BIO131-Special Project Body

Comparative Analysis of the Meiotic Activity of Representative Species under Family Asteraceae (Tridax sp., Tagetes sp., and Tithonia sp.)1

AALA, Wilson F.

ABSTRACT

Cytological studies on certain plant species can help in subsequent researches as well as in the proper taxonomy of these plant species. Family Asteraceae boasts a diverse group of plant species and certain meiotic features may help in its taxonomy. Results of the study suggest that among the parameters that were observed (meiotic rate, frequency of meiotic aberrations, and pollen fertility) meiotic rate data between Tridax sp., Tagetes sp., and Tithonia sp. could be used to discriminate between the three species. Also, even though the pollen fertility for the three species are essentially similar (for the present data only), the rate of meiosis among the three species are varying; and this variation in the rate of meiosis could actually be an isolating mechanism that keeps these species diverged from each other.

INTRODUCTION

I – Review of Literature

The Family Asteraceae (Compositae) is grouped along with Apiaceae,

Umbelliferae, and Aquifoliaceae to form the Euasterids II clade. Euasterids II, on the

other hand, is grouped along with Euasterids I clade to form the Core Asterids

(Sympetalae) (Martinez-Goss, Torreta, Nacorda, Gruezo, Buot Jr., & Cuevas, 2006).

Most, if not all, of the plant species under this family are dicots, or more

recently known as eudicots; having four or five (can also be multiples of either)

floral parts; with the vascular bundles arranged in rings around the stem; and root

hairs that is a branched taproot; while its leaves are veined or netted; and, its most

1A scientific paper submitted in partial fulfillment of the requirements in Biology 131, Section AB-1L, under Professor Neilyn O. Villa during the 2nd Semester, SY 2010-2011.

striking feature, pollen grains with three slits or pores also known as a tricolplate

pollen (Brooker, Widmaier, Graham, & Stiling, 2008; Metcalfe & Chalk, 1972).

Morphologically speaking, there are plant species under the said family that

are similar in features which would render the identification process difficult (Foster

& Gifford Jr., 1974). For this reason, cytological means can be utilized to classify

and/or delineate certain plant species. Cytologically speaking, there are various

means that could be utilized for the said purpose with which one of the easiest to

observe is cell division or more specifically, meiosis.

Meiosis is usually observed during plant gametogenesis where the plant

produces the ovule and the pollen grain in the ovaries and anthers respectively.

Between those two floral parts – anthers and ovaries – it is most practical to use the

anthers since it is easier to acquire and requires less processing (Mendioro, dela

Viña, & Villa, 2010). The cytological importance of meiosis in plant study stems from

the fact that each plant species will undergo the same process, meiosis, but at

different rates, time of the day, and forms (Hartl & Jones, 1999).

Meiosis is one of the two common paths for cell division with which the other

one is mitosis. Meiosis is divided into two phases, namely Meiosis I and Meiosis II.

The Meiosis I phase is further divided into sub-stages which are: Prophase I,

Metaphase I, Anaphase I, and Telophase I. Furthermore, the Prophase I sub-stage

has sub-phases namely: Leptonema, Zygonema, Pachynema, Diplonema, and

Diakinesis, which occurs during Prophase I in that order. The Meiosis II phase has

the same sub-stages as that of Meiosis I. Of these stages, Prophase I, Metaphase I,

and Anaphase I provide the most useful cytological characteristics pertaining to

meiotic activity (Hartl & Jones, 1999).

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One of these cytological characteristics is the species’ chromosome number

which could be observed during the Diakinesis (Prophase I) stage. With this data, it

would be easy to systematize plant classification and study since chromosome

number is a characteristic of each species. Reported chromosome numbers for

Tithonia diversifolia, Tridax procumbens, and Tagetes erecta are 2n = 34, 2n = 36,

and 2n = 24 respectively (Kobe University Library Digital Archive, 2011).

Chromosomal aberrations/rearrangements can occur during meiosis, which is

a characteristic of each plant species, and can be observed cytologically. In this

regard, there are three common configurations that can be observed: 1.) chain

configuration; 2.) ring configuration (both 1 and 2 can be observed during the

Diakinesis sub-stage of Prophase I); and 3.) laggard formation during Metaphase 1

or Anaphase I. In a similar study conducted in Rhoeo spathacea (Swatz) Stern, a

twelve-ringed chromosome configuration was reported to be the most frequent

chromosomal arrangement, while the least frequent was said to be the laggard

formation (Lin & Paddock, 1973; Lin Y. J., 1979). Such results could reflect the

meiotic profile of the target species which could provide a lot of information that

could be used for its classification.

Another cytological characteristic that is tied with meiosis is pollen fertility.

Pollen grains, as is already known, are the product of meiosis and if an atypical type

of meiosis should occur, not all of those meiocytes would become fertile pollen

grains (Erdtman, 1969; Hartl & Jones, 1999). During reciprocal translocations there

are three ways by which chromosomes can segregate during Anaphase I namely:

Alternate segregation and the Adjacent 1 & 2 segregation. Both the Adjacent 1 & 2

segregation products will yield chromosomes with deleted or duplicated segments

after Anaphase I. The gametes coming from these segregation events would

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therefore be sterile. On the other hand, the Alternate segregation products will be

chromosomes that do not contain deleted and/or duplicated segments; thus the

gametes coming from this segregation event after Anaphase I are fertile ones.

Accounting all these segregation events, gametogenesis should theoretically

produce fertile gametes one-third of the time. It should be noted, however, that the

Adjacent 2 segregation event is a rare event rendering its contribution on

gametogenesis to be insignificant. With this said, the probability of acquiring a

fertile gamete has now increased to 50% coming from the Alternate segregation

event. Pollen fertility data, when used in conjunction with the meiotic rate data,

could help shed light on the actual rate of meiosis in the plant species that will be

studied.

Studies that focus on this family (Asteraceae) are vast, though it was

observed that the representative species used for these researches are mostly

those plant species that hold economic or medicinal value, while those plant species

that have little or none of these characteristics are only chosen for taxonomic

studies. This study will determine the specific meiotic activity of Tridax sp., Tagetes

sp., and Tithonia sp. as a primer for other subsequent studies that will focus on

these plant species. This study will be conducted on Rm. C-321, Biological Sciences

Bldg., UPLB – Los Baños, Laguna and it will be conducted from February 16 to March

9, 2011.

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II – Significance of the Study

Meiotic studies, not only on family Asteraceae but on other plant groups,

could prove useful in the field of taxonomy as well as in cytogenetics. Once the

meiotic profile is determined for a target species, a great deal of information would

then be available (which includes pollen fertility, meiotic rate, and atypical meiotic

rate), which are critical in characterizing the plant species that are being studied.

This study was designed to determine and compare certain meiotic features of

Tridax sp., Tagetes sp., and Tithonia sp.

III – Objectives

This study will characterize and compare the meiotic features of Tridax sp.,

Tagetes sp., and Tithonia sp. Specifically, the study was conducted to:

1. Characterize the meiotic activity of Tridax sp., Tagetes sp., and Tithonia

sp.;

2. Compare the meiotic activity of Tridax sp., Tagetes sp., and Tithonia sp.;

and

3. Explain the correlation of meiotic aberration with pollen fertility data.

IV – Date and Place of Study

This study was conducted on Rm. C-321, Biological Sciences Bldg., UPLB – Los

Baños, Laguna and it will be conducted from February 16 to March 9, 2011.

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METHODOLOGY

A. Materials

Florets and pollen samples from the following plant species:

Tagetes sp.

Tridax sp.

Tithonia sp.

Farmer’s solution:

3 parts 95% Ethanol

1 part Glacial Acetic acid

Alcohol series:

80%, 90%, 95%, and 100% Ethanol

70% Ethanol

Xylene

Canada balsam

Acetocarmine dye

45% Acetic acid

I2KI stain

FeCl3 (mordant)

Paraffin wax

Alcohol lamp

Bent/straight needle

Forceps

Slides/cover slips

Compound microscope

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B. Methods

I. Specimen Collection

Young florets of each of the three plant species (Tithonia sp., Tridax

sp., and Tagetes sp.) will be collected at around 10:30 am to 11:30 am and

will be immersed in a freshly prepared Farmer’s solution, after which a

mordant, FeCl3, will then be added. The florets will be fixed for approximately

24 hours after immersion. Once the 24 hours has expired, the florets will be

washed with distilled water and will then be transferred in a 70% Ethanol

solution.

II. Preparation of Temporary Mounts

A floret, for each of the plant species considered, that was previously

fixed with Farmer’s solution will be obtained and dissected directly in a slide.

The floret part that is of interest would be the anthers. A light cream anther

would best serve the purposes of this experiment. Once an ample amount of

the anthers are acquired, a drop of acetocarmine dye will be added to the

slide and subsequent squashing of the anthers will be done. Once a good

squash preparation is obtained, a cover slip will be put on top of the target

area and removal of the excess stain will be done via a piece of absorbent

paper. Heating of the slide will then be carried out using an alcohol lamp. If

an overly-stained mount was obtained at this stage, a destaining agent, 45%

Acetic acid, will be applied to lighten the staining of the cells. Sealing of the

edges of the cover slip using melted paraffin wax will be carried out once an

acceptable mount is obtained.

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Labeling and documentation of the slide will be done thereafter

wherein the following parameters will be observed: 1.) stages of meiosis; 2.)

meiotic rate; 3.) rate of atypical meiosis; and 4.) chromosome number.

Meiotic rate will then be computed as follows:

meiotic rate=( number of dividing cellstotalnumber of cellsobserved )×100

The same formula will be used for the rate of atypical meiosis, only

substituting the number of atypical meiocytes for the numerator.

III. Pollen Fertility

Pollen grains will be collected from the flower of each of the

representative species and will be transferred to a clean slide. A drop of I2KI

stain will be added to the portion of the slide containing the pollen grains and

after which, a clean cover slip will be placed on top of the area containing the

sample. Labeling and documentation of the slide will be done thereafter.

Also, the percent pollen fertility would be computed via the formula:

%pollen fertility=( number of fertile pollengrains¿

total number of pollengrains observed )×100

*fertile pollen grains are ones that will appear to be darkly

stained under the microscope; sterile pollen grains will appear

collapsed and unstained.

IV. Preparation of Permanent Mounts

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Slides that were temporarily mounted will have their paraffin seal

removed via a blade. Once this is achieved, the slides will then be subjected

to an alcohol series (70% 80% 90% 95% 100%) and two changes

of xylene thereafter. Each of these steps will require 2-5 min of drying time.

After which, an ample amount of the mounting agent, Canada balsam, will be

placed on the target area and the cover slip will then be replaced on the

slide.

RESULTS AND DISCUSSION

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Various meiotic parameters were observed in this experiment mainly: meiotic

rate, frequency of meiotic aberrations, and pollen fertility for Tithonia sp., Tridax

sp., and Tagetes sp. Each prepared mount was documented and results of the

subsequent computations were summarized in tables. The succeeding sections

would make use of relevant photomicrographs or the three species mentioned and

for this reason, sample photomicrographs for the complete stages of meiosis for

each of the plant species used is summarized in Table 1.

A. Chromosome Number Identification

As was said in the previous section, the chromosome number could be

identified in meiocytes that are in the Diakinesis (Prophase I) stage. Tithonia sp. has

relatively large chromosomes which make for an easy counting of chromosomes for

this species. Almost all of the meiocytes that were observed to be at the Diakinesis

stage registered a chromosome number of 2n = 17II = 34. This is at par with the

chromosome number for this species as is reported in Carr, King, Powell, &

Robinson’s (1999) study. Tridax sp. meiocytes were quite small when compared to

Tithonia sp. and also, a good Diakinesis-stage meiocyte with well-spread

chromosomes was not found; however, a ring formation involving all the

chromosomes of Tridax sp. was obtained, and through this it was ascertained that

the chromosome number of Tridax sp. is 2n = 18II = 36. The Diakinesis-stage

meiocytes for Tagetes sp. showed that the chromosome number for this plant

species is 2n = 12II = 24, which is similar to previously reported chromosome

number for this plant species (Carr, King, Powell, & Robinson, 1999).

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Table 1. Colored photomicrographs of the various stages in Meiosis I & II of Tithonia sp., Tridax sp., and Tagetes sp.

MEIOTIC STAGEPLANT SPECIES

Tithonia sp. Tridax sp. Tagetes sp.

Meiosis I

Prophase I (Diakinesis)

Metaphase I

Anaphase I

Telophase I

Meiosis II

Prophase II -

Metaphase II

Anaphase II -

Telophase II

B. Rate of Typical and Atypical Meiosis

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Another parameter that was considered in this study is the meiotic rate.

Table 2 summarizes the data obtained for the frequency counts for the meiocytes

that are undergoing typical and atypical meiosis for the three plant species that

were used.

Table 2. The frequencies and percentages of the typical and atypical meiosis observed for Tithonia sp., Tridax sp., and Tagetes sp.

SAMPLE

NORMAL DIVISION

ATYPICAL DIVISIONRING/S CHAIN/S LAGGARD/S

FREQ

N %FREQ

N %FREQ

N %FREQ

N %

Tithonia sp.

35 5070%

2 50 4% 5 5010%

8 5016%

Tridax sp. 31 5062%

8 5016%

11 5022%

0 50 0%

Tagetes sp.

40 5080%

3 50 6% 6 5012%

1 50 2%

As can be seen in Table 2, the plant species that registered the highest

number of meiocytes undergoing typical meiosis is the Tagetes. sp., while Tridax.

sp. was observed to have the least number of meiocytes undergoing typical

meiosis.

Considering the columns for atypical meiosis, it can be seen that Tridax. sp.

has the highest frequency (the frequency considered here is the summation of the

frequency for the rings, chains and laggards), while it was the Tagetes. sp. that

showed the least frequent. It can also be seen that for Tithonia. sp., the laggard

configuration was the most frequent at 16%, while the least frequent was the ring

configuration at 4%. For the Tridax sp. the chain configuration was the most

frequent type of aberration at 22%, while the laggard configuration was not

observed at all in any one of the slides that were prepared. The chain configuration

was also the most frequent aberration observed for Tagetes sp.

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C. Chromosome Configurations during Atypical Meiosis

During atypical meiosis, abnormal behavior of the chromosomes could be

observed, which includes the formation of rings, chains and laggards. For Tithonia

sp., the laggard formation was found to be the most frequent although no reason

could be offered so as to account for this occurrence. A chain-of-thirty four

chromosome configuration with a single chiasma failure was the most frequent for

the chain configuration. Photomicrographs of the chain, and laggard formation in

Tithonia sp. is shown in Figure 1. Tridax sp. has shown a high frequency of chain

formation and a few of ring configurations. Of all the chain configurations that were

observed, the chain-of-thirty six was the most frequent, while a ring system

involving all the chromosomes was the ring configuration that was most frequent.

Figure 2 provides some sample photomicrographs of the various aberrations that

were observed for Tridax sp. In Tagetes sp., chain configurations of differing lengths

were observed to have although the most frequent is the configuration involving 24

chromosomes. Ring configurations were also observed for Tagetes sp. involving 24

chromosomes. Sample photomicrographs of the meiotic aberrations observed for

Tagetes sp. is shown in Figure 3.

Reasons as to why and how these aberrations have occurred in these three

species are unclear. Some references point to environmental influences that might

affect the frequency of atypical meiosis. In one study conducted by Cequea, de

Cequea, Imery, & Nirchio (2003), in Tridax procumbens, they have indicated that

the presence of meiotic aberrations show that these plants were heterozygous for a

paracentric inversion, which could involve one or more homologous chromosomes.

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a. b.Figure 1. Tithonia sp. undergoing atypical meiosis: a.) chain of 34 chromosomes;

b.) laggard formation.

a. b.Figure 2. Tridax sp. undergoing atypical meiosis: a.) chain of 36 chromosomes; b.)

ring of 36 chromosomes.

a. b. c.Figure 3. Tagetes sp. undergoing atypical meiosis: a.) chain formation; b&c.) ring of

24 chromosomes.

D. Pollen Fertility Data

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One of the many cytological data that are linked with meiosis is pollen

fertility since the behavior of the chromosomes during Anaphase I, and how they

would segregate, will indicate the relative amounts of sterile and fertile pollen. For

the three species, the pollen fertility was done using three replicates per species

and the results of the count data are shown in Table 4.

Table 4. The frequencies and percentages of the fertile and sterile pollen grains for Tithonia sp., Tridax sp., and Tagetes sp.

SAMPLE

REPLICATES AVERAGE%POLLEN FERTILIT

Y*

1 (N = 50) 2 (N = 50) 3 (N = 50)FERTIL

ESTERIL

E

%FERTILIT

Y

FERTILE

STERILE

%FERTILIT

Y

FERTILE

STERILE

%FERTILIT

Y

Tithonia sp.

10 40 20% 11 39 22% 18 32 36% 26%a

Tridax sp.

14 36 28% 19 31 38% 10 40 20%28.67

%a

Tagetes sp.

5 45 10% 14 36 28% 25 25 50%29.33

%a

*Values in a column with different letter superscripts are significantly different from each other at 0.05% confidence level.

As can be clearly seen in Table 4, Tithonia sp. showed the lowest average

pollen fertility rate, while Tagetes sp. yielded the highest value at 29.33%. The

average pollen fertility value obtained for each plant species was analyzed in order

to ascertain if the differences in the values are significant or not using a Two-tailed

t-Test at 0.05% confidence level. Results of the analysis revealed that the

differences in the values for the average pollen fertility of each plant species are not

significant. This result, though, does not exclude the possibility that increasing the

number of replicates per sample might give a different trend.

SUMMARY AND CONCLUSION

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This study seeks to determine and compare the meiotic activities of three

representative species under the Family Asteraceae namely: Tithonia sp., Tridax

sp., and Tagetes sp. with focus on meiotic rates, chromosome configurations, and

pollen fertility.

A critical data that could be obtained in any study that is of the same focus

as with this study would be the chromosome number elucidation. The results of this

study indicates that Tithonia sp. has a chromosome number of 2n = 34; Tridax sp.

with 2n = 36; and Tagetes sp. having 2n = 24. These data are similar to the existing

records for the chromosome numbers that were previously published.

Meiotic rate data – that of typical and atypical types – could help shed light

on the actual number of meiocytes that will develop into mature pollen grains. The

results of this study has shown that the meiotic rates for the three species varies

largely, while the atypical meiocytes that were observed among the plant species

used has shown differences on the meiotic aberration that is of highest frequency

(between rings, chains and laggards).

The pollen fertility data has shown that the differences in the values among

the three species were not significant. Combining this data with the meiotic rate

data, it can be said that even though the pollen fertility for the three species are

essentially similar (for the present data only), the rate of meiosis among the three

species are varying; and this variation in the rate of meiosis could actually be an

isolating mechanism that keeps these species diverged from each other.

RECOMMENDATIONS

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This study was conducted in order to provide initial data for the meiotic

activity of Tithonia sp., Tridax sp., and Tagetes sp. under Family Asteraceae. Further

studies could focus on a broader range of genus; or to a more narrow range – that is

to say that the subsequent researches could focus on only a single species.

The accurate elucidation of the pollen fertility data entails that a large

number of replicates – around 50 and above – should be used. It is also suggested

that a better statistical method should be employed to analyze the data even

better.

Staining techniques, protocols, as well as slide preservation methods should

also be improved in order to obtain well stained and well spread chromosomes.

APPENDICES

APPENDIX A: Statistical analysis of the pollen fertility data for Tithonia sp., Tridax sp., and Tithonia sp.

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APPENDIX B: Additional Photomicrographs

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1 2 3 Mean VarianceTithonia 20 22 36 26.00 76.00Tridax 28 38 20 28.67 81.33

Mean difference = Covariance = -2.67 78.67

l tc l = ttab =0.368 4.303

1 2 3 Mean VarianceTithonia 20 22 36 26.00 76.00Tagetes 10 28 50 29.33 401.33

Mean difference = Covariance =-3.33 238.67

l tc l = ttab = 0.264 4.303

1 2 3 Mean VarianceTridax 28 38 20 28.67 81.33Tagetes 10 28 50 29.33 401.33

Mean difference = Covariance =-0.67 241.3333333

l tc l = ttab = 0.053 4.303

l tcl < ttab : not significant



a. b. c.Figure 4. Photomicrographs of pollen grains of a. Tithonia sp.; b. Tagetes sp.; c. Tridax sp. stained with I2KI where the darkly staining pollen grains are the fertile

ones.

REFERENCES

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Brooker, R., Widmaier, E., Graham, L., & Stiling, P. (2008). Biology (International ed.). New York: The McGraw-Hill Co., Inc.

Carr, G., King, R., Powell, A., & Robinson, H. (1999). Chromomosome numbers in Compositae XVIII. American Journal of Botany , 86 (7), 1003–1013.

Cequea, H., de Cequea, D., Imery, J., & Nirchio, M. (2003). Cytogenetic study of paracentric inversions in Tridax procumbens (Compositae). Cytologia , 63 (4), 329-333.

Erdtman, G. (1969). Handbook of Palynology. Denmark: Hafner Pub. Co., Inc.

Erdtman, G. (1969). Handbook of Palynology: An introduction to the study of pollen grains and spores. Denmark: Hafner Pub. Co., Inc.

Erdtman, G. (1966). Pollen morphology and plant taxonomy: Angiosperms (An introduction to Palynology I). U.S.A.: Hafner Pub. Co., Inc.

Erdtman, G. (1966). Pollen morphology and plant taxonomy: Angiosperms (An introduction to Palynology I). U.S.A.: Hafner Pub. Co., Inc.

Faegri, K., Iversey, J., & Waterbolk, H. (1990). Textbook of Pollen Analysis (2nd ed.). Denmark: Hafner Pub. Co., Inc.

Foster, A. S., & Gifford Jr., E. M. (1974). Comparative morphology of vascular plants (2nd ed.). U.S.A.: W.H. Freeman & Co.

Foster, A., & Gifford Jr., E. (1959). Comparative Morphology of Plants. U.S.A.: W.H. Freeman & Co.

Hartl, D., & Jones, E. (1999). Essential Genetics (2nd Edition ed.). Massachusetts: Jones & BarTlett Publishers.

Kobe University Library Digital Archive. (2011, January 25). Index to Chromosome numbers in Asteraceae. Retrieved February 2011, from http://www.lib.kobe-u.ac.jp

Lin, Y. J. (1979). Chromosome distribution and catenation in Rhoeo spathacea var. concolor. Chromosoma , 109-127.

Lin, Y., & Paddock, E. (1973). Ring-Position and frequency of chiasma in Rhoeo spathacea. American Jounal of Botany , 60 (10), 1023-1027.

Martinez-Goss, M., Torreta, N., Nacorda, J., Gruezo, W., Buot Jr., I., & Cuevas, V. (2006). Botany 3: Laboratory Manual. Los Baños: UPLB.

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Mendioro, M., dela Viña, C., & Villa, N. (2010). Laboratory manual in Cytogenetics. Los Baños: GMBD-IBS-UPLB.

Metcalfe, C., & Chalk, L. (1972). Anatomy of the Dicotyledons: Leaves, stems and wood in relation to taxonomy with notes on economic uses (Vol. II). Great Britain: Oxford University Press.

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