Introduction

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Over three-quarters of the world population relies mainly on plants

and plant extracts for health care. It is estimated that world market for

plant derived drugs may account for about ` 2,00,000 crores.

Presently, The annual demand of botanical raw drugs in the country

has been estimated at 3,19,500 MT for the year 2005-06 which works

out to ` 1,069 crores (Ved and Goraya, 2007). The annual production

of medicinal and aromatic plant’s raw material is worth about `200

crores. This is likely to touch US $5 trillion by 2050 (Joy et al, 1998).

India is one of the world’s 12 biodiversity centers with the presence of

over 45,000 different plant species (Jawla et al, 2009). India’s diversity

is unmatched due to the presence of 16 different agro-climatic zones,

10 vegetation zones, 25 biotic provinces and 426 biomes (habitats of

specific species). Of these, about 15,000-20,000 plants have good

medicinal value. However, only 7,000-7,500 species are used for their

medicinal values by traditional communities. In India, drugs of herbal

origin have been used in traditional systems of medicines such as

Unani and Ayurveda since ancient times (Jawla et al, 2009).

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Some important chemical intermediates needed for manufacturing the

modern drugs are also obtained from plants (eg diosgenin, solasodine).

Not only, that plant-derived drug offers a stable market worldwide, but

also plants continue to be an important source for new drugs (Joy et al,

1998).

There are over 25,000 herbal products documented in medical

literature. According to All India Ethno-biological Survey carried out

by Ministry of Environment and Forest, Government of India, there

are over 8,000 species of plants being used by the people of India

(Sharma et al, 2008a).

The World Health Organization (WHO) estimated that 80% of the

population of developing countries still relies on the traditional

medicines, mostly plant drugs, for their primary health care needs.

Demand for medicinal plants are increasing in both developing

countries due to growing recognition of natural products, being non-

toxic, having no side effects, easily available and that too at affordable

prices.

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The Department of Biotechnology, Ministry of Science and Technology,

New Delhi has included Curculigo orchioides in the list of endangered

plants (Anonymous, 2000). Curculigo orchioides was also included in

the IUCN category of Lower risk near threatened (Sharma, 2001).

1. Curculigo orchioides Gaertn.

Curculigo orchioides is known as Talamuli in Sanskrit, Kalimusli in

Hindi and Gujarati and Nilappana in Malayalam (Kirtikar and Basu,

1988; Thomas et al, 2000). The species is a stemless perennial herb of

medicinal importance and a native of India (Wala and Jasrai, 2003).

The demand of the raw materials and derivatives of the plant for the

indigenous drug industries is satisfied mainly from the wild source,

depleting the natural population and thus the species has become near

extinct or endangered (Ansari, 1993; Augustine and Souza, 1997a).

Curculigo orchioides is reported to be available only during the

monsoon season in India, which lasts for 4 months each year. It is a

small herbaceous plant with an elongated tuberous rootstock and

lateral roots.

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1.1. Classification

According to Bentham and Hooker’s (1862-1883) system of

classification Curculigo orchioides is classified as follows:

Kingdom Plantae

Sub-kingdom Angiosperm

Class Monocotyledons

Series Epigynae

Family Hypoxidaceae

Genus Curculigo

Species orchioides

Scientific Name Curculigo orchioides Gaertn.

1.2. Distribution

It is believed to have originated in the shady forests of Asia. It is found

in all parts of India from near sea level to 2300 m altitude, especially in

rock crevices and laterite soil. It has been recorded to occur in the

subtropical himalayas from Kumaon eastwards ascending to 1800 m,

the Khasia hills, Bengal, Assam, Konkan, Kanara, the western peninsula

and Tamil Nadu extending south as far as Cape Comerin (Agharkar,

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1953; Joy et al, 1998; Gupta et al, 1994) It is also distributed in

Srilanka, Japan, Malaysia and Australia (Pandey et al, 1983). It is a

shade-loving plant and thrives well in areas that receive high rainfall.

1.3. Morphological characteristics

Roots of kali musli are straight, cylindrical, tuberous, 5–22 cm long,

and 0.5–0.8 cm thick. The external surface is brownish, marked with

loosely spaced, prominent, transverse wrinkles (Fig-1c).

Lateral roots are 5 cm or more in length, stout, fibrous, dull white in

colour, and spongy externally. The freshly cut surface of tuberous root-

stock has a starch-white color within and is mucilaginous.

Leaves are simple, sessile, narrowly linear to lanceolate, acute, plicate

or flat, crowded on the short stem with sheathing leaf bases; petiole

short to 3 cm, often absent; almost radical. They are 15–45 cm long

and 1.2–2.5 cm broad, linear or linear–lanceolate, membranous,

glabrous or sparsely soft haired (Fig-2a). The leaf tip, when in contact

with the soil, develops roots and produces adventitious buds.

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1.4. Floral biology

Flowers are epigynous, bright yellow, sessile, bisexual or unisexual,

with a lanceolate and membranous bract. Perianth is located at the top

of a slender sterile long extension of the ovary by means of which it is

exposed above the ground (Fig-1b and c).

Perianth is gamopetallus with six equal lobes of size 1.5 cm × 0.2 cm;

outer lobes are hairy on the back, while the inner ones are sparsely

hairy along nerves.

Stamens 6, filaments filiform, anthers 2 mm, ovary 3-celled, oblong to

4 mm (Fig-1c).

Ovary is tricarpellary, syncarpous, and trilocular with a fairly long

slender beak (stipe).

Ovules numerous per cell, style 2 mm, stigma-3, lobes elongate.

Fruit oblong, 1.5-2.0 cm long 8 mm broad; seeds 8, globose to 2 mm,

black, beaked, deeply grooved in wavy lines (Anonymous, 1963;

Bhaskaran and Padmanabhan, 1983; Dong and Zhang, 1998).

Flowering and fruiting occur mostly from October to January, rarely

throughout the year (Joy et al, 2004b).

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Fig- 1: Curculigo orchioides; a. Plant habit, b. Flowers, C. Underground roots and d. Marker product.

b

c d

b a

a

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2. Karyotype

Sato (1938) has compared the karyotype of the Curculigo orchioides

with other genera in the sub-family. The karyotype revealed a diploid

chromosome count of 18.

2n=18=2L+2Ms+14S ie 1 pair of the long chromosome, 1 pair of the

medium size chromosomes with secondary constrictions and 7 pairs of

short chromosomes. This study also suggest that this plant has

phylogenetic similarity with Alstromeria and Scilla.

Fig- 2: Idiogram of the haploid complement of Curculigo

orchioides

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3. Agro-technique

3.1. Propagation material

Tuber segments of 1.5–2.0 cm size, containing the apical bud are

collected during February March and used for propagation (Joy et al,

2004a).

3.2. Nursery technique

3.2.1. Raising propagules

No stock is raised in the nursery. Tuber segments of size 1.5 cm × 2.0

cm, obtained from mother plants, are planted directly in the main field

at the onset of south-west monsoon, which breaks over South India in

May–June. The tuber segments are planted at an optimum spacing of

10 × 10 cm. About 70–80 % sprouting is obtained after two months of

planting in humid tropical regions like Kerala (Joy et al, 2004a).

3.2.2. Propagule and pretreatment

The propagules required for plantation is 600–750 kg of root segments

per hectare. The tuber segments require no pretreatment before

sowing.

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3.3. Planting in the field

3.3.1. Land preparation and fertilizer application

Curculigo orchioides grows well in moist and humus-rich soils. The

land is ploughed well with the onset of monsoon. Organic manure is

mixed before planting and raised beds are prepared to prevent water

logging. Farm yard manure (FYM) at the rate of 20 t/ha is applied at

the time of land preparation. Alternatively, FYM at the rate of 15 t/ha

may be applied at the time of land preparation and NPK at the rate of

25:15:10 kg/ha can be applied as top dressing during October–

November. If available, well-decomposed poultry manure at the rate of

2.7 t/ha, instead of FYM, applied at the time of land preparation gives

better yield.

3.3.2. Planting and optimum spacing

The tuber segments are directly planted in the field in rows. About 70-

80 % sprouting of tubers takes place after two months, when planted

in humid tropical areas like Kerala. An optimum crop stand of 0.6–0.65

million is desirable for a pure crop with an optimum spacing of 10 × 10

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or 10 × 15 cm, while intercropping with a coconut gives a crop stand of

approximately 0.2 million with a spacing of 20 × 25 cm.

3.3.3. Intercropping system

The crop grows well in the shade of irrigated coconut orchards. If it is

to be raised as a pure crop, artificial shade has to be provided using

shade nets of 25 % density.

3.3.4. Interculture and maintenance practices

No additional manure is required for crop management. Manual

weeding is usually adopted. Weeding twice at two and four months

after planting is necessary for weed-free field. No special maintenance

practices are required except for regular weeding and irrigation during

dry spells.

3.3.5. Irrigation practices

The crop is grown in rain-fed area during the monsoon period. After

the monsoon ceases, it is to be irrigated fortnightly with 5 cm flooding.

3.3.6. Disease and pest control

Seedling rot is observed during the rainy season and can be controlled

by spraying and drenching the soil with bordeaux mixture (1 %). Black

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rot disease is also observed and can be controlled by spraying

tridemorph (0.05 %). Rhizomes are often eaten by rodents and hence

standard control measures may be taken for their control.

3.4. Harvest management

3.4.1. Crop maturity and harvesting

The plant starts flowering one month after planting and maximum

numbers of flowers are noted during 2-3 months of planting.

Flowering takes place throughout the year. However, fruits and seeds

are not used as drug. Roots mature in the field in seven to eight months

and may be harvested by digging.

3.4.2. Post-harvest management

Remnants of the shoot and rootlets are removed from tubers. The

tubers are cleaned of the soil particles, dried well in the shade, and

stored in gunny bags.

3.4.3. Yield and cost of cultivation

A dried tuber yield of 1000–1700 kg/ha is obtained. The estimated

cost of cultivation is ` 28, 000/ha, which does not include the cost of

planting material.

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3.4.4. Market price

The market rate of Kali musli is ` 200-300 per kg (Prajapati et al,

2003)

4. Medicinal uses

The whole plant of Curculigo orchioides is medicinaly useful.

(Bhamare, 1998; Jain, 1991). Curculigo orchioides has been used with

other plants for making number of pharmaceutical formulations in the

Indian system of medicine as a metabolic enhancer. The tuberous root

stock of Curculigo orchioides is used as a restorative, rejuvenating and

aphrodisiac drug (Porwal and Mehta, 1985; Manandhar, 1991;

Samanta, 1992). The root stock is mucilaginous, cooling, bitter,

emollient, diuretic, aphrodisiac, depurative, alterative, appetiser,

carminative, viriligenic, antipyretic and tonic (Porwal and Mehta,

1985).

The plant possesses uterine stimulant (Dhawan and Saxena, 1958;

Sharma et al, 1975; Dhar et al, 1968; Rastogi and Mehrotra, 1991),

hypoglycaemic, spasmolytic and anticancer (Dhar et al, 1968; Aruna

and Sivaramakrishnan, 1990), phagocytic (Kubo et al, 1983), immune-

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adjuvant (Oru and Kogyo, 1983), anti-neoplastic (Sharma et al, 1991),

immuno-stimulant (Saxena, 1992) and hepatoprotective (Latha et al,

1999; Rajesh et al, 2000) activities.

5. Phytochemicals

The bioactive compounds reported are three sterols viz sitosterol,

stigmesterol (Garg et al, 1989) and Yuccagenin ( Rao et al, 1978), one

triterpene of ursane series (Mehta and Gawarikar, 1991) and the

others are of cycloartene series viz cycloartenol (Garg et al, 1989)

curculigol (Misra et al, 1990), curculigenins A, B and C ( Xu et al,

1992b; Xu and Xu 1992b). Xu and co-workers (Xu et al, 1992a; Xu et al,

1992b; Xu and Xu 1992b) characterized 13 saponins from Curculigo

orchioides rhizome and named them curculigo saponin A-M

respectively. Moreover, rhizome contains curculigoside, a phenolic

glycoside characterized as 5-hydroxy-2-O-β-D-glucopyranosyl benzyl

2, 6-dimethoxy benzoate (Oru and Kogyo, 1983; Chen et al, 1989;

Mamta et al, 1995; Chen and Ni, 1999), a flavone glycoside (Dhawan

and Saxena, 1958), starch, resin, tennin, mucilage, fat, calcium oxalate

(Thakur et al, 1989) and fatty acids (Mehta et al, 1980). Two

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polyphenolics of Curculigo orchioides were isolated and characterized

as benzylbenzoate glucosides (Valls et al, 2006).

Removal of plants for medicinal and edible tuberous roots as a

substitute for safed musli, coupled with extensive denudation of

forests floor caused by cattle grazing (Jasrai and Wala, 2000; Wala and

Jasrai, 2003), poor seed setting and germination (Gupta and Chadha,

1995) are some of the major causes that contribute to the herb being

categorized as a threatened plant (Augustine and D’sousa, 1997b).

High incidence of viral and bacterial diseases poses yet another

constraint in its multiplication and strengthens the need of in vitro

techniques for multiplication (Dhenuka et al, 1999).

6. Biotechnology

The term biotechnology, initially employed in early 1920’s is rapidly

developing discipline that offers substantial opportunities in the fields

of plant regeneration, multiplication and genetic improvement. It is

applied to conventional and modern scientific techniques related to

biology for the betterment of society towards goods and services. But

one of the major challenges in experimental biology is to harness the

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modern techniques for the improved quality and quantity of plants.

Biotechnological tools have been the ultimate solution to these

problems. It holds a place of unique importance in today’s world

among scientists and agriculturist all over the globe. One of the major

branches of plant biotechnology is plant cell, tissue and organ culture.

An important aspect of plant biotechnology process is the culture of

plant cells, tissues or organs on artificial medium. The new

technologies provide a better approach for designing and manipulation

of targets. They do not replace plant breeding but provide methods

capable of achieving objectives not possible by other conventional

means. Recent progress in plant tissue culture has made this area of

research as one of the most dynamic and promising field of

experimental biology. The introduction and development of these

techniques have provided solutions to the problems which were

previously inaccessible (Ignasimuthu, 1998).

6.1. Micropropagation

Micropropagation is one of the easiest and fastest methods of

producing numerous plantlets in limited period. (Bhojwani and

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Razdan, 1996). In vitro procedures have been adopted for large scale

propagation of medicinal plants (Borthakur et al, 2000; Reddy et al,

2001; Liu et al, 2003; Aricat et al, 2004). Tissue culture techniques

have the potential to provide secure conservation method. The term

plant tissue culture broadly refers to the in vitro multiplication of

plants through plant parts (tissue, organs, embryos, single cells,

protoplasts etc) on nutrient medium under aseptic conditions.

The idea of cell and tissue to be raised in culture was coined by a

German plant physiologist Gabttlieb Haberlandt (1854-1945), who is

regarded as the father of plant tissue culture.

The mass multiplication of economically valuable plants through

micropropagation is based on the principle of totipotency. The entire

area of agriculture research is also based on the totipotency of plant

cell (Mhatre and Rao, 1998).

Micropropagation or clonal propagation is a specific aspect of plant

tissue culture dealing with the in vitro and aseptic vegetative

multiplication of plants. The application is to produce large number of

true-to-type aseptic plants in limited period of time and space. In other

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words, clonal propagation in vitro is called micropropagation. Webber

(1903) first used the word clone for cultivated plants that were

propagated vegetatively. Clonal propagation is the multiplication of

genetically identical individuals by asexual production. Muashige and

skoog (1977) outlined three major stages involved in

microprapagation.

STAGE-1: Selection of suitable explant, sterilization and transfer to

nutrient media for establishment.

STAGE-2: Proliferation or multiplication of shoots from the explant on

medium.

STAGE-3: Transfer of shoots to a rooting medium followed by the

planting into soil.

Micropropagation and plant regeneration can be grouped into to

following categories:

i. Enhanced proliferation of bud: multiplication through growth

and proliferation of existing meristems through shoot tip culture,

single node/axillary bud culture.

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ii. Organogenesis: Formation of individual organ such as shoots

and roots from the explant directly or indirectly from callus.

iii. Somatic embryogenesis: Formation of bipolar structure

containing both shoot and root meristem either directly from the

explant (de novo) or through callus.

6.1.1. Various advantages of micropropagation

i. Shoot multiplication can be achieved in small space because

miniature plantlets are produced.

ii. Propagation is carried out under sterile conditions. No damage is

caused due to the insects and diseases, and plantlets produces

are free from microbes.

iii. Through viral elimination by meristem culture, a large number

of virus free plants can be obtained.

iv. Cultures are carried out under defined conditions of

environmental, nutritional and tissue system, therefore, it is

highly reproducible system under defined set of conditions.

v. This production is unaffected by seasonal variation as uniform

conditions are maintained.

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vi. No care is required between two subcultures as compared to

conventional vegetative propagation system like watering,

weeding.

vii. Small greenhouse facilities are sufficient because of miniature

size of plantlets.

viii. Mother plant or genotype of stock plant can be stored and

maintained in vitro without damage through environmental

factors and stock plants.

ix. The plants are difficult to propagate vegetatively (recalcitrant)

by conventional methods can also be propagated by this method.

6.2. Tissue culture and its commercialization in India

In India, commercial tissue culture was established for the first time in

1987 in Kerala by Thomas and Co for cardamom, followed by Indo-

American Seed Company in 1988 at Banglore. Today there are

hundreds of companies across the country run by both government

and private organizations, where this technique is immensely used for

production of several important fruits, shrubs, flowers, forest trees

including endangered species and medicinal plants. There are 46

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commercial tissue culture units in the country with the production

capacity of 1-5 million plants per annum and their aggregate

production capacity is 180 million plantlets per annum (Anonymous,

2005). The major consumers of tissue culture plants (TCPs) are the

state agriculture department, agri export zones, sugar industry and

private farmers. The paper industry, medicinal plant industry and

State Forest Departments are using TCPs in a limited scale. In addition,

a number of progressive farmers and nurseries in the states of Andhra

Pradesh, Maharashtra, West Bengal, Karnataka, Tamil Nadu etc., are

the major consumers of TCPs particularly for flowers, banana,

sugarcane and medicinal plants. In the year 2002-2003, domestic

consumption of tissue culture raised medicinal plants in India was 1.5

million, which valued at 0.78 million. In 2007-2008, with market

projections, tissue culture raised medicinal plants have became 12.2

million with the value of ` 61.4 million (Anonymous, 2005). The

aggregate production capacity of the established commercial tissue

culture units is estimated at 150 million plants per annum.

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7. CO2 - Major GHG

Carbon dioxide (CO2) is the chief GHG that results from human

activities and causes global warming and climate change.

The concentrations of CO2 in the atmosphere are increasing at an

accelerating rate from decade to decade. According to ESRL / NOAA

the current CO2 level is 393.09 ppm approximately (Fig- 3 and 4).

The following physical and chemical properties of carbon dioxide are

important (Table-1).

Table- 1: Physical and chemical properties of CO2

Property Value

Molecular weight 44.01

Specific gravity 1.53 g at 21 °C

Critical density 468 kg/m3

Concentration in air * 392.40 ppm

Stability High

Liquid Pressure < 415.8

Solid Temperature < -78

Henry constant for

298.15 mol/ kg bar

Water solubility 0.9 v/v at 20 °C *Average saturation levels are in ppm

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Fig- 3: The yearly mean atmospheric carbon dioxide levels at Mauna Loa Observatory, Hawaii

Fig- 4: Recent monthly mean (year 2006-2010) carbon dioxide at Mauna Loa Observatory from, Hawaii.

Since the industrial revolution began in 1850, the world's population

has grown from about 800 million to over six billion people and the

CO2 content of the atmosphere has increase about 30 %.

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As a result of industrial revolution, human processes have been

causing emissions of greenhouse gasses, such as CFC's and carbon

dioxide. This has caused an environmental problem: the amounts of

greenhouse gasses grew so extensively, that the earth's climate is

changing because the temperatures are rising. This unnatural addition

to the greenhouse effect is known as global warming. The most

important greenhouse gasses are CO2, CFC's, nitrogen oxides and

methane. It is suspected that global warming may cause increase in

storm activity, melting of ice caps on the poles, which will cause

flooding of the inhabited continents, and other environmental issues.

Increasing carbon dioxide emissions cause about 50-60 % of the global

warming. In this century, carbon dioxide emissions are expected to

double and they are expected to continue to rise and cause problems.

According to the Intergovernmental Panel on Climate Change (IPCC),

capturing and storing the greenhouse gas CO2 produced by power

plants and factories before it enters the atmosphere could play a major

role in minimizing climate change.

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8. Carbon sinks

Plants are very important sinks for atmospheric carbon dioxide on

both land and in aquatic environments. On land, plants themselves

represent a global mass equivalent to about 1500 billion tonnes of

carbon at any one time. Plants utilize carbon dioxide during

photosynthesis, but also produce it during respiration. The net effect is

an uptake of carbon dioxide from the atmosphere equivalent to around

60 billion tonnes of carbon each year (Anonymous, 2011). Forests are

major carbon sinks because they cover 65 % of the terrestrial

landmass and contain 90 % of the terrestrial vegetation carbon. The

forests absorb atmospheric carbon dioxide and fix it into the soil,

through decaying plant matter (Daniels, 2012).

8.1. Global Carbon Sinks

Global carbon is held in a variety of different stocks. Natural stocks

include oceans, fossil fuel deposits, the terrestrial system and the

atmosphere. In the terrestrial system carbon is sequestered in rocks

and sediments, in swamps, wetlands and forests, and in the soils of

forests, grasslands and agriculture. About two-thirds of the globe’s

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terrestrial carbon, exclusive of that sequestered in rocks and

sediments, is sequestered in the standing forests, forest under-storey

plants, leaf and forest debris, and in forest soils. In addition, there are

some non-natural stocks like long-lived wood products and waste

dumps.

A stock that is taking-up carbon is called a sink and one that is

releasing carbon is called a source. Shifts in flows of carbon over time

from one stock to another, for example, from the atmosphere to the

forest, are viewed as carbon fluxes. Over time, carbon may be

transferred from one stock to another. Fossil fuel burning, for example,

shifts carbon from fossil fuel deposits to the atmospheric stock.

Physical processes also gradually convert some atmospheric carbon

into the ocean stock. Biological growth involves the shifting of carbon

from one stock to another. Plants fix atmospheric carbon in cell tissues

as they grow, thereby transforming carbon from the atmosphere to the

biotic system.

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8.2. Carbon sequestration

Carbon sequestration is a geo-engineering technique for the long-term

storage of carbon dioxide or other forms of carbon, for the mitigation

of global warming. Carbon dioxide is usually captured from the

atmosphere through biological, chemical or physical processes. It has

been proposed as a way to mitigate the accumulation of greenhouse

gases in the atmosphere released by the burning of fossil fuels.

CO2 may be captured as a pure by-product in processes related to

petroleum refining or from flue gases from power generation. CO2

sequestration can then be seen as being synonymous with the storage

part of carbon capture and storage which refers to the large-scale,

permanent artificial capture and sequestration of industrially-

produced CO2 using sub-surface saline aquifers, reservoirs, ocean

water, aging oil fields, or other carbon sinks.

Due to the current scenario of global warming it is a challenge to

reduce the increasing CO2 level and to form artificial CO2 sink. By

means of photoautotrophic/photomixotrophic micropropagation of

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this endangered medicinal herb, we can utilize the CO2 in creative way

to upgrade the micropropagation technique.

9. Photoautotrophic Micropropagation

Sucrose is the main source of carbon and energy in the nutrient

medium during micropropagation of plants (Thompson and Thorpe,

1987). However, its presence increases the risk of contamination and

depresses photosynthetic activity leading to mixo or heterotrophy

(Deng and Donnelly, 1993). This and other factors cause morphological

and physiological disorders in in vitro grown plants resulting into high

rate of mortality during hardening, acclimatization and field transfer

(Kozai 1991; Vasil 1991). The goal of micropropagation is to mass

propagate genetically identical, physiologically uniform and

developmentally normal plants that can be achieved by

photoautotrophic multiplication of plants under CO2 enriched

conditions.

Photoautotrophic multiplication not only reduces the loss due to

contamination but also leads to development of plants which after

transplantation to ex vitro conditions are able to acclimate quickly to

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decreased air humidity. The leaves of photoautotrophically grown

cultures have lower stomatal index (Khan et al, 2003). Such plants

show higher survival rates and better growth (Langford and

Wainwright, 1987). Improvement in survival during hardening and

acclimatization of tissue culture plantlets has been achieved in number

of herbaceous species through in vitro cultures under CO2 enriched

environment (Mousseau 1986; Fujiwara et al, 1988; Solárová and

Pospíšilová, 1997).

Photoautotrophic micropropagation is an advanced plant production

technique that emerged as an integration of biology and engineering

for practical applications. Such integration will be necessary for the

future development of transplant production systems. The outcomes of

research and development in photoautotrophic micropropagation will

contribute to improvement and problem-solving in future agriculture,

forestry and horticultural production systems (Kubota, 2002).