Preventing Extinction and Improving Conservation Status of Threatened Medicinal Plants through

Macropropagation: Adhatoda beddomei Clarke and Stereospermum tetragonum D.C.

Centre for Conservation of Natural Resources,Foundation for Revitalization of Local Health TraditionsBangalore-560106, Karnataka, India.

University School of Environment ManagementGuru Gobind Singh Indraprastha UniversitySector-16 C, Dwarka, New Delhi-110077,India.

Dr. K. HaridasanProfessor EmeritusMrs. Nandini D.

consultant

Dr. Rita SinghAssociate professor

By-Kumari Kshama AwasthiM.Sc. Biodiversity and ConservationUniversity school of environment managementGuru Govind Singh Indraprastha University

Introduction-

India is known for its rich diversity of medicinal plants (Gordon M.C and David J.N.,2001 ).

Medicinal plants are the source of obtaining variety of drugs (WHO)

Natural propagation has become critical due to changing biodiversity elements and some of the plants are threatened in their habitat.

Hence , proper propagation techniques has to be standardized to address ex-situ conservation of threatened plants.

Plant propagation is the branch of horticulture which deals with the production of new plants using various starter materials that is seeds (sexual propagation) or vegetative parts (stem,tuber,corms).

Here we focused on two important medicinal plants :

1)Adhatoda beddomei C.B Clarke (Acanthaceae) Medicinal use- fever, inflammation, bleeding, diabetes, disinfectant, jaundice, antiseptic and expectorant (Patel and Bhatt ,1984).

Included in IUCN Threatened Plants List of India (Panigrahi and Patel,2014).

2)Stereospermum tetragonum DC. (Bignoniaceae) It is used in the preparation of chyawanprash (popular Ayurvedic tonic) ,Sahachardithailam and Dhanwantharamthailam (An excellent massage oils).

It has antimicrobial, antiprotozoal and anti-inflammatory properties (Binutu et al. 1996).

Statement of the problem-

Adhatoda beddomei rarely sets the seeds (Panigrahi and Patel,2014);

Stereospermum tetragonum is usually propagated through seeds but has a very poor rate of germination (Baul, 2006).

Justification of the study-

A.beddomei and S. tetragonum are medicinal plants used in for the preparation of the medicine and is preferred by certain industries .

The study will be helpful in stabilizing the depleting species population and improve conservation status in the wild.

Review of literature- Studies has not been found on Macropropagation techniques for Adhatoda beddomei while Studies Done in By

Micro propagation of Adhatoda beddomei Using Nodal Explants

2014 Panigrahi & Patel

Conservation Strategies of Medicinal Plant-Adhatoda vasica Nees. Using in vitro and ex vitro Propagation Techniques

2013 Shabir A. Lone et al.

Role of different Revive Rooting Hormones for Ex-vitro rooting of Adhathoda vasica Nees. & Barleria prionitis L.

2011 Shabir A. Lone

High frequency In-vitro Propagation of Adhathoda vasica Nees. through shoot tip and nodal explants culture .

2008 M Khalekuzzaman et al.

Rapid micropropagation via Axillary bud Proliferation of Adhathoda vasica Nees. From nodal segments

2006 Gauri Abhyankar & V D Reddy

studies Done in By Seed germination of Stereospermum suaveolens with respect to different parameters

2014 Trivedi & Joshi

Relationship of pods and seeds trait in medicinal value tree Stereospermum suaveolens DC.

2014 Tomar, Tripathi & Kumar

In vitro plant regeneration from seedling explants of Stereospermum personatum D.C.: a medicinal tree

2009 Shukla, Shukla & Mishra

Similarly, no studies have been found on Stereospermum tetragonum .

Hypothesis-

Adhatoda beddomei will show more growth when plant cuttings will be treated with 60% RRH and with the rooting substrate garden soil: vermicompost: sand (1:1:1).

Stereospermum tetragonum will give good result when garden soil: vermicompost: sand (1:1:1) will be used as sowing substrate.

Soaking seeds of Stereospermum tetragonum in distilled water will induce highest germination.

Seed number in a pod depends on the length of the pod (applicable for S.suaveolens).

Study area -

Objectives- To standardize optimum method for successful vegetative propagation of Adhatoda beddomei.

To find out better planting substrate and pre-treatment for Adhatoda beddomei.

To find the better planting substrates and pretreatments for seed propagation of Stereospermum personatum.

Establishing a good plant stand in the field.

To study relationships of pods and seeds traits.

Selection of the mother plant

(collected from FRLHT Campus)

cuttings were made 10-12cm with at least 3

nodes

Pre-treatments Rootex and

IBA(500ppm,1000ppm, 2000ppm)

Methodology -

Adhatoda beddomei

60 cuttings

30 cuttings dipped for 1 hour

Coco peat

Garden soil: vermicompost: sand (1:1:1)

30 cuttings dipped for half hour

Coco peat

10 cuttings each in different planting substrate

Garden soil: vermicompost: sand (1:1:1)

SandSand

cuttings dipped in IBA/Rootex

Procedure followed with Indole-3-butyric acid and Rootex

For Rootex dipping for 1 minute is done

Mother plant Cuttings dipped in Rootex and planting.

Cuttings in different concentrations of IBA

Propagated seedlings of Adhatoda beddomei

The seeds were collected from

FRLHT campus and Assam

pre-sowing treatments used were-1)Cow dung slurry2)Distilled water3)Control(Normal water)

Sowing substrate used were-1)Coco peat2)Coco peat+ Soil (3:1)3)Sand

Stereospermum tetragonum

Water testing-Testing has been done for the water samples in which the seeds were soaked.

Seeds

Accession 1 (FRLHT)

Cow dung slurry

(50 seeds)

Distilled water (50 seeds)

Accession 2 (Assam)

Cow dung slurry

(50 seeds)

2 replications of 25 seeds each

Distilled water (50 seeds)

Control (50 seeds)

Control (50 seeds)

2 replications of 25 seeds each

Rooting substrate used

Coco-peat Coco-peat + Soil Sand

Seeds sowing in trays and root trainers

Mother plant of Stereospermum tetragonum, flower, pods.

Seeds sowing in tray Seeds sowing in Root trainers Seedling in cocopeat

Seedlings of Stereospermum tetragonum in sand

Cutting test for seeds of Stereospermum tetragonum-

Seeds were cut open to ensure if the embryo is present.

cut-open to see the embryo

Embryo

Seeds of Stereospermum tetragonum

Table showing the difference in parameters present in distilled water and normal water. parameters Desirable limit

as per IS:10500/2012

Permissible limit in absence of alternate source

Distilled water Normal water

Chloride (as Cl),mg/l,Max

250 1000 19.99 347.89

Fluoride (as F) mg/l, Max

1.0 1.5 0.2 0.36

Magnesium (as Mg) mg/l, Max

30 100 0.97 58.33

Sulphate (as So4) mg/l,Max

200 400 <0.5 11.52

Calcium (as Ca),mg/l,Max

75 200 3.21 138.68

Total Alkalinity(CaCo3) mg/l, Max

200 600 13.6 286

Pods and seeds traits characteristics in Stereospermum tetragonum

Pod characteristics

Seeds characteristics

•45 healthy pods were collected to make three replications containing 15 pods per replication.•length ,width and no. of seeds per pod were recorded.

•Weighing of 100 seeds for 10 replicates.•Average weight of 100 seeds.•Seed quantity estimation( amount of seeds per kg)• Plant population per hectare and quantity of seeds per hectare estimation done (using secondary data).

Statistical Analysis

To find out the relationship between pods and seeds traits.We tested for the normality of data by employing three methods:1) the skewness and kurtosis test 2) the Shapiro–Wilk W test (Shapiro and Wilk, 1965), and 3) the Shapiro-Francia W' test.In case the data is normally distributed, we used the Bartlett’s test (NIST/SEMATECH e-Handbook of Statistical Methods, 2015) to test the hypothesis of equality of variances across replications.Employed the Analysis of Variance to test whether the means from three replications are significantly different or not.To estimate degree of influence of pod length on seed number, we used regression analysis : Seed number per pod = F(pod length).Based on the R-square statistics, we selected the best fit model.

0%40%80%

120%

Germination in different substrates with IBA pre-treatment

IBA-500ppmIBA-1000PPMIBA-2000ppm

30%

42%

28%

Rootex

sand

Garden soil: vermicom-post: sand(1:1:1)

cocopeat

Results-The germination percentage was higher in garden soil: vermicompost: sand(1:1:1) when compared to sand and cocopeat in both the pre-treatments.

Rootex

IBA-50

0ppm

IBA-10

00PPM

IBA-20

00ppm

0.00%

20.00%

40.00%

60.00%

80.00%

100.00%

120.00%

Germination percentage with different pre-treatments

sandGarden soil: vermi-compost: sand(1:1:1)cocopeat

pre-treatments

germ

inat

ion

perc

enta

ge

Among two different pre-treatments (Rootex and IBA) used for the stem cuttings of Adhatoda beddomei, the best results was seen with IBA.

500ppm 1000ppm 2000ppm0

2

4

6

8

10

12

14

16

Average number and length of roots in different rooting substrate

Cocopeat

Sand

garden soil:vermicompost:sand(1:1:1)

Number of roots per cutting and the root length were affected by the different concentration of IBA used.

Distilled water Cow dung slurry Normal water0%5%

10%15%20%25%30%35%

Germination percentage with different pre-treatments

CococpeatCocopeat+soilsand

Stereospermum tetragonum-Among the different planting substrates used (cocopeat, cocopeat+soil (3:1), sand),cocopeat gave the higher germination percentage (32%) while the pre-treatment of distilled water was more effective as compared to other pre-treatments.

Results contd..

•Seed number in a pod depends on the length of the pod.•1% increase in average pod length, the average seed number per pod is estimated to increase by 0.9%.

15.119.5

22.423.1

26.528.3

28.929.4

29.630.6

32.032.3

32.434.0

34.434.8

37.338.6

39.042.0

43.944.9

48.30

10

20

30

40

50

60 Fig. 1: Effects of Pod Length on Seed Number

Actual No. of SeedsPredicted No. of Seeds

Pod length (cm)

Seed

No.

Seed No. = pod lenght (cm)^0.8918

=

=400 plants/ha.

=

Discussion –Adhatoda beddomei-500ppm-IBA pre-treatment gave the overall best results for germination, number of roots and length of roots per cutting with garden soil: vermicompost: sand(1:1:1) as the sowing substrate.

Stereospermum tetragonum-

S. tetragonum had maximum seed germination when soaked in distilled water as distilled water is having consistent content, it is free of chemicals that would otherwise harm the germination

Coco peat is found to be the suitable sowing substrate for the growth of the plant.

Pods and seeds characteristics- Healthier and larger pods has good number of seeds that has been proved by the statistical analysis for the hypothesis that number of seeds depends on the length of the pods. Plant stand estimation (using secondary data)- Plant stand is expressed as space between plants that has been determined on the basis of spacing been used for some common trees that is 5 meter square from both the sides (http://www.arborday.org/Shopping/Trees/fspacing.cfm; Moniruzzaman et.al,2007) .

Conclusion- Optimum method by means of stem cuttings for successful

vegetative propagation of Adhatoda beddomei has been standardised.

Better planting substrate (Garden soil:Vermicompost:Sand) and pre-treatment (IBA-500ppm) has been found out for Adhatoda beddomei .

Better planting substrate ( cocopeat) and pretreatments (Distilled water) for seed propagation of Stereospermum tetragonum has been found out.

Plant stand establishment has been done in nursery only due to limited time period.

Pods and seeds characteristics study proved that healthier and larger pods contains the good amount of seeds

Acknowledgements- I acknowledge my external guides Dr. . K. Haridasan, Mrs.

Nandini D. , Dr. Ganesh Babu N. M, Mrs. Geetha Suresh for their

support and help and all the members of FRLHT, Bengaluru.

I also thank my internal guide Dr. Rita Singh for her support and

guidance.

I would like to place on record my heart felt gratitude to all the

faculty members of USEM, GGSIPU for their incessant support and

encouragement.

I also thank Mr. Amit Pandey for his support and cooperation.

References-Lone S.A ,Yadav A , Badkhane Y , Sharma A.K , Bakhsh S.H , Raghuwanshi D.K , Role of different Revive Rooting Hormones for ex-vitro rooting of Adhatoda vasica Nees. &Barleria prionitisL., IJPBS, 2011;2(1).Lone A, Yadav A , Sharma A.K , Tafazul M, Raghuwanshi Yad , conservation strategies on medicinal plant-Adhatoda vasica Nees. ,using in-vitro and ex-vitro propagation techniques, IAJPR,2013,3(4),pp.3341-3360.NIST/SEMATECH e-Handbook of Statistical Methods. Available online, URL: http://www.itl.nist.gov/div898/handbook/eda/section3/eda357.htm. Retrieved May 15, 2015.Panigrahi J, Micropropagation of Adhatoda beddomei Using Nodal Explant,European Academic Research,2014,vol.2,pp.12194-121204.Shapiro, S. S.; Wilk, M. B. (1965). "An analysis of variance test for normality (complete samples)". Biometrika52 (3–4): 591–611.doi:10.1093/biomet/52.3-4.591. JSTOR 2333709.MR 205384. p. 593Tomar A, Tripathi S, Kumar A, A Relationship of pods and seeds traits in Medicinal value Tree Stereospermum suaveolens DC.,IJABE,2013,vol.7,pp.1-3.Trivedi D.R, Joshi A.G , Studies on Seed Germination of Stereospermum suaveolens with respect to different parameters, Env. & Exp. Bio.,2014,12, pp.33-37.Weisstein, Eric W. "Bonferroni Correction." From MathWorld--A Wolfram Web Resource.http://mathworld.wolfram.com/BonferroniCorrection.html. Retrieved May 15, 2015.

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