Ajit Arun Waman?Pooja Bohra?Avinash Norman

Abstract Semecarpus kurzii Engl.is an important ethnomedicinal plant used for curing tumours,inflammation,fever and pain,and is also a source of isoricinoleic acid.During the present investigation,effect of chemical pretreatments on germination and seedling growth revealed significant differences.Different pre-treatments could improve germination and treatment with KNO3(9.9 mM)was significantly superior(94%)to the untreated control(76%).Mean daily germination varied among the treatments(0.90–4.14 seeds per day),when compared with 2.27 seeds per day in control.Mean germination time was significantly reduced from 22.14 days(control)to 16.40 days(19.8 mM KNO3)and 17.16 days(1.45 mM GA3).Treatment with KNO3(19.8 mM)and GA3(1.45 and 2.90 mM)significantly reduced the time for 50% germination.Treatment with higher concentrations of GA3and KNO3 exhibited significantly faster germination initiation(11.0 and 12.0 days,respectively)over the control(17.0 days).Seedling vigour index was not improved by pre-treatments.Chemical treatments did not improve shoot growth,collar thickness,or number of seedling leaves;however,root growth was significantly improved over control.Treatment with GA3(2.90 mM)promoted leaf elongation but reduced leaf width significantly.Treatment with KNO3and GA3 could be recommended to obtain healthy seedlings for establishing new plantations of this important medicinal plant species.

Keywords Conservation·Indian system of medicines·Tribal healthcare·Wild cashew

Introduction

Semecarpus kurziiEngl.,an important species of the Anacardiaceae family,is commonly known as wild cashew in English,Chaybenin Burmese andBhallatakaorBada bhilwain Sanskrit(Nair et al.1984).The species is native to India and is found in the evergreen,semi-evergreen and moist deciduous forests of Andaman and Nicobar Islands(ANI)and Myanmar(Dagar 1989;Prasad et al.2009).In the Ayurvedic system of medicine,the species is referred to asArbudaharaowing to its antitumor properties and is known to possess a number of medicinal properties due to which it is commonly used in the preparation of formulations such asBhallataka tailaandBhallataka ghrita(Nair et al.1984).Traditionally,the leaves are used in the treatment of inflammation,fever and pain,and the leaf properties have been clinically tested in recent past(Das et al.2013).S.kurziicontains iso ricinoleic acid(9-hydroxy-cis-12-octadecenoicacid)considered as a potential renewable raw material for the oleochemical industry and a precursor for the synthesis of various antimicrobial compounds(Farooqi et al.1985).The fruit(Fig.1)is valued by the aboriginals of ANI;the Onge tribe uses it as food(Awasthi 1988)while the Shompen tribe commonly employs the species to treat various injuries(Arora 2010;Dwivedi 2015).

Fig.1 A view of the immature A and mature B fruits of S.kurzii

Like many other medicinal species,S.kurziiis collected from the wild at present.As the demand for plant-based drugs is increasing,the pressure on natural resources is growing at an alarming rate(Waman and Bohra 2016).In the absence of a sustainable replenishing strategy,rare and endemic flora is under constant threat.Existing forests have been fragmented due to anthropogenic activities which affect these species and drive them towards extinction(Prasad et al.2009).The occurrence of natural events such as cyclones, fire,high tides and Tsunami has further added to the need for conservation of economically and ecologically important species worldwide(Dam Roy and Krishnan 2005;Singh et al.2016).The devastating Tsunami that occurred in ANI during 2004 severely impacted a number of species,includingS.kurzii(Porwal et al.2012).Habitat enrichment is a viable strategy to support the conservation and sustainable use of biodiversity.Seed propagation is one of the easiest yet effective methods for regenerating medicinal plants(Butola and Badola 2004).Further,as a wild relative of commercially important species such as cashew (Anacardiumoccidentale)and marking nut(S.anacardium),possibility of usingS.kurziias rootstock should be explored.However,a literature survey revealed that there is no information on the regeneration of this species.

Considering these points,the objective of this investigation was to study the effect of chemical pre-treatments on seed germination and seedling growth to obtain a maximum number of quality seedlings.

Materials and methods

The experiment was conducted during 2015–2016 in the Division of Horticulture and Forestry,ICAR-Central Island Agricultural Research Institute,Port Blair,ANI.Nuts were collected from ripe fruits on tree located at 11°36′49.8′′N;92°43′3.6′′E in the germplasm block of the Institute.The nuts were washed,soaked in water overnight and only ones that had sank were used(Mandal 2000).

As no studies were available on the regeneration of this specific species,reports on related species were used to determine the treatments(Table 1).Seeds were soaked for 24 h in all treatments except in acetone and chloroform,wherein seeds were soaked for 1 or 2 h.Seeds that were untreated served as control.The experiment was laid out in a completely randomized design with 50 seeds in each treatment,replicated twice.Observations on germination were recorded daily and used to calculate seedling vigourrelated parameters.Standard formulae were used for the calculation of germination and seedling growth-related parameters as:

where ‘M’is the mean daily germination,‘G’the cumulative germination percentage,and ‘D’the total number of days for completion of germination

where ‘T’is the mean germination time,‘n’the number of seeds germinated on day ‘D’,and ‘D’the number of days from sowing(Ellis and Roberts 1980)

where ‘GI’is germination index,‘Gi’and ‘Gf’the number of germinated seeds at‘Di’i.e.,days of first count and ‘Df’i.e.,days of final count,respectively(AOSA 1983)

where ‘T50’is the time taken for 50%germination,‘N’the final number of germinated seeds,ni＜N/2＜nj,‘n’thecumulative number of seeds germinated by adjacent counts at time ‘t’(Coolbear et al.1984,Farooq et al.2005).

Table 1 Seed treatments used in the present study and in previous reports for formulating the treatments

SVI=L×G， （5）where ‘SVI’is the seedling vigour index,‘L’the seedling length and ‘G’the cumulative germination percentage.

The data were subjected for analysis of variance and mean separation(least significant difference)using Web Agri Statistical Package WASP 2.0(ICAR RC for Goa,Ela,India).

Results

Percent germination

Mean germination varied between 34 and 94%amongst the treatments(Fig.2).Only one treatment(9.9 mM KNO3)improved germination significantly over untreated control(76%).This treatment however,did not differ significantly from a number of other treatments.Further,treatments with chloroform at both times and with acetone(2 h)were not effective in improving germination.

Fig.2 Seed germination as affected by different treatments

Mean daily germination(MDG)

Significant differences were noticed among the treatments for MDG,and about 0.90–4.14%of the seeds germinated per day(Table 2).The highest values were recorded for seeds treated with KNO3(potassium nitrate at 19.8 mM)but did not differ significantly from those soaked in KNO3(9.9 mM),thiourea(26.2 mM),GA3(gibberellic acid at 1.45 and 2.90 mM)and in acetone for one hour.These values were,however,significantly higher than in the untreated control(2.27%seeds per day).

Mean germination time(MGT)

MGT varied between 16.40 and 28.39 days amongst the treatments.It was significantly reduced from 22.14 days(control)to 16.40 days(19.8 mM KNO3)and 17.16 days(1.45 mM GA3),while it was extended to 28.39 days in seeds treated with lower concentration of thioure a(13.1 mM).

Germination index(GI)

Treatments involving KNO3and GA3significantly improved the number of seeds germinated per day,while treatments with chloroform and acetone were unsuitable.About 1.37 seeds germinated per day(Table 2)in treatment involving KNO3(19.8 mM),which remained on par with those treated with 2.90 mM GA3(1.32),9.9 mM KNO3(1.28)and 1.45 mM GA3(1.20).All these treatments were significantly superior to untreated control(0.88)and seeds treated with water(0.76).

Table 2 Germination parameters as affected by different treatments

Time for 50%germination(T50)

Soaking seeds in KNO3(19.8 mM)and GA3(1.45 and 2.90 mM)significantly reduced the time taken for 50%germination.In contrast,seeds soaked in water or thiourea(13.1 mM)delayed this time to considerable extent.About 20.6 days were required for attaining 50%germination in untreated control(Table 2).Interestingly,the T50value was less in untreated control than seeds soaked in water.

Time taken for first germination and final germination

Seeds treated with GA3(2.90 mM)and KNO3(19.8 mM)exhibited the fastest germination(11.0 and 12.0 days,respectively),while as high as 20.5 days were required for initiation of germination in seeds treated with acetone(2 h).A number of treatments however failed to promote early germination and their values remained on par with that of untreated control.The time required for final germination ranged between 21.5 and 38.5 days amongst the treatments.

Seedling vigour index(SVI)

SVI varied significantly amongst the treatments(Fig.3);however,none of the treatments could improve this parameter over untreated control.Treatments with chloroform(both 1 and 2 h)and acetone(2 h)gave the poorest seedling vigour index values.

Fig.3 Seedling vigour index as affected by different seed treatments in S.kurzii

Seedling growth parameters

Highly significant differences were noticed among the treatments for seedling growth parameters(Table 3).Mean shoot length varied between 22.85 and 28.35 cm.However,shoot length in the control did not differ significantly from the best performing treatments.Though shoot length was observed to be superior in untreated control,root length was not promoted in this treatment.The use of chemical pre-treatments viz.GA3(2.90 mM),thiourea(13.1 mM),chloroform(2 h)and KNO3(19.8 mM)assisted in the promotion of root growth as significantly longer roots were produced by the seedlings.

Though significant variations were observed among the treatments for collar thickness,the values in untreatedcontrol did not differ significantly from those in the chemical treatments.In the case of the number of leaves produced,a similar trend was noticed where untreated control remained statistically similar to chemical treatments.Differential effect of treatments was witnessed on leaf traits.Treatment of seeds with GA3(2.90 mM),although promoted leaf elongation,reduced the leaf width significantly.Similarly,treatment with KNO3(19.8 mM)produced shorter and broader leaves.Leaf width was in the order T1＞T2＞T3＞T6.

Table 3 Seedling growth parameters as affected by different seed treatments

Discussion and conclusion

The Andaman and Nicobar group of islands in the Indian subcontinent is an important biodiversity region,exhibiting a high degree of endemism(Pandey and Diwakar 2008).The Indian System of Medicines makes use of such native diversity for treating various ailments(Kala et al.2006).Though a majority of raw material was collected from wild in ancient times,the basic principles of conservation and sustainability were never ignored when harvesting from forests(Waman and Bohra 2016).However,recently natural calamities,urbanization and indiscriminate harvesting to meet growing demands have been witnessed.This in turn has markedly affected the wild flora and threatened natural stocks(Maiti and Geetha 2014).

Seed propagation is one of the effective mechanisms to create and maintain the diversity in nature.A number of treatments have been found to promote the seed germination process;however,success varies with species and thus standardization is required(Butola and Badola 2004).Further,germinability alone is insufficient to judge the efficacy of a treatment and hence quantitative parameters are required to be studied to understand the performance of various treatments(Ranal and de Santana 2006).Germination percentage indicates the degree of success of a treatment,while MGT is the weighted mean of the germination time which considers that the number of seeds germinated each day varies(Labouriau 1983).Time for 50%germination(T50)is a measure of the central tendency of the germinating seeds and is the overall speed of germination(Ranal and de Santana 2006).SVI expresses vigour of the regenerated seedlings.

Significant variations were noticed among the treatments for germination,growth and vigour-related parameters.Shoot length,collar thickness and number of leaves were not improved significantly with the use of any chemical pre-treatment.On the other hand,root length and leaf parameters were positively affected with various treatments.In general,the treatment of seeds with gibberellic acid and potassium nitrate helped produce seedlings with better growth parameters.

During the present investigation,potassium nitrate,thiourea and gibberellic acid promoted seedling growth and the response was found to be dose-dependent.Seeds treated with KNO3and thiourea produced seedlings with shorter shoot lengths than the control,but the reverse was observed for root length.A higher concentration of KNO3had lower shoot length(22.85 cm)but higher root length(11.58 cm)than those observed with lower concentration of KNO3(27.65 and 7.55 cm,respectively).A chemical is known to produce desirable results based on the concentration employed and its interaction with the seed and environment.Both suboptimal and supra-optimal concentrations are not desirable as they may lead to undesirable results.

Potassium nitrate is a chemical which has been documented to improve the germination process in a number of species(Shim et al.2008;Samir et al.2015).It may also serve as a nutrient source(Hegazi et al.2011)and thereby help in the promotion of growth in plant tissues.Gibberellic acid is known to enhance the synthesis of a hydrolase enzyme(Prasad and Prasad 2009)and control the mobilization of starch required for breakdown during respiration,thereby promoting cell elongation(Shah 2007).Gibberellins also assist in increasing mineral availability that promotes the germination process.Improvement of seed germination and seedling vigour using gibberellins has been well-documented in a number of economically important species(Sfairi et al.2012;Samir et al.2015;Khan 2015).Hence,the superiority of these chemicals in improving seed germination in the present study is justified.

An earlier report on cashew suggested that pre-soaking seeds in chloroform and acetone could give rapid,synchronized germination(Subbaiah 1982).This was attributed mainly to the removal of a waxy pericarp by these solvents which facilitated imbibition process.However,for unknown reasons,seed germination was adversely affected by these treatments during this study.

In general,delayed germination in cultivated cashew has been reported to be due to slow water imbibition through a dry seed coat(Subbaiah 1982).Soaking in water is a common method of improving germination.However,during the present investigation,seeds soaked in water for 24 h took longer for completing 50% germination(27.5 days)than untreated control(20.6 days).Oyewole and Koffa(2010)suggested significant interaction amongst seed size,soaking duration and seed storage.They observed delayed onset of germination in medium-sized seeds stored for 2 weeks and soaked for 12 h(31.0 days)than those in untreated control(25.0 days).Hence,detailed studies inS.kurziion similar aspects could help in understanding the reasons for this occurrence in the present study.

Medicinal plants are the main source of primary healthcare in a number of developing countries and their potential has been exploited by major pharmaceutical giants in developed countries.The promotion of underutilized species such asS.kurziirequires systematic efforts,and present study demonstrates the possibility of regeneration in a basic way.Wild relatives of crop plants have been valuable resources as they provide opportunities for the identification of useful genes for improvement programmes(Samir et al.2015).They could also be used for obtaining quality seedlings for use as rootstocks for grafting commercially important species including cashew and marking nut.

Appreciable germination was observed in the untreated control which suggests that the species could regenerate on its own under natural conditions,provided the seeds remain protected in the soil.Use of pre-treatments such as potassium nitrate or gibberellic acid could help improve germination and produce vigorous seedlings.Large number of seedlings produced following pre-treatments could be ideal for afforestation programmes for the conservation of this species,for broadening the resource base to obtain raw material for traditional medicines,and for possible exploitation as rootstocks to related species.

AcknowledgementsThe authors are grateful to the Director of the Institute for providing the necessary facilities and constant encouragement.Authors are also thankful to Dr.Sanjay Mishra,Botanical Survey of India,Andaman and Nicobar Islands for his help in the identification of the species.