S

Ep

BS

a

ARRAA

KAKTGS

1

tk2iJC2aiciatd2eiH(

b

0d

Industrial Crops and Products 34 (2011) 1241– 1244

Contents lists available at ScienceDirect

Industrial Crops and Products

journa l h o me page: www.elsev ier .com/ locate / indcrop

hort communication

ffect of temperature on seed germination parameters in Kalmegh (Andrographisaniculata Wall. ex Nees.)

irendra Kumar ∗, Sanjeet K. Verma, H.P. Singheed Quality Lab, Central Institute of Medicinal and Aromatic Plants (CSIR), P.O. CIMAP, Lucknow 226015, India

r t i c l e i n f o

rticle history:eceived 31 January 2011eceived in revised form 7 April 2011ccepted 11 April 2011vailable online 10 May 2011

eywords:ndrographis paniculata

a b s t r a c t

Kalmegh (Andrographis paniculata Wall. ex Nees) is a medicinal herb of tropical south east Asia and nativeto India and Sri Lanka. It has been used herb for liver ailment in all prevailing systems of medicine viz.Ayurvedic, Unani, Homeopathic and modern throughout most of the south East Asian countries viz. China,India, Sri Lanka, Indonesia, Thailand, Burma, and Vietnam, etc. Commercially cultivation of Kalmegh isdone through seeds. Optimal germination potential, temperature and first as well as final count day arethree primary parameters for developing the seed quality standards. Germination of Kalmegh variety‘CIM-Megha’ was carried out at six constant temperatures at an interval of every ‘5 ◦C’ from ‘15 to 40 ◦C’

◦

almeghemperatureerminationeed quality

temperatures coupled with 16 h light and 8 h dark photo period. The temperature of ‘25 C’ was foundoptimally suitable with ‘94.6’ and ‘23.6’ percentage of germination and germination energy, respectively,while the temperature at ‘40 ◦C’ was deleterious with no germination. Significant decrease in percentageof germination and germination energy was observed at ‘15 ◦C’, ‘20 ◦C’, ‘30 ◦C’ and ‘35 ◦C’ of temperaturein comparison to ‘25 ◦C’. The study further revealed that days 5-6 and days 7-9 after seed sowing were

l cou

the ideal for first and fina

. Introduction

Kalmegh (Andrographis paniculata Wall. ex Nees. Family: Acan-haceae) is an important indigenous medicinal plant commonlynown as ‘King of Bitters’ (Saraswathy et al., 2004; Chauhan et al.,009; Gomathinayagam et al., 2009). Kalmegh grows abundantly

n south eastern Asian countries viz. India, Sri Lanka, Pakistan,ava, Malaysia, and Indonesia but cultivated extensively in India,hina, Thailand, East and West Indies and Mauritius (Mishra et al.,007; Kanokwan and Nobuo, 2008; Niranjan et al., 2010; Katakynd Handique, 2010). It thrives well in tropical climatic conditions.e. hot and humid. However, it can be cultivated in subtropi-al regions during the monsoon season. The extract of Kalmeghs used as anti-pyretic, anti-periodic, antibacterial, anti-malarial,nti-inflammatory, anti-thrombogenic, blood purifier, hepatopro-ective, besides the treatment of jaundice, dermatological diseases,yspepsia, febrifuge and anthelmintic disorders (Saraswathy et al.,004; Chauhan et al., 2009; Gomathinayagam et al., 2009; Kapadit al., 2010; Niranjan et al., 2010). Consumption of Kalmegh herb

s estimated to be 250 tones (Shrama et al., 2008; Kataky andandique, 2010). The demand of Kalmegh is increasing day by day

Chauhan et al., 2009). Since the plant is seed propagated, it is essen-

∗ Corresponding author. Tel.: +91 9450095841.E-mail addresses: birendrak67@rediffmail.com, b.kumar@cimap.res.in,

iren k67@yahoo.com (B. Kumar).

926-6690/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.indcrop.2011.04.008

nt, respectively for seed germination of Kalmegh.© 2011 Elsevier B.V. All rights reserved.

tial to assess the seed quality for ensuring the crop stand and herbyield which depend on quality seed.

Germination of a seed in a laboratory test is the emergence anddevelopment of a seedling to a stage where the aspect of its essen-tial structures (root system, shoot axis, cotyledons, terminal buds)indicates whether or not it is able to develop further into a satisfac-tory plant under favourable soil conditions (ISTA Rule, 2006). Sincegermination is a complex biological process and at a point of timeseveral factors have to enact simultaneously the resultant effectis reflected, in the form of emergence of seedling, after a certainperiod of time. The percentage of germination happens to be one ofthe most important characteristics of the seed to be used for cultiva-tion. Germination energy is a measure of the speed of germinationand hence, it is assumed, to be responsible for the vigor of the seedand of the seedling which it produces (Czabator, 1962). Only thoseseeds which germinate rapidly and vigorously under the favourablesituation under controlled conditions are likely to be capable ofproducing vigorous seedlings in field conditions. The period duringwhich maximum number of seedlings could be obtained is calledas germination period (Czabator, 1962). Effective stand after thegermination (associated germination parameters like germinationenergy, germination period, etc.) is another important character-istic that gives an idea about the final population. Thus, it would

be desirable to have information regarding these parameters forproducing good quality seed.

Temperature is one of the most critical factors affecting thegermination of the seed (Bewley and Black, 1994; Verma et al.,

1 s and Products 34 (2011) 1241– 1244

2pttte1asptutp

2

2

c‘erRata‘pwdawasspc

ad(p

3

ap

242 B. Kumar et al. / Industrial Crop

010). In many agricultural and vegetable species effect of tem-erature on seed germination has been well depicted, elucidatinghe critical lower and upper temperatures for germination, and theemperature at the highest rate of germination and its germina-ive capacity (Wagenvoort and Bierhuizen, 1977; Garcia-Huidobrot al., 1982; Covell et al., 1986; Ellis et al., 1987; Mwale et al.,994; Jensen, 2001) but in case of Kalmegh there is no such reportvailable till date. A systematic study to generate information ontandardization of germination parameter is essential for any seedropagated crop. Keeping this in view, the present study was under-aken with the objectives to standardize germination parametersnder varying temperature conditions and to elucidate suitableemperature conditions to optimize maximum seed germinationercent in Kalmegh (Cv. ‘CIM-Megha’).

. Materials and methods

.1. Germination test in Petri dish

The experiment was conducted during July–August 2010 at sixontrolled and constant temperature regimes i.e. ‘15 ◦C’, ‘20 ◦C’,25 ◦C’, ‘30 ◦C’, ‘35 ◦C’ and ‘40 ◦C’ with the seeds of the Kalmegh vari-ty ‘CIM-Megha’ collected in the month of December 2009 from theesearch farm of Central Institute of Medicinal and Aromatic Plants,esource Centre, Hyderabad, India. The experiment was conductedt constant controlled temperature in different seed germina-ors having temperatures viz., ‘15 ◦C’ × ‘180 lx’, ‘20 ◦C’ × ‘183 lx’nd ‘25 ◦C’ × ‘180 lx’, ‘30 ◦C’ × ‘180 lx’, ‘35 ◦C’ × ‘180 lx’ and

40 ◦C’ × ‘183 lx’ with16 h light and 8 h dark regime. Seeds werelaced on the top of the filter paper (TP) (15 cm diameter) soakedith sterile distilled water in Petri plates (16 cm diameter × 3 cmeep). The treatments (temperature) were replicated eight timesnd each Petri dish contained 100 seeds. Counts of germinationere checked daily for normal (bearing both root and shoot)

nd abnormal (lacks either root or shoot or stunted growth)eedlings from first day of germination till the day of maximumeed germination (Kumar et al., 2008a,b, 2009, 2010). Germinationercentage, germination energy (%) and germination period werealculated using the formula cited by Czabator’s index (1962);

Germination percentage

= Total number of seeds germinatedTotal number of seeds in all replicates

× 100

Germination energy

= 1/4 of maximum number of seeds germinated in a dayTotal number of seeds in all replicates

× 100

Germination period(GPD) = Days from seeding to when

maximum number of seeds germinated

At the end of experiment, data was subjected to analysis of vari-nce (ANOVA) and mean separation. The statistical analysis wasone using GenStat® Release 7.21. The least significant differenceLSD) at 5% level was used to compare the means of different testarameters under different temperature conditions.

. Results and discussion

The temperature regimes and number of days to countingffected the germination of Kalmegh seeds. Variation due to tem-eratures, number of days to counting and their interactions were

Fig. 1. Mean germination percentage of Kalmegh seeds at different temperatureswith standard error.

highly significant. The percent germination and days to germina-tion varied with temperatures. The first emergence of seedlings wasobserved on 3rd day at ‘30 ◦C’, 4th day at ‘25 ◦C’ and ‘35 ◦C’, 5th dayat ‘20 ◦C’ and 9th day at ‘15 ◦C’ temperatures. The maximum seedgermination was found on 7th day at ‘25 and 30 ◦C’, 9th day at ‘20and 35 ◦C’, and 16th day at ‘15 ◦C’ temperatures.

The mean seed germination percentage over the temperaturesfor number of days to counting varied from 17.55 (‘15 ◦C’) to 71.14(‘25 ◦C’) (Table 1). Among the temperatures ‘25 ◦C’ had the highestmean germination percentage (71.14) followed by ‘30 ◦C’, ‘20 ◦C’,‘35 ◦C’ and ‘15 ◦C’ while among the number of days to counting,days 16 and 15 were at par (77.47 and 77.45) while day 14 wassignificantly different from them with slightly lower mean germi-nation percentage for remaining count days i.e. 13–3 which werein descending order (Table 1). A similar trend was observed for ger-mination energy percent too (Table 1). Overall, ‘25 ◦C’ was the besttemperature in respect to germination percentage and germina-tion energy percent followed by ‘30 ◦C’, ‘20 ◦C’, ‘35 ◦C’ and ‘15 ◦C’.Considering these two factors simultaneously i.e. number of daysto counting and temperatures, ‘25 ◦C’ temperature was found thebest with maximum mean germination percentage and germina-tion energy percent (94.62 and 23.65) at day 7 and was followed by‘20 ◦C’ (89.88 and 22.47) at day 9, ‘30 ◦C’ (87.50 and 21.87) at day 7,‘35 ◦C’ (79.50 and 19.87) and ‘15 ◦C’ (53.75 and 13.44) at day 16 ofconstant and controlled temperatures (Figs. 1 and 2).

Thus, it seems that temperature is a critical factor in thegermination of Kalmegh seeds as in Tagetes minuta and Cymbo-pogon martinii (Forsyth and Van Staden, 1983; Verma et al., 2010).Chauhan et al. (2009) conducted seed germination experiment onKalmegh using different substratum under ‘25 ◦C’ constant tem-perature and found 72, 75 and 78 germination percentage in filterpaper, soil and sand, respectively. Among the tested temperatures,since 95 percent seeds germinated under ‘25 ◦C’, it seems to bethe optimum for germination of Kalmegh. Significant reductionin percentage of germination and germination energy at above orbelow at 25 ◦C temperature is an indication of threshold high andlow cut-off between studied range of temperature i.e. ‘15–40 ◦C’.Optimum germination temperature was reported to be ‘30 ◦C’ forKalmegh seed (Chaudhary, 1975; Baskin and Baskin, 2001). In a pre-vious study with Kalmegh seed, day 18 was found to best for final

count day with 84 percent germination (Saraswathy et al., 2004).The present study revealed that the first and final count day was5–6 and 7–9 days, respectively at ‘25 ◦C’. The germination period(i.e. the period during which maximum number of seedlings could

B. Kumar et al. / Industrial Crops and Products 34 (2011) 1241– 1244 1243

Table 1Mean germination percentage (G) and germination energy percent (GE) of Kalmegh seeds at different temperatures and number of days to counting.

Number of days to counting Temperature Mean

15 ◦C 20 ◦C 25 ◦C 30 ◦C 35 ◦C

G GE G GE G GE G GE G GE G GE

1 0 0 0 0 0 0 0 0 0 0 0.00 02 0 0 0 0 0 0 0 0 0 0 0.00 03 0 0 0 0 0 0 20.62 5.15 0 0 4.12 1.034 0 0 0 0 29.25 7.31 69.62 17.40 13.62 3.40 22.50 5.625 0 0 12.38 3.09 85.38 21.34 81.50 20.37 29.38 7.34 41.73 10.436 0 0 53.25 13.31 93.38 23.34 86.75 21.69 55.25 13.81 57.73 14.437 0 0 75.25 18.81 94.62 23.65 87.50 21.87 65.50 16.37 64.58 16.148 0 0 88.75 22.19 94.62 23.65 87.25 21.81 77.75 19.44 69.67 17.429 13.38 3.34 89.88 22.47 94.25 23.56 86.62 21.65 79.50 19.87 72.72 18.1810 15.88 3.97 89.25 22.31 93.12 23.28 85.62 21.40 79.00 19.75 72.58 18.1411 27.88 6.97 89.25 22.31 93.00 23.25 85.38 21.34 78.50 19.62 74.80 18.7012 33.62 8.40 88.62 22.15 93.00 23.25 85.00 21.25 77.38 19.34 75.53 18.8813 38.50 9.62 88.50 22.12 92.62 23.15 84.00 21.00 76.50 19.12 76.03 19.0114 46.50 11.62 88.00 22.00 92.00 23.00 82.88 20.72 75.50 18.87 76.97 19.2415 51.38 12.84 87.25 21.81 91.75 22.94 82.12 20.53 74.75 18.69 77.45 19.3616 53.75 13.44 86.88 21.72 91.25 22.81 81.62 20.40 73.87 18.47 77.47 19.37Mean 17.55 4.39 58.58 14.64 71.14

LSD (P = 0.05) = temperature: 2.19 (G), 0.55(GE); number of days to counting: 1.16(G), 0.2

Fa

b‘(

untgmcrn

4

pmpa

on health and its major diterpenoid constitute andrographolide. J. Health Sci.

ig. 2. Mean germination energy percentage of Kalmegh seeds at different temper-tures with standard error.

e obtained) was found to be 7–9 day under four (‘20 ◦C’, ‘25 ◦C’30 ◦C’ and ‘35 ◦C’) controlled and constant temperature conditionsTable 1).

The differential behavior of the Kalmegh variety ‘CIM-Megha’nder different temperature regimes indicated that the germi-ation capacity of the seeds of the variety was affected byemperature. Temperature can affect the percentage and rate ofermination through its effects on loss of dormancy and the ger-ination process itself (Roberts, 1988). Reduced temperatures and

ritical high temperature would be expected to retard the metabolicate to the point where pathways essential for the onset of germi-ation would cease to operate (Verma et al., 2010).

. Conclusion

Temperature affected time to germinate and germinationercent in Kalmegh. The seed of Kalmegh showed maximum ger-

ination at ‘25 ◦C’ constant and controlled temperature with 95

ercent of germination potential and days 5-6 and days 7-9 as firstnd final count day, respectively.

17.78 69.16 17.29 53.53 13.38

9 (GE); temperature × number of days to counting: 3.29 (G), 0.82 (GE).

Acknowledgements

The authors are highly grateful to Prof. (Dr.) Ram Rajasekharan,Director, Central Institute of Medicinal and Aromatic Plants (CSIR),Lucknow, India for providing necessary help during investigation,Dr DD Patra, Scientist ‘G’, CIMAP for critically examining and valu-able suggestions in upgradation of manuscript and Dr KP Shastry,SIC, CIMAP RC, Hyderabad, India for providing seed material. Theyare also grateful to Prof. Dennis T. Ray and anonymous reviewersfor valuable comments and improving the manuscript. This studywas financially supported by Council of Scientific and IndustrialResearch, New Delhi, India.

References

Baskin, C.C., Baskin, J.M., 2001. Seed: Ecology, Biogeography and Evolution of Dor-many and Germination. Academic Press, An Imprint of Elsevier, CA, USA.

Bewley, J.D., Black, M., 1994. Seeds: Physiology of Development and Germination,2nd ed. Plenum Press, New York.

Chaudhary, R.L., 1975. Ecological observations on Andrographis paniculata Nees.Botanique 6, 103–108.

Chauhan, J.S., Tomar, Y.K., Singh, N.I., Ali, S., Badoni, A., Debarati, Rana, A., 2009.Assessment of compatible substratum for Andrographis paniculta standard seedgermination testing. J. Am. Sci. 5, 70–75.

Covell, S., Ellis, R.H., Roberts, E.H., Summerfield, R.J., 1986. The influence of temper-ature on seed germination rate in grain legumes. J. Exp. Bot. 37, 705–715.

Czabator, F.J., 1962. Germination value: an index combining speed and completenessof pine seed germination. Forest Sci. 8, 386–396.

Ellis, R.H., Hong, T.D., Roberts, E.H., 1987. Comparison of cumulative germination andrate of germination of dormant and aged barley seed lots at different constanttemperatures. Seed Sci. Technol. 15, 717–727.

Forsyth, C., Van Staden, J., 1983. Germination of Tagetes minuta L. I. Temperatureeffects. Ann. Bot. 52, 659–666.

Garcia-Huidobro, J., Monteith, J.L., Squire, G.R., 1982. Time, temperature and germi-nation of pearl millet (Pennisetum typhoides S & H.) I. Constant temperature. J.Exp. Bot. 33, 288–296.

Gomathinayagam, M., Anuradha, V.E., Zhao, C., Ayoola, G.A., Jaleel, C.A., Anneer-selvam, R.P., 2009. ABA and GA3 affect the growth and pigment composition inAndrographis paniculata Wall. ex Nees., an important folk herb. Front. Biol. China4, 337–341.

International Seed Testing Association (ISTA) Rule, 2006. International Rules for SeedTesting. International Seed Testing Association, Bassorsdorf, Switzerland.

Jensen, M., 2001. Temperature relations of germination in Acer platanoides L. seeds.Scand. J. Forest Res. 16, 404–414.

Kanokwan, J., Nobuo, N., 2008. Pharmacological aspects of Andrographis paniculata

54, 370–381.Kapadi, C., Patel, B., Katkar, K., Suthar, A., Chauhan, V.S., 2010. A quantitative estima-

tion of 14-deoxy-11, 12-didehydroandrographolide in Andrographis paniculataby HPTLC. Int. J. Pharm. Sci. 2, 49–52.

1 s and

K

K

K

K

K

M

M

244 B. Kumar et al. / Industrial Crop

ataky, A., Handique, P.J., 2010. A brief overview on Andrographis paniculata (Burm.f) Nees., a high valued medicinal plant: Boon over synthetic drugs. Asian J. Sci.Technol. 6, 113–118.

umar, B., Gupta, A.K., Verma, A.K., Dubey, A., 2008a. Comparative seed germinationof Tagetes minuta. J. Trop. Med. Plants 9, 149–151.

umar, B., Gupta, A.K., Verma, A.K., Saini, R.K., Khanuja, S.P.S., 2008b. Comparativegermination kinetics and efficiency in Marigold (Tagetes erecta L.) accessionsover storage. J. Med. Arom. Pl. Sci. 30, 142–145.

umar, B., Verma, A.K., Ram, G., Singh, H.P., Lal, R.K., 2009. Genotype-independentgermination-testing procedure for Isabgol. J. New Seeds 10, 196–205.

umar, B., Verma, A.K., Ram, G., Singh, H.P., Lal, R.K., 2010. Seed germination of fiveelite genotypes of Papaver somniferum. J. Trop. Med. Plants 11, 107–112.

ishra, S.K., Sangwan, N.S., Sangwan, R.S., 2007. Andrographis paniculata (Kalmegh):a review. Pharmacog. Rev. 1, 283–289.

wale, S.S., Azam-Ali, S.N., Clark, J.A., Bradley, R.G., Chatha, M.R., 1994. Effect oftemperature on the germination of sunflower (Helianthus annuus L.). Seed Sci.Technol. 22, 565–571.

Products 34 (2011) 1241– 1244

Niranjan, A., Tewari, S.K., Lehri, A., 2010. Biological activities of Kalmegh (Androgra-phiz paniculata Nees.) and its active principles – a review. Indian J. Nat. Prod.Resour. 1, 125–135.

Roberts, E.H., 1988. Temperature and seed germination. In: Long, S.P., Woodward,F.L. (Eds.), Plants and Temperature. Symposia of the Society of ExperimentalBiology, vol. 42. Company of Biologists Ltd., Cambridge, pp. 109–132.

Saraswathy, S., Manavalan, R.S.A., Subramanian, S., 2004. Seed germination studieson Kalmegh (Andrographis paniculata Nees.). Madras Agric. J. 91, 226–229.

Shrama, A., Shanker, C., Tyagi, L.K., Singh, M., Rao, C.V., 2008. Herbal medicine formarket potential in India: an overview. Acad. J. Plant Sci. 1, 26–36.

Verma, S.K., Kumar, B., Ram, G., Singh, H.P., Lal, R.K., 2010. Varietal effect on ger-mination parameter at controlled and uncontrolled temperature in Palmarosa

(Cymbopogon martinii). Ind. Crops Prod. 32, 696–699.

Wagenvoort, W.A., Bierhuizen, J.F., 1977. Some aspects of seed germination in veg-etables II. The effect of temperature fluctuation, depth of sowing, seed size andcultivar on heat sum and minimum temperature for germination. Sci. Hortic. 6,259–270.