CHAPTER 2

LITERATURE REVIEWS

2.1 Rice (Oryza sativa L.)

Rice is an annual plant, relating to grass. The rice plant produces the grain
known as rice. It is a staple food for a large part of the world’s population. Rice plant
composes of four main parts including root, stem, leaf, and panicle (Fig. 2.1).

2.1.1 Taxonomy and classification

Rice (Oryza L.) is a cereal grain belonging to the grass family. Rice is related
to other grass plants which produce grain for food such as wheat, oats and barley. The
rice plant belongs to the division Angiospermae, class Monocotyledoneae, order
Glumiflorae, family Poaceae ("true grass") and genus Oryza. Originally, just two
species, Oryza sativa and Oryza glaberrima, were cultivated. Oryza sativa is native to
tropical and subtropical southeastern Asia and Oryza glaberrima is native to Africa
and less cultivated (Crawford and Shen, 1998). The Oryza Sativa is the most
important and originated three subspecies: Indica (long grain), Japonica (round grain)
and Javanica (medium grain).
Rice is rich in genetic diversity. Thousands of varieties are cultivated
throughout the world. Oryza satia appears to have been domesticated from the crop
wild relative (Asian) Oryza rice, Oryza rufipogon around the foothills of the
Himalayas, with Oryza sativa var. indica on the Indian side and Oryza sativa var.
japonica on the Chinese and Japanese side. Based on the geographical analysis, Oryza
sativa indica was domesticated within a region of south of the Himalaya mountain
range, likely eastern India, Myanmar, and Thailand, whereas Oryza sativa japonica
was domesticated from wild rice in southern China (Londo et al., 2006).
The difference histories have led to different ecological niches for the three
main types of rice. Indica are mainly lowland rices, grown mostly submerged,
 10

throughout tropical Asia. Indica rice is widely grown in the warm climate belt, from
Indochina, Thailand, India, Pakistan, Brazil and Southern USA. While japonica are
usually cultivated in dry fields, in temperate East Asia, upland areas of Shoutheast
Asia and high elevations in South Asia (Oka, 1988). Japonica type is mostly grown in
colder countries such as Japan, Korea, northern China and California, whereas
Javanica is only grown in Indonesia. More than 140,000 varieties of cultivated rice
(the grass family Oryza sativa) are thought to exist but the exact number remains a
mystery. Three main types of rice had developed in Asia over the centuries,
depending on the amylose content of the grain. Indica is high in amylose and cooking
to fluffy grains to be eaten with the fingers. Japonica is low in amylase and cooking
to sticky masses suitable for eating as clumps with chopsticks. And javanica is
intermediate amylose content and stickiness. Current genetic analysis indicates that
Oryza sativa would be best divided into five groups, labeled indica, aus, aromatic,
temperate japonica and tropical japonica. The same analysis suggests that indica and
aus are closely related, as are tropical japonica, temperate japonica, and aromatic
(Garris et al., 2005).
Further analysis of the genetic material of various types of rice indicates that
japonica was the first cultivar to emerge, followed by the indica, aus, and aromatic
groups, whose genome did show significant differences in age. Within the japonica
group, there is some genetic evidence that temperate japonica is derived from tropical
japonica. Other studies have suggested that the three groups of Oryza sativa cultivars:
the short-grained "japonica" or "sinica" varieties are exemplified by Japanese rice; the
long-grained "indica" varieties are exemplified by Basmati rice; and the broad-grained
"javanica" varieties, which thrive under tropical conditions are also exemplified by
the medium grain “Tinawon” and “Unoy” varieties, which are grown in the high-
elevation rice terraces of the Cordillera Mountains of northern Luzon, Philippines
(Zohary and Hopf, 2000). The earliest find site for the japonica variety, dated to the
5th millennium BCE, was in the earliest phases of the Hemudu culture on the south
side of Hangzhou Bay in China, but was found along with japonica types.
 11

Figure 2.1 Rice plant comprises four main parts: root, stem, leaf, and panicle.
(Virmani et al., 2008)

2.1.2 Importance of rice as a food

Rice is the major cereal crop that is primarily consumed by humans directly as
harvested, and only wheat and corn are produced in comparable quantity. Rice
provides more than 20% of the calories consumed by humans. Rice is a very
nutritious grain, especially brown rice. It has high fiber, vitamin B, carbohydrate,
protein etc. Rice is a great source of complex carbohydrates, which is an important
source of the energy of our bodies. Rice is healthful for what it does not contain. Rice
has no fat, no cholesterol and is sodium free. Rice is an excellent food to include in a
balanced diet. Rice is gluten free so it is nonallergic, making rice the essential choice
for people with gluten free dietary requirements. Rice is a good source of vitamins
and minerals such as thiamine, niacin, iron, riboflavin, vitamin D, calcium, and fiber.
Whole grains (such as brown rice) contain high amounts of insoluble fiber-the type of
fiber that is believed to help protect against a variety of cancers. It is also a source of
protein containing eight amino acids. Rice is vital to more than half the world’s
 12

population. It is the most important food grain in the diets of hundreds of millions of
Asians, Africans, and Latin Americans living in the tropics and subtropics. More than
90 percent of the world's rice is grown and consumed in Asia. In these areas,
population increases are high and will likely remain high at least for the next decade.
Rice will continue to be primary source of food in asia. In America, American
consumption, although increasing, is still only about 11 kg per person annually, as
compared with 90–181 kg per person in parts of Asia. And in Europe, the average
European eats only 5 kg of rice seeds (Encyclopedia-rice-1, 2008).

2.1.3 History of rice cultivation

Throughout history rice has been one of man's most important foods. Today,
this unique grain helps sustain two-thirds of the world's population. It is life for
thousands of millions of people. It is deeply embedded in the cultural heritage of their
societies. About 80% of the world's rice is produced by small-scale farmers and is
consumed locally. Rice cultivation is the principal activity and source of income for
about 100 million households in Asia and Africa. From an early history in the Asian
areas rice has spread and is now grown on all continents except Antarctica.
Descended from wild grasses, historians believe that rice was first domesticated in the
area covering the foothills of the Eastern Himalayas (north-eastern India), and
stretching through Burma, Thailand, Laos, Vietnam and Southern China. Remains of
early cultivated rice have been found in the Yangtze valley dating to about 8500 BC
(Rice-1, 2008).
Chinese records of rice cultivation go back 4000 years. Most believe the roots
of rice come from 3000 BC in India, where natives discovered the plant growing in
the wild and began to experiment with it. Cultivation and cooking methods are
thought to have spread to the west rapidly and by medieval times, southern Europe
saw the introduction of rice as a hearty grain. In several Asian languages the words
for rice and food are identical. African rice has been cultivated for at least 3000 years
(The history of rice in West Africa, 2008).
In the Middle East and Mediterranean Europe, it started around 800 BC. Rice
spread throughout Italy and then France, after the middle of the 15th century, later
 13

propagating to all the continents during the great age of European exploration. In
1694, rice arrived in South Carolina; it was probably originating from Madagascar.
The Spanish brought rice to South America at the beginning of the 18th century (Rice-
2, 2008)
Rice cultivation has been carried into all regions having the necessary warmth
and abundant moisture favorable to its growth, mainly subtropical rather than hot or
cold. The crop was common in West Africa by the end of the 17th century. It is
thought that slaves from that area who were transported to the Carolinas in the mid-
18th century introduced the complex agricultural technology, thus playing a key part
in the establishment of American rice cultivation. Their labor then insured a
flourishing rice industry. Modern culture makes use of irrigation, and a few varieties
of rice may be grown with only a moderate supply of water. Methods of growing
differ greatly in different localities, but in most Asian countries the traditional hand
methods of cultivating and harvesting rice are still practiced. The fields are prepared
by plowing (typically with simple plows drawn by water buffalo), fertilizing (usually
with dung or sewage), and smoothing (by dragging a log over them). The seedlings
are started in seedling beds and, after 30 to 50 days, are transplanted by hand to the
fields, which have been flooded by water. Irrigation is maintained by dike-controlled
canals or by hand watering during the growing season. The fields are allowed to drain
before cutting (Encyclopedia-rice-2, 2008).

2.1.4 World rice production

World production of rice has risen steadily from about 200 M MT of paddy
rice in 1960 to 600 M MT in 2004 (Rice-3, 2008). In the year 2004, the top three
producers were China (26% of world production), India (20%), and Indonesia (9%).
In 1997, world paddy production was over 570 M MT, or about 350 M MT milled
rice basis. Of this, Asia shared over 90% of the production. Paddy is usually kept by
farmers for household consumption or for next season seed or emergency saving,
what is left is sold to local market for extra cash. Rice is a unique commodity, most
production is consumed in farmer’s home or village. World trade figures are very
different, as only about 5–6% of rice produced is traded internationally. The world's
 14

leading rice-producing countries are China, India, Indonesia, Bangladesh, Vietnam,

and Thailand (Table 2.1). The largest three exporting countries are Thailand (26% of
world exports), Vietnam (15%), and the United States (11%), while the largest three
importers are Indonesia (14%), Bangladesh (4%), and Brazil (3%). Although China
and India are the top two largest producers of rice in the world, both of countries
consume the majority of the rice produced domestically leaving little to be traded
internationally.
World rice production in 2007 was approximately 645 M MT. Today, at least
114 countries grow rice and more than 50 countries have an annual production of
100,000 MT or more. Asian farmers produce about 90% of the total, with two
countries, China and India, produce more than half the total crop. In March to May of
2008, the price of rice rose greatly due to a rice shortage. Rice prices hit 24 cents a
pound that was twice of that of the seven months earlier in late April 2008 (Rice-3,
2008).
Thailand is the major rice exporter in the world market. Thailand currently
exports around 7-9 M MT of milled rice per year. The exports continue to grow
despite the stagnation in domestic production because of the declining trend in the
domestic demand for rice. Thailand has a reputation for high-quality, long-grain white
rice, which usually commands a substantial price advantage over lower grades. On the
30th of April, 2008, Thailand announced the project of the creation of the Organisation
of Rice Exporting Countries (OREC) with the potential to develop into a price-fixing
cartel for rice (Rice-3, 2008). In Thailand, rice is the most important crop of the
country. Even though declining in relative importance, it still occupies about 55% of
the total arable land. Rice farmers in the northeast, the main rice-growing region and
the home of the famous Jasmine rice, are generally subsistence farmers, selling only
their excess production. The main surplus production is from the central region and
the north, where the average farm size is three times larger than in the northeast, and
the production environment is favorable.
 15

Table 2.1 World rice production during 1999-2003. (World Wide Rice Production,
2008)

World Rice Production (in '000 MT)

Country 1999/2000 2000/01 2001/02 2002/03
China 138,936 131,536 124,320 123,200
India 89,700 84,871 91,600 80,000
Indonesia 33,445 32,548 32,422 32,500
Bangladesh 23,066 25,086 25,500 26,000
Vietnam 20,926 20,473 20,670 20,500
Thailand 16,500 16,901 16,500 16,500
Burma 9,860 10,771 10,440 10,440
Philippines 7,772 8,135 8,450 8,300
Japan 8,350 8,636 8,242 8,200
Brazil 7,768 7,062 7,480 7,600
United States 6,502 5,941 6,764 6,457
Korea, South 5,263 5,291 5,515 5,300
Egypt 3,787 3,965 3,575 3,800
Pakistan 5,156 4,700 3,740 3,500
EU 1,751 1,567 1,620 1,792
Taiwan 1,349 1,342 1,245 1,197
Australia 787 1,259 930 965
Others 28,282 27,270 27,575 28,156
World Total 409,200 397,354 396,588 384,407

Source: USDA, Foreign Agricultural Services (FAS).

2.1.5 Three rice growth stages

The growth of the rice plant is divided into three phases.

Rice is generally a short living plant, with an average life span of 3-7 months; its life
span depends on the climate and the variety. The life span of rice comprises three
basically distinct sequential growth stages that are vegetative growth stage,
reproductive growth stage, and ripening growth stage (Yoshida, 1981). The vegetative
stage is a period from germination to an initiation of panicle primordia; the
reproductive stage is from panicle primordial initiation to flowering (heading); and
the ripening period is from flowering to mature grain. In the tropics, the reproductive
phase is about 35 days and the ripening phase is about 30 days. The differences in
growth duration are determined by changes in the length of the vegetative phase. For
example, IR64 which matures in 110 days has a 45-day vegetative phase, whereas
 16

KDML 105 which matures in 140 days has a 75-day vegetative phase. An example of
a 120-day rice variety planted in a tropical environment, spends around 60 days in the
vegetative growth stage, 30 days in reproductive growth stage, and 30 days in the
ripening growth stage, was shown in Fig. 2.2.
The 3 growth stages (phases) could also be subdivided into a series of 10
distinct stages (Growth stage of the rice plant, 2002). These stages are numbered and
described as follows. Stage 0 is from germination to emergence. Stage 1 is called
seedling. Stage 2 is tillering and stage 3 is stem elongation. These first 4 stages make
up the vegetative phase, the first phase of rice plant growth. Stage 4 is panicle
initiation to booting. Stage 5 is heading or panicle exsertion and stage 6 is flowering.
Stages 4, 5, and 6 constitute the reproductive phase, the second phase of rice growth.
Stage 7 is the milk grain stage. Stage 8 is the dough grain stage and stage 9 is the
mature grain stage. Stages 7 through 9 correspond to the ripening phase, the last phase
in the development of the rice plant.

2.1.5.1 Vegetative Stage

Vegetative growth stage comprises germination, emergence, seedling

establishment, and tillering. This stage ends when the internodes begin to elongate.
Germination is a continuous phase that occurs within a short time after a seed
is placed in soil or water. Germination occurs as a consequence of very active
metabolic changes during the activation stage (Takahashi, 1965). At the end of the
activation stage, the white tip of the coleoptile emerges from the ruptured seed coat.
Subsequently, the coleoptile grows and the first leaf appears. Under aerated
conditions the seminal root is the first to emerge through the coleorhiza from the
embryo, and this is followed by the coleoptile. Under anaerobic conditions, however,
the coleoptile is the first to emerge, with the roots developing when the coleoptile has
reached the aerated regions of the environment. If the seed develops in the dark and
when seeds are sown beneath the soil surface, a short stem (mesocotyl) develops,
which lifts the crown of the plant just below the soil surface. After the coleoptile
emerges it splits and the primary leaf develops. Seedling emergence is the time when
the tip of a seedling emerges from the soil or water surface and, thus, includes both
 17

germination and postgermination growth. Seedling emergence is an appropriate term

for the beginning of the rice growth in the field. A tiller and roots emerge from the
same node at the same time. A tiller and roots start emerging from the third node
when the sixth leaf emerges.

Figure 2.2 Life history of a 120-day rice variety grown in the tropics under
the transplanting cultivation system (http://www. Knowledgebank .irri. org/RiceIPM,
2008, September 12).
 18

2.1.5.2 Reproductive Stage

The reproductive stage is noticed when the nodes (solid joints) and internodes
(hollow areas between nodes) develop. These make up the culm (stem) in jointing
stage. The number of nodes per culm ranges from 13 to 16 and is related to the length
of growing season. Normally, only four to five internodes elongate. The upper
internode is usually the longest and bears the head, called the "panicle."
The reproductive growth stage is characterized by the elongation of internodes
(which resulting in increasing of the plant height), emergence of the flag leaf (the last
leaf at the top), booting, heading, and flowering. Initiation of panicle primordia
usually dates back to about 30 days before flowering.
Panicle initiation is a stage about 30 days before flowering when the panicle
has grown about 1 mm long and it can be recognized visually or with a magnifying
lens. Rice plant initiates panicle primordial when its certain physiological and
environmental conditions are met. Although the duration from panicle primordial
initiation to heading is slightly affected by variety and weather conditions, it is
considered almost constant, about 30 days under most conditions. Variations in length
of time from germination to heading are attributed to variations in duration from
germination to panicle primordial initiation. Thus, early-maturing varieties initiate
panicle primordial earlier than late-maturing varieties.
Internode elongation usually starts around the initiation of panicle primordial
and continues until flowering. The top five internodes are usually elongated at
heading. The panicle grows fast and moves upward in the flag leaf sheath as the
internode elongate after the flag leaf emerges about 18 days. About 6 days before
heading, the leaf sheath thickens indicating that a panicle is enclosed. The 6 days are
called the booting stage.
Internode elongation is closely associated with growth duration (Hosoda and
Iwasaki, 1960, Vergara et al., 1965). Internode elongation usually starts around
panicle primordial initiation in early-and medium-maturing varieties. Whereas in late-
maturing varieties, it starts before panicle primordial initiation. In photoperiod-
sensitive rice varieties, extended photoperiod increase the number and total length of
 19

internodes. Whereas in photoperiod-insensitive varieties, there is no influence of

photoperiod on their internode elongation.
Flowering (heading) refers to the panicle exsertion of rice plant. Spikelet
anthesis (or flowering) begins with panicle exsertion, or on the following day. Rice
plant takes 10-14 days to complete heading. Pollen grain are viable for only 5 minutes
after emerging from the anther, whereas the stigma can be fertilized for 3-5 days. It
takes 7-10 days for all the spikelets within the same panicle to complete anthesis;
most of the spikelets complete anthesis within 5 days. Within the same field it takes
10-14 days to complete heading because panicle exsertion varies within tillers of the
same plant and between plants in the same field. Hence, it takes about 15-20 days for
all the spikelets of a crop to complete anthesis. The time of day when anthesis occurs
varies with weather conditions. Under normal weather conditions in the tropics, most
rice varieties (Oryza sativa) begin anthesis at about 0800 and end at about 1300 hours.
When temperatures are low, anthesis may start late in the morning and continue into
the late afternoon. On rainy days, pollination may occur without the opening of the
lemma and palea. Pollen grains are shed from the anthers and fall onto the feathery
stigmas. The pollen that reaches the stigma germinates and forms a pollen tube that
carries the male nuclei inside the ovary for fusion with the egg nuclei. The complete
process from pollination to fertilization takes from 18 to 24 hours.

2.2.5.3 Ripening stage

The ripening period is characterized by grain growth-increase in size and

weight, changes in grain color, and senescence of leaves. At the early stages of
ripening, the grains are green; they turn yellow as they mature. The texture of the
grains changes from a milky, semifluid stage to a hard solid. On the basis of such
changes the ripening period is subdivided into milky, dough, yellow ripe, and
maturity stages. The rice kernel reaches its maximum length in 12 days, its maximum
width in 2 days, its maximum thickness in 28 days, and its dry weight in 35 days after
flowering. Before and during kernel growth, several factors may influence its
development. In some cases, the hulls and kernel will be distorted, whereas in others,
 20

only the kernel is affected. The affected kernels may be throughout the panicle or only
on a specific portion of the panicle.

2.1.6 Plant description

The rice plant composes of four main principle parts: a fibrous root system,
culm (stem), leaves, and panicle (head). The roots, culm, and leaves make up the
vegetative part of the plant. The floral part consists of the panicle, spikelets, and
flower.

2.1.6.1 Roots

Rice roots, which occupy an important part of a plant, play vital roles in
growth and grain production. Their functions include maintenance of the aerial
organs, water and nutrient absorption, and synthesis of hormone-like substance
(Tanaka, 1961b; 1962). The fibrous root system (Fig. 2.3) of the rice plant begins to
develop both outward and downward from the plant's base after seed germination.
Finely-branched adventitious roots arise from the lower nodes of the stem (culm). The
size and length of the roots vary. Drained conditions are best for good root
development that is proportionate to top growth. Maximum root development is
reached at the peak of the tillering stage, then begins to decrease, and toward the
ripening stage, almost ceases.
The rice root system consists of two major types: crown roots (including mat
roots) and nodal roots. In fact both these roots develop from nodes, but crown roots
develop from nodes below the soil surface. Roots that develop from nodes above the
soil surface usually referred as nodal roots. Nodal roots are often found in rice
cultivars growing at water depths above 80 cm. Most rice varieties reach a maximum
depth of 1m or deeper in soft upland soils. In flooded soils, however, rice roots
seldom exceed a depth of 40 cm. That is largely a consequence of limited O2 diffusion
through the gas spaces of roots (aerenchyma) to supply the growing root tips. Rice has
fibrous roots which consists of rootlets and root hairs (Fig. 2.4). The seedlings first
have the embryonic roots and later the adventitious roots that are produced from the
 21

underground nodes of the young culms. On germination primary root develops from
the base of the grain, quickly followed by two additional roots, all subsequently
giving rise to short lateral roots. The main rooting system of the plant, however
develops from the nodes of the stem below ground level. In the "floating rices",
whorls of adventitious roots are formed from the first three very short nodes, giving
rise to whorls of permanent adventitious roots. Tillers are produced at the nodes and
adventitious roots are produced from lower nodes of these culms, so that the plant
quickly develops a mass of adventitious roots. Under normal conditions the root
system is fairly compact, the roots tending to develop horizontally rather than
vertically, the plant therefore draws its nutrients from nearby surface of the soil. Root
development is influenced by soil texture, cultivation, water and air in the soil, the
amount of available food supply and by the system of transplanting.

2.1.6.2 Culm (Stem)

The stem of rice plants is called the culm (Fig. 2.3). In rice plants, the culm is
an important organ closely associated with lodging resistance, and eventually
influencing grain yield. The culm is the jointed rice stem that develops from the
plumule (primary bud of the seed embryo) and is composed of solid centers and
hollow internodes. Culm height varies according to management practices and
varieties. The main axis of the stem is differentiated from the growing point of the
embryo, enclosed at first by the coleoptile. The ultimate height of the stem depends on
the number of internodes and environmental conditions. Early strains of short
maturation period have lower internodes than those with a long maturation period.
The number of internodes may vary from about ten to twenty. The culm is more or
less erect, cylindrical, and hollow except at the nodes, and varies in thickness from
about 6-8 mm. Nodes are clearly defined by the presence of a distinct thickening, the
pulvinus, immediately above the node. The pulvinus may be coloured, varying in
intensity from a "touch" of purple to a deep uniform purple. The colour of the
pulvinus is always associated with colours in the leaf sheath. The internodes may be
green coloured. The pigment in the coloured form may be different in the epidermis
or in the parenchyma or confined to the bundle sheaths. A bud may form in the axil of
 22

each leaf of the main stem, but normally only the lowermost bud from the crowded
nodes at ground level develop into branches, thus a typical tillered plant develops.

Figure 2.3 Rice plant (http://www.ikisan.com, 2008, September 29).

Each stem of rice is made up of a series of nodes and internodes. The

internodes vary in length depending on variety and environmental conditions, but
generally increase from the lower to upper part of the stem. Each upper node bears a
leaf and a bud, which can grow into a tiller. The number of nodes varies from 13 to 16
with only the upper 4 or 5 separated by long internodes. Under rapid increase in water
level some deepwater rice varieties can also increase the lower internode lengths by
over 30 cm each. The leaf blade is attached at the node by the leaf sheath, which
encircles the stem. Where the leaf blade and the leaf sheath meet is a pair of claw like
appendages, called the auricle, which encircle the stem. Coarse hairs cover the surface
for the auricle. Immediately above the auricle is a thin, upright membrane called the
 23

ligule. The tillering stage starts as soon as the seedling is self supporting and generally
finishes at panicle initiation.

Figure 2.4 Part of a young rice seedling (Reano, 2007).

2.1.6.3 Leaves
The mature leaf has four main parts: the sheath, blade, ligule, and auricles.
The leaf varies in length, form, and tightness, and covers the stem. These mature
leaves are flat and vary in length and width according to variety and production
practices. The collar is the junction of sheath and blade. The swollen zone at the
sheath base where it joins the culm is the pulvinus. The leaves are long and narrow,
usually pubescent or hispid, with a distinct midrib, but varying considerably in length.
The leaves of many varieties are coloured, the colour being usually concentrated in
 24

the midrib region and on the margins, though occasionally the whole leaf is coloured.
The uppermost leaf or "flag" (Fig. 2.4) of the axis posseses a blade always shorter and
broader than the lower leaves. As the panicle emerges from the sheath, its blade is
nearly parallel to the panicle axis. After the panicle has emerged the blade falls.
Ultimately the panicle is either at an acute angle to the axis, more or less horizontal
then it leads definite drooping.

2.1.6.4 Panicle (Head) and flower

Panicle (Fig. 2.5) formation starts when all nodes have been formed and
internodes begin to elongate. Usually panicles form 3 to 4 weeks before they are
noticeable in the field. It takes about 25 to 33 days from internode elongation until the
panicle emerges from the plant. The initiation of panicle primordial stars about 30
days before heading; it corresponds to the time when the fourth leaf from the top
begins to elongate.
The major structures of the panicle are the base, axis, primary and secondary
branches, pedicel, rudimentary glumes, and the spikelets (Fig. 2.6). The panicle axis
extends from the panicle base to the apex; it has 8-10 nodes at 8 to 4 cm interval from
which primary branches develop. Secondary branches develop from the primary
branches. Pedicels develop from the nodes of the primary and secondary branches; the
spikelets are positioned above them. Since rice has only one fully developed floret
(flower) per spikelet, these terms are often used interchangeably. The inflorescence is
a spikelet borne on a long peduncle. The rachis bears branches, either single or in
pairs, from which arise the spikelets. The number of spikelets on panicle varies
considerably with the variety from a few to some hundreds.
The spikelet, or flower is borne on a short stalk, the pedicel. There are two
short, rudimentary, bristle like outer glumes , and the flower is enclosed in two inner
glumes of boat shaped. Flowering glumes or lemma are provided with fine nerves and
the palea is similar in size and texture to the lemma but is three nerved. Both the
lemma and palea may be awned or awnless. The flower consists of two small, oval,
thick, and fleshing bodies, the lodicules situated at the base of the axis. Unlike other
cereals, paddy has six well developed and functioning stamens provided with short
 25

filaments and a pistil with a plumose stigma. The stigma is some what longer than
broad, smooth and bears two styles and sometimes a short, rudimentary third. The
three some times growing together at the base.

Figure 2.5 Rice panicle (Polato, 2007).

The flower includes all the components and parts enclosed by the lemma and
palea (hulls). Flowering starts the first day the panicle emerges (usually 25-33 days
from internode elongation). Flowering begins at the tip of the panicle and moves
progressively downward to the panicle base. Depending on weather and variety,
flowering normally lasts from 6 to 10 days. Flowering usually occurs from mid-
morning to shortly after noon. Rice is normally self-pollinated, with pollen being shed
just before or at the time flowers open. Flowering is delayed or stopped during
cloudy, rainy, or cool weather. The period from first heading when 10 to 20 percent of
the panicles have emerged from the boot until maturity is usually 30 to 40 days.

2.1.6.5 Seed

Rice is harvested from field in form of paddy (Fig. 2.8). Paddy is a complete
seed of rice, one grain of paddy contains one rice kernel. Each paddy consists of many
layers. Outermost layer is rice shell called hull (husk). The hull comprising of
 26

interlocked half shells, each of them protects one half side of the paddy. The hull
consisted mostly of silica and cellulose. Next layers are all call bran layers. Each layer
is of very thin bran film. Bran is mainly fiber, vitamin B, protein and fat, the most
nutritious part of rice. At the base of each grain is embryo which will grow to actual
new plant. The innermost part is rice kernel, consisting mainly starch. Rice starch
consists of mainly 2 types of starch, amylase and amylopectin. A mixture of these 2
starches determine cooking texture of rice.
The rice grain (Fig. 2.9), commonly called as seed, consists of the true fruit or
brown rice (caryopsis) and the hull, which encloses the brown rice. Brown rice
consists mainly of the embryo and endosperm. The surface contains several thin
layers of differentiated tissues that enclose the embryo and endosperm. The palea,
lemmas, and rachilla constitute the hull of indica rices. The hull of indica rice
comprises palea, lemma, sterile lemmas, and rachilla constitute. The lemma is larger
than the palea, and covers about two-thirds of the surface area of matured brown rice.
The edges of the palea fit inside those of the lemma so that the hull closes tightly (Fig.
2.9). In japonica rices, however, the hull usually includes rudimentary glumes and
perhaps a portion of the pedicel. A single grain weighs about 10-45 mg at 0%
moisture content. Grain length, width, and thickness vary widely among varieties.
Hull weight averages about 20% of total grain weight.

2.1.6.6 The sheath

The sheath (Fig. 2.3, Fig. 2.10) is always present as a whole or part of the
internode from the pulvinus upwards. As the base of the sheath tend to exceed the
length of the internode and consequently enwrap the base of the succeeding sheath to
a variable extent, from the tenth leaf upwards. However, the internodes are longer and
the sheaths are relatively and progressively shorter than the internodal length. Growth
of the sheath is mainly from the base and may continue after the blade has attained its
maximum length. The sheath splits at the base, is finely ribbed, and is more or less
glabrous.
 27

Figure 2.6 Rice spikelet (Reano, 2007).

2.1.6.7 Ligule

The ligule (Fig. 2.10) is a small, white, triangular scale that looks like a
continuation of the sheath. The ligule is present in all most all varieties of paddy and
its membranous, and tends to split as it develops. The ligule may be colour less or
coloured, a faint pink or purple. A coloured ligule is always associated with colour in
the sheath.
 28

2.1.6.8 Auricles

The articles (Fig. 2.10) are situated at the junction of the sheath and blade and
are sickle shapped. Long slender teeth are normally present on the convex face of
each ligule colour if present, is always assoicated with colour in the pulvinus. If the
auricles are coloured, so also is the sheath, but the converse is not true. Some strains
of paddy are devoid of auricles. Rice plants have both auricles and ligules which
make a distinguishing character of rice to differentiate from Echinochloa spp., (a most
common weed in rice fields).

Figure 2.7 Rice flower (Polato, 2007).

2.1.6.9 Collar

At the base of the blade, i.e. around the junction between the blade and sheath,
there is a white band called the collar.
 29

Figure 2.8 Rough rice seeds.

2.1.6.10 Tiller

Tillering usually begins with the emergence of the first tiller when seedlings
have five leaves. This first tiller develops between the main stem and second leaf
from the base of the plant. Subsequently when the 6th leaf emerges the second
tillerdevelops between the main stem and the 3rd leaf from the base. Tillers growing
from the main stem are called primary tillers. These may generate secondary tillers,
which may in turn generate tertiary tillers. These are produced in asynchronous
manner. Although the tillers remain attached to the plant, at later stages they are
independent because they produce their own roots. Varieties and including spacing,
light, nutrient supply, and cultural practices.
 30

Figure 2.9 Cross section of rice seed: showing compositions of rice seed.
(http://www.ikisan.com, 2008, August 12)

Figure 2.10 Ligure, auricle (Polato, 2007).

 31

2.1.7 Rice improvements

2.1.7.1 Green revolution

The story of the Green Revolution in rice actually began in India, moved to
the Philippines and then throughout Southeast Asia (Ganzel, 2007). As early as 1949,
the new United Nations Food and Agriculture Organization (FAO) set up a rice
breeding program in Cuttack, India. Rice has traditionally been the most important
food source across Asia. Both rice and wheat are members of the grass family. So,
when the Mexican dwarf wheat program produced such spectacular results, rice
breeders took notice. The search for semidwarf rice varieties that could produce high
yields under fertilization began at Cuttack. In India, local scientists crossed japonica
rice with taller indica, a tropical local variety. They were able to produce two good
strains known as ADT-27 and Mahsuri that yielded well and were adapted to the
Indian environment. ADT-27, in particular, created the first phase of the Green
Revolution in rice.
In 1949, Dee-geo-woo-gen, a semidwarf indica, was crossed with Tsai-yuan-
chung, a tall, disease-resistant variety. Taichung Native I (TN1) was selected from
this cross and released in 1956. TN1 responds to high levels of nitrogen. It produces 6
t/ha, on the average, with the record yield of 8.1 t/ha. Thus, TN1 is considered the
first high yielding indica variety. It demonstrated that increasing the yield potential of
indica rices can be achieved by improvements within indica rices. Thus, it pointed the
way for tropical rice breeding (Khush, 1987)
U.S. scientists thought even better rice varieties could be produced. The Ford
and Rockefeller Foundations played their attention to the rest of Asia where famine
threatened. In 1960, they established and funded the IRRI, the International Rice
Research Institute, in the Philippines. The IRRI team recognized that rice had the
same problems as wheat with "lodging" or falling over when heavy heads were
supported by tall, spindly stalks. So, they imported seeds for over 10,000 varieties of
rice from around the world. Many of these were dwarf varieties to cross with taller
varieties. They also borrowed the "shuttle breeding" program from the Mexican
program – rice would be grown in one region during the winter months and then in
 32

another region during the summer, effectively cutting in half the time it took to
develop new varieties.
In 1962, IRRI began research in Philippines. At the time, there was fear that
Asia's population growth was outstripping food production and widespread famine
was inevitable. IRRI plant breeder crossed Dee-geo-woo-gen and peta at IRRI. Peta,
originating in Indonesia and popularly grown in the Philippines, is a tall, high-tillering
variety. From the cross IR8 was selected and released in 1966. IR8 matured in 130
days, compared to 160-170 days of the traditional varieties. Traditional varieties were
averaging around one ton of rice per hectare of land (Ganzel, 2007). IR8 is erect
leaved, high tillering, photoperiod insensitive. It responds well to nitrogen and
produces about 6 t/ha in the wet season (in-season cultivation) and 9 t/ha in the dry
(off-season cultivation). IR8 is considered the first high yielding indica rice variety
adapted to troical climates. IR8 gave double the yield of previous rice varieties when
grown in irrigated conditions, had greater resistance to diseases and insects, and was
more responsive to fertilisers. With good management IR8 have enormous potential to
boost rice yields in Asia. Their impact was elegantly described as Dwarf rice-a giant
in tropical Asia (Chandler, 1968). In 1966, IR8 was tested under different fertilizer
conditions. It was found that the IR8 rice produced around 5 t/ha with no fertilizer and
rose to almost 10 tons with 120 kg of nitrogen per hectare. That was 10 times the
traditional rice yield. IR8 was hailed as "Miracle Rice" and helped to spark what is
now known as the Green Revolution.
In the 1960s, high-yielding, lodging-resistant, and fertilizer-responsive rice
varieties helped initiate the world wide expansion in food production called Green
Revolution. Additional rice improvement provided varieties that were resistant to
several major diseases and insect pests and required growth periods of only 3.5-4
months. New production practices were designed to fit these modern rices into a wide
range of environments, providing small-scale farmers in developing countries with
higher yields and potential for multi cropping.
In the mid-60s, there were numerous crossing of IR8 with at least 13 other
varieties from six nations. Eventually, IR36, a semidwarf variety the proved highly
resistant to a variety of pests and diseases and produced the slender rice grain
preferred in many countries was developed. IR36 matured rapidly – in 105 days
 33

instead of the 130 days of IR8 and 170 days for traditional varieties. That meant that
many regions could finally grow two crops a year, instead of one. By the 1980s, at
least 11 million hectares were planted with IR36 around the world. In 1990, IRRI
produced IR72 which out produced even IR36. From its base in the Philippines, IRRI
sent its modern varieties of rice around the world. India, Pakistan, Bangladesh, China,
Brazil, Argentina, Sri Lanka, Taiwan, Malaysia, Korea, Burma, and even the U.S.
used IRRI varieties and agricultural techniques (Ganzel, 2007).
Improved rice varieties undoubtedly helped avert a world rice crisis. But the
source of the problem - unchecked population growth - has continued, and today
experts are again warning of a world crisis in food production. It was estimated that
by 2020 the world population will be around 8 billion people - 5 billion of whom will
be rice consumers. Today, only around 3 billion people consume rice, so world rice
production must increase by 60% in the next 12 years to meet the needs of the 2020
population. However unlike the Green Revolution around 46 years ago, there is
virtually no more tillable-land available to grow rice. Future gains must be made
solely by improving rice yields, and on top of that, there's an imperative to use fewer
harmful chemicals as fertilisers and for pest control. IRRI is currently working on a
new plant type to address some of the shortfalls of the once prized IR8 variety. It has
optimistically been dubbed "Super Rice". IRRI started work on this highly productive
rice 11 or 12 years ago. They sought new parents, or sources of germplasm, to build a
plant with an entirely different architecture. It has fewer but stronger stems or tillers
and there are many more seeds on each rice flower or inflorescence. In the old IR8
plant, half of the plant's weight is grain and half is straw, whereas the new Super Rice
plant is 60% grain and 40% straw. So more energy goes into grain production,
increasing yield potential by about 20%. The Super Rice also has a vigorous root
system, and IRRI scientists are working on improving its resistance to disease and
insects. However, developing a new rice plant to save half the world from starvation
is not all smooth sailing. The scientists at IRRI have encountered many problems in
the development of Super Rice. For instance, most current high-yielding rice varieties
produce around 100 grains per panicle. The prototype Super Rice, on the other hand,
produced 250 to 300 grains per panicle, which was too many (Malcolm, 1998). The
plant simply could not supply enough carbohydrates and nutrients to fill the grains.
 34

The breeders overcame the problem by reducing the number of grains back to 200,
which still makes Super Rice twice as productive as older plant types. Scientists are
also trying to improve the Super Rice grain quality by incorporating genes for disease
and insect resistance.

2.1.7.2 High-yielding varieties

The High Yielding Varieties are a group of crops created intentionally during
the Green Revolution to increase global food production. Rice, like corn and wheat,
was genetically manipulated to increase its yield. Yields from experimental rice fields
in the tropics have increased dramatically with the advent of new high yielding
varieties. When nitrogen is applied at rates exceeding 40 kg/ha, many traditional
indica varieties tiller profusely and grow excessively tall, lodge early, and yield-less
(Chandler, 1963).
Traditional tropical varieties are late maturing, photoperiod sensitive, and high
tillering. They have tall, weak, thick culms; long drooping, thin, pale-green leaves;
and relatively large lax panicle. These varieties are grown under suboptimal water and
weed control at moderately wide spacing.
High yielding rices, such as japonica rices have short, narrow, erect, dark-
green leaves; thin but short and sturdy culms; and short, dense panicles. They respond
to increased application of nitrogen, and produce high yield (Jennings, 1964). After
World War II the improvement of native indica rices was started (Huang et al., 1972).

2.1.7. 3 Plant-type concept of high-yielding rice varieties

The term “plant type” refers to a set of morphological characters associated

with the yielding ability of rice varieties. The plant-type concept in rice, similar to the
wheat ideotype (Donald, 1968), emerged gradually from many observations and
comparisons between low and high yielding varieties in Japan (Baba, 1954, 1961;
Tsunoda, 1964).
Better varieties of crop species must have been selected for higher yields in
the past. In practice, the high yields have been achieved by increased inputs,
 35

particularly nitrogen fertilizers. Hence, varieties must have been selected on the basis
of their response to nitrogen fertilizers. Tsunoda (1964) compared low and high
yielding varieties of rice, sweet potato, and soybean, and summarized the
morphological characteristics:
-Low nitrogen responders have long, broad, thin, drooping, pale-green leaves,
and tall, weak stems.
-High nitrogen responders have erect short, narrow, thick, dark-green leaves,
and short, sturdy stems.
Tsunoda postulated from physiological knowledge of crop photosynthesis that
the thick, dark-green leaves lose less light through reflection. Reduced leaf size are
erect habit permit uniform light distribution to all leaves and reduce respiration.
Consequently, dry matter and yield increase even under low light conditions. The
uniform light penetrations and the short, sturdy culm minimize lodging; thus there is
little or no yield loss once panicles are well developed (Tsunoda, 1964; Jennings,
1964). Experimental data for tropical varieties clearly demonstrate that the certain
morphological characters are indeed associated with yielding ability (Tsunoda et al.,
1964; 1966). Work on plant type has been well summarized and the concept appears
to have been accepted by rice breeders in many countries as a guide for breeding high
yielding varieties (Ishizuka; 1971; Jennings, 1964; Yoshida, 1972).
In this study, morphological characters deserve particular attention of high-
yielding rice varieties (mutants of KDML 105) are, dwarf (short with stiff culm), elect
leaves, high tillering, and photoperiod insensitive.

2.1.7.4 Thai Rice improvement

The International Rice Research Institute (IRRI), has worked in cooperation

with Thailand since the establishment in the years 1960. In 1966 an IRRI new rice
variety named "Miracle Rice, IR-8" was created and distributed to various countries
to grow. Farmers in Asian countries multiplied the seed and adopted IR8 to solve
problems on rice shortages. Thailand did not release IR8 because its grains were
shorter than Thai rice and were chalky. However, Thailand has crossed IR8 with
many Thai rice varieties and resulted in developing of several premium varieties such
 36

as RD1 and many other RD related names. There have been numerous joint-
cooperation projects between Thailand (the Royal Thai government agencies) and
IRRI especially research and training such as the Joint Research and Training
Program for Deepwater Rice. In this agreement Thailand accepted the role as a
teamleader in developing the deepwater rice and was in charge of most of the IRRI
research projects relating to the deepwater rice, to be conducted in Thailand. Thailand
has also conducted many other research and development (R&D) projects, in
cooperation with IRRI, such as the studies of methane release in rice, the draught
resistant selection project, the Rainfed Lowland Rice research project, the Upland
Rice research project, the Rice Biotechnology project, and etc. (Sriwatanapongs, no
date)
The joint projects in the first phases emphasized on the varieties improvement
and training. There were exchanges of rice germplasm to be used in the rice varietal
improvement project. Many Thai recommended varieties and local cultivars such as
Gam Pai 15, Leuang Yai 34, Nahng Man S-4, Leuang Hawm were sent to IRRI to be
used as the parents in IRRI. And many new developed rice varieties were obtained
such as IR64 (GP15/TN1), IR113 (LY34/BP176), IR141 (NMS-4/ MAS), IRI.56
(Leuang Hawm/TN1) and IR253 (GP15*2/TN1) KDML105 variety was in the IR841
cross (IR262-43-8-11/ KDML105). In this period, the important cross was Leuang
Tawng (off season) and IR8 (LT/IR8) which its hybrid progenies were selected. RD 1
and RD3 were the hybrid progenies of LT/ IR8 that produced high yields non-
glutinous rice. They were capable of solving problems on green leafhoppers that
carried the yellow-orange leaf virus disease. The differences of RDI and RD3 are
maturity dates, harvesting, flag leaf exertion and husk color. RD2, IR253-4-1-2-1, was
a progeny from cross between GPI5 (Thai glutinous rice) and a semidwarf plant
variety, Tai Chung Native 1 (TN1). The parentage and the former breeding lines of
these 3 varieties prior to prosing for approval are including: RDI (Leuang
TawngllR8), RD2 (GP 15*2/TN1), and RD3 (Leuang Tawng/IR8). There were many
breeding lines from IRRI used in the rice varietal improvement program in Thailand.
RD4 (Leuang Tawng/IR8//W12521RD2) was glutinous rice with highly resistant to
gall midge. RD7 (C 4-63/Gow Ruang 88//Sigadis) and RD9 (Leuang Yai
34/TN1//W1256///RD2) were semidwarf type rice producing high yields, with
 37

disease and insect resistance quality. They were approved and released in 1975
(Sriwatanapongs and Somrith, no date).
The approved rice varieties during 1977-1981 were short type hybrid rice,
Thai hybrid rice, genetic mutation breeding rice and selected upland rice. In 1979, two
short type varieties that could prolong stems according to water level were approved.
They were RDI7 and RDI9. The approval and introduction of RD rice varieties had
been done continuously and had not been limited to improved varieties by
hybridization only. Recommended varieties during this period were therefore varied,
consisting of approved rice varieties in different years are shown in Table 2.2.
In 1990-1991 there were epidemics of brown plant hoppers and once again in
1997-1998 (Sriwatanapongs and Somrith, no date). Therefore, the rice variety
improvement project in the following years, especially in irrigated rice, would
emphasize on variety breeding that is resistant to brown plant hoppers as a key
objective. Following the approval of Suphan Buri 90 was the approval of Chai Nat 1,
Phrae 1, Suphan Buri 1 and Suphan Buri 2 for that purpose. Due to the increased
quantity of exporting fragrance rice, the government greatly stipulated policies on
premium quality rice to be exported, focusing on the production of KDML105 and
RD15 varieties. The Rice Research Institute proposed the approval and introduction
of two fragrance rice varieties that are photoperiod insensitive to be grown in the
Irrigated area. They are Klang Luang 1 and Hawm Suphan Buri varieties. These 2
varieties were the outcome of the improvement program for exporting premium
quality rice. Furthermore, rainfed rice and deep-water rice varieties had been
proposed for approval and introduction. Approved and recommended rice varieties
during 1993 up to 1998 are shown in Table 2.3.
 38

Table 2.2 Approved rice varieties in 1977-1981.

(Sriwatanapongs and Somrith, no date)

Variety Name Original Line Female/Male Parent

1977
Rice Division 6 KDMLlO5'65G2U-68-
Gamma-ray irradiated KDML1O5
(RD6) 254
Rice Division 11
WP153 IR661-1-140-3/KDML1O5
(RD11)

1978
Rice Division 8
KKN6721-5-7-4 NSPT*2/IR262-24-3-1
(RD8)
Rice Division 13
BKN6402-352 Nahng Phaya 132/Pak Sian 39
(RD13)
Rice Division 15
KDML105’65G,U-45 Gamma-ray irradiated KDML105
(RD15)

1979
Rice Divison 17
BKN6986-66-2 IR262-24-3-1/Pin Gaew 56
(RD17)
Rice Divison 19
BKN6986-147-2 IR262-24-3-1/Pin Gaew 56
(RD19)
Developed from Chiang Rai
Sew Mae Jan (SMJ)
Collection
Goo Meuang Luang Developed from Southern region
(GML) upland rice Collection
Dawk Payawn Developed from Southern region
(DPY) upland rice Collection

1981
Rice Division 10
RD1’69NF,U-G-6-6 Fast neutron ray irradiated RD1
(RD10)
Rice Division 25 BKNLR75091-CNT-
KDML105/IR2061//KDML105/IR26
(RD25) B3-RST-40-2-2
 39

Table 2.3 Approved and recommended rice varieties during 1993 up to 1998
(Sriwatanapongs and Somrith, no date)

Variety Name Original Line Female/Male Parent

1993
Chai Nat 1 1R13146-158-1/1RI5314-43-2-
CNTBR82075-43-2-1
(CNT1) 3-2//BKN6995-16-1-1-2
1994
Phrae 1 KKNLR75052-PRE-40-
lR2061-214-2-14-8/RD4
(PRD1) 1-1-1
IR25393-57-2-3//RD23//IR2731
Suphan Buri 1
SPR85163-5-1-1-2 6-96-3-2-2I//SPRLR77205-3-2-
(SPR1)
1-1/SPRLR79134-51-2-2
Suphan Buri 2
SPR83260-143-1-3 RD23/IR60
(SPR2)
Plai Ngahm
Pure-line selection from
Prachin Buri Traditional var.
Plai Ngahm var.
(Plai Ngahm PCR)
Leb Nok Pattani Pure-line selection from
Traditional var.
(Leb Nok PTN) Leb Nok var
Look Daeng
Tissue culture technique
Pattani Traditional var.
from Look Daeng var.
(Look Daeng PTN)
(Look Daeng SC2-4)
Pure-line selection from
Chiang Phauhalung Traditional var.
Ghiang var.
1997
Klong Luang 1
KLG83055-1-1-1-2-1-4 NMS-4/IR841-85-1-1-2
(KLG 1)
Hawm S.uphan SPR84177-8-2-2-2-1/SPR85091-
SPR89111-17-2-2-2-2
Burl(HSPR) 13-4//KDML105
1998
Niaw Ubon 2 IR43070-UBN-501-2-1-
SPT7149-429-3/lR21848-65-3-2
(NUBN2) 1-1

2.1.8. Thai jasmine rice (Oryza sativa L. cv. KDML105)

Thai jasmine rice (Oryza sativa L. cv. KDML 105, jasmine rice, Thai Hom
Mali rice, Khao Dawk Mali 105, KDML 105) is a long grain rice variety (indica);
known as fragrant, aromatic, or scented rice. It has a naturally distinctive scent
released during the cooking process and is similar to a flowery perfume. Thai jasmine
 40

is a photoperiod-sensitive rice variety with the critical photoperiod of 11 hours 52

minutes. Thai jasmine rice is praised for its whiteness and silkiness. When cooked, it
is soft, white and fluffy. The taste of this rice enhances the traditional spices of
oriental cookery. The official name is “Thai Hom Mali Rice” or “Khao Hom Mali”
rice is recognized world wide as Thailand’s specialty.
Due to its particularly high quality, jasmine rice today enjoys huge popularity
and a worldwide reputation, its consumption increasing with global economic growth
and rising prosperity. Leading buyers of jasmine rice currently include China, Hong
Kong, Singapore, Malaysia, EU and the USA, along with many countries in Asia,
Europe and the Middle. Export markets have also opened up in Africa. Annual export
of Thai jasmine rice from Thailand was 2.25-2.57 M MT during 2003-2006 (Rice-4,
2007). Thai jasmine rice is the most famous in the world rice market, highly favored
by Thai people and consumers in foreign countries.
During the past decade, Thai rice in particular jasmine rice has been a target of
copying and modification in western countries, especially the United State of
America, in order to develop aromatic rice varieties that can complete with jasmine
rice and ultimately the world market. Recently, Dr. Chris Deren of the University of
Florida (USA) announced that he was transforming jasmine rice using a basic gene
mutation method involving gamma rays in order to develop new strains that can be
grown in USA and are suitable to be harvested easily by machines (photoperiod
insensitive and dwarf phenotype) (Hargrove, 2003). This project is carried out in the
framework of the “Stepwise Program for Improvement of Jasmine Rice for US”,
supported by the US government. The original strain of jasmine rice, Khao Dok Mali
(KDML105) was obtained from the International Rice Research Institute (IRRI).
 41

2.2 Ion Beam

2.2.1 Ion Beam Technology

Since the 1950’s, studies on the interaction between energetic ions and
materials have resulted in large-scale applications of low-energy ion beams in
semiconductor industry and material science. Small, low-energy ion accelerators have
also been rapidly developed as the basic technology of the applications. An
accelerator is equipment that uses man-made technology to produce charged particles
and accelerate them to certain energy. It generally consists of three main components:
ion (or electron) source, accelerating tube and beam line, and target chamber. Ions are
produced in the ion source, accelerated, bent and focused in the beam line, and finally
introduced into the target chamber to irradiate experimental samples (Nastasi,1996).

2.2.1.1 The basic structure of a beam line

Although processing of bio-samples call for some special requirements of the

ion beam facility, the basic structure of a bioengineering ion beam facility is little
different from that of an industrial ion implanter. The ion beam line normally consists
of the following basic subsystems (Fig. 2.11): (1) ion source, (2) focusing system, (3)
mass analysis system, (4) accelerating system, (4) measurement system, and (5) target
chamber and vacuum system. The components and their positional sequence may vary
for different beam lines. For example, some beam lines adopt pre-acceleration and
post-analysis. The focusing system may be installed at different positions of the beam
line according to the ion-optical requirements to reduce losses of ion beam. The
working principle of the accelerator is as follows. In the ion source, discharge of gas
or vapor produces plasma. Macroscopically plasma is electrically neutral, i.e. the total
charges of ions and electrons are basically the same. The ions in the plasma are
extracted by a biased electrode and transported into the mass analyzer. The ions of the
same species having the same charge selected by the mass analyzer from ions of
 42

Duoplas-
matron Accelera- Small Main
ting tube target Sample
ion target holder
source (150 kV) chamber chamber

Power Diffu- Turbo Diffu-

supply & sion mole- sion
HV pump cular pump
generator pump

Figure 2.11 The photograph (above) and the corresponding schematic setup (below)
of ion implanter CMU-2.

different masses or charges come to the acceleration section (for the post-acceleration
type). The acceleration system accelerates the ions to the required energy and
bombard them into the sample inside a target chamber. To guarantee normal operation
of the beam line and prevent the ion beam from being scattered and lost during
transportation, every component of the beam line should meet certain requirements.
 43

For monitoring the beam parameter, corresponding measurement systems such as

inspectors of the beam profile and implantation fluence should be installed at
locations concerned on the beam line.

2.2.1.2 Facility – CMU-2

The facility, as shown in Fig. 2.11, is a versatile ion implanter, which has been
developed for high-current ion beam treatment of materials. The ion source is a
duoplasmatron source (Fig. 2.12), which is able to produce high ion-beam currents of
nitrogen ions coming from dual-volume-compressed plasma that is generated by
ionization of the source gas due to filament-emitted electron impacts. The ions are
extracted and further accelerated by a high voltage as maximum of 150 kV. The
accelerating voltage is adjustable so that the ion energy can be varied. At the ion
source a magnet allows focusing or defocusing of ion beam. In order to obtain a large
beam cross section for large area treatment, the beam can be defocused up to about 10
cm in diameter at the target. As the ion implanter does not have a device such as a
magnet for ion mass analysis, the ion beam consists of both atomic and molecular
nitrogen ion species. This offers two advantages. The first, the ion beam current can
be high so that the ion beam treatment time can be shortened, and the second, the ions
have two energies so that a broad implanted ion depth profile can be achieved. The
extracted and accelerated ion beam is directly introduced into target chambers for ion
implantation or bombardment of target materials. There are two target chambers
installed at the beam line for various treatment purposes. The small chamber is used
for material treatment that requires short time as the chamber can be pumped down
fast. The big main chamber is used for normal treatment of samples which may be in
big size or complex shape. Inside the main chamber a special target holder is installed
for complicated treatment operation. The holder is able to rotate horizontally with
360° and translate up and down by many centimeters. It is able to hold samples on its
four sides to carry out ion beam bombardment simultaneously. It has an ability of
water cooling to prevent the target from over heating. For ion beam mutation,
biological samples such as rice seeds are placed in standard petri dishes using sticker
tape for fixing or in specially designed seed holders. On a seed holder, made from
 44

copper with 10 cm in diameter, hundreds of small holes are made and each hole holds
a seed. The seed holders are then installed onto the sides of the target holder for
bombardment. A powerful diffusion pump beneath the main chamber pumps the
chamber for about two hours to reach the required low pressure, typically about 1 – 5
× 10-5 torr. The ion beam bombardment of biological samples is operated in a pulse
mode normally with beam exposure time of five minutes and beam cutoff five
minutes respectively to avoid overheating the samples. The ion fluence, number of
ions per square centimeter, is controlled by the product of the ion beam current and
the bombardment time.

2.2.1.3 Ion bombardment

The ion beam system of CMU-2 comprises of a high-current duoplasmatron

ion source, a broad beam transport system and versatile target chambers. Under a low
pressure, 10-3-10-4 Pa, nitrogen ions (N+ + N2+) is produced in the ion source with a
beam current of about 2 mA, accelerated by an electrostatic field, defocused by
magnetic lens and finally introduced into a target chamber to bombard rice seeds in a
special target holder (Fig. 2.13). The adjusted beam with a spot size of 10 cm in
diameter was able to bombard 800 rice seeds altogether.

2.2.1.4 Equipment requirements for ion implantation of biological samples

Up to now, in applications of low-energy ion beam in genetic modification,

successful examples have included ion beam induced mutation for breeding and ion
beam induced gene transfer. In the former case, the ions are required to penetrate into
and slow down in the genetic substance and finally stop in the substance, so that they
can cause displacements of the molecules of the genetic substance and subsequent
reorganization. In the latter case, only ion sputtering and absorption are considered
but not the ion penetration range. In other words, ion sputtering or etching reduces the
surface thickness of the biological object so that existing channels can be linked to
form pathways for gene transfer. Actually, ion implantation and etching occur
 45

simultaneously. An increase in the permeability of the biological material surface

enhances deeper implantation for late-coming ions to pass through the channels.
Therefore, the ion energy in ion beam biotechnology is not required to be very high
(Yu, 1999).

Figure 2.12 Schematic of duoplasmatron ion source.

For ion beam mutation breeding of rice for example, many seeds have to be
treated in the same chamber. This is not a problem for high-penetration mutation
sources such as γ-rays. However, in ion bombardment, due to the limited ion range,
the ions must be exactly aimed at the embryo part of each seed. If a beam is 30 cm in
diameter, about 3,000 seeds of rice or wheat at maximum can be put in this
bombarding zone for a very delicately designed sample holder. If the beam size is
small, mechanical movement is needed for large-area homogeneous bombardment,
but this can cause perturbation of the sample alignment and orientation. Electric
scanning of the beam is also not practical for a large implantation area. Therefore, the
ion beam used for mutation breeding purposes should be as large as possible as well
as highly homogeneous (Yu,1999).
For studies on ion-beam biological effects, ions are required to be implanted
into cells accurately and quantitatively. Mass spectra of fast ion analysis and
absorption or photon emission of the ion impact may be applied for measurement of
the cell fine structure. Particular ion beams may be used for cell surgery or grafting.
All of these require the ion beam to have a high spatial resolution. Therefore, ion
 46

beams applied in life science should be developed in the two directions of high-
current broad beams and micro beams (Yu,1999).
In order to avoid loss or scattering of ions due to collisions with gas
molecules, the production and acceleration of ions should be operated in vacuum.
Biological samples, except dry crop seeds, contain a large amount of water. This
introduces a serious problem. Evaporation of a large amount of water causes the
vacuum to degrade as well as rendering the samples unviable due to rapid cooling or
over-loss of water. In order to avoid damage of the bio-samples by freezing, the
design of the vacuum system should be considered carefully and the samples should
be pre-treated. Additionally, pollution from organic substances in the vacuum system
and the cathode (if a hot-cathode ion source is used) is inevitable and should be taken
into account in the design of the ion source (Yu,1999). A solution to the conflict
between the beam line vacuum and the survival of the bio-samples can be resolved by
transport of the ion beam out of the high vacuum region. In this case the beam should
have an energy of at least 2 MeV (Yu,1999). The bio-samples are directly exposed to
the ion beam during ion bombardment. Thus the sample chamber should also
guarantee sterile conditions for the plant cells and microbial samples while they are
transported into and out of the chamber (Yu,1993).
 47

e-

N+ + N2+

N2

Figure 2.13 Ion beam technology in 150 kV non-mass analyzed CMU-2 ion
implanter. The implanter comprises of ion source, transport system and target
chamber. Under the pressure of 10-5-10-6 torr, the mixed ions of atomic- (N+) and
molecular-nitrogen (N2+) were produced in ion source, adjusted to be 10 cm in beam
spot size by magnetic lens, accelerated to have the designed energy in accelerating
tube, and transported to bombard bio-sample in target chamber.

2.3 Application of ion beam in Biological research works

In the mid-1980’s, enlightened by ion implantation modification of material

surface, physicists started to apply the low-energy ion implantation technique for
breed improvement of agricultural crops and subsequently found biological effects of
ion implantation in rice (Yu, et al., 1989; Yu,1999). Since then, ion implantation as a
new genetic modification method has increasingly been applied to breeding of crops
and microbes. In 1988, the etching effect of ion beam on the cell surface was
discovered, then the idea of ion beam processing of the cells for gene transfer was
proposed, and thereafter gene transfer in rice was achieved using the ion beam
technique (Yu,1993). In past ten years, work on research and applications in this field
has been rapidly developed and particularly active in China, Japan and USA (IBBE-
Proc., 2002). In China, the benefit brought by applying this technology has been
counted up to an order of one billion US dollars. The key laboratory of ion beam
 48

bioengineering in China has received the support of a hundred million US dollars per
year from both the government and private enterprise (Yu, 2002).
Scientists in Thailand have actively joined this international tide for several
years. The research group at Chiang Mai University, consisting of physicists,
biologists and agriculturists from various departments and faculties and collaborating
with scientists from USA, China and Australia, has made its special contributions.
They have built an ion beam bioengineering (IBBE) laboratory, equipped with a
specialized bioengineering ion beam line, an atomic force microscope (AFM),
biological experimental equipment, and a bio-clean housing compartment (TRF,
2001). All of these have significantly facilitated their research. They have
successfully carried out experiments on ion-beam induced DNA and exogenous
macromolecule transfer into both bacterial and plant cells as well as on mutation of
local rice. From these investigations, They have achieved a number of international
publications (Vilaithong et al., 2000; Anuntalabhochai et al., 2001; Yu et al., 2002;
Phanchaisri et al., 2002; Vilaithong et al., 2002; Yu et al., 2003; Appavatjrut et al.,
2003; Vilaithong, et al., 2004).

2.3.1 Ion Beam Induced Gene Transfer

Plant genetic transfer, often simply called gene transfer, is an important

component of genetic engineering. Genetic engineering involves processes in which,
according to needs, exogenous genes from separation and cloning are assembled into
a specific kind of carrier to be transferred into a receptor cell using physical, chemical
or biological means to realize expression (at the levels of cell, organs, and whole
system) and inheritance in the new genetic background. It provides not only genetic
expression, adjustment and control, and inheritance studies with an ideal experimental
system, but also more importantly future oriented plant improvement and molecular
breeding are provided with an effective route.
In plant gene transfer, researchers attempt to discover simpler and more
effective means for realizing gene transfer of walled cells so as to bypass the
complicated process of plasmogen culturing. Some physical methods have been
developed, and considerable progress has been accomplished. Relatively successful
 49

methods include electroporation, particle gun or microinjectile bombardment,

microinjection and macroinjection, microlaser, and ultrasonication. Ion-beam-induced
gene transfer is the most recently developed physical method. GUS gene transfer into
rice cell suspension (Yu, 1993) and tobacco pollen (Hase, 1998), and plasmid DNA
transfer into bacteria E. coli (Anuntalabhochai et al., 2001) have been achieved. All of
these methods involve perforation of the cell wall to provide exogenous genes with
passages to enter the receptor cell (Yu, 1999).
Possible mechanisms for ion-beam-induced gene transfer into cells that have
been proposed include enhanced permeability of the ion-beam-etching induced
thinning of the cell wall, and physical channels in the cell envelope created by ion
beam bombardment. It is now an open question as to which mechanism is dominant
or whether both are. A fuller understanding of the detailed mechanisms involved in
ion-beam-induced gene transfer will greatly facilitate control of the process and will
expedite applications of the technique.

2.3.2 Ion-Beam-Induced Exogenous Macromolecule and DNA Transfer

into Plant and Bacterial Cells at Chiang Mai University

2.3.2.1 Ion-beam-induced exogenous macromolecules transfer into

plant cells
Although the technology of ion-beam-induced gene transfer into either
plant or bacterial cells has been successfully established, the mechanisms involved are
not yet understood. The aim of the work was to study the process of induction and
thus to develop applications of ion beam bioengineering. Cells of various kinds of
plant tissue were bombarded in vacuum with argon and nitrogen ion beams at
energies of 15 – 30 keV with fluence ranging from 5 × 1014 to 3 × 1016 ions/cm2. The
ion bombardment effects on tissue viability, and Trypan blue and neutral red dye
molecule transfer into the cells through the cell envelope, were investigated. The
results showed that the characteristics of tissue survival subsequent to ion
bombardment and penetration of dye molecules into the cells through the cell
envelope depended on ion species, energy and fluence. For 30-keV argon-ion
bombardment at a fluence of 2 × 1015 ions/cm2, the dye molecules entered the cells
 50

without fatal injury, whereas under other conditions, the dye either did not enter the
cells or stained the nuclei. Furthermore, ion-bombardment-induced crater-like
structures were observed on the cell envelope surface. Calculations indicated that
exogenous molecule transfer into living plant cells can be achieved by ion beams
having the appropriate physical parameters such that the ion range and the radiation
damage range lie within the solid cell wall thickness (Vilaithong et al., 2000;
Apavatjrut et al., 2003).

2.3.2.2 Ion-beam-induced plasmid DNA transfer into bacteria E.

coli
The research group at Chiang Mai University was the first research group in
the world to report, in one of the most influential international journals, the
observations of the interaction of energetic ions with bacterial cells, inducing direct
DNA transfer into E. coli (Anuntalabhochai et al., 2001). In the experiment, Ar- and
N-ion beams were used to bombard the bacteria E. coli strain DH5α in a vacuum with
energy of 26 keV and fluence in the range 0.5 – 4 × 1015 ions/cm2. Three DNA
plasmids, pGEM2, pGEM-T easy and pGFP, carrying different marker genes, were
subsequently transferred (separately) into the appropriately ion-bombarded bacteria
and successfully expressed. The results of this study indicate that ion beams with an
energy such that the ion range is approximately equal to the cell envelope thickness,
at a certain range of fluence, are able to generate pathways for macromolecule transfer
through the envelope without irreversible damage. The dependence of the DNA
transfer on the plasmid DNA size, ion fluence and incubation time was studied. The
results showed that the smaller the DNA, the closer to 2 × 1015 ions/cm2 the fluence,
and the longer the incubation time, the easier the DNA transfer (Phanchaisri et al.,
2002).

2.3.2.3 Ion penetration depth in the plant cell wall

The investigation of the depth of ion penetration in plant cell wall material has
been carried out. Based on the biological structure of the plant cell wall, a physical
model was proposed, which assumed that the wall was composed of randomly
 51

orientated layers of cylindrical microfibrils made from cellulose molecules of

C6H12O6. Based on this model, the research group derived multiplication factors of
2.43 for ion range and 7 for sputtering yield that then allowed calculation of both of
these parameters from the conventional ion implantation programs. Using these
correction factors, it is possible to apply common ion implantation programs to
estimate the ion penetration depth in the cell for bioengineering purposes. These
estimates have been compared with experimentally measured data and good
agreement is achieved (Yu et al., 2003).

2.3.2.4 Observation of micro-hole or crater formation on the ion-

bombarded cell surface

In the course of elucidating the mechanisms involved in ion-bombardment-

induced direct gene transfer into plant and bacterial cells, observations of micro-hole
or crater formation on the ion-bombarded cell surface were reported (Apavatjrut et al.,
2003; Yu et al., 2002; Phanchaisri et al., 2002). Various ion species such as N, O, Ar,
Mg, Fe, and Xe ions were used to bombard onion skin and E. coli strain DH5α cells
at energies around 20 – 30 keV to fluences in the range 0.5 – 4 × 1015 ions/cm2. On
the surfaces of the bacterial cells bombarded with Ar ions at 26 keV and the plant
cells bombarded with heavier ion species such as Ar, Fe and Xe, micro-craters or
holes were observed with scanning electron microscopy (SEM). The research group at
Chiang Mai University have proposed that these microstructures induced by ion
bombardment play the role in acting as the pathways for exogenous DNA
macromolecules to pass through the cell wall for transfer to the cell interior.

2.3.3 Ion Bombardment Induced Genetic Mutation

2.3.3.1 General

Genetics is the study of biological inheritance and mutation. From a cell

genetics viewpoint, chromosomes are the major genetic base. A chromosome
functions based on its basic unit – the gene. Thus, in the final analysis, genetic
 52

mutation stems from changes in the chromosome and gene, and this is mutation.
Ion-bombardment-induced chromosomal aberration has not only complicated
types and generation transmission but also its own characteristics.
There are several types of chromosomal structure aberrations. These include
deletion, repetition, reciprocal translocation, and exchange. N-ion bombardment can
induce all four types of chromosomal structure aberrations.
Ion-bombardment-induced chromosomal quantity aberration has a low
frequency; nevertheless, aberrations have been found in ion-bombarded wheat
Premebi, black wheat AR1 and quadruple black wheat. The chromosome delayed and
unequal separation, triple separation, and single-body early separation, result in an
increase or a decrease in the number of the chromosome in the child-cell. Ion
bombardment induced chromosomal quantity aberrations are significant for selection
and breeding of non-whole-number bodies of crops.
There is a complete maintenance system in the biological body to repair
injuries caused by external factors. If damage to the genetic substance (DNA) is
repaired, mutations cannot occur. High plants have a higher ability to repair
ionizating-radiation-induced damage. It has been reported that γ-ray-induced breaks
of single chains of the carrot DNA can be repaired by 50% within 5 minutes. Similar
reports have also been made for young wheat embryos. In the process of genetic
mutation, only those unrepaired or mistakenly repaired can produce molecular-level
mutations in the DNA copying. Since mutation occurrence has the feature of a single
cell event, the mutated cells compete with normal cells in the fission and reproduction
process and some are eliminated. Even those uneliminated cells are only divided into
body cells in the individual growth and development process. The body cells die and
disappear as the individuals age. Only when these mutated cells transmit the
mutations to the male-female pairs or one of them and include the mutations into the
joined cells through the insemination process to form young embryos, can the
mutations be maintained and transmitted, allowing mutated individuals to be formed.
Practice has demonstrated that aberrations of the chromosome structure, behavior and
quantity are transmitted from M1 generation to M2 generation according to the above-
described process. Ion-bombardment-produced initial damage is different from that
brought about by conventional ionizating radiation. The latter causes breaking of
 53

single and double strands, whereas the former results in a large number of atoms of
the genetic substance being displaced and rearranged.

2.3.3.2 Mutation breeding

The primary purpose of mutation breeding is to increase genetic variability

available to plant breeders. It is generally assumed that mutation breeding is most
appropriate when a specific trait of interest is not found in the germplasm that can be
used for hybridization.
Since the discovery of x-ray-induced genomic mutation in 1927 (Muller et
al.,1927), many types of mutagenic sources, for example, gamma-ray, UV irradiation,
chemical agents, neutron, and ion beam bombardment, have been developed and
applied in crop mutation. High-energy ion beam irradiation at MeV energy levels was
applied as a mutagenic source in Japan and succeeded in induction of mutation in
many plant species such as Arabidopsis (Hase et al., 2000), rice (Maekawa et al.,
2003), carnation (Okamura et al., 2003), and rose (Yamaguchi et al., 2003). A number
of mutants induced by ion bombardment in many kinds of plants were reported (Mei
et al., 1994). In China, low-energy ion beam bombardment at energy levels lower
than 100 keV , as a new mutagenic source and DNA transferring, has been intensively
carried out since 1986. During 1986-1987, 11 new lines of rice mutants with higher
yield rate, broader disease resistance, and shorter growing period but higher quality
were bred in China (Yu et al., 1991). By using N+ ion implantation into rice, Yu et al.,
(1991) discovered genetic effect on rice in M2 generation, leading to extent research
work in the low-energy ion beam mutation. A higher mutation rate and wider
mutational spectrum with less damage in low-energy ion implanted bio-materials
were reported (Yu et al.,1991).
In a reported experiment (Yu, 1999), 30-keV N, H and Ar ions were used to
bombard cotton seeds. H-ion bombardment had an exciting effect on the root tip cell
division (fluence level ~5 × 1016 ions/cm2). Ar-ion bombardment had similar exciting
effect on the cell division at high fluence levels. However, N-ion bombardment
generally showed a restraining effect on the cell division. The abnormal nuclei
induced by all three kinds of ion bombardment were more than the control. At low
 54

fluence levels, H- and Ar- ion-bombardment-produced cells with abnormal nuclei

were more than that obtained by N-ion bombardment. At high fluence levels (4 – 5 ×
1016 ions/cm2), H-ion bombardment induced the most abnormal nuclei. Generally, N-
ion bombardment induced the least abnormal nuclei.
For the ion-bombardment induced chromosomal mutation frequency, H ions
induced the highest effect and the ratio of the bridge in the mutation types was higher
than for N and Ar ions. The N- and Ar-ion bombardment induced delayed
chromosomes were more than with H-ion bombardment. The chromosomal mutation
frequencies induced by the three ion species as a function of ion fluence show similar
behaviors. Roughly speaking, at both low and high fluences, the frequencies are low,
whereas at medium fluences such as about 5 × 1016 ions/cm2 the frequencies are the
highest. In a certain fluence range, the order of the biological effectiveness is H > N >
Ar, which is inversely proportional to the heavy-to-light order of these ion masses.
The reason may be that the non-inert ions can produce double damage in both
structure and energy of the chromosome (Yu,1999).