Varieties of Brassica oleracea and the Intrinsic Similarities of Plant Structures Therein

 

 

 

 

 

 

 

by:  

The Green Giants

Tara Esshaki

Rachel Disipio

Matthew Ravenscroft

 

 

 

 

 

 

 

 

 

 

 

LBS 145 Cell and Molecular Biology

Section 3

 Joseph Maleszewski

10-03-02

 

ABSTRACT: Tara Esshaki, Revised by Matthew Ravenscroft

Second revision by Rachel Disipio

 

            Two varieties of Brassica oleracea, broccoli and cauliflower were put through several tests to determine their carbohydrate content.   The results were reducing sugars, mono- and disaccharides, aldehyde and ketone, furanose and hexose ring type sugars.  A photosynthesis test was done to determine the present pigments and the absorption spectrum of each plant. Finally, an enzyme test was performed to determine the effectiveness of salivary amylase on each sample.  In the lab, the question asked how similar the Brassica oleracea varieties, broccoli and cauliflower are to each other.  It is known that the nutritional value of broccoli and cauliflower are very close to one another with regards to the amounts of protein, fat and vitamin C (Robertson, 2002).  Given the tests we preformed, the only differences between the Brassica olercea varieties became evident only when Bial’s carbohydrate analysis and photosynthetic characterists were compared. 

 

 

DISCUSSION: Matthew Ravenscroft

                                                            Revised by: Rachel Disipio

 

            The vegetables broccoli and cauliflower are members of the Brassica oleracea species.  The parts of the plant that humans consume are the immature inflorescence, the flower buds, pedicels and their stalk.  A hypothesis suggested by the group was that, on presentation, the broccoli inflorescences are in no way similar to the colorless cauliflower.  Despite this, we suggested that the broccoli was close in composition to the cauliflower.  The immature inflorescence of broccoli exhibits the most striking difference to that of the cauliflower when tested for pigment content.  Due to the lack of distinction in the carbohydrate values, as well as inconclusive results with respect to salivary amylase potency, the main evidence of relation lies in the pigmentation of the two Brassica varieties.

            Carbon is the very key to living existence.  Since carbon carries so much importance for every plant, carbohydrate (a combination of carbon and hydrogen) similarities between the vegetables would be crucial in determining the closest relative to the broccoflower plant.  Unfortunately, the carbohydrate tests were sincerely unfruitful considering that the three species exhibit very similar carbohydrate content. 

            Barfoed’s test, the first experiment, was run to test for the presence of reducing sugars, specifically those that are monosaccharides.  In this particular test, a positive result would yield a red precipitate at the bottom of the test tube.  Among the varieties of Brassica preliminary results seemed to disprove our hypothesis.  Though all the tests resulted in the formation of a precipitate, only the sample of cauliflower tested was red (figure 1).  The broccoli yielded a yellow precipitate.  The broccoli precipitate, upon first observation, was not the same as that of cauliflower.  The readings from the spectrophotometer negated our original thoughts.  Though different color precipitates, the absorbance readings were very similar, varying only slightly (table 5), to each other and to that of the control 1% glucose.  This led us to the conclusion, the varieties of Brassica all contained monosaccharide reducing sugars.  The broccoli’s carbohydrate structure cannot be differentiated from that of the cauliflower, they resemble each other.       Benedict’s, the next experiment carried out, yielded similar results, in the way that the data was interpreted.  Again, upon the examination of the tubes themselves preliminary results were drawn.  Because all tests yield precipitate of different colors, none of which were red, the precipitate color of the positive control, (figure 3), it was concluded that all three varieties had slightly different carbohydrate structures.  They all contained free aldehyde or ketone reducing sugars, just in different proportions.  Again, however, the first ideas were determined to be incorrect upon examination of the spectrophotometer (table 5).  The absorbance readings were very close to one another.  Thus the absorbance values, this indicated that there was a shared carbohydrate structure for the two tested Brassica varieties.  Since the two exhibited a mixture of aldehyde and ketone content, as well as a scattering of mono- and disaccharides, these two tests drew no distinction between the varieties of flower buds and pedicels. 

            Bial’s test, the last carbohydrate experiment performed, like the proceeding two tests, the colors the liquids turned upon completion of the experiment were analyzed first.  The tubes for the cauliflower turned a different color, than that of the broccoli (figure 4).  The spectrophotometer absorbance readings for this particular experiment supported our initial conclusions, the furanose ring structure composition for cauliflower, differs from that of broccoli.  Cauliflower contains furanose (five-member ring) sugars whereas broccoli contains hexose (or higher) furanose.  The readings taken were obviously higher for that of the yellow-green cauliflower solution.  The cauliflower than was absorbing less light that of the olive-brown broccoli, demonstrating that different colored solutions found in the Brassica varieties was caused by different carbohydrate structures.

            Pigments are the chemical compounds most important in the conversion of light energy in photosynthesis (Malezski et al., 2002).  The photosynthetic pigment test, through paper chromatography, provides the basis for the hypothesis.  The pigments found in the broccoli where unmistakably different from those of the colorless cauliflower (figure 5).  The cauliflower contains only slight traces of carotene and xanthophylls, whereas the broccoli exhibited four distinct pigments, chlorophyll a and b, xanthophyll and carotene.  The absorbance values are very similar to the pigment test results.  The broccoli has absorbance values very different to that of the cauliflower (table 5).  The absorbance readings from the broccoli were much greater at all wavelengths when compared to that of the cauliflower.  This trend was expected upon the analysis of the paper chromatography test.  The broccoli exhibited more pigments, each with higher Rf values (for the pigments found in both Brassica varieties) than that of the cauliflower.  These results have demonstrated the most notable, and perhaps the most recognizable differences between the two Brassica oleracea verities.

            Salivary amylase is the major enzyme found in the saliva.  The enzyme is secreted from glands within the mouth; its function is to dissolve starches into smaller chains of mono- or disaccharides.  In the final test we examined and compared the conditions of starch breakdowns in the two vegetable varieties.  In the test of water and vegetable extract, the iodine displayed a slight yellow color, revealing that starches had either been completely broken down into their component mono- or disaccharides, or starch was never present in the inflorescence to begin with.  Although the tests yielded identical results, they were in fact deemed inconclusive.  Even though starch was not detected, it could not be attributed to break down by salivary amylase, due to lack of background evidence.  An original trial of the iodine test was never preformed; therefore there was nothing to base the salivary amylase experimental results off of.

Sources of errors in the experiment can be attributed, for the most part, to human error.  Time sensitive experiments, particularly those of the carbohydrate tests were not followed as accurately as they could have been.  Utilizing a stop watch would have given us a more precise timing, than that of solely relying on the wall clock.  Other sources of error include the use of different sample products and how they were treated.  The life of the broccoli and cauliflower samples did not span the length of the experiment.   At times frozen samples were used as opposed to fresh; at others brand new samples were used.  Also due to lack of availability, it was not always possible to purchase the same brand of vegetable to use.  A constant sample could not be kept because of the length of the study.  There were concerns with the effectiveness of some of the experiments.  The prediction was supported only on the basis of pigmentation and a single carbohydrate analysis; other tests are necessary to further support the hypothesis.  If money and available technology was not a limiting factor a more thorough break down of the components of both the broccoli and cauliflower could be compared.  Especially with regards to an analysis of the enzymes that are found in both vegetable, and how, if at all, salivary amylase affects the two varieties.    

 

 

INTRODUCTION: Tara Esshaki, Revised by Matthew Ravenscroft

Second revision by Rachel Disipio

 

            Wild Brassica oleracea is indigenous to the Atlantic seaboard of Europe (e.g. coastlines of Britain and the Bay of Biscay) and the Mediterranean basin. Six main vegetables have been derived from wild stock through selection of cultures: It is professionally supported that one of the reasons it has been possible for people to produce so many vegetables from this one species is because it is geographically diverse so that there is a great genetic diversity from which each environment or people can draw good attributes through selective breeding (Sauer, 1993). 

Use of Brassica oleracea can be traced back to Greek and Roman times, at least 2500 years ago. The Greeks were using non-heading Kale-like vegetables. The Romans in addition were using a loose-headed cabbage as well as a possible precursor to broccoli. However, it is only from about 1600 years ago and onwards that the main varieties (except Brussels sprouts) are clearly referred to in writings. Modern day cultivars of Brassica oleracea have been produced to be more pest resistant and which have solid heads that can withstand mechanical harvesting, shipment and storage. The various forms of Brassica oleracea are mainly cooked or used in salad.  Brassica contain substances called goitrins, which can interfere with the body’s uptake of iodine and encourage goiter (enlargement of the thyroid).  This condition is only a problem in people who have a persistent iodine deficiency (Sauer, 1993).

Organic compounds are a major component in all living things.  They give the living organism its energy to function and survive. For example, sugars are common carbohydrates and are organic because they contain combinations of carbon, hydrogen, and oxygen.  Testing for carbohydrates and their structures is fairly simple because the main task is to identify color change in a reaction (Maleszewski et al. 2002). To compare the structure of a carbohydrate, a sugar test can be performed to identify a reducing sugar, which means it might have a free aldehyde or ketone. Benedict’s test, which is the sugar test for reducing sugars, is one amongst several simple tests that are done to compare the structure of a carbohydrate.  Barfoed’s, Selivanoff’s, Bial’s, Mucid Acid, and Iodine tests are some of the other tests done to check for reducing sugars that are monosaccharides, furanose rings, galactose, or the presence of starch respectively. 

            The testing of photosynthetic contents in living structures is also a valuable comparison device, because all living things require the transformation of light energy to chemical energy and inorganic carbon to organic carbon, which is an essential process in photosynthesis (Maleszewski, et al, 2002).  Pigments are the single most important chemical compounds that assist in the transferring of light to chemical energy.  These pigments can be found within the cells of plants, some of which depend upon the green pigment, called chlorophyll.  By using a solution of blended inflorescence buds from each sample, pigment identification can be performed.  Different plants vary in the number and kind photosynthetic pigments, which is why this can be a useful test in comparing the two bud samples.  The other test was done to determining of the absorption spectrum of the Brassica pigments.  This test will help determine the relative absorption of the pigments at different wavelengths of light, and how the different wavelengths of light are used by the chloroplasts will be analyzed (Maleszewski, et al, 2002).

                        Enzymes are defined as proteins that act as biological catalysts to speed up chemical reactions (Marieb, 1998).  They cause chemical changes under certain limits, they, however, are not the changes themselves.  Enzymes are very sensitive, often only functioning under very narrow limits.  Given their function, enzymes are very important; they make it possible for reactions to take place that might otherwise require biologically extreme temperatures, pH or pressures (Maleszewski et al. 2002).  Salivary amylase is the active enzyme found in saliva.  It begins the chemical digestion of starch in the mouth.  Starch digestion continues until amylase is in activated by stomach acids and inadvertently broken apart by enzymes produced by the stomach (Marieb, 1998).  Given the importance of enzymes, salivary amylase in particular, to relate them to one aspect of the study.  We felt it necessary to compare the way the enzyme salivary amylase breaks down each of the Brassica varieties, broccoli and cauliflower.  Under different temperature conditions (room, a one minute boil and a three minute boil), the enzyme’s ability to break down the respective vegetable sample was tested.  This test will help determine if the rate of break down via salivary amylase differs between the two Brassica oleracea varieties. 

Both the broccoli and the cauliflower are nutritionally important foods, known for their numerous health benefits.  Upon first appearance the two plants seem to have nothing more in common beyond both being members of the same species.  One reason this project was preformed was to see how similar these seemingly opposite plants really were.  Perhaps even more significant was to determine how similar these plants really are so that broad speculation could be made about the study of genetically engineered hybrid of these two vegetables, broccoflower.  The topic of genetic engineering is a sensitive political subject that is becoming more important to study as the production and creation GMOs (genetically modified organisms) is intensified.  The goal of the study is to give more solid background information that may, in the future, serve as a stepping-stone in both the research and developments of new GMOs. 

 

 

                       

 

 

 

Figure 1:  The stock solutions of broccoli (right) and cauliflower (left).  The solutions were prepared by blending 20g of each vegetable with 40mL-distilled water.  The resulting solutions were then filtered through eight layers of cheesecloth to remove the large, pieces.