J. CYTOL. GENET. VOL. 17 (NS), NOS 1 & 2, 2016 1

MITOTIC INDEX AND PHASE INDEX IN AGRIFOUND WHITE AND YAMUNASAFED VARIETIES OF ALLIUM SATIVUM LINN.

SUKESHINI D. DEOGADEI AND P. N. NASARE2*IDepartment of Botany, Hislop College, Civil Line, Nagpur 440 001

2Department of Botany, Nilkanthrao Shinde Science and Arts College, Bhadrawati 442 902*For correspondence. Email: pnnasare@rediffmail.com

(Received 16 January 2016, revised accepted 10 February 2016)

SUMMARY

Mitotic cell division was studied in root tips of Agrifound white and Yamunasafed varieties of Allium sativum. In Agrifound white, maximum metaphases (9.1%)and anaphases (5.15%) have been observed in root tips fixed at 9.00–9.30 a. m. andin Yamuna safed, maximum metaphases (10.1%) and anaphases (7.3%) have beenobserved in root tips fixed at 9.30–10.00 a. m. The data on mitotic and phase indicesat different time intervals have been analysed.

Keywords: Allium sativum, mitosis, mitotic index, phase index.

INTRODUCTION

The genus Allium (Amaryllidaceae) comprises about 700 species. Several of them arecultivated for vegetables, spices and medicinal purpose. A. sativum (garlic) is a popular condimentcultivated all over the world.

Bulbs of Agrifound white (G-41) variety of garlic are compact and silvery white withcreamy flesh. Each bulb of 3.5–4.5 cm diameter has 20–25 elongated cloves. This variety has beenrecommended for Maharashtra and Madhya Pradesh.

Bulbs of Yamuna safed are creamy white measuring 4.5–6 cm in diameter. The number ofcloves per bulb is 15–16. It is recommended for Madhya Pradesh, Maharashtra, Haryana, Gujarat,Punjab, Rajasthan, Uttar Pradesh and Chhattisgarh.

A number of plant species have been subjected to mitotic studies and phase indices. Theyinclude Foeniculum vulgare (Jahagirdar 1975), Coriandrum sativum (Gaikwad 1975), Carumcopticum (Khanolkar 1977), Solanum nigrum (Kothekar 1978), Trigonella foenum-graecum (Devi1990), pearl millet (Bansikar & Srivastava 1992), Linum usitatissimum (Karpate 1995), Cyamopsistetragonoloba (Kanaklata 1995), Lathyrus sativus (Girhe 1999), Brassica napus (Landge 2000),Lens culinaris (Yasar & Ahmed 2006), Triticum aestivum (Sanjaykumar et al. 2010), Capsicum

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J. CYTOL. GENET. VOL. 17 (NS), NOS 1 & 2, 20162

annuum (Yadav & Yadav 2010), Ocimum sanctum (Nasare & Choudhary 2010), Hibiscusrosa-sinensis (Ozmen 2010), Treculia africana (Osuji & Ower 2010), Saccharum (Shrivastava& Jain 2011), Phaseolus vulgaris and Raphanus sativus (Truta et al. 2011), Vigna unguiculata(Amirthalingam et al. 2013) and A. cepa (Paul et al. 2013, Udo et al. 2014, Pankaj et al. 2014).

The present study was designed to study the mitotic activity and phase index in the rootmeristems of var. Agrifound white and var. Yamuna safed of A. sativum to find out the maximummitotic activity at specific time for each variety.

MATERIALS AND METHODS

Agrifound white and Yamuna safed varieties of A. sativum were procured from Dr. Punjabrao Deshmukh KrishiVidyapeeth, Akola (M.S.) and National Horticulture Research Development and Foundation, Nashik (M.S.) respectively.Healthy cloves of A. sativum varieties Agrifound white and Yamuna safed were selected and kept for germination onthe jars filled with water. After 4 d, root tips were harvested at different intervals of time from8–11 a. m. and fixed in Carnoy’s fixative (1:3, glacial acetic acid:absolute alcohol) for 24 h. Fixed root tips werewashed with distilled water and macerated in 1N HCl for 10–15 min at 60o C in hot air oven. Macerated roottips were transfered to 2% acetocarmine for 20 min. Stained root tips were then squashed in acetocarmine and sealedwith paraffin wax. The chromosome spreads were then observed microscopically. The cells were scored for the differentcell division stages, number of total dividing cells, number of prophases, metaphases, anaphases and telophases. Duringmitotic cycles of the cell division at different intervals of time, data were prepared to calculate mitotic index andphase indices by following formulae of Bhatta & Sakya (2008).

The different phases of mitosis were counted to calculate the mitotic index and phase indices by adopting thefollowing method:

Mitotic Index = TDC x 100/TCPI% = prophase cells x 100/TDCMI% = metaphase cells x 100/TDCAI% = anaphase cells x 100/TDCTC% = telophase cells x 100/TDC,Phase index (PI) = TC x 100/TDCwhere

TC = total number of cells (dividing and non-dividing) and TDC = total dividing cells.

OBSERVATIONS

The mitotic activity occurring in a span of 3 h between 8 and 11 a. m. in the root tips ofAgrifound white and Yamuna safed varieties of A. sativum has been recorded and the periodicevents recorded (Tables 1, 2, Figs 1, 2).

DEOGADE & NASARE:

1

2

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DEOGADE & NASARE:

TABLE 1: Mitotic index and phase index in root tip of A. sativum var. Agrifound white.

Time (a. m.) TC TDCProphase Metaphase Anaphase Telophase Mitotic Phase

index index index index index ± SE index

8.00–8.30 1683 413 11.17 6.18 4.63 2.73 24.54 ± 0.20 407.50

8.30–9.00 1667 440 11.82 8.33 4.08 2.10 26.39 ± 0.19 378.86

9.00–9.30 1670 441 10.54 9.10 5.15 2.40 26.41 ± 0.17 378.68

9.30–10.00 1628 407 7.31 8.85 5.10 3.75 25.00 ± 0.13 400.00

10.00–10.30 1691 413 6.86 8.28 4.90 4.02 24.42 ± 0.17 409.44

10.30–11.00 1657 393 6.46 7.97 5.07 4.47 23.72 ± 0.14 421.63

SE, Standard error.

TABLE 2: Mitotic index and phase index in root tips of A. sativum var. Yamuna safed.

Time (a. m.) TC TDCProphase Metaphase Anaphase Telophase Mitotic Phase

index index index index index ± SE index

8.00–8.30 1601 418 10.31 8.81 4.43 2.44 26.11 ± 0.47 383.01

8.30–9.00 1571 422 11.84 8.98 4.39 1.97 26.86 ± 0.26 372.27

9.00–9.30 1511 418 10.52 9.07 4.90 2.45 27.66 ± 0.08 361.48

9.30–10.00 1567 435 7.85 10.15 7.21 2.62 27.76 ± 0.19 360.23

10.00–10.30 1528 394 7.73 8.44 5.56 4.25 25.79 ± 0.19 387.82

10.30–11.00 1471 405 8.84 8.91 5.03 4.69 27.53 ± 0.18 363.21

SE, Standard error.

Between 8.30 and 9.00 a. m. a higher percentage cells at prophase cells has been observedand between 9 and 10 a. m. there is an increase in the number of cells showing meta- andanaphases. By 11 a. m. the cells become predominantly telophasic.

In both varieties, the mitotic indices are at their peak in root tips fixed at 9–10 a. m. InAgrifound white, the highest index of 26.41 has been observed in root tips fixed at 9.00–9.30a. m. but in Yamuna safed, the mitotic index is at its peak (27.76) in root tips fixed at 9.30–10.00a. m. The phase index reaches its peak (421.63) by 11 a. m. in Agrifound white and in Yamunasafed, it reaches peak level of 387.82 slightly earlier at around 10.30 a. m. However, thedifferences in indices seen between the varieties appear insignificant (Figs 1, 2).

DISCUSSION

Nasare & Choudhary (2010) reported the effect of physical and chemical mutagens onmitotic activity in root tip cells in O. sanctum. They observed that the frequency of mitoticallydividing cells was found to decrease with an increase in the dose/concentration of the physical

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as well as chemical mutagens. Osuji & Owei (2010) observed in T. africana that cell division tookplace the whole day and night but it is more in the day time than at night. The most intense periodof mitosis was between 2.00 and 6.00 p. m. with a peak at 4.00 p. m. (Nigerian time). Mitoticcounts showed that the number of prophase cells decreased when that of metaphase increased asthe day progressed. The peak of anaphase was during the early hours of the night at 8.00 p. m.Srivastava & Jain (2011) reported a reduction of 85.92 and 95.10% in mean root length at 40 and80 ppm chromium dosage along with 61.25 and 82.5% reduction in mean root number/noderespectively in sugarcane cultivar CoLk8102. Mitotic index of root tip cells of treated sets declinedand the frequency of aberrant mitotic phases increased pari passu to the increasing chromiumconcentration. Truta et al. (2011) studied the cytogenetic effects induced by 2 caffeineconcentration (0.1%, 0.5%) in root meristematic cells of plants P. vulgaris and R. sativus. Theyobserved that caffeine has genotoxic potential. The maximum tested concentration (0.5% caffeine)provided the most complex pattern of ana-telophase aberrations, especially in radish. R. sativusgenotype showed a higher sensibility to the caffeine action. Amirthalingam et al. (2013) observedin V. unguiculata that the drastic reduction in mitotic index was observed due to cadmium toxicity.Paul et al. (2013) observed the effect of blitox on root mitosis of A. cepa and Pankaj et al. (2014)studied the effect of oxytocin on mitotic cell division in the same species and concluded that thesechemicals act as mitotic depressers as well as mutagenic agents.

Mitotic and phase indices will be help during the karyotypic analysis of Agrifound white andYamuna safed varieties of A. sativum.

ACKNOWLEDGEMENTS

Authors thank Dr. Mousmi Bhowl and Dr. K. J. Cherian Department of Botany, Hislop College for providingfacilities. Thanks are also due to to Dr. Dipti Christian, Principal, Hislop College for help.

REFERENCES

AMIRTHALINGAM T, VELUSAMY G & PANDIAN R 2013 Cadmium induced changes in mitotic index andgenotoxicity on Vigna unguiculata (Linn) Walp J Environ Chem Ecotox 5 57-62

BANSIKAR V & SRIVASTAVA A K 1992 Mercury induced chromosomal inconstancy in pearl millet J Cytol Genet27 79-90

BHATTA P & SAKYA S R 2008 Study of mitotic activity and chromosomal behavior in root meristem of Alliumcepa L treated with magnesium sulphate Ecoprint 15 83-88

DEVI P 1990 Cytological effects of chemical mutagen on Trigonella foenum-graecum J Cytol Genet 25 117-119

GAIKWAD P D 1975 Cytogenetic studies in Coriandrum sativum Linn Ph D Thesis RTM Nagpur University Nagpur

GIRHE S 1999 Mutational Studies in Lathyrus sativus L Ph D Thesis RTM Nagpur University Nagpur

JAHAGIRDAR H A 1975 Cytogenetic studies in Foeniculum vulgare Mill Ph D Thesis RTM Nagpur University Nagpur

MITOTIC AND PHASE INDICES IN GARLIC

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KANAKLATA 1995 Mutational studies in Cyamopsis tetragonoloba Taub Ph D Thesis RTM Nagpur University Nagpur

KARSPATE R R 1995 Mutational studies in Linum usitatissum L Ph D Thesis RTM Nagpur University Nagpur

KHANOLKAR S M 1977 Cytogenetic studies in Carum copticum Ph D Thesis RTM Nagpur University Nagpur

KOTHEKAR V S 1978 Mutational studies in Solanum nigrum L Ph D Thesis RTM Nagpur University Nagpur

LANDGE S P 2000 Induced mutations in Brassica napus L cv Westar Ph D Thesis RTM Nagpur University Nagpur

NASARE P N & CHOUDHARY A D 2010 Effect of physical and chemical mutagens on mitotic activity in root tipsof Ocimum sanctum Int J Mendel 27 53-55

OSUJI J O & OWEI S D JNR 2010 Mitotic index studies on Treculia fricana Decne in Nigeria Australian J AgrlEngi 1 25-28

OZMEN A 2010 Cytotoxicity of Hibiscus rosa-sinensis flower extract Caryologia 63 157-161

PANKAJ P P, KUMARI N & PRIADARSHINI A 2014 Evaluation of cytotoxic in Allium cepa L root tips cell IntJ Pharm Clin Res 6 36-39

PAUL A, NAG S & SINHA K 2013 Cytological effects of blitox on root mitosis of Allium cepa L Int J Sci ResPub 3 1-7

SANJAYKUMAR, ARYA S K, ROY B K & SINGH A K 2010 The effects of 2 4-dichlorophenoxy acetic acid andisoproturon herbicides on the mitotic activity of wheat (Triticum aestivum L) root tips Turk J Biol 3455-66

SRIVASTAVA S & JAIN R 2011 In-situ monitoring of chromium cytotoxicity in sugercane J Environ Biol 32759-763

TRUTA E, ZAMFIRACHE M M & ZENOVIAOLTEANU 2011 Caffeine induced genotoxic effects in Phaseolusvulgaris L and Raphanus sativus L Bot Ser 35 49-54

UDO I J, AKPAN G A& ESENOWO I K 2014 Cytotoxic effects of 5 medicinal plants on mitosis in Allium ceparoot tips Cur Res J Bio Sci 6 71-75

YADAV H & YADAV P K 2010 A study of mitotic cell division in Capsicum annuum induced by Chilli Mottle Virusdisease Asian J Exp Bio Sci 1 445-447

YASAR K & AHMED 2006 The effect of cadmium on seed germination root development and mitotic of root tipcells of Lentil (Lens culinaris Medik) World J Agrisci 2 196-200

DEOGADE & NASARE: MITOTIC AND PHASE INDICES IN GARLIC

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J. CYTOL. GENET. VOL. 17 (NS): 7–14 (2016)

IN VITRO SHOOT REGENERATION FROM NODAL EXPLANTS OF VITEXAGNUS-CASTUS L.

L. RAJANNA, P. VIJAY RAGHAVAN* AND G. S. SHAILAJA SHARMADepartment of Botany, Bangalore University, Jnanabharathi, Bengaluru 560 056

*For correspondence. Email: vijayragh@gmail.com

(Received 27 November 2015, revised accepted 28 February 2016)

SUMMARY

An efficient micropropagation protocol has been standardized for the shootregeneration from the nodal explants of Vitex agnus-castus, an important aromaticmedicinal plant. Nodal segments were cultured on MS medium supplemented withvarious concentrations of plant growth regulators. Among them, MS + 2.5 mg/l IBAor MS + 3.0 mg/l IBA and MS + 2.0 mg/l IAA + 1.0 mg/l KN and MS + 2.0 mg/l NAA + 1.0 mg/l KN were found to be the best for inducing multiple shoots undersingle hormone and with different combinations respectively. Under these hormonaltreatments, the regenerated plantlets were able to develop rooting in the same mediaand were successfully hardened and planted in the field within 2 months from the dateof inception of the experiment.

Keywords: Vitex agnus-castus, nodal explants, micropropagation.

INTRODUCTION

Vitex agnus-castus, commonly called “Chaste tree” or “Vitagnus” is an important aromaticmedicinal plant. V. agnus-castus a deciduous shrub native to European, Mediterranean and CentralAsian countries, has long, finger-shaped leaves and displays fragrant blue-violet flowers inmidsummer and bears purple black berries that are yellowish inside, resembling peppercorn withan aromatic odour. Upon ripening, the berry is picked and allowed to dry. The berries were usedby monks during the Middle Ages to suppress sexual desire; hence, its common names – monk’spepper and chaste tree. The twigs of this shrub are very flexible and were used for furniture inancient times. References to V. agnus-castus go back to more than 2000 y, describing it as ahealing herb. Ancient Egyptians, Greeks and Romans used it for a variety of health problems. In400 BC, Hippocrates recommended chaste tree for injuries and inflammation. Later, Greek botanistDioscorides recommended it specifically for inflammation of the womb and lactation (Artz 2007,Roemheld-hamm 2005, Sarkar 2009).

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MICROPROPAGATION OF VITEX AGNUS-CASTUS

MS medium used for shoot induction contained full strength of MS inorganic salts and vitamins, 3% sucrose(w/v) and 0.8% agar (w/v). The medium was fortified with either single hormone (2,4-D or IAA or IBA or NAA orBAP or KN) or combination of hormones (one of the auxins with one of the cytokinins). Medium without hormonewas used as control. In both the cases the pH of the medium was adjusted to 5.8 before autoclaving for 15 min.Explants were cultured in either 25 × 150 mm borosil test tubes with 15 ml medium in each tube having single explantor cultured in 250 ml glass bottles with 35 ml medium in each bottle consisting of 3 explants. For in vitro shootinduction and root induction, the cultures were incubated at 25 ± 2° C with 16 h illumination under light intensityof 3000 lux provided by cool florescent lamps.

The shoot induction experiment was carried out with either 12 replicates (test tubes) with each replicate having1 explant or with 6 replicates (bottles) with each replicate having 3 explants. The experiments were repeated thrice.The shoot length and number of shoots were recorded after 45 d of inoculation. The data was subjected for meanlength of shoot, mean number of shoots per explant and percentage of shoot and root induction and shoots survivalrate after hardening. The photographs were taken using Nikon coolpix S6700 camera.

OBSERVATIONS

Multiple shoots of V. agnus-castus were proficiently produced from nodal explants. Theconcentrations of auxins and cytokinins used in the present investigation ranges from 0.5 to 4.0mg/l and the results are presented in Table 1. They were used either singly or in combinations.The nodal explants were able to produce multiple shoots on MS media fortified with all the testedauxins and BAP. However, no response was observed when KN was used singly. Among theauxins, when 2,4-D was added with MS at the concentrations of 0.5 to 1.5 mg/l it did not triggershoot initiation. However, in MS + 2 to 3.5 mg/l (2,4-D), shoot emergence (0.5 to 1.5 cm inlength) has been observed in 72.2% of the cultures. In case of IAA, maximum of 6 shootsdeveloped at 4 mg/l and in case of IBA and NAA, at the same concentration, highest number ofshoots (8) have developed.

The most effective combinations were MS + 2.0 mg/l IBA + 1.0 mg/l BAP, MS + 3.5 mg/l IBA + 3.0 mg/l BAP, MS + 3.5 mg/l IBA + 1.5 mg/l BAP, MS + 2.0 mg/l IAA + 1.0 mg/l BAP,MS + 2.0 mg/l IAA + 1.0 mg/l KN, MS + 2.0 mg/l 2,4-D + 1.0 mg/l KN and MS + 2.0 mg/lNAA + 1.0 mg/l KN. The nodal explants of V. agnus-castus showed initial shooting responses justafter 3 d of innoculation in presence of these combinations and also produced roots in the samemedia. However, the growth rate, number of multiple shoots produced and the duration requiredfor rooting by the explants varied in presence of different combinations. In case of combinationsinvolving IBA and BAP, the highest number of 15 multiple shoots were produced, however, in caseof MS + IAA + BAP, only 3 multiple shoots were seen. In contrast, when 1.0 mg/l KN was usedwith IAA, as many as 40 multiple shoots developed showing faster growth rate. Similar responseswere observed when 2,4-D and NAA are combined with 1 mg/l of KN

Regenerated shoots obtained from explants on MS + 2.0 mg/l IBA + 1.0 mg/l BAP exhibitedslow growth rate and were ready for hardening after 65 d. The nodal explants of V. agnus-castus

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RAJANNA ET AL.:

TABLE 1: Effect of auxins and cytokinin on shoot induction in V. agnus-castus.

Hormones (mg/l) Shoot induction No. of shoots induced Shoot length in cmused with MS (%) per explant (Mean ± SE) (Mean ± SE)

2,4-DUp to 1.5 Nil

2 38.89 0.33 ± 0.21 0.56 ± 0.042.5 72.22 2.0 ± 0.0 1.11 ± 0.053 33.33 1.33 ± 0.21 1.05 ± 0.07

3.5 16.67 0.33 ± 0.21 0.58 ± 0.054 Nil

IAA0.5 Nil1 16.67 0.33 ± 0.21 0.5 ± 0.05

1.5 33.33 0.66 ± 0.42 0.73 ± 0.062 77.78 1.66 ± 0.21 1.6 ± 0.06

2.5 77.78 3 ± 0.25 2.35 ± 0.083 83.33 3.5 ± 0.22 3.7 ± 0.09

3.5 94.44 4.8 ± 0.16 5.33 ± 0.074 77.78 6.0 ± 0.0 3.1 ± 0.13

IBA0.5 72.22 1 ± 0.36 1.55 ± 0.091 77.78 1.66 ± 0.21 1.78 ± 0.07

1.5 83.33 2.33 ± 0.33 2.63 ± 0.072 94.44 4.0 ± 0.0 3.15 ± 0.07

2.5 100.00 5.16 ± 0.3 5.55 ± 0.163 100.0 8.16 ± 0.16 3.78 ± 0.10

3.5 83.33 10.0 ± 0.0 2.41 ± 0.154 77.78 8.83 ± 0.65 1.65 ± 0.10

NAAUp to 2 Nil

2.5 50.00 3.66 ± 0.33 1.65 ± 0.093 77.78 4.33 ± 0.21 2.75 ± 0.12

3.5 83.33 5.5 ± 0.34 4.33 ± 0.144 72.22 6.0 ± 0.0 3.11 ± 0.10

BAPUp to 0.5 Nil

1 33.33 1.66 ± 0.21 1.81 ± 0.191.5 38.89 3.66 ± 0.33 2.65 ± 0.262 61.11 5.83 ± 0.30 2.98 ± 0.29

2.5 100.00 8.0 ± 0.0 3.35 ± 0.093 100.00 6.0 ± 0.0 3.76 ± 0.12

3.5 77.78 6.16 ± 0.47 2.41 ± 0.074 38.89 5.66 ± 0.21 1.6 ± 0.05

produced 13–15 multiple shoots and rooted on the same media after 35 and 25 d, and were readyfor hardening after 48 and 35 d in presence of MS + 3.5 mg/l IBA + 3.0 mg/l BAP and MS +3.5 mg/l IBA + 1.5 mg/l BAP respectively. MS + 2.0 mg/l IAA + 1.0 mg/l BAP influenced theexplants to produce 2–3 multiple shoots, which rooted in the same media after 50 d and wereready for hardening after 70 d. In case of MS + 2.0 mg/l 2,4-D + 1.0 mg/l KN the explantproduced 35–40 multiple shoots, rooted in the same media after 35 d and were ready for hardeningat about 48 d after inoculation. The explants showed faster growth rate in presence of thecombinations MS + 2.0 mg/l IAA + 1.0 mg/l KN and MS + 2.0 mg/l NAA + 1.0 mg/l KN. Underthe influence of these combinations the explants produced large number of multiple shoots