adhathoda beddomei and stereospermum tetragonum plants

1. 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
Traditions
Bangalore-560106, Karnataka, India.
University School of Environment Management
Guru Gobind Singh Indraprastha University
Sector-16 C, Dwarka, New Delhi-110077,India.
Dr. K. Haridasan
Professor Emeritus
Mrs. Nandini D.
consultant
Dr. Rita Singh
Associate professor
By-
Kumari Kshama Awasthi
M.Sc. Biodiversity and Conservation
University school of environment management
Guru Govind Singh Indraprastha University

2. 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).

3. 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).

4. 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.

5. 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.

6. 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 .

7. 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).

8. Study area -

9. 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.

10. 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,1000
ppm, 2000ppm)
Methodology -
Adhatoda beddomei

11. 60 cuttings
30 cuttings dipped for 1 hour
Coco
peat
Garden
soil:
vermico
mpost:
sand
(1:1:1)
30 cuttings dipped for half hour
Coco
peat
10 cuttings each in different planting substrate
Garden
soil:
vermico
mpost:
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

12. Mother plant Cuttings dipped in Rootex and planting.
Cuttings in different
concentrations of IBA
Propagated seedlings of Adhatoda beddomei

13. The seeds were
collected from
FRLHT campus
and Assam
pre-sowing
treatments used
were-
1)Cow dung slurry
2)Distilled water
3)Control(Normal
water)
Sowing substrate
used were-
1)Coco peat
2)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.

14. 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

15. Mother plant of Stereospermum tetragonum, flower, pods.

16. Seeds sowing in tray Seeds sowing in Root trainers Seedling in cocopeat
Seedlings of Stereospermum tetragonum in sand

17. 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

18. 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(CaC
o3) mg/l, Max
200 600 13.6 286

19. 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).

20. 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.

21. 0%
20%
40%
60%
80%
100%
120%
sand Garden soil:
vermicompost:
sand(1:1:1)
cocopeat
Germination in different substrates with IBA pre-treatment
IBA-500ppm
IBA-1000PPM
IBA-2000ppm
30%
42%
28%
Rootex
sand
Garden soil:
vermicompos
t: 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.

22. 0.00%
20.00%
40.00%
60.00%
80.00%
100.00%
120.00%
germinationpercentage
pre-treatments
Germination percentage with different pre-
treatments
sand
Garden soil:
vermicompost:
sand(1:1:1)
cocopeat
Among two different pre-treatments (Rootex and IBA) used for the
stem cuttings of Adhatoda beddomei, the best results was seen with
IBA.

23. 0
2
4
6
8
10
12
14
16
500ppm 1000ppm 2000ppm
Average number and length of roots in different
rooting substrate
Cocopeat
Sand
garden
soil:vermicompost:sa
nd(1:1:1)
Number of roots per cutting and the root length were affected by
the different concentration of IBA used.

24. 0%
5%
10%
15%
20%
25%
30%
35%
Distilled water Cow dung slurry Normal water
Germination percentage with different pre-
treatments
Cococpeat
Cocopeat+soil
sand
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.

25. 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%.
0
10
20
30
40
50
60
15.1
18.5
19.5
20.5
22.4
22.9
23.1
25.9
26.5
27.5
28.3
28.4
28.9
29.0
29.4
29.5
29.6
29.7
30.6
31.9
32.0
32.1
32.3
32.3
32.4
32.7
34.0
34.1
34.4
34.7
34.8
36.0
37.3
37.3
38.6
38.7
39.0
40.0
42.0
42.5
43.9
43.9
44.9
47.0
48.3
SeedNo.
Pod length (cm)
Fig. 1: Effects of Pod Length on Seed Number
Actual No. of Seeds
Predicted No. of Seeds
Seed No. = pod lenght (cm)^0.8918

26. =𝟗𝟐𝟑𝟑𝟔. 𝐬𝐞𝐞𝐝𝐬
=400 plants/ha.
= 𝟏𝟑. 𝟓 𝒈𝒓𝒂𝒎𝒔 𝒐𝒇 𝒔𝒆𝒆𝒅𝒔

27. 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.

28. 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) .

29. 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

30. 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.

31. 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. 593
Tomar 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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ago and the
Second best time is now