study on 24 hour root tip cell division mitotic and …1)16/20.pdfdividing of root tips were...

American-Eurasian J. Agric. & Environ. Sci., 16 (1): 172-183, 2016ISSN 1818-6769© IDOSI Publications, 2016DOI: 10.5829/idosi.aejaes.2016.16.1.12821

Corresponding Author: Sanjay Kumar, Department of Botany, Nagaland University, HQs: Lumami 798627, Nagaland, India.

172

Study on 24 Hour Root Tip Cell Division Mitotic andMitotic Phase Index of Allium chinense

Sanjay Kumar and Tsipila Thonger

Department of Botany, Nagaland University, HQs: Lumami 798627, Nagaland, India

Abstract: The plant material (Allium chinense) was collected from kitchen garden of Lumami village (Zunhebotodistrict), Akuluto village (Zunheboto district) and from vegetable market of Mokokchung main town(Mokokchung district), Nagaland (North East India). The meristematic mitotic cells dividing as well as non-dividing of root tips were recorded per hour for 24 hours to characterize mitotic index and mitotic phase indicesin A. chinense. The maximum values of all the indices analyzed were presented as mitotic index, MI(59.85%)and mitotic phases index,MPI [interphase, II (28.19%),prophase PI, (81.25%),metaphase, METAI(22.66%),anaphase, AI (5.23%), telophase, TI (7.99%), cytokinesis, CI (5.52%)]. The correlation between MI andMPI was recorded such as [MI was correlated with METAI (0.601**) at p=0.01, PI and TI was correlated withCI (0.428* and 0.522**) and time (0.631**) at p=0.05 and p=0.01, respectively]. ANOVA has not shown anysignificant differences among the indices at p=0.05]. The goodness of fit regression line (MI, II, PI, METAI, AI,TI and CI) was found as follows R =0.239, 0.0414,.089, 0.2011, 0.0772, 0.568 and 0.4345]. The average movement2

of mitotic and phase index along with residuals (with not much variations) were recorded for the species (perhour/24 hour).

Key words: Mitotic stages Mitotic index Mitotic phase index Correlation Regression Residuals

INTRODUCTION in Himalayan region. Presently, A. chinense was grown in

Allium chinense and A. bakeri Regel are known as and cultivated in field in entire North East regionsynonyms to each other and both belong to the Alliaceae especially Nagaland, Manipur, Arunachal Pradesh,family [1-2]. It has been reported that A. chinense Tripura, Mizoram, Meghalaya, Assam, Sikkim (Westsupports sub-genus cepa in the section of saccuniferum Bengal), Darjeeling (West Bengal). In North East of India[3]. It has been suggested that A. chinense is an edible A. chinense has an important place in mount agricultureuncultivated species of genus Allium [4]. A. chinense was and used as vegetables [8-10],spices and condimentsrecorded with different vernacular names around India [11-13], pickles [14] flavors in various food (soups,such as morkhem (Sangtam tribe of Nagaland), tilhou curries) preparations [15], medicine [16] to cure ailments(Meitei tribe of Manipur), rynsun (khasi tribe of by local healers [17] and ornamental plant [18,12]. It wasMeghalaya) and the world as Chinese Allium, Chinese reported that a quantity of uncultivated species of Alliumscallion, Japanese scallion or oriental onion. It has been were used to introduce new desirable traits to fabricatereported that A. chinense is a tetraploid (2n=4x=32) transgenic plants against pest resistance [19]. The wholeplant but some other plants with deviation in chromosome plant (A. chinense) was sold by the poor farmers andnumbers (2n=3x=24; 2n=24 and 2n=33) were also reported family in the confined local markets for generating incomeby Dutta, M. and M. Bandyopadhyay [3], Srivastava [4], and their livelihood in the regions of North East India. TheDubouzet et al. [5] and Gohil and Kaul [6]. market value and the availability of whole plant, fibrous

In nature, the occurrence and gardening of roots, fresh leaves and inflorescence depends upon theA. chinense was accounted from eastern Asia [7]. budding time and positive climatic environment of theNormally, in India, A. chinense was found in wild in nature plant life.

almost each and every one’s household kitchen garden

Number of dividing cells of phasesMitotic Phase Index % 100Total number of dividing cells

= ×

Total number of dividing cellsMitotic Index % 100Total number of dividing cells + non-dividing cells

= ×

Am-Euras. J. Agric. & Environ. Sci., 16 (1): 172-183, 2016

173

The literature survey on the species showed that The total number of interphase, prophase,some extent of taxonomical, distribution, morphological metaphase, anaphase, telophase and cytokinesisand ethno-botanical work has been reported by Fritsch were recorded from the five different slides forand Friesen [21]. Although a few karyomorphological each hour up to 24 hour (number of stages/h/24 h).studies were reported by Sharma and Gohil [22] and The mitotic index (MI) and other mitotic phase indices,Mukherjee and Roy [23], but different authors suggested MPI (interphase index, prophase index, metaphasethat data were not sufficient for complete description of index, anaphase index, telophase index and cytokinesisthe plant [13]. There was no report available on the index) were calculated from the mean number of stagesphysiological genetics, morphogenetic life cycle, cell from five slides up to 24 hours. Mitotic index and otherdivision cycle, phase indices, nuclear and plant cell indices were calculated according to the formulae givenmorphology in A. chinense which were important belowcharacteristics of a plant cell and helpful to understandthe life cycle of a particular plant.

The aim of the present paper is to understand thecell’s life cycle (per hour index/24hour) of A. chinense interm of mitotic and phase indices (mitotic index,interphase index, prophase index, metaphase index,anaphase index, telophase index and cytokinesis index).

MATERIALS AND METHODS supported by statistically such as Mean, Standard

The material of whole plant A. chinense was collected Regression, ANOVA, two periods average movementfrom kitchen garden of Lumami village, Akuluto village line at a time and Residual graphs using MS Excel, SPSSand from vegetable market of Mokokchung town. The and curve expert.plants were checked at regular interval of time (5 d) forgrowth of the root tips (2-3 cm) long. Root tips were RESULTSstained with 2% carmine (30 minutes), squashed usingconventional root tip squash technique and observed The results of mitotic index (MI), mitotic phaseunder microscope (Leica) for various mitotic stages indices [interphase index (II), prophase index (PI),(interphase, prophase, metaphase, anaphase, telophase metaphase index (Meta I), anaphase index (AI), telophaseand cytokinesis) and capture them with the help of digital index (TI) and cytokinesis index (CI)] in A. chinense wereLeica microscope. presented in Figures 1-20 and Tables 1-2.

The mean number of MI and MPI was

Error, Bar graphs, homogeneity test, Correlation,

Fig. 1: Mitotic Index (%) for 24 hours in A. chinense

A.chinense Residuals

Time (per hour )

Mito

tic In

dex

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.011.012.013.014.015.016.017.018.019.020.021.022.023.024.0-59.85

-49.85

-39.85

-29.85

-19.85

-9.85

0.15

10.15

20.15

30.15

40.15

50.15

60.15


174

Fig. 2: Goodness of fit regression line (df=4) of mitotic index for 24 hours of A. chinense.

Fig. 3: Two periods at a time movement average line of total mean of mitotic index for 24 hours of A. chinense.

Fig. 4: Residual graph of total mean of mitotic index (%) for 24 hours in A. chinense


175

Fig. 5: Interphase Index (%) for 24 hours in A. chinense

Fig. 6: Prophase Index (%) for 24 hours in A. chinense

Fig. 7: Metaphase Index (%) for 24 hours in A. chinense


176

Fig. 8: Anaphase Index (%) for 24 hours in A. chinense

Fig. 9: Telophase Index (%) for 24 hours in A. chinense

Fig. 10: Cytokinesis Index (%) for 24 hours in A. chinense


177

Fig. 11: Goodness of fit regression line (df=4) among interphase, prophase and metaphase indices for 24 hours ofA. chinense.

Fig. 12: Goodness of fit regression line (df=4) among anaphase, telophase and cytokinesis indices for 24 hours ofA. chinense.

Fig. 13: Two periods at a time movement average line of total mean of interphase, prophase and metaphase indices for24 hours of A. chinense.


Time (per hour)

Inte

rpha

se In

dex

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.011.012.013.014.015.016.017.018.019.020.021.022.023.024.0-24.12

-19.12

-14.12

-9.12

-4.12

0.88

5.88

10.88

15.88

20.88

25.88


Time (per hour)

Prop

hase

Inde

x

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.011.012.013.014.015.016.017.018.019.020.021.022.023.024.0-81.25

-61.25

-41.25

-21.25

-1.25

18.75

38.75

58.75

78.75


178

Fig. 14: Two periods at a time movement average line of total mean of anaphase, telophase and cytokinesis indices for24 hours of A. chinense.

Fig. 15: Residual graph of total mean of interphase index for 24 hours in A.chinense

Fig. 16: Residual graph of total mean of prophase index for 24 hours in A.chinense


Time (per hour)

Met

apha

se In

dex

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.011.012.013.014.015.016.017.018.019.020.021.022.023.024.0-22.66

-17.66

-12.66

-7.66

-2.66

2.34

7.34

12.34

17.34

22.34


Time (per hour)

Ana

phas

e In

dex

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.011.012.013.014.015.016.017.018.019.020.021.022.023.024.0-5.23

-4.23

-3.23

-2.23

-1.23

-0.23

0.77

1.77

2.77

3.77

4.77


Time (per hour)

Telo

phas

e (p

er h

our)

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.011.012.013.014.015.016.017.018.019.020.021.022.023.024.0-7.99

-5.99

-3.99

-1.99

0.01

2.01

4.01

6.01

8.01


179

Fig. 17: Residual graph of total mean of metaphase index for 24 hours in A.chinense

Fig. 18: Residual graph of total mean of anaphase index for 24 hours in A.chinense

Fig. 19: Residual graph of total mean of telophase index for 24 hours in A.chinense


Time (per hour)

Cyt

okin

esis

Inde

x

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0-5.52

-4.52

-3.52

-2.52

-1.52

-0.52

0.48

1.48

2.48

3.48

4.48

5.48


180

Fig. 20: Residual graph of total mean of cytokinesis index for 24 hours in A.chinense

Table 1: Pearson correlation of indices (%) and time for 24 hours in A.chinense.Mitotic Interphase Prophase Metaphase Anaphase Telophase Cytokinesis

Items index index index index index index index TimeMitotic Index 1 .063 .057 .601** .229 .190 .053 .160Interphase Index 1 .164 .043 .373 .078 .043 .136Prophase Index 1 .063 .051 .241 .428* .164Metaphase Index 1 .172 .103 .024 .044Anaphase Index 1 .215 .343 .187Telophase Index 1 .522** .631**Cytokinesis Index 1 .323Time 1**. Correlation is significant at the 0.01 level (2-tailed).*. Correlation is significant at the 0.05 level (2-tailed).

Table 2: Analysis of variance (ANOVA) among indices (%) for 24 hoursof A.chinense.

Items Sum of squares df Mean squareMitotic indexBetween groups 1232.969 23Within groups .000 0Total 1232.969 23 53.607Interphase indexBetween groups 986.186 23Within groups .000 0Total 986.186 23 42.878Prophase indexBetween groups 6373.739 23Within groups .000 0Total 6373.739 23 277.119Metaphase indexBetween groups 739.145 23Within groups .000 0Total 739.145 23 32.137Anaphase indexBetween groups 69.688 23Within groups .000 0Total 69.688 23 3.030Telophase indexBetween groups 107.009 23Within groups .000 0Total 107.009 23 4.653Cytokinesis indexBetween groups 51.242 23Within groups .000 0Total 51.242 23 2.228

DISCUSSION

The plant cell’s life cycle were described anddiscussed in the form of mitotic and mitotic phases indexper hour per 24 hour in A. chinense.

The growing root tip of a seedling i.e. the number ofdividing meristematic cells may be associated with theactual time period of the mitosis in the cell division cycle.Also, the number of the mitotic cells in one of the divisionphases is related to the time used by this phase in relationto the total length of the mitosis. Therefore, mitotic index(number of cells in division as a percentage of the totalmeristematic cells) and phase index (percentage of thecells in the several phases of division) was used as ameasure to know the actual time period and time utilizedby the phase in mitosis in relation to the total length ofcell division cycle.

The total mean number of mitotic index (%) werecalculated (mitotic index/hour/24 hour) and maximum(59.85±) and minimum (30.14±) values at (6 AM) and (4AM) were recorded with approximate mitotic index range(=30 % mitotic index =60%), respectively. The meannumber of mitotic index were analyzed for thehomogeneity of the data collection (mitotic index/hour/24


181

hour) and it was observed that all the data were maximum (28.19, 81.25, 22.66, 5.23, 7.99 and 5.52) andhomogeneous and similar (not diversed) (alphabets, a, b,c). The high mitotic index suggests that cells weredividing at very fast rate and highly active (6 AM) ormoderate rate at other time. The high mitotic index alsosupports the increase in number of mitotic cells whichcontributes to the growth and development of the planttissues as well as whole plant (Fig. 1). The results are inagreement with those reported by Khodjakov and Rieder[24]. The total mean number of mitotic index (mitoticindex/hour/24 hour) was correlated with other mitoticphase indexes and it was moderately correlated (0.601**)with metaphase index at p=0.05 which supports thenumber of metaphase stage available at all the 24 hourtime and active participation of metaphase index formitotic index in cell division cycle (Table 1). The analysisof variance (ANOVA) of total mean number of mitoticindex was found non significant with mean square value(53.607) and sum of square values between (1232.969) andwithin (0.00) group at p=0.05 respectively (Table 2). Thegoodness of fit regression line (df=4) was drawn andmean number of mitotic index have shown the fitness(23.99 %) of the line (y=0.001 x -0.084x +1.265x -4 3 2

6.68x+53.88). The less fitness of the regression line withmitotic index values may be due to the diverse nature ofthe dividing and non dividing mitotic cells of the differentstages (interphase, prophase, metaphase, anaphase,telophase and cytokinesis) of cell division cycle (Fig. 2).The two period’s average line at a time movement wasdrawn and the movement of the mitotic index was mixed aslow (1-5 AM), high or elevated (5-8 AM) and sometimesalmost constant. The constant nature of mitotic indexmovement supports that most of the mitotic cells were inresting stage (G , G , S and G ) and almost constant for the0 1 2

synthesis and preparation for the next cell division cycle(resting stage/hour/24 hour) (Fig. 3). The residual graphshowed that most of our collected data (mitotic index perhour per 24 hour) more or less near to the residual line andshowed constancy or similarity in data collection. Theresiduals of mitotic index were less disturbed or moreacceptable as all the residual points were very near orclose to the residual line. Also, some more standardizeddata collection method required to convert into perfectstandardized data which may be very near or close oroverlap to the residual line (Fig. 4).

The total mean number of mitotic phase indexes(interphase index, prophase index, metaphase index,anaphase index, telophase index and cytokinesis index)were calculated (mitotic phase index/hour/24 hour) and

minimum (3.38, 5.09, 3.46, 0.0, 0.0 and 0.0) percent valuesat (3 AM, 12 AM, 4 AM, 2 AM, 9 AM and 9 AM) and (2PM, 9 AM, 1 AM, 4&5 AM, 4&5 AM and 5 AM) wererecorded, respectively. The mean number of mitotic phaseindexes were analyzed for the homogeneity of the datacollection (mitotic phase index/hour/24 hour) and it wasobserved that all the data were homogeneous and similar(not diversed) with little or negligible variation (alphabets,a, b, c). The high mitotic phase index (Prophase index)suggests that mitotic cells were dividing at very fast rateand highly active (12 AM) and low mitotic phase indexes(anaphase index, telophase index and cytokinesis index)showed that mitotic cells were not dividing at very fastrate or not highly active which gives the lower index (4and 5 AM). The high mitotic phase index also supportsthe increase in number of mitotic cells which contributesto the growth and development of the plant tissues aswell as whole plant [25] (Fig. 5-10). The total mean numberof mitotic phase indexes (interphase index, prophaseindex, metaphase index, anaphase index, telophase indexand cytokinesis index) were correlated and it wasobserved that prophase index moderately correlated withcytokinesis index (0.428*) at p=0.05, while telophase indexmoderately correlated with cytokinesis index (0.522**)and time (0.631**) at p=0.01. The correlation suggestedthat number of cytokines is cells (production of twodaughter cells) has significant role for prophase index aswell as telophase index. Also, the maximum number oftelophase cells may be observed all the time whichcontributes for the telophase index (telophasestage/hour/24 hour). It also ensures that once prophasemitotic cells progress through the mitotic cell cycle stagesand achieves telophase stage it must undergo forcytokinesis and which completes the mitotic cell cycle(Table 1). The analysis of variance (ANOVA) of totalmean number of mitotic phase indexes (interphase index,prophase index, metaphase index, anaphase index,telophase index and cytokinesis index) was found non-significant with mean square value (42.878, 277,119,32.137, 3.03, 4.653 and 2.228) and sum of square valuesbetween (986.186, 6373.739, 739.145, 69.688, 107.009 and51.242) and within (0.00) group at p=0.05, respectively(Table 2). The goodness of fit regression line (df=4) wasdrawn and total mean number of mitotic phase indexes(telophase index) have shown maximum fit (56.80 %) of theline (y=0.0002x -0.0125x +0.2546x -1.5837x+4.7969)4 3 2

followed by cytokinesis index (43.45%), metaphase index


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study on 24 hour root tip cell division mitotic and …1)16/20.pdfdividing of root tips were...

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