Journal of Medicinal and Aromatic Plant Sciences 32 (3) (2010) 239-244

Sprouting and survival of Coleus forskohlii stem cuttings by hormonal pretreatment

RAJ KUMAR SINGH TIWARI* AND KUNTAL DAS Department of Plant Pathology, T.C.B. College of Agriculture and Research Station (I.G.K.V.), Bilaspur, Chhattisgarh, India. Received 5th January, 2010

ABSTRACT
Experiments were conducted to evaluate the growing behavior of stem cutting of Coleus forskohlii Briq. Percentage of cuttings sprouted, nodes sprouted and survival were recorded to assess the effect of cutting length, diameter and pretreatment with exogenous hormones viz., indole 3 acetic acid, -naphthalene acetic acid and indole 3 butyric acid. The data revealed the significant effect of these factors on sprouting and survival of C. forskohlii stem cuttings. Maximum sprouting, node sprouting and survival were recorded when cuttings were treated with IBA @ 100 ppm concentration as liquid and in powder formulation irrespective of diameter and length comparable with control and water dip treatment. Powder formulations of all hormones were found to be more effective than liquid formulations in relation to sprouting and survival of cuttings. Sprouting and survival of thin cuttings of four nodal length were highest compared to other treatments. Key words: Coleus forskohlii, sprouting, hormones, stem cutting

This herbaceous species is not only hard to propagate sexually but also show complexities and show undesirable characters if propagated through sexual means. Vegetative propagation therefore, is the most vital and sole method to reproduce these plants species conserving the innate desirable characters [12]. Establishment and growth rate of the cutting depends upon many factors like seasonal and age variations, portion and diameter of branch, growing media, moisture level, nutrient status and temperature etc. [11]. In addition, the use of plant growth regulatory hormones - auxins, plays a vital role in influencing the important phases of plant growth and development. Auxins are a class of plant growth substances often called phytohormones or plant hormones. Among the auxins Indole 3 Acetic Acid (IAA), - Naphthalene Acetic Acid (NAA) and Indole 3 Butyric Acid (IBA) are typically the principal auxins used for rooting and sprouting of cuttings and majority of plant species are responsive for rooting [7, 10]. These chemicals are available in commercial preparations, dispersed in concentrated liquid (liquid formulation), or in talc (powder formulation), that can be diluted with water (aqueous solution) to proper strength. The present investigation was conducted in view of the vegetative propagation of Coleus forskohlii Birq. by means of stem cuttings. The objectives were set, to study the effect of length, diameter and pretreatment by growth regulatory hormones of stem cuttings, on its sprouting and survival. MATERIALS AND METHODS The experiment was conducted at the research farm of T.C.B. College of Agriculture and Research Station, Bilaspur (Chhattisgarh), India during September 2008. Disease free equally matured C. forskohlii plants were collected from the medicinal plant nursery established in this centre. Stem cuttings were prepared with two length sizes i.e. with two nodes, with four nodes and each type had three different diameter types viz. thick (0.65 cm), medium (0.52 cm), thin (0.43 cm).

INTRODUCTION Coleus forskohlii Birq., an indigenous medicinal herb with tuberous root has been recorded to produce a root drug. In Ayurvedic Materia Medica under the Sanskrit name the plant was refered as Makandi and Mayani [13]. It is widely used in various disorders viz. antiaging, antioxidant and a remedy for heart, abdominal and respiratory disorders [2, 6]. It grows on dry, barren hills and is cultivated in India for its roots, which contains the diterpenoid forskohlin, the presence of which is exclusive to this particular species [1, 14]. The pharmaceutical industry is largely dependent upon wild populations for supply of plant material for forskolin. In fact, it is now listed as one of the plant species in India vulnerable to extinction due to indiscriminate collection of the plant from forests [15]. Thus, it is necessary to develop methods for the conservation of this threatened species which is endangered and can be propagated in vitro.

Author for correspondence; E mail: rkstiwari@rediffmail.com

239

Ganesh\Final\13

Three plant growth regulators i.e. Indole 3 acetic acid (IAA), -naphthalene acetic acid (NAA) and indole 3 butyric acid (IBA) were used in two (liquid and powder) formulations. As a liquid formulation IAA and NAA were applied at a concentration of 500 ppm, 100 ppm, 50 ppm and 10 ppm whereas, concentration of IBA was 1000 ppm, 500 ppm and 100 ppm as per the usual recommendation [a]. Powder formulation was prepared at a concentration of 100 ppm and 500 ppm for all the three growth regulators. For preparation of powder formulations, inert talc powder was mixed proportionately with the growth hormones to get the desired concentration. Water dipping (cuttings dipped in sterile distilled water) and control (without any treatment) treatments were added to compare the effect of growth regulators. Dilute solution dip method was used to apply the exogenous hormone formulations onto stem cuttings [9]. In brief, stem cuttings were dipped in the respective hormone preparations for overnight at room temperature and planted next day in the nursery bed. Irrigation water was applied for 24 hours by overhead sprinklers during the experimental period. After 25 days of planting the number of sprouted cuttings, number of node sprouted were counted whereas, their survival was recorded at 60 days after plantation. All the recorded data were expressed in percentage. The experimental design was randomized complete block design with ten replications for each treatment. Data gathered were analyzed statistically using analysis of variance (ANOVA) and CD were calculated at 5% to separate the treatment means. RESULTS AND DISCUSSION Presented data indicated that the growth hormones at different concentrations used as both formulations (liquid and powder) along with the length and diameter of stem cuttings had significant (Pd0.05) effect on sprouting, nodes sprouting and per cent survival (Tables 1 and 2). Data presented in table indicated that some of the concentrations of growth hormones were found to increase the sprouting significantly however, results were not consistent on the application of different formulations. Moreover, irrespective of all treatments, exogenous hormone application induced mean sprouting, node sprouting and survival superiorly in powder formulations as 44.7%, 22.9%, 83.7% than liquid formulations as 47.4%, 24.4%, 89.4% (Tables 1 and 2). Among growth hormones, IBA @ 100 ppm was found superior (Pd0.05) effectivity than the control, in enhancing the growth as can be observed by the mean per cent sprouting, per cent node sprouting and per cent survival of 53.5%, 24.6% and 100.0% respectively in liquid formulation and 54.6%, 26.3% and 100% respectively in powder formulation. Similarly, in powder formulation of IAA @ 500 ppm, the growth of cutting was superior to control (Figures 2 and 3). IBA were in conformity with the reports of its effectiveness as compared

to several naturally occurring auxins in promotion of adventitious roots [9]. Moreover, Butola and Badola (2004) have recommended IAA and IBA as promising treatments to improve rooting, growth and biomass in A. glauca and Heracleum candicans [3]. Hormone application mean in liquid formulation irrespective of other parameters revealed that IAA @ 100 ppm stimulated per cent sprouting (44.4%) and per cent node sprouting (18.4%) comparable to water treatment and control whereas, for IBA @ 500 ppm and 1000 ppm per cent node sprouting (16.7%, 20.8%) and per cent survival (95.8%, 100.0%) was comparable with control (Figure 1). In case of powder formulation application, per cent node sprouting and per cent survival were comparable with control treatment for NAA @ 1000 ppm (26.5%, 100.0%) NAA @ 500 ppm (26.7%, 100.0%). Securedex B was least in enhancing the sprouting and survival of the stem cuttings compared to other treatments (Figure 2). Chaudhry and Khan (2000) reported that IAA is known to promote the expansion of roots in cutting thereby increasing the survival rate. Similarly, Choudhury and Rashid (2000) reported IAA and IBA increase stem diameter whereas, Sun et al. (1998) evaluated the effects of plant growth regulators (NAA, IAA, IBA) on sprouting of stem cuttings [5, 16].
100 90 80 70 60 50 40 30 20 10 0
pp m NA A5 0p pm NA A1 0p pm IBA 10 00 pp m IBA 50 0p pm IBA 10 0p pm Wa t er di p pi n g pm pm pp m pp m pp m 50 0p 10 0p A5 00 A1 00 Co nt r 50 10 ol

Mean value (%)

IAA

IAA

IAA

IAA

NA

NA

Treatments

Figure 1. Bars represents mean per cent sprouting ( ), per cent node sprouting ( ) and per cent survival ( ) of stem cutting of Coleus forskohlii irrespective of length, diameter of cutting after hormone application in liquid formulation
Mean values (%)
120 100 80 60 40 20 0
10 00 pp m 00 0p pm 00 pp m pp m pm m dip pi n g A1 00 0p NA A5 00 Co 00 ntr ol pp

IBA 5

IAA 5

NA

Treatments

Fig. 2:

Bars represents mean per cent sprouting ( ), per cent node sprouting ( ) and per cent survival ( ) of stem cutting of Coleus forskohlii irrespective of length, diameter of cutting after hormone application in powder formulation.

240

IBA 1

Wa ter

IAA

Table 1.

Effect of length of cuttings, diameter of cuttings and exogenous growth hormones as liquid formulations on per cent sprouting, node sprouted and survival of stem cuttings of Coleus forskohlii

4 Node ppm Dm Cutting sprouted (%) 500 D1 D2 D3

Indole Acetic acid

2 Node Plant survival (%) 50.0 50.0 100.0 42.5 50.0 50.0 100.0 100.0 50.0 100.0 100.0 100.0 47.5 40.0 47.5 100.0 100.0 100.0 100.0 100.0 100.0 100.0 73.3 25.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 0.0 100.0 Cutting sprouted (%) 32.5 50.0 32.5 33.3 58.3 58.3 33.3 33.3 58.3 16.7 29.2 58.3 41.7 25.0 29.2 29.2 50.0 29.2 29.2 29.2 58.3 16.7 16.7 50.0 58.3 25.0 41.7 25.0 25.0 66.7 41.7 58.3 100.0 41.7 29.2 41.7 0.0 25.0 66.7 Node sprouted (%) 10.4 25.0 10.4 12.5 29.2 29.2 18.8 12.5 29.2 10.4 10.4 20.8 16.7 14.6 14.6 10.4 14.6 10.4 10.4 12.5 29.2 8.3 10.4 25.0 29.2 12.5 20.8 12.5 10.4 41.7 10.4 29.2 62.5 20.0 10.0 30.0 0.0 10.0 40.0 Plant survival (%) 42.5 50.0 42.5 47.5 100.0 100.0 45.0 47.5 100.0 100.0 100.0 100.0 40.0 45.0 50.0 50.0 100.0 50.0 100.0 100.0 100.0 100.0 100.0 50.0 100.0 100.0 100.0 100.0 100.0 75.0 100.0 100.0 100.0 100.0 100.0 100.0 0.0 100.0 100.0

Mean (2 Node & 4 Node) Cutting sprouted (%) 37.5 41.7 49.6 33.3 45.8 54.2 37.5 45.8 45.8 29.2 43.7 40.6 37.5 25.0 28.3 43.7 45.8 27.1 29.2 47.9 58.3 20.8 26.7 50.0 58.3 25.0 41.7 27.1 25.0 62.5 41.7 54.2 64.6 50.0 35.4 50.0 29.2 25.0 75.0 41.3 Node sprouted (%) 10.4 16.7 13.3 10.4 20.8 24.0 14.6 13.5 19.8 10.4 10.4 15.6 13.5 14.6 12.5 12.5 12.5 9.4 10.4 14.4 21.9 9.4 10.4 18.8 29.2 12.5 20.8 11.5 10.4 28.1 10.4 27.1 36.5 20.0 10.0 27.5 7.5 7.5 30.0 16.1 Plant survival (%) 46.3 50.0 71.3 45.0 75.0 75.0 72.5 73.8 75.0 100.0 100.0 100.0 43.8 42.5 48.8 75.0 100.0 75.0 100.0 100.0 100.0 100.0 86.7 37.5 100.0 100.0 100.0 100.0 100.0 87.5 100.0 100.0 100.0 100.0 100.0 100.0 50.0 50.0 100.0 81.5

Node sprouted (%) 10.4 8.3 16.3 8.3 12.5 18.8 10.4 14.6 10.4 10.4 10.4 10.4 10.4 14.6 10.4 14.6 10.4 8.3 10.4 16.3 14.6 10.4 10.4 12.5 29.2 12.5 20.8 10.4 10.4 14.6 10.4 25.1 10.4 20.0 10.0 25.0 15.0 5.0 20.0

42.5 33.3 66.7 33.3 33.3 50.0 41.7 58.3 33.3 41.7 58.3 22.9 33.3 25.0 27.5 58.3 41.7 25.0 29.2 66.7 58.3 25.0 36.7 50.0 58.3 25.0 41.7 29.2 25.0 58.3 41.7 50.0 29.2 58.3 41.7 58.3 58.3 25.0 83.3

100

D1 D2 D3

50

D1 D2 D3

10

D1 D2 D3

500
a-Naphthalene acetic acid

D1 D2 D3

100

D1 D2 D3

50

D1 D2 D3

10

D1 D2 D3

1000
Indole Buteric acid

D1 D2 D3

500

D1 D2 D3

100

D1 D2 D3 D1 D2 D3 D1

Water Dipping

Control Mean

D2 D3

241

Table 2.

Effect of length of cuttings, diameter of cuttings and exogenous growth hormones as powder formulations on per cent sprouting, node sprouted and survival of stem cuttings of Coleus forskohlii

4 Node ppm Dm Cutting sprouted (%)

Indole Acetic acid

2 Node Plant survival (%) 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 62.5 52.5 100.0 50.0 67.5 75.6 Cutting sprouted (%) 40.0 37.5 60.0 20.0 90.0 80.0 32.5 60.0 60.0 10.0 20.0 60.0 22.5 20.0 50.0 17.5 30.0 45.0 30.0 30.0 70.0 20.0 35.0 42.5 Node sprouted (%) 30.0 20.0 40.0 10.0 32.5 40.0 13.5 47.5 45.0 10.0 10.0 45.0 10.0 15.0 45.0 10.0 15.0 45.0 15.0 25.0 35.0 15.0 15.0 30.0 Plant survival (%) 100.0 87.5 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 50.0 50.0 60.0 60.0 20.0 57.5

Mean (2 Node & 4 Node) Cutting sprouted (%) 30.0 58.8 62.5 35.0 90.0 85.0 31.3 45.0 75.0 20.0 35.0 65.0 22.5 50.0 55.0 36.3 55.0 72.5 25.0 35.0 60.0 20.0 32.5 42.5 47.4 Node sprouted (%) 17.5 26.3 32.5 12.5 32.5 37.5 12.5 28.3 38.8 30.0 16.3 33.8 10.0 20.0 33.8 15.0 21.3 42.5 12.5 20.0 28.8 21.3 13.8 28.8 24.4 Plant survival (%) 100.0 93.8 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 56.3 51.3 80.0 55.0 43.8 66.6 89.4

Node sprouted (%) 5.0 32.5 25.0 15.0 32.5 35.0 11.5 9.0 32.5 50.0 22.5 22.5 10.0 25.0 22.5 20.0 27.5 40.0 10.0 15.0 22.5 27.5 12.5 27.5

1000

D1 D2 D3

20.0 80.0 65.0 50.0 90.0 90.0 30.0 30.0 90.0 30.0 50.0 70.0 22.5 80.0 60.0 55.0 80.0 100.0 20.0 40.0 50.0 20.0 30.0 42.5

500

D1 D2 D3

Indole Buteric acid a-Naphthalene acetic acid

1000

D1 D2 D3

500

D1 D2 D3

1000

D1 D2 D3

500

D1 D2 D3 D1 D2 D3 D1 D2 D3

Water Dipping

Control

Mean

Table 3. CD at 5% level of different treatments for hormone application in liquid and powder formulations

C.D. at 5 % (liquid formulation) Treatments Hormones Diameter Number of nodes Hormones x Diameter Hormones x Nodes Diameter x Nodes Hormones x Diameter x Nodes Sprouting 9.5 4.1 5.7 16.9 13.8 5.8 23.9 Node sprouting 5.6 2.7 1.9 9.5 7.7 5.4 13.4 Survival 13.7 7.4 ns 15.7 12.8 5.4 22.2

C.D. at 5 % (powder formulation) Sprouting 9.6 9.4 3.5 12.9 10.5 6.1 18.2 Node sprouting 11.6 4.6 3.8 13.8 11.3 6.5 19.5 Survival 4.9 5.1 2.6 9.5 7.7 4.5 13.4

242

100 90 80 70 60 50 40 30 20 10 0

Mean value (%)

79.4

82.0

82.6

formulations 64.7%, 34.5%, 93.3% were noticeably higher than liquid formulations 49.8%, 21.4%, 82.6% respectively (Fig. 3 & 4). Coleus forskohlii is propagated by stem tip cuttings, described as herbaceous cuttings [8]. Market demand of these species for pharmaceuticals and ethno-medicinal utility, are met through harvesting from wild populations. Thus, plant growth regulators can be widely used in vegetative propagation to improve sprouting, rooting and survival of cuttings. From the present study it can be concluded that Indole 3 Acetic Acid (IAA), - naphthalene acetic acid (NAA) in powder formulation are applicable to thin stem cuttings of Coleus forskohlii for better sprouting and absolute survival. ACKNOWLEDGEMENT The authors are thankful to Chhattisgarh Council of Science and Technology (CGCOST) for providing the requisite funds to carry out the research work which is part of the project entitled Conservation of indigenous medicinal plant of Chhattisgarh forest with special reference to growing behavior of commercially important species. REFERENCES
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49.8 36.5 37.5 21.4 13.1 13.9

Thick

Medium

Thin

Branch cutting diameter (cm)

Fig. 3:

Bars represents mean per cent sprouting ( ), per cent node sprouting ( ) and per cent survival ( ) of stem cutting of Coleus forskohlii at three different cutting diameter (thick= 0.65 cm, medium= 0.52 cm, thin= 0.43 cm) irrespective of cutting length after hormone application in liquid formulation.

100 90 80 70 60 50 40 30 20 10 0

88.9

93.3 86.1 64.7 50.2 34.5

Mean values (%)

27.5 16.4

22.3

Thick

Medium

Thin

Brach cutting diameter (cm)

2. 3.

Fig. 4:

Bars represents mean per cent sprouting ( ), per cent node sprouting ( ) and per cent survival ( ) of stem cutting of Coleus forskohlii at three different cutting diameter (thick= 0.65 cm, medium= 0.52 cm, thin= 0.43 cm) irrespective of cutting length after hormone application in powder formulation.

4.

5.

Mean values of per cent sprouting, per cent node sprouting and per cent survival irrespective of hormone type, concentration and length of cutting revealed that the thin stem cuttings were significantly more inducible by the application of hormones in both liquid and powder formulations whereas, growth of thick cuttings were least inducible (Figures 3 and 4). For example, mean per cent sprouting was significantly higher in thin cuttings (49.8%) than thick (36.5%) after liquid formulation application. Similarly, after application of liquid formulation sprouting mean was considerably higher in thin cuttings (64.7%) than thick cuttings (27.5%). Same trends were followed for mean per cent node sprouting and mean percent survival of stem cuttings (Figures 3 and 4). Exogenous hormone applications in power formulations were more effective than liquid formulations on average. For example, the average per cent sprouting, node sprouting and survival of thin stem cuttings in powder

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