ADALYA JOURNAL ISSN NO: 1301-2746

Effect of Fungal Elicitors on Growth in the in vitro Cultures of umbellata L.

Author - S. Saranya Research Scholar, PG and Research Department of Botany Government Arts College (Autonomous), Karur 639 005, Tamil Nadu - INDIA E-mail: [email protected]

Co-author - P. Velayutham Research Supervisor, Krishna College of Arts and Science, Kolluthannipatti, Karur Dt., Tamil Nadu - INDIA E-mail : [email protected]

Abstract

Improvement strategies on plant growth are appreciable for commercially important medicinal due to

over exploitation. Oldenlandia umbellata L. is one such yielding medicinal plant with the wide range of

application in pharma and textile industry. An attempt was made in the present study to enhance the growth of

O.umbellata in the in vitro by three fungal elicitors, namely, Aspergillus niger, Mucor prayagensis and Trichoderma

viride. Among the three fungal elicitors A. niger derived elicitors showed the better callogenic response, shooting

and rooting. The optimum concentration for each elicitor was studied. The maximum number of 79 shoots and 47

roots were obtained in 100 µg l-1 A. niger elicited callus.

Keywords: Oldenlandia umbellata L., suspension culture, fungal elicitors, callogenesis, regeneration, rooting,

hardening.

Introduction

Plant cell, tissue, and organ culture is one of the promising technologies for commercial production of

secondary metabolites when natural resources are limited. The stimulation of defined compounds within carefully

regulated in vitro cultures provides better understanding of metabolic pathways under highly controlled growth

conditions[1]. The industrial application of plant cell cultures has limited success and only few high-value natural

products such as shikonin, paclitaxel, resveratrol, artemisinin, ginsenosides, and ajmalicin have been

commercialized [2] so far due to the low content of the desired metabolite, recalcitrant nature and slow growth rate,

genotypic variations, chemical instability, and uneconomical downstream processing. Several strategies have been

developed to overcome these issues, such as medium optimization, elicitation, precursor feeding, immobilization, in

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situ product removal, genetic transformation and bioreactor engineering[3]. Elicitation is an attractive strategy

employed to induce growth of the plant and associated metabolite production due to the addition of trace amounts of

elicitors in a plant in vitro culture systems. Plants show a wide range of morphological and physiological responses

with respect to the elicitors. It involves numerous molecular changes inside the plants concerning for the particular

stress conditions. Fungal elicitors have been widely employed to increase natural product formation in plant cell

cultures and this strategy has been effective in stimulating the growth and metabolite production.

Oldenlandia umbellata L. is one of the important members of known for its dyeing and

medicinal properties. This plant is used in traditional medicine and Siddha for its styptic property[4]. The leaf and

root extracts were considered as good expectorants and used for treatment of asthma, bronchitis, and bronchial

catarrh[5]. The decoction prepared from its leaves is used as a rinse to treat poisonous bites[6] and also used as a

febrifuge. A novel pH indicator dye was reported from this plant[7]. Extract of the whole plant shows significant

antitumor activity[8]. The major dyeing property depends on anthraquinone contents of roots and used to impart red

colour to the textile materials[9,10]. The multipurpose usage has made increased usage of this plant and a reliable

protocol was developed for enhanced growth of the plant through tissue culture technique. So, the present study

aims to improve the plant growth by using fungal elicitors in addition to the plant growth regulators.

Materials and Methods

Plant Material and Fungal Strains

The leaf derived callus of O. umbellata was obtained from previously reported protocol [11]. Three fungal

strains such as Aspergillus niger, Mucor prayagensis and Trichoderma viride, were procured from the Institute of

Microbial Technology, Chandigarh, and used for elicitation

Preparation of Fungal Elicitors

The fungal strains were grown separately in 250 ml conical flasks containing 100 ml SD (Sabrouds

dextrose) broth. The flasks were incubated at room temperature under static conditions. At stationary phase, after 21

days, the flasks were autoclaved and the fungal mats were separated from the culture medium. Fully grown mycelia

with spores were homogenized with mortar and pestle and centrifuged at 4000 rpm and the supernatants were

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collected. The collected supernatant was autoclaved for 20 min at 121°C. The autoclaved fungal extracts were used

as elicitors for in vitro culture system. The fungal extracts were stored at 4°C for elicitation purpose.

Treatment of Fungal Elicitors

All three fungal elicitors of four different concentrations, viz., 25, 50, 100 and 200 µg l-l (Equalent

polysaccharide content) were added separately into the standardized MS liquid medium for callus culture (10 µM

NAA and 30 g l-l sucrose) and the well-developed callus was aseptically transferred to the respective liquid medium.

Cell suspension cultures were established and maintained on the above liquid medium at a subculture interval of 7

days. All the cultures were maintained in an orbital shaker at 120 rpm min-1 for 30 days.

The treated calli were then transferred to shooting medium fortified with 6 µM BAP in combination with 2

µM Kin and respective fungal elicitors. The well grown shoots were separated and transferred to rooting medium of

6 µM IBA. Fully matured plantlets with well developed roots were transferred and allowed for the acclimatization

process.

Determination of growth parameters

The different parameters were recorded such as number of shoots, shoot length, number of roots per shoot,

and root length were recorded.

Statistical analysis

All the experiments were repeated three times and each experiment consisted of one explant per tubes and

five replicates. Statistical analysis was carried out using Analysis of Variance (ANOVA) comparing the treatments

and using Duncan’s Multiple Range Test (DMRT) at the 5% probability and analysed using SPSS for Windows,

version 21.

Results

In the present study, callus induced from the leaf explants of O. umbellata was treated with different fungal

elicitors in varying concentrations to screen the suitable elicitor and its concentration for enhanced plant growth.

Callus Culture

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Callus was induced from the leaf explants on MS medium fortified with different concentrations of IAA,

IBA, NAA and 2,4-D. Maximum rate of 100 per cent callus induction with green compact organogenic callus was

observed on MS medium supplemented with 10 µM NAA.

Effect of Fungal Elicitors on Callus Growth

The calli were grown on suspension media containing 10 µM NAA and four different fungal elicitor

concentrations, namely, 25, 50, 100 and 200 µg l-1 of the three elicitors to optimize for higher production of cell

biomass (Fig.1.a-d). The treated calli were further subcultured on solid media with respective fungal elicitors and

obtained large amount of calli. Of the three fungal elicitors treated, large amount of green compact callus was

obtained on medium treated with the extract of A. niger followed by T. viride. However, the callus treated with M.

prayagensis showed an equal response for callus growth to that of control at all tested concentration.

Regeneration of shoots from fungal elicited calli

The well developed calli were grown on regeneration medium containing 6 µM BAP + 2 µM Kin with

respective fungal elicitors. The shoot regeneration response was varying among the various elicitor treatments with

different concentrations (Table 1; Fig.2.a-d). The maximum shoot regeneration response was obtained from 100 µg

l-1 A. niger elicitor treated calli. Maximum number of 78.8 shoots with shoot length of 11.4 cm was achieved on the

callus treated with A. niger. The M. prayagensis elicitor treated calli at 50 µg l-1, concentration produced 72.6

shoots with 10.2 cm shoot length, where as T.viride at 25 µg l-1 concentration produced an average number of 68.2

shoots with 9.8 cm shoot length. The shoot regeneration frequency and number of shoots were decreased when the

calli were treated with higher dose.

Rooting and Hardening

The healthy shoots obtained from all the treatments showed better rooting on standardized rooting medium.

The rooting response was accompanied with shooting frequency with same concentrations (Table 2; Fig.3.a,b). The

maximum root regeneration response was obtained from 100 µg l-1 A. niger elicitor treated shoots followed by 50 µg

l-1 M. prayagensis and 25 µg l-1 T. viride with an average number of 46.6, 28.6 and 30.8 respectively from root

induction medium containing 6 µM IBA. The root regeneration frequency and number of roots were decreased when

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the shoots were treated with above optimal concentration. Similar to organogensis, during elicitation process also the

plantlets developed in vitro flowers in umbels (Fig.3.b). The rooted plants were hardened successfully on paper cups

(Fig.3.c).

Discussion

In the present study, the growth response and morphological parameters were hiked up due to elicitation

process. These results were adding an advantage to the current research on enhancement of desired compounds

through elicitation in tissue culture. Several plant elicitors have been found to stimulate defence responses, while

others stimulate growth responses, leading to increased morpho-physiological and yield parameters [12].

Elicitors cause metabolic changes in plant and result in altered level of morphological development and

bioactive compound production[13]. Poly- and oligosaccharides are the most studied signalling molecules for

elicitation pathways because these compounds can effectively induce similar plant defence responses to pathogen

invasion [13] and some workers stated the molecular dialogue between the fungal species and the plant[14]. One such

response is the production of phytoalexins from the infected plant. The increase of phytohormone would enhance the

biomass weight of the plant. In the present study also fungal elicited callus proliferated faster than that of the

untreated callus in the suspension culture as well as in the solid media treated with fungal elicitors.

Elicitor concentration plays a very important role in elicitation process. High dosage of elicitor has been

reported to induce hypersensitive response leading to cell death, whereas, an optimum level was required for

induction [15,16,17]. In the present study also, growth responses were decreased in increasing concentration of fungal

elicitors above the optimum concentration.

Of the three fungal strains studied, A. niger derived elicitors showed the maximum response in callus, shoot

and root formation in O. umbellata. These observations were supported by the results of Chakraborty and

Chattopadhyay[18] in the stimulation of menthol production and biomass accumulation in the cell suspension culture

of Mentha piperita treated with A. niger. Similarly, the best eliciting effects of A. niger and T. virens on Salvia

fruticosa was also reported for growth and production of oleanolic acid and ursolic acid[19].

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However, when compared to other fungal strains, M. prayagensis showed a little poor response in

callusing, shooting and rooting. But in contrast to this result, increased biomass was reported on elicitation of cell

suspension cultures of Peganum harmala by Mucor sp. against the control [20].

Trichoderma viride derived elicitor at its lower concentration showed significant results in this study.

Certain Trichoderma sp. has beneficial effects on plant growth and enhances resistance to both biotic and abiotic

stresses. Early work revealed that some Trichoderma sp. promotes growth responses in radish, pepper, cucumber

and tomato [21,22]. Further studies demonstrated that Trichoderma also increases root development and crop yield, the

proliferation of secondary roots, and seedling fresh weight and foliar area [23].

When comparison made with all three fungal elicitors, the parameters of plant growth varies differently.

The best elicitor system at its optimal concentration for enhanced plant growth was fixed for O. umbellata plant

system. The maximum shooting and rooting was recorded in 100µg l-1 concentration of A. niger than other two

fungal elicitors. The optimal dose for each elicitor was also reported in this study as 50 µg l-1 concentration for M.

prayagensis and 25µg l-1 for T. viride elicitor. This result was highly significant when compared to that of control.

So the present study concludes that A. niger derived fungal extracts can be a suitable elicitor in terms of

morphological development of the plant. References

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Fig. 1. Suspension culture of leaf derived callus of Oldenlandia umbellata L. on MS liquid medium treated with different fungal elicitors. a. Control; b. Aspergillus niger; c. Mucor prayagensis d. Trichoderma viride.

Fig. 2. Regeneration of plantlets from the control and fungal treated calli of Oldenlandia umbellata L. on MS solid. a. Control; b. Aspergillus niger; c. Mucor prayagensis d. Trichoderma viride.

Fig. 3. Rooting and hardening of Oldenlandia umbellata L. plantlets treated with fungal elicitros. a,b. rooting of isolated shoots derived from fungal elicited plantlets and in vitro flowering (b); e. hardening of rooted plantlets on paper cup

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Table 1. Shoot regeneration from the control and fungal treated callus of Oldenlandia umbellata L.

Concentrations of Fungal elicitors Mean No. of shoots Mean shoot length (cm) (µg/l) Control 65.4 ± 1.62hi 9.4 ± 1.09fg Treated with A. niger 25 67.2±1.65fg 9.8±0.17de 50 71.8±1.35cd 10.6±0.24b 100 78.8±1.28a 11.4±0.14a 200 68.8±1.39e 10.4±0.19bc Treated with M. prayagensis 25 72.8 ± 1.20b 9.7 ± 0.07ef 50 72.6 ± 1.28bc 10.2 ± 0.13cd 100 64.2 ± 0.86ij 9.2 ± 0.14gh 200 55.8 ± 1.39l 6.4 ± 0.17k

Treated with T. viride 25 68.2 ± 0.96ef 9.8 ± 0.09de 50 66.4 ± 1.63gh 9.2 ± 0.15gh 100 68.2 ± 1.48ef 8.8 ± 0.16hi 200 60.4 ± 1.74k 7.9 ± 0.07j

Values are Mean of 5 replicates recorded after 28 days of culture. Values in the last two columns are Mean ± SE of Mean followed by the letters within the column indicating the level of significance at P<0.05 by Duncan’s Multiple Range Test.

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Table 2. Rooting of isolated shoots of control fungal treated plantlets of Oldenlandia umbellata L.

Concentrations of Fungal elicitors Mean No. of shoots Mean shoot length (cm) (µg/l) Control 65.4 ± 1.62hi 9.4 ± 1.09fg Treated with A. niger

25 67.2±1.65fg 9.8±0.17de 50 71.8±1.35cd 10.6±0.24b 100 78.8±1.28a 11.4±0.14a 200 68.8±1.39e 10.4±0.19bc Treated with M. prayagensis

25 72.8 ± 1.20b 9.7 ± 0.07ef 50 72.6 ± 1.28bc 10.2 ± 0.13cd 100 64.2 ± 0.86ij 9.2 ± 0.14gh 200 55.8 ± 1.39l 6.4 ± 0.17k Treated with T. viride

25 68.2 ± 0.96ef 9.8 ± 0.09de 50 66.4 ± 1.63gh 9.2 ± 0.15gh 100 68.2 ± 1.48ef 8.8 ± 0.16hi 200 60.4 ± 1.74k 7.9 ± 0.07j

Values are Mean of 5 replicates recorded after 28 days of culture. Values in the last two columns are Mean ± SE of Mean followed by the letters within the column indicating the level of significance at P<0.05 by Duncan’s Multiple Range Test.

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