individual glucosinolate (GSL) content of leaves of vegetable turnip rape (Brassica rapa L. var. rapa) was determined in a set of 45 varieties consisting in early, medium and late types grown at two locations in northwestern Spain. The objectives were to determine the diversity among varieties in GSL content and to relate that variation with earliness and plant habit. Eight GSL were identified, being two aliphatic GSL, gluconapin (84.4?% of the total GSL) and glucobrassicanapin (7.2?% of the total GSL) the most abundant. Indolic and aromatic GSL content were low but also showed significant differences among varieties. Differences in total and individual GSL content were found among varieties, plant habit groups, and earliness groups. Total GSL content ranged from 19 to 37.3???mol?g^?1?dw in early and extra-late groups, respectively, and from 19.5 to 36.3???mol?g^?1?dw for turnips and turnip greens groups, respectively. These differences were consistent to values found for gluconapin content where the turnip group had the highest values (31.8???mol?g^?1?dw) and the turnip top group had the lowest (15.7???mol?g^?1?dw). Two varieties, MBG-BRS0429 and MBG-BRS0550 (from turnip greens and extra-late groups) and MBG-BRS0438 (from turnips and late groups), stood out as they had the highest total GSL content and could be used as a good source of these beneficial bioactive compounds. Elucidation of genetic diversity among crops can provide useful information to assist plant breeders to design improved breeding strategies in order to obtain varieties rich on GSL.     

Effects of soil fumigation with Telone C on soil and plant nitrogen,verticillium wilt and yield of Norland and Norgold potatoes

Two fields in Weld County, Colorado were fumigated in the fall or spring with Telone C in an attempt to restore their productivity. The soils were Julesburg sandy loams and had been cropped to either corn or potatoes for many years. Observations were made over three years on the following: soil ammonium and nitrate nitrogen carry over, seasonal changes in these two forms of nitrogen, petiole nitrate concentration,Verticillium population, verticillium wilt expression,Verticillium stem infection and potato yields of two varieties. No significant differences occurred between control and fumigated blocks in the carry over of soil ammonium or nitrate nitrogen. During the growing season of the first year, no significant differences in soil nitrate concentrations occurred, while during the second and third year at certain times, all fumigated blocks contained significantly less nitrate nitrogen than the control block. Significant seasonal differences measured between blocks in soil ammonium concentration were not related to fumigation. Petiole nitrate concentrations while similar during the first year, were significantly higher in most fumigated treatments the second year and generally lower than the controls in the third year. Verticillium populations were not consistently affected by fumigation. Fluctuations not associated with fumigation prevented drawing definite conclusions. Both spring and fall fumigation reduced wilt severity in the two varieties. Effects of spring fumigation carried through two seasons while fall fumigation did not. Plant infection, determined by culturing stem sections, corresponded with the wilt severity ratings. Fall fumigation increased market yields of Norland potatoes but the increase was not statistically significant. Usually, both fall and spring fumigation significantly increased market yield of Norgold potatoes and over three years, spring fumigation was significantly better. Yield increases resulting from fumigation were considered greater than could be explained from alteration in the soil ammonium to nitrate ratios and/or verticillium wilt control as measured in this study.     

Sioux: A scab resistant red potato variety

Sioux is a uniform round, red, slightly scaly russet variety of medium maturity. The variety is resistant to scab (Streptomyces scabies) and susceptible to Fusarium, Verticillium and late blight. In Nebraska, Sioux produces moderate to high yields, but higher yields of U.S. #1 potatoes when compared with the standard red table varieties Norland and Bounty. The specific gravity of Sioux is higher than that of the standard red table and white chipping varieties Haig, Platte and Kennebec. The variety is suitable for chipping and the production of frozen products. When grown as a late summer crop, Sioux is of good color and carries to market in better condition than the standard red varieties. In the 12 States in the North Central Regional Trials, Sioux was equal to the standard variety Red Pontiac in total yield and superior in yield of U.S. #1 potatoes. The specific gravity of Sioux was higher than that of Irish Cobbler or any of the other standard varieties. The yield of Sioux was as high and the specific gravity was higher than any of the standard varieties in Texas and Alabama trials.     

Albinism of potato spindle tuber viroid-infected Rutgers tomato in continuous light

Potato spindle tuber viroid (PSTV) caused albinism in the new growth of Rutgers tomato under continuous light. The albinism response was obtained with 4 isolates causing severe symptoms in tomato and with 1 mild isolate which is normally symptomless. Rutgers tomato inoculated with the 4 severe strains of PSTV developed typical ‘bunchy top’ symptoms in 12 to 16-hr day lengths. Symptoms developed slower and much less distinctly on plants incubated in 6-hr days. New growth in PSTV-infected plants was green in short days (12-hr illumination) and chlorotic to white in continuous light. Infectivities of extracts from white portions of plants were from 3 to 10 times greater than those from green portions of infected plants. Albino symptoms also developed in other tomato varieties when inoculated with PSTV and kept under continuous light. Albinism was most pronounced under continuous light at 30°C, somewhat less intense at 24°, and at 16°, new growth of infected leaves and stems remained green. Albinism did not develop in otherSolanum species which are symptomless hosts of PSTV nor in seedling potato plants grown in continuous light.     

Purple top roll disease of potato

A graft transmissible disease of potato termed purple top roll is prevalent in several potato varieties grown in the hilly tracts of India. The common symptoms are rolling of the basal parts of younger leaves, sometimes accompanied by pink or purple pigmentation. The plants are stunted with profuse growth of axillary branches and aerial tubers. The losses in yield varied from 40 to 70%. Five to 20% of the tubers from diseased plants developed hairy sprouts. Tuber transmission of the pathogen varied from 6 to 33%, depending on the variety.Datura, tomato and white clover are among susceptible plants.N. rustica was tolerant or a symptomless carrier. Sunnhemp, Petunia andSesamum were immune to the pathogen, which was transmitted by the leaf hopper,O. albicinctus Distant but not byCuscuta spp. or by sap inoculation. Application of Chloramphenicol, Tetracycline and Antiamoebin resulted in remission of symptoms and increased tuber yields, indicating possible association of a mycoplasma-like organism.     

Variability of Phytonutrient Content of Potato in Relation to Growing Location and Cooking Method

Potato has a defined complement of metabolites that contribute to the human diet. Among these are the carotenoids and anthocyanins. Carotenoids are found in all potatoes in the flesh. White-fleshed varieties have 50 to 100 μg per 100 g fresh weight (FW), while moderately yellow-fleshed varieties will generally possess from 100 to 350 μg per 100 g FW. The more intensely yellow-fleshed genotypes, which may look orange, at the higher extremes are at levels above 1,000 μg per 100 g FW. The highest level published is 2,600 μg per 100 g FW in diploid germplasm derived from South American Papa Amarilla cultivars. Potato generally has predominantly lutein, a xanthophyll which is also found in the human retina, and must be obtained in the diet. The genotypes with extremely high levels of total carotenoids have zeaxanthin, an isomer of lutein, which is also present in the human retina. Anthocyanins are present in red- or purple-skinned and fleshed varieties. Total anthocyanins range from 1.5 mg to 48 mg per 100 g FW in a solidly pigmented purple-skinned, purple-fleshed breeding line. The degree of pigmentation in the flesh appears to be under polygenic control. Anthocyanins are potent antioxidants and anti-inflammatory substances. The level of total anthocyanins is correlated with antioxidant level (r?=?0.94, P?<?0.001). Several methods of cooking interacted with genotypes in the antioxidant level remaining after cooking compared to raw potatoes. No method of cooking completely eliminated antioxidant activity, while boiling appeared to increase it compared to raw potato in the case of the most highly pigmented clone.     

双季早稻养分积累分配特征及其与产量构成的相关性

三熟制模式下南方双季稻茬口衔接季节紧张是协调双季稻周年增产中存在的主要问题。虽然前人在三熟制模式下水稻品种搭配以及肥料利用率方面做了大量研究,但是三熟制模式下南方双季早稻产量及其养分积累分配相关性的研究鲜有报道。为了比较南方双季早稻养分积累分配特征,本文以15个水稻早稻品种为研究对象,对不同水稻品种的秧苗素质、基本苗及分蘖动态、经济性状、营养元素积累及分配、产量及产量构成因素进行比较分析。结果表明,不同品种间秧苗素质存在显著差异,‘株两优819’的叶龄、苗高、百株鲜重等多项指标优于其他品种,秧苗素质表现佳。分蘖动态调查表明不同品种达到分蘖峰值的时间不同,其中以‘株两优929’的高峰苗最大,1 hm²达616.65万茎蘖数,‘株两优929’与‘中早39’高峰苗差异最为显著,‘株两优929’较‘中早39’显著增加33.62%。氮、磷、钾总养分的积累方面,‘株两优929’和‘陵两优942’养分积累量显著高于其他品种,分别为311.88 kg/hm²和376.35 kg/hm²。‘陵两优942’的地上部干物质积累量最高,为12 028.09 kg/hm²。‘株两优929’的实际产量最高,为7873.01 kg/hm²。成熟期水稻品种养分积累量与子粒产量的相关性分析表明,氮、磷积累与有效穗均呈显著正相关（r=0.62^*,r=0.50^*）,株高与千粒重之间呈极显著正相关（r=0.80^**）,有效穗数与每穗粒数之间呈极显著负相关（r=-0.76^**）,结实率与千粒重之间呈极显著负相关（r=-0.68^**）。通过关联度分析表明,‘陵两优942’和‘株两优929’的综合表现明显优于其他品种。本研究对南方多熟制稻区早稻品种筛选及高效栽培具有重要意义。     

Genetic Improvement of Rice for Bacterial Blight Resistance: Present Status and Future Prospects

The production and productivity of rice has been challenged due to biotic and abiotic factors. Bacterial blight (BB) disease, caused by Xanthomonas oryzae pv. oryzae, is one of the important biotic stress factors, which reduces rice production by 20%–50%. The deployment of host plant resistance is the most preferred strategy for management of BB disease, and breeding disease resistant varieties remains a very economical and effective option. However, it is difficult to develop rice varieties with durable broad-spectrum resistance against BB using conventional approaches alone. Modern biotechnological tools, particularly the deployment of molecular markers, have facilitated the cloning, characterization and introgression of BB resistance genes into elite varieties. At least 46 BB resistance genes have been identified and mapped from diverse sources till date. Among these, 11 genes have been cloned and characterized. Marker-assisted breeding remains the most efficient approach to improve BB resistance by introducing two or more resistance genes into target varieties. Among the identified genes, xa5, xa13 and Xa21 are being widely used in marker-assisted breeding and more than 70 rice varieties or hybrid rice parental lines have been improved for their BB resistance alone or in combination with genes/QTLs conferring tolerance to other stress. We review the developments related to identification and utilization of various resistance genes to develop BB resistant rice varieties through marker-assisted breeding.     

Experimental host range of the potato spindle tuber ‘virus’

Two hundred thirty-two plant selections (species and varieties) were tested for susceptibility to the potato spindle tuber ‘virus’ (PSTV). One hundred thirty-eight selections were found to be susceptible to PSTV but no ‘virus’ was recovered from the remaining 94. Susceptible plants were found in the families Boraginaceae, Campanulaceae, Caryophyllaceae, Compositae, Convolvulaceae, Dipsaceae, Sapindaceae, Scrophulariaceae, Solanaceae, and Valerianaceae. Most of the susceptible selections were symptomless carriers of PSTV. Visible symptoms were produced by both mild and severe strains of PSTV inLycopersicon esculentum cv. Allerfruheste-frëiland,Scopolia anomala, S. corniolica, S. lurida, S. sinensis,S. stramonifolia, S. tangutica, Solanum aviculare, andS. avicular var.albiforme; and only by the severe strain inGynura aurantica, Petunia hybrida var. Burpee Blue, andSolanum depilatum. Temperature of 21.1-22.8 C (70-73 F) with a light intensity of about 400 ft-c favored local lesion development inScopolia sinensis. S. sinensis appeared to be more susceptible than otherScopolia species.     

Evaluation of potato varieties and selections for resistance to bacterial soft rot

Potato varieties and selections were evaluated for resistance to bacterial soft rot caused byErwinia carotovora subsp. carotovora and Erwinia carotovora subsp. atroseptica in mist chamber. A randomized complete block design with a single treatment and 3 replications was used in this study. Potato lines were inoculated with bacterial suspension (10 tubers / line) using the puncture inoculation method and incubated for 4 days in a mist chamber at 21 C and 100 % relative humidity in the dark. Although there were differences in incidence of soft rot and in amount of weight reduction, most of the potato lines were susceptible to tuber soft rot. Mean incidence (%) of soft rot ranged from 10 to 100%. Mean reduction of tuber weight (%) ranged from 0.13 to 16.43 %. Among the varieties and selections tested, AF 522-5 was the most resistant to tuber weight loss, while AF 1424-7 was most susceptible. There were also significant differences between years, and a variety by year interaction. Testing over a period of years is recommended. A significant correlation coefficient of soft rot incidence (%) and reduction of tuber weight (%) was obtained (r=0.65; P=0.05), indicating that incidence was a fair measure of soft rot potential. However, the greater variability in results for incidence make weight loss a better measure of soft rot reaction. There was a significant difference among varieties in the reduction of tuber weight when potato lines were inoculated either 1 month or 2 months after harvest, but the interaction of time by year prohibits a conclusion as to which time gives a better test reaction. Late maturing varieties had slightly less weight loss than earlier maturing varieties.     

Relation between susceptibility to wheat bug attack and digestibility of glutenin

Two trials of winter wheat, embracing 17 varieties and 4 breeding lines, harvested in 2003 from locations in the semi-arid eastern part of Austria, were severely infested by naturally occurring bugs (Eurygaster sp.). In these trials bug damage was determined by selecting and weighing the infested kernels. Glutenin degradation was determined by the addition of meal from bug-infested kernels to meal from sound kernels from the same trials to reach sample mixtures representing a degree of bug attack of 6%. These mixtures and blanks made from sound meal were incubated in buffer solution (pH=8.5) at 37 °C for 45 min. The reduction in glutenin content due to the activity of bug proteinases was measured by RP-HPLC analysis of the glutenin fraction and comparison of the results of damaged and sound samples. The results showed good correlations between the degree of bug attack and the digestibility of glutenin (location 1: R²=0.69, P<0.01; location 2: R²=0.36, P<0.01). Both parameters seem to be variety specific. The determination of digestibility of glutenins was repeated in material from a trial harvested in 2005 in another location in eastern Austria, with eight varieties being the same as used in the 2003 trials. A correlation with the degree of bug attack in location 1 from the 2003 harvest was found again (R²=0.61, P<0.01).     

Rate of Cooling Alters Chip Color,Sugar Contents,and Gene Expression Profiles in Stored Potato Tubers

When stored at temperatures below 10 °C, potatoes accumulate sucrose and the reducing sugars glucose and fructose. This process, cold-induced sweetening, has been studied extensively because potatoes with elevated reducing sugar contents produce undesirable, dark-colored products and acrylamide, a suspected carcinogen, during high-temperature cooking. Potatoes in commercial storages are cooled slowly, but many research studies have used potatoes cooled rapidly. In this study, effects of cooling rate and variety on chip color, sugars, and gene expression were examined. Sucrose and reducing sugar contents were substantially lower in slowly cooled than in rapidly cooled tubers of ‘Snowden’ and “MegaChip’ for the first 11 weeks after cooling to 3 °C began. Differences in gene expression for VInv, β-amylase, SPS, AGPase and GBSS were observed between cooling treatments and varieties. Overall, the data showed that cooling rate, time in storage, and variety influenced multiple aspects of cold-induced sweetening.     

Phytophthora erythroseptica in Peru: Its identification and pathogenesis

EightPhytophthora isolates cultured from diseased potato plants and tubers in Peru were identified asPhytophthora erythroseptica Pethyb. var.erythroseptica. This pathogen was not previously reported from South America. The isolates were pathogenic to potato plants but they differed in virulence. Injuring roots prior to inoculation enhanced infection and disease development but all isolates infected noninjured roots. Continuous high soil moisture during incubation favored disease development. Under these conditions, disease developed at soil temperatures from 10 C to 30 C, but most rapidly at 25 C and 30 C. Plants grown 60 days before inoculation developed symptoms earlier and more rapidly than plants grown 15-, 30-, and 45 days. Four concentrations of mycelial-suspension inocula did not alter disease development, but zoospore inoculum induced symptoms and plant death more rapidly than mycelial suspensions at 25 C.     

Evaluating cultivars in unbalanced on-farm participatory trials

Data from on-farm participatory varietal trials are often highly unbalanced. Using statistically robust techniques we showed that such data are amenable to sophisticated analyses. Data from 38 varieties of wheat tested in 44 mother trials (all test varieties compared) and 663 baby trials (single entries compared with local checks) over 6 years in Lunawada, Gujarat, India, were analysed. Two combined analyses for grain yield were performed by using either a fixed effects model within general linear modelling (GLM) or a mixed effects model using the restricted maximum likelihood (REML) procedure. Both analyses made substantial adjustments over the raw means and the estimated mean values from the two analyses for the fixed set of tested varieties were highly correlated (R² = 0.89) as were the t-value that compared test cultivars with the check variety (R² = 0.96). The GLM detected 15 varieties and REML detected 12 varieties that were significantly superior to the check cultivar with 11 varieties in common. Both methods were similar in identifying the top-most promising varieties but REML was more conservative (i.e. more subject to Type II error) with larger standard errors (S.E.s) of variety means for n > 17. However, for both procedures the S.E.s increased sharply when the number of trials decreased below 17.     

The Origin and Spread of Early-Ripening Champa Rice: It’s Impact on Song Dynasty China

Often overlooked is the importance of early-maturing rice varieties with their ability to escape droughts, avoid floods, and in some localities, open up the opportunity for double cropping. Most varieties grown in the tropics until the last half century matured in 150 to 180 days or longer and were photoperiod sensitive. However, non-photoperiod sensitive one-hundred-day varieties were grown in the Champa Kingdom in what is now Central Vietnam centuries ago. How these varieties moved into Song Dynasty China and the impact of the early-ripening rice on population growth in South China is well documented. However, the origin of the Champa varieties is less clear. According to a recent DNA test (see below), the Champa varieties belong to the aus sub-population. The aus have the characteristics of both indica and japonica and originated in the hill areas of what are now Eastern India, Bangladesh, and Myanmar. Because the Champa Kingdom adopted the Indian language and religion, at least in the higher courts, it seems logical that the Champa rice must have been disseminated from the hilly areas in the Indian sub-continent to Central Vietnam and eventually to China—first Fujian and later to the Yangtze region during the Song Dynasty in the eleventh century.     

Antioxidant Capacity and Antimutagenic Activity of Anthocyanin and Carotenoid Extracts from Nixtamalized Pigmented Creole Maize Races (Zea mays L.)

Nixtamalization process is the first step to obtain maize based products, like tortillas; however, in both the traditional and commercial processes, white grain is generally preferred. Creole maize races, mainly pigmented varieties, have increasingly attention since these are rich in anthocyanins and carotenoids. The aim of this investigation was to evaluate the antioxidant and antimutagenic activity of rich anthocyanins and carotenoids extracts from creole maize races before (grain) and after (masa and tortilla) the nixtamalization process. Most anthocyanins and carotenoids were lost during nixtamalization. Before nixtamalization, blue and red genotypes contained either higher antioxidant capacity and anthocyanin contents (963?±?10.0 and 212.36?±?0.36 mg of cyanidin-3-glucoside eq/100 g, respectively) than the white and yellow genotypes. However, the highest carotenoid levels were displayed by red grains (1.01?±?0.07 to 1.14?±?0.08 μg of β-carotene eq/g extract). Anthocyanins losses were observed when the blue grains were processed into masa (83 %) and tortillas (64 %). Anthocyanins content correlated with antiradical activity (r?=?0.57) and with 2-aminoanthracene -induced mutagenicity inhibition on TA98 and TA100 (r?=??0.62 and r?=??0.44, respectively). For white grains, nixtamalization also reduced carotenoids (53 to 56 %), but not antioxidant activity and 2-Aa-induced mutagenicity. Throughout the nixtamalization process steps, all the extracts showed antimutagenic activity against 2-aminoanthracene—induced mutagenicity (23 to 90 %), displaying higher potential to inhibit base changes mutations than frameshift mutations in the genome of the tasted microorganism (TA100 and TA98, respectively). The results suggest that even though there were pigment losses, creole maize pigments show antioxidant and antimutagenic activities after nixtamalization process.