RiceOryza sativa L.

2n=2x=24

Progenitor: Oryza rufipogonO. nivara

Top 20 Rice Producers by Country—2011 (million metric ton) People's Republic of China 202.6

India 155.7

Indonesia 65.7

Bangladesh 50.6

Vietnam 42.3

Thailand 34.5

Burma 32.8

Philippines 16.6

Brazil 13.4

Cambodia 8.7

Japan 8.4

United States 8.3

South Korea 6.3

Pakistan 6.1

Egypt 5.6

Madagascar 5.0

Nigeria 4.5

Nepal 4.4

Sri Lanka 3.8

Iran 3.2

Source: Food and Agriculture Organization

Total world production in (million metric tons) : 722.8

Four major ecosystems where rice is grown in India

• Irrigated

• Rainfed lowland

• Rainfed upland

• Flood prone

Origin• Area of greatest diversity

– Assam-meghalaya area in India to mountain ranges in mainland of Southeast Asia and Southwest China

• O. sativa domesticated from O. rufipogon and O. nivara in Asia and•O. glabirrima from O. barthii in Africa

Classification• Family: Graminaea• Tribe: Oryzeae• Genus: Oryza

Types:•Indica type•Japonica type•Javanica type

Indica• Tall stature• Heavy tillering with many

unproductive tillers• Adapted to tropics• Drought tolerant• Disease and insect resistance• Lodging resistant• Low grain to straw ratio• Seed dormancy• Lacks cold tolerance

Japonica• Short to intermediate stature• Medium tillering but tillers remain

productive • Adapted to temperate zones• Susceptible• Susceptible• High ratio• No seed dormancy• Cold tolerant

Indica and Japonica biotypes of rice

Rice genetics

• The haploid rice genome consists of 3.9 x 108bp with 28,236 genes

• Recessive dwarfing genes have been identified (d1, d2, etc.), but not useful as they lead to reduction in the kernel size.

• Semi-dwarf gene ‘sd1’ found in semi-dwarf cultivar “dee-geo-woo-gen” from Taiwan and

• Played a major role in bringing about the green-revolution in rice as sd1 not only confers short stautre but also leads to high yield with the increase in tillering.

Male sterility systems in rice

• Cytoplasmic genetic male sterility

• Environment-sensitive genic male sterility

• Chemically induced male sterility

Cytoplasmic genetic male sterility

• Male sterility is controlled by the interaction of a genetic factor S present in the cytoplasm and nuclear gene

• Factor S located in the mitochondrial DNA.

• A line is male sterile when the male sterility- controlling factor S in the cytoplasm and recessive alleles (rf ) of fertility-restoring genes are present in the nucleus.

• The maintainer line (B line) is iso-cytoplasmic to the CMS line since it is similar to it for nuclear genes but differs in cytoplasmic factor (N), which makes it self-fertile

B line A line

rf rf

rfrf rfrf

B line (fertile) A line (Male sterile)

N S

N S

Environment-sensitive genic male sterility

• This male sterility system is controlled by nuclear gene expression

• Influenced by environmental factors such as temperature, daylength, or both.

• First observed in pepper by Martin and Crawford in 1951 and subsequently in different crops.

• Exploited commercially only in rice because of the pioneering work of Chinese scientists

Advantages and disadvantages of the EGMS system

• There is no need for a maintainer line for seed multiplication, thus making seed production simpler and more cost-effective.

• Negative effects of sterility-inducing cytoplasm are not encountered.• The EGMS trait is governed by major genes, thus enabling their easy

transfer to any genetic background.• Since there is no need for restorer genes in the male parents of two-line

hybrids, this system is ideal for developing indica/ japonica hybrids because most japonica lines do not possess restorer genes.

• Since the sterility trait is conditioned by environmental factors, any sudden change such as temperature fluctuation because of a thunderstorm, typhoon, etc., will influence the sterility of EGMS lines.

• The multiplication of EGMS lines and hybrid seed production are restricted by space and season. This means that an EGMS line is used in a given region and season.

Classification of the EGMS system

1. TGMS: Temperature-sensitive genic male sterility2. rTGMS: Reverse temperature-sensitive genic male

sterility3. PGMS: Photoperiod-sensitive genic male sterility4. rPGMS: Reverse photoperiod-sensitive genic male

sterility5. PTGMS: Photo thermo sensitive genic male sterility

TGMS

• Sensitive to the temperature for the expression of male sterility or fertility.

• For example, – most TGMS lines remain male sterile at high

temperature (day temperature >30 ºC/night >24 ºC) and

– they revert back to partial fertility at a lower temperature (day <24 ºC/>16 ºC night),

– for example, 5460S, IR68945, H89-1, and SA2.

Reverse TGMS (rTGMS)

• Sensitive to low temperature (<24 ºC day/>16 ºC night) for the expression of male sterility

• Whereas, at a higher temperature (>30 ºC day/24 ºC night), they become male fertile

• Reverse of the TGMS system• For example,

– JP 38, Dianxin 1A, and IVA

PGMS

• Sensitive to the duration of daylength for the expression of sterility or fertility.

• For example,– most PGMS lines remain male sterile under long-day (>13.75

h) conditions and – revert back to fertility under short-day (<13 h) conditions, f– or example, N9044S and N5088S.

• PGMS lines that express sterility under short daylength and fertility under long daylength are known as reverse PGMS (rPGMS).

• This category is yet to be found.

PTGMS

• Sensitive to both photoperiod and temperature.• Temperature is the key factor

– PTGMS lines become completely male sterile or fertile beyond a particular temperature range

– That is, >30 ºC or <24 ºC, without any influence of photoperiod. – Within this temperature range (24–32 ºC), photoperiod influences

the PTGMS lines– Longer photoperiod hours will enhance male sterility at lower

temperatures vis-à-vis a shorter photoperiod – i.e., 14 h at 30 ºC will make the PTGMS line sterile in comparison

with 13 h at 30 ºC– For example, Nongken 58S, Xinguang S, and Miai 64S.

Chemically induced male sterility• Early 1970s, attempts have been made to identify and use potential chemical hybridizing

agents (CHAs) for hybrid rice seed production. • Chemicals used

– ethylene- releasing compounds, – highly carcinogenic arsenic compounds, and – growth hormones

• China is probably the only country where gametocides are used in commerical hybrid seed production.

• Rice hybrids developed by using CHAs have been tested along with 3-line bred hybrids and were reported to give consistently comparable and often higher yields.

• Over the years, seed yields have increased from 0.4 t ha¯¹ with 40–60% seed purity to 1.5 t ha¯¹ with 80–90% seed purity. CHAs must be able to selectively induce total male sterility.

• The effectiveness of CHAs is highly stagespecific – i.e., these should be applied at the stamen and pistil primordia formation stage or stage IV)– genotype-specific (i.e., the gametocidal effect varies from variety to variety).

• In India, oxanilates, when sprayed at stage IV (meiotic stage) of rice development, were found to be effective and variety Pusa 150 was sterilized more effectively by the gametocidal spray than other varieties, thus indicating genotype specificity (Ali 1993).

Properties of an ideal CHA

1. Wide-spectrum action to induce sterility in successively emerging panicles.

2. Selective and total sterilization of stamens without affecting ovular fertility.

3. Be less phytotoxic, non-carcinogenic, and without residual toxicity that could harm human beings and animals.

4. Be easy to apply and economical.

Disadvantages of CHAs

1. Production of impure hybrid seeds if the CHA is not effective because of unfavorable weather conditions or non-synchronized tillering and growth.

2. Health hazards of some CHAs (such as zinc methyl arsenate or sodium methyl arsenate).

3. High cost of the chemicals.

Rice ideotype

• The concept of plant type was first introduced by Matsushima in 1957, and later by Jennings (1964). In 1969, Chandler proposed the ideotype of ‘dwarf rice’ as follows:1. Shorter culm length (100cm or less).2. Greater culm diameter, which increases culm strength.3. Lower relative internode elongation under heavy nitrogen

application.4. Shorter erect leaves of medium width.5. High tillering capacity; this does not reduce yield potential in

rice.6. More panicles/m2.7. High (55% or more) harvest index.

IRRI NPT• Simulation models predicted that a 25% increase in yield potential was

possible by modification of the following traits of the current plant type (Peng et.al., 2008):

1. Enhanced leaf growth combined with reduced tillering during early vegetative growth.

2. Reducef leaf growth and greater foliar N concentration during late vegetative and reproductive growth, a steeper slope of the vertical N concentration gradient in the leaf canopy with a greater proportion of total leaf N in the upper levels.

3. Increased carbohydrate storage capacity in stems.4. A greater reproductive sink capacity and an extended grain-filling period.5. Low tillering capacity (3-4 tillers when direct seeded) few unproductive tillers; 200-

250 grains per panicle.6. A plant height of 90-100 cm thick and sturdy stems, leaves that are thick, dark green

and erect.7. A vigorous root system.8. 100-130 days growth duration.9. Increased harvest index.

Breeding objectives

• High yield and stability• Photoinsensitivity • Diversification of maturity groups according to

the need of cropping sequence and systems• Resistance to diseases

– Blast, Helminthosporium, Stem rot, Sheath blight, Bacterial leaf bilght, Rice tungro virus, Grassy stunt virus

Breeding objectives ----cntd

• Insect resistance– Stem borer, Gall midge, Brown plant hopper, Rice gundhi

bug, Rice mealy bug• Improved aroma and quality (Basmati type for

export)• Resistance to stresses

i. Droughtii. Cold (high altitude)iii. Alkalinity-salinityiv. Water logging

Breeding methods• Introduction based on germplasm collection• Hybridization followed by pedigree method• Backcross breeding• Development of F1 hybrid cultivars• Mutation Breeding• Marker assisted breeding• Biotechnology and Genetic engineering

Introduction based on germplasm collection

• Seeds of improved strains collected from one ecological area are transported and tested in another ecological area.

• Used either directly as variety or introduced into the crosses.

• Played an important role in distributing rice germplasm from its centre of diversity (Asia)

• In early years, selection utilized to isolate pure lines from mixed landraces or natural populations.

Hybridization followed by pedigree method

• Principal method for developing improved cultivars.• Pedigree method of selection, or its modifications, utilized, as rice

plants can be space planted far enough to permit observation of individual plants.

• Jennings et.al. (1979) suggested multiple crosses involving both tall and dwarf parents and production of large number of F1 plants from these multiple crosses and advancing only short plants from F1 to F2.

• Singh et.al. (1980,1981) identified Bala, Saket 3, Krishna, FH109 and C8585 as the desirable lines based on combining ability analysis for number of tillers per plant, number of ear bearing tillers, panicle length, number of grains per panicle, 500 grain weight and grain yield per plant, and Bala, Cauvery, Krishna, FH 109 and Sona based on combining ability analysis for days to flowering, plant height, days to maturity, flag leaf length, flag leaf width and grain yield per plant.

Pedigree selection• In this, selection among individual plants and their progenies during

inbreeding following crosses among selected donors. • While making crosses due consideration is to be given to per se

performance and the general combining ability effects of the parental lines for economic traits

Backcross breeding• Utilised in rice to :

• Transfer genes for specific characteristics, such as – Disease– Insect resistance or– Dwarfing genes, into otherwise desirable varieties

• Not extensively exploited in rice

Hybrid rice

• Heterosis is exploited.• The crosses showing heterosis in descending order are

indica × japonica > indica × javanica > japonica × javanica > indica × indica > japonica × japonica > javanica × javanica.

• Heterosis can be positive or negative. • Positive heterosis for yield and negative heterosis for growth

duration.• Farmers tend to use a lower seed rate for hybrids than for

conventional varieties because of their better seed quality and higher seed cost.

• However, it is necessary to purchase fresh seeds every season to raise a commercial crop.

Types of heterosis

Heterosis is expressed in three ways1.Mid-parent = F1 – mid-parent × 100

heterosis (%) Mid-parent 2.Heterobeltiosis (%) = F1 – better parent × 100

Better parent 3.Standard = F1 – check variety × 100

heterosis (%) Check variety

Methods of developing hybrids

Three-line method• Based on cytoplasmic genic male sterility and• The fertility restoration system • Involves three lines—

– the CMS line (A), – maintainer line (B), and – restorer line (R)—for the commercial production of rice hybrids.

• The seed of the male sterile line is multiplied by crossing A and B lines in an isolation plot.

• Hybrid seed is produced by crossing the A line with an R line in isolation in another plot.

• Seed production techniques are now developed to produce– up to3 t ha–1 (mean 1.2 t ha–1) of hybrid seed in the tropics and – Up to 6 t ha–1 (mean 2.7 t ha–1) in subtropical and temperate regions of China.

• Some of the CMS lines produced are IR46826A,IR46827A,IR 54785A,Madhu A,HR 7017A etc.

B line A line

rf rf

rfrf rfrf

B line (fertile) A line (sterile)

Rf

R line

RfRfRfrf

Hybrid (fertile) R line (fertile)

Three line scheme

N S N

N S S S

Two-line method• The two lines are involved in a cross for hybrid rice seed production. • One is a male sterile line in which male sterility controlled by recessive

genes, – the expression of which is influenced by environment (temperature,

photoperiod, or both) and• The other is any inbred variety with a dominant gene for that locus• Another two-line approach for hybrid rice seed production is by spraying

chemical hybridizing agents (CHAs)—– ethrel, ethyl 4 fluoro oxanilate, or sodium methyl arsenate′

• Selectively sterilize the male reproductive organs of any one parent and planting the other line (not sprayed) close to the pollinator rows.

• China is the only country that used CHAs such as sodium methyl arsenate and zinc methyl arsenate on a commercial scale.

• Two-line hybrids produced in China are : Pei ai 64s x Shanquing 11, Shulianyou1 etc.

Male sterility systems and their utilization in hybrid seed production

One-line method

• The one-line method involves the use of apomixis to develop F1 hybrids.

• This represents true breeding so that farmers can use the harvest from the hybrids as seed for the next crop as with any inbred rice variety.

• Attempts to discover apomixes have not succeeded so far

• However, research is still under way at IRRI, in China, and in some other countries – using genetic engineering techniques.

Mutation breeding• It has considerable significance in rice improvement • LD50 for various characteristics has been shown to vairy from 10 to 50 KR for gamma

rays and 500-800 R for fast neutrons• The doses recommended by FAO on the use of induced mutations for rice

improvement are

* Seeds pre soaked in water for 20 hrs treated in the mutagen solution for 8 hours at 28±2°C.• IIT-48, IIT-60 and Jagannath are few mutant varieties of economic importance in

India.

Mutagen* Indica Japonica

EMS 0.5-1.25% 0.5-1.00%

NMH 0.01-0.02% 0.01%

NEH 0.01-0.02% 0.01%

EI 0.02-0.04% 0.03%

EO 0.30% 0.2%

Marker assisted breeding in Rice• DNA (or molecular) markers has irreversibly changed the disciplines

of plant genetics and plant breeding.• While there are several applications of DNA markers in breeding,

the most promising for cultivar development is called marker assisted selection (MAS).

• MAS refers to the use of DNA markers that are tightly-linked to target loci as a substitute for or to assist phenotypic screening.

• By determining the allele of a DNA marker, plants that possess particular genes or quantitative trait loci (QTLs) may be identified based on their genotype rather than their phenotype.

• The major QTL’s which have been exploited in rice are sub1A (for submergence tolerance), Xa21 (for bacterial blight resistance), Saltol (for salinity tolerance) and waxy gene.

Advantages of marker-assistedselection

• Greatly increase the efficiency and effectiveness for breeding compared to conventional breeding.

• The fundamental advantages of MAS compared to conventional phenotypic selection are:

– Simpler compared to phenotypic screening– Selection may be carried out at seedling stage – Single plants may be selected with high reliability.

• Leading to 1) Greater efficiency or 2) Accelerated line development in breeding programs.

• DNA markers may be more cost effective than the screening for the target trait. • Another benefit from using MAS is that the total number of lines that need to

be tested may be reduced. – Since many lines can be discarded after MAS at an early generation, this permits a more

effective breeding design.

• Background markers may also be used to accelerate the recovery of recurrent parents during marker-assisted backcrossing

Importance of QTL mapping for MAS

• The identification of genes and quantitative trait loci (QTLs) and DNA markers that are linked to them is accomplished via QTL mapping experiments.

• QTL mapping represents the foundation of the development of markers for MAS.

• Previously, it was generally assumed that markers could be directly used in MAS.

• Factors influencing the accuracy of QTL mapping– population size and type,

– level of replication of phenotypic data,

– environmental effects and

– genotyping errors.

MAS schemes in plant breeding

Marker assisted backcrossing

• Three levels of selection in which markers may be applied in backcross breeding.

• In the first level– Markers may be used to screen for the target trait, which may be useful for traits that have

laborious phenotypic screening procedures or recessive alleles.

• The second level – Involves selecting backcross progeny with the target gene and tightly-linked flanking markers in

order to minimize linkage drag. – We refer to this as ‘recombinant selection’.

• The third level – Involves selecting backcross progeny – That have already been selected for the target trait with ‘background’ markers. – In other words, markers can be used to select against the donor genome, accelerating the

recovery of the recurrent parent genome.

• With conventional backcrossing, it takes a minimum of five to six generations to recover the recurrent parent. Data from simulation studies suggests that at least two but possibly three or even four backcross generations can be saved by using markers.

1 2 3 4

Target locus

1 2 3 4

RECOMBINANT SELECTION

1 2 3 4

BACKGROUND SELECTION

TARGET LOCUS SELECTION

FOREGROUND SELECTION BACKGROUND SELECTION

P1 x F1

P1 x P2

CONVENTIONAL BACKCROSSING

BC1

VISUAL SELECTION OF BC1 PLANTS THAT MOST CLOSELY RESEMBLE RECURRENT PARENT

BC2

MARKER-ASSISTED BACKCROSSING

P1 x F1

P1 x P2

BC1

USE ‘BACKGROUND’ MARKERS TO SELECT PLANTS THAT HAVE MOST RP MARKERS AND SMALLEST % OF DONOR GENOME

BC2

Marker assisted pyramiding

• Pyramiding is the process of simultaneously combining multiple genes/QTLs together into a single genotype.

• Possible through conventional breeding – but extremely difficult or impossible at early generations.

• DNA markers may facilitate selection because – DNA marker assays are non-destructive and – Markers for multiple specific genes/QTLs can be tested using a

single DNA sample without phenotyping. • The most widespread application for pyramiding has been

for combining multiple disease resistance genes in order to develop durable disease resistance.

F2

P2

F1

P1 x

P1 P2 F1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

P1 P2 F1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

R S R R S R R S R R R R S R R R R R S R

R1

R2

S

S

S R S R R R R S R S R R R R R S S R R R

Line number

Phenotype Race 1

Phenotype Race 2

Resistant to Race 1

Susceptible to Race 2

Susceptible to Race 1

Resistant to Race 2

MARKER 1

MARKER 2

Early generation marker assisted selection

• One of the most intuitive stages to use markers to select plants is at an early generation (especially F2 or F3).

• The main advantage is – Many plants with unwanted gene combinations, especially

those that lack essential disease resistance traits and plant height, can be simply discarded.

• This has important consequences in the later stages of the breeding program because the evaluation – For other traits can be more efficiently and cheaply designed

for fewer breeding lines (especially in terms of field space).

P1 x P2

F1

PEDIGREE METHOD

F2

F3

F4

F5

F6

F7

F8 – F12

Phenotypic screening

Plants space-planted in rows for individual plant selection

Families grown in progeny rows for selection.

Preliminary yield trials. Select single plants.

Further yield trials

Multi-location testing, licensing, seed increase and cultivar release

P1 x P2

F1

F2

F3

MAS

EARLY GENERATION SELECTION MARKER ASSISTED SELECTION

F4Families grown in progeny rows for selection.

Pedigree selection based on local needs

F6

F7

F5

F8 – F12Multi-location testing, licensing, seed increase and cultivar release

Only desirable F3 lines planted in field

Biotechnology and genetic engineering in rice

Biotechnology in rice

• Basic biotechnological approaches include– tissue culture techniques to create somaclonal variation and

selection of desirable types, – regeneration of haploid plants and doubling of chromosome number

and – plant transformation and creation of transgenic plants.

• Haploid breeding has been successfully utilized in rice improvement.

• While plants have been regenerated from both male and female gametes

• Male gametes have proved to be more useful in regenerating large number of haploid and double haploid plants

• Varieties developed by anther culture in China : Hua Yu 1 and 2

Genetic engineering in rice

• Genetically modified rice are types of rice that have been genetically modified (also called genetic engineering) for agricultural purposes.

• The rice genome is usually modified using particle bombardment via the use of a gene gun or more commonly, a process known as Agrobacterium mediated transformation.

• Rice plants can be modified in DNA to be herbicide resistant, resist pests, increase grain size, generate nutrients, flavors’ or even produce human proteins.

• The natural movement of genes across species, often called horizontal gene transfer or lateral gene transfer, can also occur with rice through gene transfer mediated by natural vectors.

---cntd• Scientists are genetically modifying rice for several purposes

– rice resistant to herbicides, diseases, and pests, increasing nutritional value, eliminating rice allergies, producing human blood protein, increasing yield; improving tolerance to drought and salinity; and enhancing nitrogen use efficiency.

• In 2000, the first two GM rice varieties both with herbicide-resistance, called LLRice60 and LLRice62, were approved in the United States.

• Later, these and other types of herbicide-resistant GM rice were approved in Canada, Australia, Mexico, and Colombia. However, none of these approvals resulted in commercialization.

• Reuters reported in 2009 that China had granted biosafety approval to GM rice with pest resistance, but it hasn't been commercialized either.

• As at December 2012 GM rice had not yet become widely available for production or consumption.

Examples of transgenic rice produced at IARI

Trait Transgene Status

Bacterial blight Xa21 Field tested in India, China, Philippines

Stem borer resistance Bt (cry 1A, 1B, 1C etc.) Field tested in China

Sheath blight resistance Chitinase Transgenics show increased tolerance to sheath blight

Abiotic stress tolerance DREB Transgeneics under evaluation

Golden rice (ß-carotene) Psy, cry1, 1cy Many varieties, transformed, backcrossing used to introduce into other varieties

International and national institutions and programmes

• International Rice Research Institute (IRRI), Manila, Philippines

• Consultative Group on International Agricultural Research (CGIAR)

• West African Rice Development Association (WARDA), West Africa

• Central Rice Research Institute, Cuttak• All India Coordinated Rice Improvement Project,

1965 by ICAR with headquarters at Hyderabad, later elevated to Directorate of Rice Research in 1975

Important varieties• In India: IR 8, Jaya, Sona, Ratna, Haryana Basmati, VL Dhan

163, Pant Dhan 6• Hybrids: APHR1, APHR2, Pant Sankar Dhan 1, Sahyadri,

Narendra Sankar Dhan 2• In H.P.:

– Himdhan (has field tolerance to major diseases and insect pests), – Nagar Dhan (tolerant to major diseases),– Bhrigu Dhan (field tolerance to low temperature, diseases and

insect pests), – Varun Dhan (early maturing, semi-dwarf, lodging resistant and cold

tolerant with good cooking quality)– Others: Himalaya 1, 2, 741, 2216, Kasturi, Palam Dhan 957 etc.