Biodiversity is life ComplIed by DorriIIIque D\met, IITA and AdenIyi JayaoIa. University of Ibadan

www.iita.org

Biodiversity is life

Compiled by

Dominique Dumet, IITA and Adeniyi Jayeola, University of Ibadan

May 2010

Notes This pamphlet was produced by the IITA Genetic Resources Center (GRC) for the use of students participating in the Biodiversity Week, 24-27 May 2010, at I1TA as part of the celebration of the UN International Year of Biodiversity.

© International Institute of Tropical Agriculture (I1TA), 2010.

To Headquarters from outside Nigeria: I1TA, Carolyn House 26 Dingwall Road, Croydon CR9 3EE, UK

Within Nigeria: PMB 5320, Oyo Road Ibadan, Oyo State

Prepared and printed by the Communication Office, IITA.

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Contents

1, Introduction, .................. , ........ , ....................... , ......... , ........ 1

2. In situ conservation ............................................................ 4

3. Ex situ conservation .............................................. " ... " ....... 7

a. How do we store the diversity in a genebank? ." ................ 8

b. Field bank .......................... " ...................................... 14

c.In vitro bank ...................... " ... " .. " .............. " ............. 14

d. Cryoconservation and Cryobanking." ......... " ........ " ........ 18

e. How do we use the diversity of a genebank7 " ....... " ... " ... 19

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1. Introduction

The International Year of Biodiversity

• 2010 has been declared the International Year of Biod iversity (IYB) by the United Nations General Assembly­the year that celebrates the diversity of life on Earth, including every plant, animal, and microorganism

• The major IYB 2010 partners are listed in the box. • In Nigeria, IITA is organizing this IYB 2010 event in

response to the global call to promote the understanding of biodiversity during this special year.

What is biodiversity? • EJiQs. is a Greek word, meaning life. • Diverse is an English word, meaning of different kinds . • Biodiversity, therefore, refers to the variety of life on earth,

animals, plants, insects, and microorganisms (Figure 1).

Why is biodiversity important? There are two types of benefits :

Direct services or benefits. These are visible and we can see and may be able to quantify such benefits .

• It provides many of our needs such as shelter, food, medicines, fuel , and building materials.

Figure 1. Samples of plant biodiversity in the JlTA forest.

E: Trumpet flower, Pararistofochia gofdieana (Hook.f) Hutch.& Dalz. Conservation status : Vulnerable (according to IUCN Red List of Threatened Species)

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Indirect services or benefits. We cannot normally see these benefits because they take place naturally.

• It regulates the Earth's climate. • It controls erosion. • It recycles nutrients and enriches the soil. • It maintains oxygen in the air, purifies the water, and

protects against flood and storm damage (trees reduce wind speed).

• It helps in fermentation (various fermented foods and drinks are known: ogi, iru, Wara).

• It also helps in bioremediation (use of microorganisms or fungi to break down harmful, complex organic molecules into simpler, less harmful units, e.g., sewage management/treatment).

The part of the biodiversity that feeds and maintains people is called agricultural diversity or agrobiodiversity. This is the foundation for food security and human well-being. For hundreds of years, farmers have deliberately selected and improved plants and animals for their favorite and/or most suitable characteristics. For example, a farmer living in a region with short rainy season will have selected crops able to grow and produce quickly.

Unfortunately, biodiversity is disappearing very fast. This is due to two categories of factors: biotic factors such as diseases and pests, and abiotic factors such as climatic conditions (tsunami, flood, drought etc.), crop replacement/adoption (local crops replaced by one with higher yields), urbanization, pollution, etc.

Biodiversity and climate The temperature of the Earth has been increasing slowly in the last 100 years because of the gradual but steady accumulation on the earth's surface of such heat-absorbing and surface-warming gases as water vapor, carbon dioxide (CO,), and methane (CH4 ).

These gases are called greenhouse gases. Human activities such as burning of fossil fuel (wood, charcoal, petroleum, kerosene) lead to increases in the levels of the greenhouse gases on the earth's surface. Scientists suggest that the observed small increases in the global temperature may be connected to the increases noted in the global emission of greenhouse gases.

Consequences of global warming The rise in the earth's temperature has resulted in a warmer world with the following consequences reported in the last 100 years:

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• global mean sea level rose by 10-20 cm. • decrease of up to two-thirds of the glacier cover in

Switzerland. • Mt Kenya lost 92% of its ice mass, while Mt Kilimanjaro

lost 82%. • Extinction of golden toad and the Monteverde harlequin

frog, the first casualties of climate change. • two communities (Lateu in Pacific Island and Shishmaref

in Alaska) have become refuges when the rising sea level submerged their settlements.

• several plant species also lost their habitats, exposing them to severe environmental threats.

Plants and regulation of the earth's climate Green plants are autotrophic, meaning that they synthesize their food from their environment making use of CO, (a greenhouse gas), water, and energy from sunlight. The more green plants we have globally, the less CO, we accumulate and the less the burden of a warmer world and its attendant uncertainties.

Plants for food and agriculture The total number of botanical plant species cultivated as agricultural or horticultural crops is estimated at almost 7,000. However, only 30 major crop species feed the world. Among the latter are wheat, rice, sorghum, potato, tomato, maize, cassava, sweetpotato, and banana. It is important to maintain the diversity of major crops for further genetic improvement, but it is equally important to maintain the so-called underused crops (those not grown worldwide such as yam or cowpea) for future food security.

Biodiversity can be maintained in either in situ or ex situ condi­tions.

2. In situ conservation In situ conservation is the keeping of biological diversity safe in the original habitat or natural environment. This is the plant community structure of the !ITA forest:

• Under-storey: forest floor consisting of you ng trees and herbs; litter or piles of dead leaves, twigs, and fruits; worm casts; microbes helping to decompose the litter into reusable nutrients. Nutrient cycling is taking place here. The forest floor is dim because of low light

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penetration. The litter covers the soil surface, thus reducing water loss through evaporation.

• Middle stratum: small trees and shrubs, climbers, epiphytes, and parasites.

• Upper stratum: emergents or tall trees of different species, usually of large diameters (> 1 m) e.g., Iroko, Agbalumo, Arere, and Araba.

The IITA forest is one of the last remnants of rainforests in Southwestern Nigeria. Hunting, farming, and harvesting of plants are not permitted in the arboretum or nursery. But for research purposes, some plant parts may be removed with permission. These measures are taken to protect and conserve biodiversity in situ (Figures 2 and 3).

Methods for plant identification • Morphology of the plant (all observable characteristics).

Characteristics of any part of the plant can be used. These include the characteristics of the flower, fruit, leaf, and stem. When a characteristic can be measured (cm, g), it is called a quantitative characteristic. When it is not measured, it is called a qualitative characteristic, e.g., color of the flower. Morphological characteristics enable us to quickly recognize a plant.

• Senses of smell and touch combined. These are examples of qualitative characteristics. These characteristics observed from uSing senses other than sight have proved valuable for people with eyesight problems.

• The specialist in the herbarium can identify the plant. A herbarium is a collection of plants already preserved and identified for reference purposes.

When the use of morphological characteristics fails to provide best results in identification, other methods can be used.

• Histological method. All organisms are contracted from basic units called "cells." The method takes the search for characteristics of plants from the surface to the internal parts or organs. Cell arrangement, number, location, shape, and size have provided useful qualitative and quantitative characteristics.

• DNA barcoding is a scientific method that gives a plant its own ID. This latest method relies on gene sequence technique to identify plants since all living things can be distinguished on the basis of their genes.

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Figure 2. Mutually beneficial interaction : plant pollination and pollen gathering.

Figure 3. Animal plant interaction.

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3. Ex situ conservation Ex situ conservation is the conservation of biological diversity outside its natural habitat, i.e., seed-bank, in vitro bank, field bank, arboretum.

What is a genebank? A genebank is a place where genes are kept securely, like money in a bank. A gene is a hereditary unit consisting of a sequence of DNA (deoxyribonucleic acid) that occupies a specific location on a chromosome (a linear strand of DNA and associated proteins in the nucleus of superior cells) and determines a particular characteristic in an organism. The total of all genes in a population of a particular species is called a gene pool. A genebank is a center where gene pools (in other words, the diversity) are maintained out of their natural conditions. It is ex situ conservation.

The purpose of IlTA's Genebank is to capture and safeguard agrobiodiversityand make it easily accessible for further genetic improvement (improvement of an animal or plant by breeding to enhance its use).

UTA's Genebank was created in the early 1970s. It presently maintains about 28,000 samples called accessions. An accession is a distinct, uniquely identifiable sample maintained in storage for conservation and use. IlTA maintains seven main collections: cowpea (15,000), wild Vigna species (1815), maize (880), soybean (1742), yam (3200), cassava (3500), and banana (290).

The totality of the genetic resources is called germplasm.

How do we distinguish/manage accessions in a genebank? Traditionally, an accession is characterized by its passport and agromorpholog ical characters.

Passport data Just like human travelers, each accession is given a passport number that provides detailed information. In addition to the passport number (which can be compared to our name and surname), common passport data include:

Genus Species Common name Location and date of collection

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A genus is a group of related species exhibiting similar characteristics (genera is the plural form).

A species is a group ranking below a genus and consisting of related organisms capable of interbreeding (reproduction). Both genus and species are written in Latin.

A common name (also called vernacular name) is used within a community and it varies from one place to another.

The table below gives you an example of genus, species, and common names of crops maintained in UTA's Genebank.

Genus Species Common name

Vigna unguiculata Cowpea/Niebe/Black eyed pea

Vigna 5ubterranea Bamoara groundnut I Pois Bambara

Dioscorea rotundata Yaml igname

The location is the exact place where the sample was collected. Somehow we could compare this information with the place of birth on your passport. Each single dot on the map represents a location where a cowpea accession was collected in Africa (Figure 4). When looking for a cowpea able to tolerate drought, you preferably select those collected in dry areas. In contrast, if flooding is what the breeders want, they will preferably take accessions collected in the most humid zones.

Description data Traditionally, plants are described using agromorphologial 'plant descriptors'. For example, in the case of cowpea, there are 52 known descriptors. Among them are color of the seed coat (black, brown, white, with stripes, etc.) (Figure 5), the shape of the leaves, the color of the stem, the number of days to produce a pod (that can vary from 49 to 129 days!), the resistance towards diseases etc. Biotechnology now offers new descriptors called molecular descriptors. They are based on DNA sequence of the accession.

a. How do we store the diversity in a genebank?

Plants are classified into two categories based on their agronomical multiplication (i.e., multiplication for agricultural use).

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Figure 4. Projection of cowpea collecting points in Africa .

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Seed crops: Plants are multiplied via botanical seeds such as cowpea, 8ambara groundnut, maize, soybean, and rice. A botanical seed is composed of at least one embryo surrounded by various storage and protective tissues . The surrounding tissues will allow the embryo to develop to a full seed ling once it is placed in the right conditions for germination .

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Figure 5. Cowpea seed coat diversity.

Clonal crops: Plants are multiplied via vegetative tissues (called vegetative multiplication) such as cassava, cocoyam, yam, banana, sweetpotato, apple, and pineapple. (Vegetative tissues used for clonal crop propagation include tubers (yam), cuttings (cassava), (Figure 6) and suckers (banana). All these tissues contain at least one meristem. A meristem is an undifferentiated tissue from wh ich new cells are formed, and is able to regenerate a full plant.

Crops multiplied by seeds are maintained in a seed-bank (Figure 7), whereas crops multipl ied vegetatively are traditionally maintained in a field-bank (Figure 8) and in vitro bank (Figure 9). Whatever the bank, its aim is to maintain the plant or plant tissues for as long as possible with minimum input. In other words, the objective is to reduce the metabolism of the plant or plant tissues to a minimum to increase its lifespan.

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Figure 6. Cassava cuttings that are ready to plant.

Figure 7. Seed bank stores .

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Figure 8. Banana germ plasm maintained in field -bank.

Figure 9. Germplasm maintained in in vitro bank.

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Seed-bank In a seed-bank, accessions are mainta ined as botanical seeds. Seeds are classified into two categories based on their tolerance for dehydration and low temperatures:

Orthodox seeds: Those seeds naturally undergo a drying period when they are mature and they can withstand further artificial dehydration. They are also tolerant of cold temperatures. Cowpea, soybean, maize, rice, and African yam bean produce orthodox seeds (Figure 10).

Recalcitrant seeds: Those seeds do not undergo natural dehydration as they mature and are extremely sensitive to both artificial dehydration and low temperatures. Avocado, litch i, jackfruit, and mango are examples of recalcitrant seeds. Such seeds cannot be stored in seed-banks; their ex situ conservation involves field- or in vitro-banking.

Figure 10. Harvest of orthodox seeds for long -term conservation .

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Orthodox seeds can be stored for a long time when they are dehydrated to the best water content and stored in conditions with low humidity, oxygen, and tem perature. When entering a seed­bank, seeds are ideally processed as followed:

1. Sanitation certification (check for diseases and pests) 2. Mechan ical cleaning 3. Identity check (visual comparison with seed file) 4. Dehydration to optimal water content (7 to 8% of water on a

fresh weight basis for IITA's crops) 5. Water content determination (to check optimal water

content is reached before storage) 6. Germination test (to check if seeds are alive) 7. Weighing and counting (to manage the stock and plan

distribution and regeneration) 8. Packaging

At I1TA, seeds of cowpea, maize, soybean, wild Vigna and Bambara groundnut are stored at 5 and -20 degrees Celsius. Samples maintained at 5 degrees are used for distribution while the samples in the -20 degrees store are maintained for long-term conservation.

b. Field bank

As mentioned above, crops multiplied vegetatively can be maintained in a field-bank. For yam this involves planting the entire collection every year and every 2 years for cassava. Field-banking is the most vulnerable approach in ex situ conservation. While maintained in the field, germplasm is subjected to biotic stresses (termites, viruses, rats) and abiotic stresses (drought, wind).

As a consequence, field-banking requires close monitoring (check for signs of deterioration so action can be taken). Another limiting factor of field-banking is the fact that germplasm cannot be maintained clean which causes problems for international distribution (see section on distribution).

c. In vitro bank

In vitro banking requires in vitro culture. In vitro culture is the technique or process of keeping tissue alive and growing in a sterile artificial environment. Sterile means free from live bacteria or other microorganisms.

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The artificial environment has three main components: a. The culture medium b. The light regime c. The temperature

The culture media provides all chemical/biochemical compounds required for the tissues to grow. In IITA's in vitro collection, the essential compounds are the minerals (iron, zinc, aluminum, etc.) growth regulators (which will, for example, order the meristem to elongate or the root to grow), a solidifying agent (generally agar which replaces the soil), sugars (to provide energy to the plant), and various vitamins, amino acids, etc.

The light regime is defined by the photoperiod (photoperiod is the duration of light and absence of light over a period of 24 hours) and the light intensity.

In the case of yam and cassava culture, the optimal temperature varies from 18 to 28 degrees Celsius depending on the in vitro culture step (see below) while the light intensity is around 38 IJmol/m 2 /s and the photoperiod is 12 hours.

In vitro genebanking involves three main steps (see below) which have to be performed in a sterile environment in order to avoid the growth of unwanted organisms, such as bacteria or fungi. For this reason, all plant tissue manipulations are done under a sterile laminar air flow (Figure 11).

a. In vitro introduction of the ex plants b. Subculture = In vitro multiplication c. In vitro storage

In vitro introduction. This step involves cleaning the explants from bacterial contamination and placing them onto a medium which will allow growth. At !ITA's Genetic Resources Center or Genebank, explants used for in vitro banking are either meristems (an undifferentiated tissue from which new cells are formed) (Figure 12) or nodal cuttings (at least one bud on a stem; note that the bud contains one meristem) (Figure 13). The advantage of using meristems not nodal cuttings is that this part of the plant is often free from disease (viruses and bacteria) so the material maintained in vitro is kept clean.

Subculture: Once the meristem has elongated into a stem with at least one node it is subcultured. Each bud of the newly developed stem is transferred to a fresh medium and will produce a new plant. If one stem contains three buds, three new seedlings

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Figure 11. Genetic resources center staff working under sterile conditions Le., under sterile laminar flow.

Figure 12. Banana meristem newly cut from a sucker.

Figure 13. Yam nodal cutting.

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can be produced . During the in vitro introduction and subculture steps, in vitro cultures are maintained at a high temperature (above 26 degrees Celsius) to promote their growth.

In vitro storage: Once seedlings have developed a shoot and a root, they are transferred in the in vitro bank (i.e., storage room) . During in vitro storage they are maintained at low temperature to reduce their growth (below 21 degrees) . As the seedl ing develops, it uses the different compounds present in the media until they are all used up. Once all minerals and other compounds have been used, the plant starts showing signs of senescence (senescence means ageing) such as the leaves yellowing or dropping . When plants are at that stage, they need a new subculture. The faster a plant grows in vitro, the more expensive is its in vitro conservation.

Figure 14 shows the in vitro development of one cassava meristem from excision to full seedling development

Figure 14. In vitro development of one cassava meristem from excision to full seedling development.

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d. Cryoconservation and Cryobanking

Cryopreservation is the conservation at very low temperature, generally -196 degrees Celsius (the temperature of liquid nitrogen) . At such temperature all metabolisms are stopped and a tissue can theoretically be stored forever. The idea is to maintain tissues at such a temperature and take them out only when they are needed.

In th is system, samples are frozen in liquid nitrogen. The main challenge for cryobanking is the removal of the freezable water from the plant t issues before freezing. Indeed, as the temperature decreases water changes into crystals (it solidifies) that can kill the plant tissue. Various processes have been developed to avoid lethal ice formation; they are based on the dehydration of the plant tissue using chemical compounds (called cryoprotectants) and/or evaporation and various freezing rates (Figures 15 and 16). IITA has developed such processes for the long-term conservation of cassava and is presently developing a similar system for yam .

Figure 15. Meristem expasure ta cryapratectant far safe cryapreservation .

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Figure 16. Cryopreservation of cassava meristem at UTA.

e. How do we use the diversity of a genebank?

The main objective of a genebank is to maintain crop diversity for its use today and in the future. To do so, all data related to each accession (passport and characterization) need to be made available to potential users (breeders, biotechnologists, teachers, etc.

IITA has developed an on-line searching and ordering tool (www. iita.org). People from all over the world can select their germplasm on-line and send us their request via email or regular mail, or visit to IITA campus. Whenever a plant goes out of Nigeria, it requires a phytosanitary certificate certifying that the plant is disease free. Like humans, plants carry a lot of viruses and bacteria and it is important not to spread these diseases into other countries. Specific tests are performed on plants to check their health status.

Next time you see a tree, a seed or a tuber, think about its conservation!

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