Manual on

Identification of Large Cardamom Variety

in Bhutan
 Manual on

Identification of Large Cardamom Variety

in Bhutan
Surendra Prasad Rijal & Jimba Rabyal

Food and Agriculture Organization of the United Nations

Thimphu, 2020
Required citation:
FAO, 2020. Manual on Report on Identification of Large Cardamom Variety in Bhutan.
Thimphu.

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ACKNOWLEDGEMENTS

I acknowledge FAO Bhutan for providing me an opportunity to prepare

this manual for all interested farmers, researchers and extension workers
involved in the value chain of Large Cardamom production.

The manual is an outcome of various research works done by different

researchers across the world.

Furthermore, I would like to thank National Commercial Agriculture

Research Program (NCARP), Pakhribas, Dhankuta, Nepal; ICIMOD;
National Spice Crop Development Program, Khumaltar; Cardamom
Development Center, Ilam; and Spice Board of India for the research
work on Large Cardamom, whose research became important guidelines
for this task.

Lastly, I would like to thank Mr. Bipul Kharel and my wife Mrs. Sunita
Rijal, and all of the unknown divine factors that may have contributed to
the success of this effort.

i
 TABLE OF CONTENTS

Acknowledgements ..........................................................................................i
Introduction .....................................................................................................1
Botanical Classifications .................................................................................3
Other cultivated species .................................................................................3
Cardamom parts........................................................................................5
Large cardamom variety identification ...................................................10
a. Morphological traits ..................................................................10
b. Electrophoresis ..........................................................................10
Morphological characteristics of large cardamom varieties ...................10
c. Damberse ...................................................................................10
Variety Maintenance ...............................................................................11
d. Jirmale .......................................................................................12
e. Seremna......................................................................................13
f. Madhuse ....................................................................................13
g. Ramala .......................................................................................14
h. Bharlange ...................................................................................14
i. Ramse ........................................................................................15
j. Golse ..........................................................................................16
k. Chibese ......................................................................................17
l. Sawane .......................................................................................17
m. Zongu-Golse ..............................................................................18
Electrophoresis........................................................................................21
Molecular Methods..................................................................................22
a) DNA bar coding..........................................................................22
b) Restriction Fragment Length Polymorphism (RFLP)................22
c) Complete Polymerase Chain Reaction (PCR)............................23
Reasons of use of molecular markers in large cardamom.......................23
Molecular cardamom variety identification in Nepal (Methodology).....23
Reference.......................................................................................................26

iii
INTRODUCTION

Large Cardamom is a perennial herb with subterranean rhizomes and 50-

140 aerial leafy shoots. Each shoot has a height of 1.7 to 2.6 meters and
possesses 9 to 13 leaves in each tiller. Leaves are glabrous on both sides
with a prominent mid-rib. The inflorescence is a condensed spike with
yellowish perianth. Each spike has 10-15 fruits. The Fruit is round or oval
shape, capsule with reddish-brown color. Each capsule is tri-locular with
many seeds (Spice Board of India).

Large cardamom is world’s third expensive spice crop after saffron and
vanilla. Saudi Arabia, India, Pakistan, Bangladesh, UAE, Europe, U.S.A,
and Australia are its market. Major large cardamom producing countries
are Indonesia, India, Nepal, China, and Bhutan but the species of the
cardamoms differ geographically. India, Nepal, and Bhutan produce
Ammomum subulatum based varieties whereas China produces varieties
of the genus Ammomum other than subulatum species. Indonesian large
cardamom varieties are based on Ammomum compactum. Java Island is a
famous large cardamom producing area of Indonesia. Nearly 80 percent
of large cardamom production of India is from Sikkim. Nepal’s large
cardamom production is concentrated in eastern Nepal i.e. Taplejung,
Sankhuwasabha, Panchthar, Illam, Dhankuta, and Bhojpur. However, in
recent years, its coverage is escalating towards western Nepal because of
its lucrative price.

Large cardamom is an integral part of biryani in most of the Pakistani

and Indian restaurants. It is also used as a kind of dessert after meal for
fighting against bad breath. In Europe, it is used for bakery purposes. Large
cardamom has several medicinal uses mostly in herb-based medicines. Its
average productivity in Nepal is around 0.540 ton per hectare. In the year
2016, the total large cardamom production in Nepal was 6521 tons, 5623
ton in India, and 2245 tons in Bhutan.

ICIMOD’s publication unveils that Nepal is the largest producer of large

cardamom in the world. The price of large cardamom in Delhi market was
INRs 1560 per kilogram in February 2019. But Siliguri based market sold
cardamom at Rs. 1079/ kg in 2016.

1
There are several varieties of large cardamom. Some of the most popular
varieties of large cardamom are Ramala, Seremna, Damberse, Zhangu-
Golse, Golse, Bharlange, Jirmale, Madhuse and Ramse.

The India’s Arunachal Pradesh originated varieties are Bebo dark red, Bebo
light red, Boklok, Tali, Jaker, and Belak. Each variety exhibits different
characteristics i.e. color of the pseudo-stem, height, leaf structure, bearing
habit, the size of capsule, disease tolerance and drought tolerance.

Large cardamom is highly cross-pollinated crop. It is a challenge to

maintain true to the type varieties of large cardamom unless maintained
under separate facilities with proper management. This manual is
developed as a guidebook for all professionals in research and extension
system for identification of different varieties of large cardamom.

2
BOTANICAL CLASSIFICATIONS

Large cardamom is one of the popular spices found in the Zingiberaceae

family. It is a tall, perennial, evergreen, herbaceous monocot plant (Kumar
et al., 2012). The height of this plant is 1.5 to 3.0 m (Bisht et al., 2011)
and on the upper part of the stem, there are leaves. The rhizomes are of
dull red color and the flower buds protrude from the base of the rhizome.
Spring is the flowering period of large cardamom. Short peduncle and
buds covered with tight red bracts. The individual flowers remain open
for three days or more. At the same time, new ones are opened. Flowering
remains intact with the flowers for one month (Sharma et al., 2000). There
are many classification systems but the botanical classification system is
the one most frequently used.

Large cardamom has been botanically described as follows:

Kingdom Plantae
Division Spermaphyta
Sub-division Angiospermae
Class Monocotyledonae
Order Scitaminea
Family Zingiberaceae
Genus Amomum
Species Subulatum

OTHER CULTIVATED SPECIES

Other cultivated species in China is Amomum sps. It is bigger in size and

is considered to be inferior to Amomum subulatum Roxb. Indonesian large
cardamom variety is based on Ammomum compactum. (Emperor Akbar,
2016).
1. Amomum delbatum 4. Amomum longiforme
2. Amomum aromaticum 5. Amomum corinostachyum
3. Amomum plauciforum 6. Amomum kingii

3
 There are other wild species
1. A. spiceum 7. A. vivale 13. A. uticolosum
2. A. xanthophlebium 8. A. testaCellln 14. A. ochrellm
3. A. macranthunt 9. A. sqllarrosllnt 15. A. uphalotes
4. A. lIlacrodells 10. A. biflorum 16. A. leppaceum
5. A. hastilabiulll 11. A. maeroglossa 17. A. aeilleatum
6. A. eylindraceum 12. A. citrinll17l 18. A. uligillosllm

There are various wild species of cardamom found in the forest areas. The
small cardamom is different from large cardamom. The Scientific name of
small cardamom is Elettaria cardamom Maton. Guatemala produces the
highest amount of small cardamom. Its genome 2n is 48. Large cardamom
variability is also reported with 2n = 26, 34, 42 and 44 (Sharma and
Bhattacharya, 1959) as mentioned by (Pradhan et al 2014).

Leaf

Pseudo stem

Bud

Rhizome
Root
Figure 1: Various parts of Large Cardamom
Figure 1: Various parts of Large Cardamom

m parts 4
nnial evergreen monocotyledon plant, which has six parts.
 Rhizome
Root
Figure 1: Various parts of Large Cardamom

Cardamom parts
Cardamom parts evergreen monocotyledon plant, which has six parts.
It is a perennial
It is1.a perennial
Rhizome: evergreen
It is a monocotyledon
vital organ ofplant, which has
cardamom, six parts.
which has roots below
and apical
1. Rhizome: It is agrowing tipsofabove.
vital organ It has
cardamom, nicehas
which aroma. The figure
roots below below
and apical growing tips
illustrates
above. rhizome.
It has nice aroma. The figure below illustrates rhizome.

Rhizome

Root

Figure 2Figure 2 : and

: Rhizome Rhizome
roots and roots

2. Pseudo stem: It is made of bundles of leaves wrapped around each

other. The tips which do not bear flowers develop into pseudo stem
2. Pseudo stem: Itemerge
and leaves is made of
outbundles
of it. of leaves stems
Pseudo wrappedarearound eachwith
covered other.leaves
The tips which do
bearbase
flowers developThe
or sheath. intocolor
pseudoofstem and stems
pseudo leaves emerge
dependsoutonofthe
it. Pseudo stems are cove
varieties.
with
Its height is around 1 to 2 meters; and one bush possesses 20 to 70 Its heigh
leaves base or sheath. The color of pseudo stems depends on the varieties.
around 1 to 2 meters; and one bush possesses 20 to 70 pseudo-stems. The number of pse
pseudo-stems. The number of pseudo stems depends on soil type
stems depends on soil type and variety - Sawane, Ramse and Golse possess many tillers whe
and variety - Sawane, Ramse and Golse possess many tillers whereas
Damberse and Chibse give lesser tillers.
Damberse and Chibse give lesser tillers.

Figure
3: Pseudo-stem & Spike Figure 4: Emerging Buds of
Pseudo-stem and spikes

5
 a) Golse: Green colored and medium
b) Ramse: Monorish red and long
c) Saune: Green, long
d) Jirmale: Green and medium sized
e) Madhuse: Monorish red colored and dwarf
f) Damberse: Monorish red colored and short
g) Bharlange: Monorish red colored and long

3. Leaf: Leaves emerge from pseudo stems. Each pseudo stem has 5 to 13
leaves, depending upon the variety. Leaves are long, tapering towards
tip, green and without hairs. Middle portions are wide and tapering
towards tip. It has a long main vein in the center right from the base to
the tip and secondary veins initiated from the main vein. Basal part of
the leaf is covered by sheath. Leaves emerged from alternate directions.
Cardamom leaves are droopy (Seremna) and vertical depending on the
variety. Its green chlorophyll play role in photosynthesis and stomata
beneath transpire and keep the plant healthy.

Table 1: Varieties and their leave characteristics

Variety Leaves
Seremna Droopy and vertical
Ramala Broad and slightly elongated
Damberse Leaver are short and erect the veins of leaves
Jirmale Green leaves, length 24 cm width 5.05 cm
Chibese Light green leaves, sword type leaves
Leaves having wavy margins, length 32.67 cm width 5.92
Bharlange
cm
Ovate and board leaves length 29.34 cm and width
Saune
6.66cm
Golse Narrow and erect leaves 27 cm long and 5.38 cm wide
Ramse Green colored narrow, 25.7 cm long and 6.47 cm wide
Madhuse Length 25cm and width 6.12 cm

6
4. Inflorescences: Two types of buds emerge from the rhizomes; one
that develops into pseudo stem and another develop into flowering
buds. The flowering buds become flowers after attaining 1 to 1.5
cm in height; and the Inflorescence is 10 to 16 cm long. One pseudo
stem contains one to four inflorescences. Flowers emerge from this
inflorescence. One inflorescence contains 20 to 25 flowers. Flowers
will be light yellow and vary in size, according to varieties. Cross-
pollination is essential for fruits though there are both male and female
parts in the flower. The pollination is mostly by bumble bees, bees,
and other insects. The seed formation takes place after successful
pollinations. The pictures below illustrate the Inflorescence.

Figure 5: Inflorescences, Flowers, Flowering buds and pseudo stems

5. Capsules: Each inflorescence possesses 15 to 12 capsules. The sizes

of the capsules are 2.3 cm long and 1.5 cm wide. The color of the
capsules is light red to brown. A dried capsule weighs 0.5 to 1.45
grams. The color and size of the capsule differ with the variety to
variety.

Figure 6: Dried capsules

7
Table 2: Varieties and their capsule characteristics
Variety Capsule character
Long shaped, medium sized and medium quality. Dry
Ramse
weight of 20 capsules is 15.4 g
Big and round, plump and weighty Dry weight of 20
Golse
capsule 14.5 g
Jirmale Big and round
Big grey colored triangular in tip and round at base but
Sawane
smaller than Bharlange. Dry weight 20 capsules is 12.1 g
Chibese Small capsule.
Zongu-Golse Big and bold capsules Dry weight of 20 capsule is 17.2 g
Seremna Medium sized weighty capsules
Round medium sized, dark pinkish in color. Dry weight
Ramala
of 20 capsules is 16.1 g
Damberse Big sized and tasty
Bharlange Big sized long capsule
Madhuse Dry weight of 20 capsule is 18.3 g

Table 3: Physical properties of cardamom capsule

Particulars Minimum Average Maximum
Length in cm 2.25 2.2 2.3
Width in cm 1.35 1.1 1.35
Ratio of Length/Width 1.82 1.70 2.01
Bark % 36.14 32.5 38.8
Seed % 60.11 58.11 62.11
Pulp % 2-3 - -

8
6. Seed: Seeds are always inside the capsules. One capsule possesses 25
to 80 seeds. The Immature capsule possesses white seeds. It turns red
in color as the capsule matures and finally it will attain black color.
The seed is covered with sticky pulp. Dried large cardamom possesses
61 % seed and 37 % bark and the remaining part will be pulp.

Figure 8: Fresh capsule Figure 7: Dried capsule with seed

Table 4: Varieties and 1000 seed weight

Variety Seed weight 1000 seeds in gram
Ramse 18.3
Golse 16.3
Zongu-Golse 10.9
Sawane 15.09
Madhuse 15.8
Ramala 14.5

Chemical properties of large cardamom are presented below.

Table 5: Chemical properties of large cardamom
Ingredients Amount in percentage
Water 8.49
Volatile oil 2.0
Protein 6.0
Total ether extract 5.31
Nonvolatile ether extract 2.31
Volatile ether extract 3.00
Crude fiber 22.00
Carbohydrate 43.21

9
Ingredients Amount in percentage
Alcohol extract 7.02
Ash 4.57
Water soluble ash 2.15
Water soluble alkaline ash 0.09
Acid soluble ash 0.90
Source: Cardamom cultivation, NSCDP, Lalitpur

Large cardamom variety identification

Variety Identification in large cardamom can be done through two methods;

a. Morphological traits
Large cardamom varieties can be identified through careful observation
of its morphological traits such as color, number and height of its pseudo-
stem; size, shape and bearing habit of leaves; and size, shape of capsule,
number of seeds per capsule and color of capsule.

b. Electrophoresis
Through electrophoresis, based on the protein bands computer can identify
the variety. Its use is seen in case of small cardamom but there have been
no reports available in case of large cardamom.

Morphological characteristics of large cardamom varieties

In order to distinguish large cardamom types depending upon its
morphological characters, understanding the character of different
varieties is very important. Different characters for different varieties are
described in this section.

c. Damberse
Its capsules are bigger in size. It is less tillering, dwarf, reddish green stalk
(pseudo stem), and big-sized capsules with more seed inside the capsule,
tasty and fetch a better price in the market. It is cultivated at 700 to 1200
meters from sea level. Its leaves are upright and short. It ripens from
September to October.

10
Variety Maintenance
Special farmers must be selected at least in four locations that can exert
extra energy to save it from crosspollination with other unwanted varieties.
Green net of fifty percent shade can be used in maintenance of the variety.
It will save cardamom plant from pollination with other unwanted variety
in the one hand while in the other hand it will save from the attack of
aphid-like vectors, which transfer viral diseases.

Only selected personnel should be allowed to enter the plant areas in

disinfected clothes and after dipping boots or legs inside lime water. No
one should allow smoking in the vicinity of the area. Adequate manure
applications, adequate health care must be practiced. Daily inspection of
the mother plant must be practiced. The infected or diseased plant must
be taken out and incinerated in the fire. The equipment used must undergo
sterilization after use otherwise diseases will easily transfer upon the use
of such equipment which is used in cutting or digging of the diseased
plant. Fire sterilization is recommended for the equipment. Irrigation
by sprinkler method can be installed for offering adequate moisture and
humidity.

The area must be small for keeping the plants in ICU (Intensive Care
Unit). The Government should offer a special grant for the purpose of
variety maintenance. Interested people should collect seed and other
planting materials from such stocks.

The periphery must not possess banana plants, peach plants and maize
plants. These plants are alternate hosts of the Chirkey and Foorkey
virus diseases. The aphid Pentalonia nigronervosa, Myzuspersica and
Ropalosiphum maidis attack cardamom and transfer Foorkey and Chirkey
viral diseases.

Fungal diseases mainly from Collechtrochium geosporides is becoming

very serious which damage the crop very fast which put farmers at the risk
of a higher rate of economic injuries. Periodic inspection of the cardamom
field and regular sprays of the bordeux mixture in each 15 days interval
can reduce chance of fungal disease incidence. Taking care of mother
stock is very important because it will be the future of entire cardamom
industry.

11
This variety maintenance principle will be applicable in other varieties
too.

Figure 9: Damberse Variety of Large cardamom

Distinguishing Character
• Dwarf
• Less tillers
• Lower altitude (700-1200 masl)
• High quality bigger size capsules
• Tasty capsule

d. Jirmale
Its leaves and pseudo-stems are green in color. It produces a large number
of tillers. It gives good production in low water availability conditions
too. Its season of ripening is from August to September. In one spike,
22 to 37 capsules are available and each capsule possesses 56 seeds. It
is somehow –tolerant to most diseases. This variety is also known as
dryland cardamom. This variety is dwarf in size and is cultivated at 700
to 1,000 meters from sea level. Its capsule possesses 63 percent bark and
37 percent internal pulp and seed. It is also known as Salakpure. Heavy
manure application is required each year.

Distinguishing characters
• Long green leaves and pseudo stem
• Disease tolerant
• Round & Big capsules

Figure 10: Jirmale variety of large cardamom

12
e. Seremna

Figure 12: Pseudo-stem of Seremna Figure 11: Seremna showing

Source: ICIMOD drooping leaves
Source: Pakhribas
It has drooping leaves which are suitable for 1200 to 800 meters from
mean sea levels. It is tolerant of Chirkey and Foorkey diseases. Regular
application of water and manure will make it productive for 15 to 20 years.
It is famous for its production. Plant height is 1.5 to 2 meters. It can be
identified with its green colored leaves and pseudo stem. Capsules are big
and long possess 65 to 70 grains. One plant gives 2 to 3 spikes. It is Indian
variety from Sikkim area.

Distinguishing character
• Drooping leaves
• Tolerant to Chirkey and Foorkey disease
• Capsules are long and bigger in size

f. Madhuse
This variety is not commercially cultivated in Nepal but cultivated in
Kalimpong, India. Flowerings of this variety resemble that of Turmeric
flower that grows from the soil surface. Local scientists and farmers of
India found this variety as little bit resistance to Chirke-Furke diseases.
NCARP, Pakhribas, Dhankuta started research in Madhusai for its
suitability in Nepal and for disease resistance capacity. Stem color is
red. Each Capsule size is 72.5mm. This variety performs well in lower
altitudes (700-1000msl).

13
Distinguishing characters
• Dwarf variety
• More information about this variety is presented in table 7.

g. Ramala
This variety resembles Ramse and is cultivated above 1500 meters from
sea level. The average capsules per spike is 65 to 70 and per capsule seed
rate is 65 to 70 grams. It gives 2 to 3 spikes per plant. Per spike capsule
rate is 10.

Figure 13: Ramala variety

Distinguishing characters
• Capsules are dark pink in color with 30-40 seeds
• Maroonish tillers
• Ready to harvest in October
• Leaves are broad and slightly elongated.

h. Bharlange
• This cultivar grows in low, medium and high altitude areas in South
Regu (East Sikkim) and at high altitudes at Gotak (Kalimpong
subdivision in Darjeeling district of West Bengal
• Its yield performance is exception ally high at higher altitude areas
i.e. 1500 m and above.
• It is a robust type and total tillers may range from 60 to 150.
• Color of tillers is maroon-ish green to maroon towards collar zone;
girth of tillers is more than that of Ramse.
• Each productive tiller on an average produces almost three spikes
with an average of 20 capsules/spike

14
 • Size of capsules is bigger and bold with 50-65 seeds. Harvesting
begins in the last week of October
• This cultivar is also susceptible to Foorkey and Chirkeys disease
(NCARP, 2018)

Figure 14: Bharlange Large Cardamom Variety

Distinguishing characters
• Big and bold capsules hold 50-70 seeds
• Slightly elongated capsules
• Flowering is from June to July depending upon
altitude Fruit bearing tillers and spikes ratio are
relatively high Plant height is around 2.4 m.
• ICIMOD recommends it in higher altitude above
1500 m to 2200m

i. Ramse

Figure 15: Left Leaves of Ramse middle Pseudo-stem of Ramse variety,

right spikes of Ramse variety

15
Distinguishing characters
• Short spike length and difficult to harvest
• Grows in steep slopes
• Performs well in higher altitudes above 1500 m
• Harvesting in September-October
• Smaller capsules, each bearing 25-40 seeds per capsule Leaves are
confined in the apical areas only.
• Red pseudo-stem

j. Golse
It has short & thick pseudo-stem and short leaves. Leaves emerged from
basal part to apical part. Capsules of this variety are big, round and
yellowish in color. It performs well in the elevation 1200 to 1600 meters.
In Sikkim, it is cultivated in 600 to 1,000 meters in altitude. The pseudo
stems are smaller than that of Ramse. The leaves initiation starts right
from the basal part of the plant. The leaves are erect. Its leaf base beaks
easily compared to the other variety. Its inflorescence is longer compared
to Ramse. Its inner pulp and seeds are tasty and longevity is comparatively
high. Harvesting starts from last September. This variety is seen to be
infected with the Chirkey and Foorkey viruses.

Figure 16: Golse Large cardamom plant.

Distinguishing characters
• Bigger size capsules
• Spike length is high and easy to harvest
• Pseudo stem is smaller
• Leaf initiation is right from the base to the top

16
 • It can be cultivated in 1200 -1600 mm altitudes
• Susceptible to viral diseases
• It has round and big sized capsules

k. Chibese
Farmers from the altitude 1,300 to 1800 meters can choose Chibese. A
single fruit bearing tiller bears 2 to 4 spikes, each bearing around 10 to
20 capsules. This is a productive variety with good market potential. The
figure below shows Chibese.

Distinguishing characters
• Smaller size capsules
• High production potential
• Single tiller bears 2-4 spikes
• Each spike bears 10-12 capsules

Figure 17 : Chibese Variety

l. Sawane
This variety is grown at lower elevations (900 to 1,500m) in all cardamom
growing areas of Sikkim and Kalimpong of India and Taplejung of Nepal.
Plants are 1.5 to 2m in height and robust in nature. Leaves are ovate
and broad with maroonish tillers. Capsules are relatively smaller than
Bharlange and contain only 30 to 50 seeds.
Flowering starts with on onset of rain, in the
late March to May, and harvesting is done
in September to October.

This variety is highly susceptible to disease.

Bhutan should omit this variety. Sikkim 1
and Sikkim 2 are other varieties released in
India which are selected from this variety. Figure 18: Sawane variety

17
Distinguishing characters
Capsules are smaller than Bharlange containing
35-50 seeds
• Susceptible to diseases and pests
• Flowering starts at late March to May,
harvesting follows during September to
October.
• Its plants are longer like Ramse and leaves
are smaller and wider like that of Golse
• Leaves color are dark green
• Pseudo stems are light green Long pseudo-stem,
• It is harvested in August Short leaves

m. Zongu-Golse
This variety does well in the altitude range of 1,000 meters from mean
sea level. It starts flowering from April, and capsule formation takes place
from October. It is from the place Zhangu of Northern Province Sikkim.
The pseudo stems are similar to that of Ramse. It is tolerant to diseases like
local varieties. Leaves are narrow and tapering. Pseudo stems are green in
color. Studies were conducted to assess the performance of eight varieties
of large cardamom at mid-altitudes (660 meters MSL) in Arunachal
Pradesh. Out of the eight varieties tested, Ramla, Sawane, Madhuse
and Ramsey performed better and could be introduced for commercial
cultivation at mid-altitude region of Arunachal Pradesh.

Distinguishing characters
• Capsules are big and bold with 50-
70 seeds
• Disease tolerant cultivar
• Tillers are similar to that of Ramse
Adaptive to lower elevations.

The following tables will elaborate latest Figure 19: Zongu-Golse

varietal descriptions based on the latest variety
research at Arunachal Pradesh and Sikkim in India, and Pakhribas, Nepal.

18
Careful study of the table will further help to understand the varieties.

Table 6: Agro-morphological evaluation of large cardamom germplasm at

Pakhribas, Nepal (NCARP, 2018) and ICIMOD 2017

SN Traits Ramse Golse Saune Madhusai Damberse Bharlange Jirmale

Plant height
1 65.8 52 51.4 45 62.8 51.4 44.8
(cm)
2 Plant vigour Good Good Good Good Good Good Good

3 Stem color Red Green Green Red Red Red Green

Leaf length
4 25.7 27 29.34 25 32.67 32.67 24
(cm)
Leaf breadth
5 6.47 5.38 6.66 6.12 6.68 5.92 5.05
(cm)
6 Leaf color Green Green Green Green Green Green Green
Number of
7 adults tillers 2 2 3 2 2 3 3
per bush
Number of
8 new tillers 7 7 5 9 9 6 7
per bush
Lower Leaf
Susceptibility Fungal Viral
9 Elevation streak Medium Medium Low
to Diseases disease disease
Yes Viral
Number of
10 7 5 7 5 7 8 7
Leaves
Altitude of Low to Low to Low to
11 High Middle High Low
cultivation Middle Middle Middle

Based on such data we have to differentiate the varieties of large cardamom.

Frequent cross-pollination and seed originated saplings can deteriorate
varietal characters. Thus, variety maintenance practices must be carried
out to find true to the type variety. Altitude based recommendation is
presented in the table.

19
Table 7: Arunachal Pradesh based study on large cardamom
Variety Mean Plant Height Number of sucker/Plant
Four year Cm Four year Mean
Ramsey 100.1 17.52
ZonguGolse 86.44 12.08
Golse 107.2 13.68
Madhuse 126.8 16.53
Sawane 123.6 19.08
Ramala 130.9 21.21
Bebo Red 196.6 9.7
Bebo light red 166.5 9.3

Table 8: Comparative analysis of cardamom varieties

Fresh Weight of Dry Weight of Weight of
Variety 20 capsules in 20 capsules in Seed capsule 1000 gram
gram gram seed

Year 1999 2000 1999 2000 1999 2000 1999 2000

Ramse 67.6 67 15.5 15.4 41.6 42.3 18.3 18.3
ZonguGolse 65.66, 65.9 17.3 17.2 43’.6’0 39.7 10.8 10.9
Golse 56.34 55.9 14.3 14.5 29 33.4 16.6 16.3
Madhuse 70.24 72.5 18.7 18.3 84.8 87.4 15.5 15.8
Sawane 54.92 54.9 11.8 12.1 27.2 28.7 14.9 15.09
Ramala 74.58 81 16.5 16.1 59.2’0 58.3 14.9 14.5
Bebo Red 166.2’0 165 17.9 17.8 54.2 57.1 13.7 13.2
Bebo light red 198.1 201.2’0 19.7 19.9 52.2’0 55 20.2 20.6
CD at 5% 20.68 20.5 3.19 2.98 10.4 9.88 1.07 1.04
(Yadav & Dubey, 2001)

20
Table 9: Large cardamom cultivar diversity in Sikkim Himalaya
Capsule/Seed
S.N Variety Morphological Traits Altitude
Characteristics
Ramsey Tall & robust with large
(cultivated number of tillers. Plant height Size Small
1 Above 1500
on steep 1.5 to 2.0 meters, Tiller color 25-40 seeds
Slopes) Maroonish, narrow leaves.
Sawane,
Tall & robust, Plant height: 1.5 Size: Bigger (975-1500m
Widely
2 to 2.0 meters, Leaves: Ovate and bold, 35 - masl) and high
adapted
and broad 50 seeds (> 1500 m
cultivar
Plant height 1.5 to 2.0 meters
The productive
Tiller color: Maroonish Bold size
3 Bharlange tiller and spike
Leaves: Narrow leaves with Seeds 50-70
ratio is high
wavy margins
Plant height: 1.5 to 2.0 meters Color dark
4 Ramla Tiller color: Maroonish pinkish, 30-40 1000-1500m
Leaves: Broad and long seeds
1000-1500 m,
on an average
Plant height: 1.5 to 2.0 meters 2-3 spike in each
Seremna
5 Tiller color: Green 65-70 seeds productive tiller
Leaves: Mostly drooping type with an average
of 10 capsules in
each spike
Leaves narrow and erect,
50-70 seeds
6 Golse Green tiller color, 1-1.5 m 1000 -1500 m
Bigger &Bold
plant height, not robust.

Electrophoresis
In this method, proteins are extracted from the grain, and the protein
composition is used to identify the variety. In India, electrophoresis
methods were in use at ICAR based facilities; we can use the facility and
calibrate them. In small cardamom (Elettaria cardamomum), there are
reports of its use but are not seen in large cardamom. This device can be
useful in other cases too. Its use can detect varietal purity effectively and
accurately because of the computer added technology associated with it.

21
Molecular Methods
Molecular tools like PCR, RFLP, DNA Bar coding are recently used tools
in the variety identification process.

Plant variety and cultivar identification are some of the most important
aspects in the agricultural system. The large number of varieties or landraces
among crop plants have made it difficult to identify and characterize
varieties solely on the basis of morphological characters because they are
non-stable and originate due to environmental and climatic conditions,
and therefore phenotypic plasticity is an outcome of adaptation. To
mitigate this, scientists have developed and employed molecular markers,
statistical tests, and software to identify and characterize the required
plant cultivars or varieties for cultivation, breeding programs as well as
for cultivar right protection. (Nicholas et al 2012).

a) DNA bar coding

DNA barcodes allow non-experts to objectively identify species – even
from small, damaged, or industrially processed material. Just as the unique
pattern of bars in a universal product code (UPC) identifies each consumer
product, a “DNA barcode” is a unique pattern of DNA sequence that
identifies each living thing. Short DNA barcodes, about 700 nucleotides
in length, can be quickly processed from thousands of specimens and
unambiguously analyzed by computer programs.

With DNA barcodes, students can help discover and catalog biodiversity
on our planet using tools developed at the DNA Learning Center.

b) Restriction Fragment Length Polymorphism (RFLP)

RFLP is a difference in homologous DNA sequences that can be detected
by the presence of fragments of different lengths after digestion of the
DNA samples in question with specific restriction endonucleases. RFLP,
as a molecular marker, is specific to a single clone/restriction enzyme
combination.

22
c) Complete Polymerase Chain Reaction (PCR)
PCR fragment analysis requires a method that supplies sufficient separation
resolution. Despite this requirement, PCR fragments are commonly
analyzed by slab gel electrophoresis. Though readily available in many
laboratories slab gel electrophoresis is incapable of providing researchers
with high-resolution separations of PCR fragments. Furthermore, agarose
gel electrophoresis does not scale to effectively meet the needs of high-
throughput environments, leading researchers to feel frustrated and out of
time.

Reasons of use of molecular markers in large cardamom

Molecular markers which detect variation at the DNA level overcome
most of the limitations of morphological and biochemical markers hence,
the potential application of RAPD markers has been demonstrated in many
crops for a long time (Bowditch et al., 1993 and Zhang et al., 2011).A
comparative analysis of the different methods of variety identification in
terms of accuracy, cost and time will be essential to follow the techniques
according to the need.

Molecular cardamom variety identification in Nepal (Methodology)

The following methodology was used to access the cardamom varieties in
Nepal by molecular method.

The sixteen plant materials (Amomum subulatum) used in this study were
collected from Taplejung, Panchthar and Ilam district of Eastern Nepal
during 2012. Immature leaf samples were collected from various eco-
geographical locations of different altitude irrespective of wild as well
as cultivated genotype. The samples were transported to the laboratory as
soon as possible and stored at -400C until DNA extractions. Geographical
situations of collected samples were recorded with the help of GPS
machine (GPS Garmin). Each sample was reduced to fine powder through
mortar and pestle with the addition of liquid N2 and transferred to a 2.0 ml
eppendorf tube. DNA extraction was performed using a modified CTAB
method (Doyle and Doyle, 1990), where modification was done for CTAB
buffer reaction time at 650C for1 hour. DNA concentration was estimated
by Q5000 UV vis Spectrophotometer (Quawell).

23
RAPD amplification was performed with random decamers (Kit
from Operon Technologies, Alameda, CA, USA). On preliminary
experimentation sixty primers were tested but only twenty five arbitrary
RAPD primers showed reproducible banding pattern and were chosen for
the analysis based on highly readable and polymorphic bands as described
by Pandiyan et al., 2010.

DNA amplifications were performed in 15 µl reaction volume containing

50ng template DNA, 2X Green Go Taq® Reaction Buffer (pH 8.5),400μM
dATP,400μM dGTP, 400μM dCTP, 400μM dTTP and 3mM MgCl2.
(Promega) and 0.6picomole of primer. The mixture was gently mixed
and centrifuged in shaker for 60 seconds prior to adding two drops of
mineral oil. After addition of mineral oil it was transformed to thermo-
cycler, All the amplifications were performed in the same thermo-cycler
(MULTIGENE OPTIMAX, Lab net International, Inc.) programmed for
1 cycle of 95° C for 5 min followed by 40 cycles of 95° C for 60 sec,
36°C for 1 min 30 sec, 72°C for 2 min, and final 1 cycle of 72°C for 10
min. Samples were kept at 20C until further analysis. 12 µl aliquots of
amplification products were loaded in a 1% (w/v) agarose gel (Bioneer)
for PCR product separation in 1X TAE buffer (Bioneer) in an electrical
field (120 V) for 90 minutes. Gels was stained with Ethidium Bromide
(0.5µg/ml for 30 min) and visualized under exposure of UV light under
gel doc system (UVDI, Major Science). For scoring band and to confirm
consistent amplifications during the whole experiment standard 1 kb
Ladder (Bioneer) was used.

RAPD profiles of 16 different cultivars were scored on binary system as

1 for presence of the band and zero for absence of band. Only the bands
that are clear and reproducible were scored. Polymorphic Information
Content (PIC) for each primer were calculated as described by Roldan
Ruiz et al., (2000) as PIC= 2Pi (1-Pi), Where Pi is the frequency of ith
null allele. Scored bands were subjected to study the Dice similarity
coefficient with the help of NTSYS pc versions 2.01 and based on genetic
distance UPGMA genetic maps were created. To confirm the clustering
(Chaudhary, Raju et al, 2016)

24
For details, please consult the paper GENETIC DIVERSITY IN LARGE
CARDAMOM (Amomum subulatum Roxb.) DISSECTED USING RAPD
MARKERS. By Raju Chaudhary, Surya Kant Ghimire, Bal Krishna Joshi,
Bisnu Raj Ojha) International Journal of Advanced Research 4(6):1443-
1451 · June 2016

25
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 DoA
Royal Government of Bhutan Department of Agriculture

FAO Country Office

UTF/BHU/013/BHU: FAO Technical Assistance
Food Security and Agriculture Productivity Project (TA-FSAPP)
UN House, Thimphu: Bhutan
Tel# 02-322424/334570