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44 Conservation of Podophyllum hexandrum

rate,

it may eventually face the threats of genetic drift, population bottlenecks and

inbreeding depression. Therefore, among these populations, screening and selection of

podophyllotoxin

Fig. 3.1. Effect of various concentrations of IAA and BAP on zygotic embryo cultures of P.

hexandrum. A and B: two and six week old cultures, respectively (MS + 0.5 μM IAA + 2.0 μM

BAP); C and D: three and four week old cultures, respectively (MS + 1.0 μM IAA + 0.5 μM

BAP); E and F: four and three weeks old cultures, respectively (MS + 1.0 μM IAA + 2.0 μM

BAP). R = roots.

Conservation of Podophyllum hexandrum 45

rich elite clones and their mass propagation using micropropagation protocols can be a

helpful tool for the conservation of this species. Once such clones are selected from

naturally growing populations and multiplied using micropropagation, a cultivation

package can be developed so that the farmers in the upper Himalayan mountain ranges can

start cultivating it as a cash crop which will reduce the pressure of extraction on this

species from its natural habitats.

Fig. 3.2. Potted P. hexandrum plants after hardening and transfer to soil.

46 Conservation of Podophyllum hexandrum

The authors are highly grateful to the Director of the High Altitude Plant Physiology

Research Center, Srinagar- Garhwal, U.P., India for providing research facilities.

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©CAB International 2010. Medicinal Plant Biotechnology 48

(ed. Rajesh Arora)

Chapter 4

Traditional and Biotechnological Strategies for

Conservation of Podophyllum hexandrum Royle

– A Case Study

Amit C. Kharkwal, Dharam Singh, Om Prakash, Deepshikha

Pande Katare, Ajit Varma, Amita Bhattacharaya and Paramvir

Singh Ahuja

Introduction

Podophyllum hexandrum Royle (English: common name Indian Podophyllum and

Himalayan Mayapple) is a small herbaceous monotypic genus of the family Berberidaceae

with a diploid chromosome number 2n=12; predominantly occurring in the North

Temperate Zone and discontinuously distributed in eastern North America, insular East

Asia and Hindu Kush-Himalayas (Good, 1974; Xiang et al., 2000; Arora et al., 2008).

The roots and rhizomes of Podophyllum yield a drug known as podophyllin.

Podophyllin consists of podophyllotoxin, podophyllic acid, podophylloresin and quercitin.

It is a cholagogue, alterative, emetic, and a strong purgative (Singh et al., 1999). The

podophyllotoxins are lignans, which have anti-cancer, anti-mitotic, and immunostimulatory

activities (Kaplan, 1942; Loike et al., 1978; Pugh et al., 2001) and whose semi-synthetic

derivatives etoposide (VP-16-213), teniposide (VM-26) and etopophos are approved drugs

for the treatment of testicular and small cell lung cancer (Stahelin and Warburg, 1991).

Ever increasing demand for this drug in modern medicine, coupled with its existing use

in traditional systems has resulted in ruthless uprooting of the underground parts leading to

intense collection coupled with lack of organized cultivation. Presently, the rate of its

overexploitation far exceeds its natural regeneration potential. Consequently P. hexandrum

has been declared an endangered species (Airi et al., 1997).

In the present study, survey, standardization of propagation techniques, seed biology

study, DNA polymorphism of plants using RAPD and quantification of podophyllotoxin

using HPLC was done in the samples procured from five geographically distinct

populations in the state of Himachal Pradesh (HP) India.

Indian Podophyllum: Traditional and Modern Conservation Strategies 49

Survey and distribution

A survey was made for collection of proximal P. hexandrum populations of HP from five

locations, namely Great Himalayan National Park (GHNP) district Kullu, Koksar and

Kukum-Seri of the District of Lahul and Spiti, Pangi and Chamba regions of district

Chamba. GPS (Magnell Inc., USA) data were obtained for altitude, latitude, longitude and

population size (Table 4.1) from these five populations.

Table 4.1. P. hexandrum population size, location and geographic coordinates.

Geographical location

of populations

Total no. of

individuals

Altitude

(msl)

Longitude Latitude

Chamba 600 2200 32º02'.786N 76º01'.052E

Pangi 150 3600 33º05'.950N 76º26'.046E

Koksar 250 3200 32º24'.868N 77º14'.080E

Kukum-Seri 800 2800 32º42'.335N 76º40'.557E

Great Himalayan

National Park (GHNP)

40 2400 31º45'.389N 77º22'.510E

Collection of plant materials

Full-grown rhizomes collected in the months of May and September from five different

populations of Himachal Pradesh were used for podophyllotoxin estimation and vegetative

propagation respectively. Mature and immature pods were also collected from five

different regions.

Characterization

Chemical characterization

Variation in total percentage of resin content and podophyllotoxin was recorded between

the five accessions (Table 4.2). No correlation was found between the amount of resin and

podophyllotoxin content of the rhizomes.

Table 4.2. Total resin and podophyllotoxin content of five different populations.

Region (Himachal Pradesh) Extract (%) Resin (%) Podophyllotoxin (%)

Chamba 25.3 4.29 1.29

Pangi 39.4 11.8 1.10

Koksar 28 9.4 1.96

Kukumseri 23.4 10.2 5.22

GHNP 25.7 8.57 0.70

Cytological characterization

Cytological studies on five populations revealed no variation in chromosome number and

structure among the populations. The representative karyotype is shown in Fig. 4.1. Across

the five populations it was observed that the Kukum-Seri population exhibited the breakage

of chromosome (Fig. 4.2) and Pangi showed doubling of chromosome followed by

cytomixes (Fig. 4.3 a–d).

50 Indian Podophyllum: Traditional and Modern Conservation Strategies

Fig. 4.1. Karyotype of P. hexandrum (100X); Fig. 4.2. Kukum-Seri population showing

breakage of chromosome (100X).

Fig. 4.3. a–d. Pangi population showing cytomixis and chromosome doubling (100X).

Genetic diversity characterization by using randomly amplified polymorphic DNA

(RAPD)

Eighty random 10-mer primers were screened for the five populations (9–10

plants/population). Only 11 primers, which gave three or more polymorphic bands, were

finally selected for producing consistent, scorable and reproducible results and therefore

taken for further analysis. Shannon’s index of phenotypic diversity was used to partition

the diversity into intra- and inter-population components (Table 4.3). The genetic diversity

present within populations, H

pop

, compared with that between populations, H

pop

, revealed an average diversity of 62% within populations. However, the

distribution of diversity did vary between and within populations with different primers.

Indian Podophyllum: Traditional and Modern Conservation Strategies 51

Phenotypic frequencies detected with 11 primers were calculated and these data were

used in estimating the diversity (H

) within the five populations of P. hexandrum. The

Kukum-Seri population exhibited lowest intra-population variability as compared to

Chamba population, which exhibited highest variability followed by GHNP, Koksar and

Pangi populations (Table 4.4).

The principal co-ordinate analysis displayed the relationship of the individuals within

the populations. Within each population, most individuals clustered in a single group (Fig.

4.4).

Exceptionally, the GHNP population did not show clear population structure. The

narrow genetic base of Kukum-Seri and Pangi populations were displayed in a PC plot

where they form a narrow distinct group. Though individuals of Chamba population form a

distinct group even then they are distributed throughout the axis due to a broad genetic

base. Some of the individuals of Koksar population were mingled with other populations.

But the individuals of GHNP populations were distributed randomly throughout the axis

suggesting a broad genetic base as well as relationship with other populations. The

evaluation of genetic variability distribution within and among populations was measured

by AMOVA (analysis of molecular variance). AMOVA values obtained by RAPD data

show that 74% of total variation is attributed to the individuals within a population while

26% is due to differences among populations (Table 4.5).

Based on the RAPD data the genetic distances were calculated among populations. The

GHNP and Chamba populations showed more genetic similarity with all other populations.

The maximum genetic similarity was observed between Pangi and Kukumseri populations,

followed by GHNP and Chamba. Though overall genetic distances among populations

were below 21%, they form a distinct entity except few individuals. This result can also be

attributed with allele similarity in the adjacent population. The Nm value displays the gene

flow existing between the populations. The maximum Nm value (Table 4.6) was found

between GHNP and Chamba (1.510) followed by Koksar with Chamba (0.913) and Koksar

with GHNP (0.903). Based on binary RAPD data, the Nei genetic distances were calculated

between the populations. These values were used to construct a dendrogram by the

UPGMA method to display the relationship between the populations. There were two

distinct clusters formed in the dendrogram (Fig. 4.5); one of them with GHNP, Kukumseri

Primer H

pop

-H

pop

OPA-05 2.87 1.61 0.56 0.43

OPA-07 1.83 1.22 0.66 0.33

OPA-08 3.14 1.64 0.52 0.47

OPA-14 1.87 1.05 0.56 0.43

OPA-15 1.26 0.87 0.69 0.31

OPA-16 2.6 1.8 0.69 0.31

OPA-18 2.35 1.51 0.64 0.35

OPE-02 2.29 1.45 0.63 0.36

OPG-11 1.94 1.33 0.68 0.31

OPM-04 2.32 1.43 0.61 0.38

OPM-15 1.72 1.07 0.62 0.37

Average 2.19 1.36 0.62 0.36

Table 4.3. Partitioning of genetic diversity into within and between populations for 11

primers.

52 Indian Podophyllum: Traditional and Modern Conservation Strategies

and Pangi populations and another with Koksar and Chamba. Though GHNP is associated

with the former population it is distantly placed in that cluster.

Table 4.4. Estimates of genetic diversity (H

) within five populations of P. hexandrum.

Primer GHNP Koksar Kukum-Seri Pangi Chamba

OPA-05 1.29 1.71 1.69 1.84 1.51

OPA-07 1.04 0.83 1.08 1.49 1.64

OPA-08 1.86 2.01 1.72 0.32 2.3

OPA-14 0.92 1.13 0.32 1.94 0.93

OPA-15 0.98 0.52 0.85 0.85 1.18

OPA-16 1.65 1.86 1.16 2.16 2.16

OPA-18 2.01 1.71 0.94 1.08 1.83

OPE-02 1.56 1.5 1.35 1.74 1.08

OPG-11 1.52 1.86 0.78 1.08 1.41

OPM-04 2.01 1.25 1.46 0.93 1.49

OPM-15 1.28 0.99 0.32 0.93 1.83

Average 1.46 1.39 1.06 1.30 1.57

Fig. 4.4. Principal co-ordinate analysis of P. hexandrum based on 11 random primers.

Table 4.5. AMOVA in 48 individuals among five populations of P. hexandrum.

Source of

variation

d.f. SS MS Est.

Variance

% of

variation

Among

population 4 145.767 36.442 2.930 26% 0.001

Within population

43 358.233 8.331 8.331 74% 0.001

d.f. – degree of freedom, MS – mean squared deviation, SS. sum of squares, P–

significance of the variance components after 999 random permutations

Indian Podophyllum: Traditional and Modern Conservation Strategies 53

Fig. 4.5. Cluster analysis of P. hexandrum based on 11 random primers.

Table 4.6. Estimates of genetic distances (Nei) and gene flow (Nm) between the populations

of P. hexandrum in Western Himalayas.

Population Population Genetic

Distance (Nei)

Gene

Flow (Nm)

GHNP Koksar 0.125 0.903

GHNP Kukum-Seri 0.101 0.827

Koksar Kukum-Seri 0.122 0.598

GHNP Pangi 0.109 0.849

Koksar Pangi 0.207 0.376

Kukum-Seri Pangi 0.081 0.794

GHNP Chamba 0.105 1.510

Koksar Chamba 0.141 0.913

Kukum-Seri Chamba 0.166 0.547

Pangi Chamba 0.167 0.602

Propagation

Sexual method

Seed propagation through direct seed germination and excised embryo culture was

attempted in the present study for attaining quick and high germination of P. hexandrum

seeds.

Seed biology

The present study on seed biology of P. hexandrum indicates the existence of differential

seed dormancy based on responses to various treatments and ontogenic variability in the

five populations of HP.