hybrid rice-way forward

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Hybrid Rice Status and Way Forward
Dr. Ramlakhan Verma Scientist
CENTRAL RICE RESEARCH INSTITUTE CUTTACK, ODISHA
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Heterosis ?
Superiority of F1 over either parent

• Shull (1908) first described this phenomenon
  after observing the stimulation of growth, and
  other physiological characters in maize (Zea mays
  L.) in the hybrid
• The word heterosis was coined by him in 1914.

With this idea of heterosis or hybrid vigour,
plant and animal breeders have capitalized on its
occurrence to make genetic improvements.
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Shulls concept of Heterosis

• Conclusions based on his investigation
• Self-fertilization purifies the strains and
  releases new variation
• Self fertilized plants are weaker than cross
  fertilized one
• Generations of close breeding reduces the strains
  into simple constituent biotype which are weaker
  than the hybrid combinations
• Crossing the weakened pure strain would cancel
  the effects of inbreeding

Interpretation of increased vigour, size,
fruitfulness, resistance to diseases and insect
pests or climate rigour of any kind manifested by
cross-bred organism as compared with
corresponding inbreds, as the specific result of
unlikeness in the constituents of the uniting
parental gametes Shull
(1952)
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Some Landmarks
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Rice Hybrids

• Yuan Longping started the research of indica
  hybrid in 1964
• Erjiunan1A, Zhenshan 97A and V20A CMS-WA lines
  were developed during 1972.
• Taiyin1, IR4 and IR1, promising restorers were
  identified (1973)
• Nanyou 2, first indica hybrid rice released in
  1974
• China became the first country to produce hybrid
  rice.

Yuan Longping is the first scientist who
successfully altered the self-pollinating
characteristic of rice and realized large-scale
farming of hybrid rice. This earned him the title
"Father of Hybrid Rice".
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Development of hybrid rice in China
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Hybrid Rice Area in Major Rice Countries (000
ha) (2012)
Country Total Rice Area Hybrid Rice Area Potential area (000 ha) that can be covered under hybrid rice
Bangladesh 12000 700 5.83 3000
India 44100 2000 4.54 15000
Indonesia 13201 650 4.92 3000
Philippines 4537 177 3.90 2000
Myanmar 8038 78 0.97 2000
Pakistan 2500 250 10.00 1000
Vietnam 7652 595 7.78 3000
USA 1204 439 36.46 400
L. America 5047 70 1.39 2000
Others 100 2000
Subtotal 3428 32400
China 30311 15600 51.47
Total 19028
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Changes of hybrid rice area in countries outside
China
Hybrid rice area increased in the major hybrid
rice countries/regions
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Hybrid Rice Research in India

• A systematic goal oriented network mode project
  on hybrid rice development was initiated by ICAR
  in December, 1989
• This programme had financial support from ICAR,
  FAO, UNDP, Barwale Foundation and technical
  backstopping from IRRI, Philippines
• Private sector has also been playing key role in
  hybrid rice research and development

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Major activities under Hybrid Rice Network

• Development and evaluation of hybrids
• Development and Improvement of parental lines
• Optimizing packages for hybrid rice cultivation
  and seed production
• Use of molecular tools for improving efficiency
• Technology dissemination
• Public-Private partnerships
• Coordinating the research activities

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Hybrid Rice Growth - India
At present 2.3 mha Area (2012-2013), 5.2 of
total rice cultivated area in India
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Region wise
Hybrid rice seeds produced (2012)
Total 34,500 tones seed
Institution wise
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Evaluation and Release of Rice Hybrids in India

• Total hybrids evaluated 3500
• Hybrids Released 66
• Public Sector hybrids 31
• Private Sector hybrids 35
• Central Releases 43
• State Releases 23

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Advantages of Hybrid Rice Technology

• Higher yield potential due to heterosis higher
  returns
• Hybrids have better tolerance to abiotic stresses
  specially to drought at early veg. Stage
• Hybrid seed production is profitable
  (Rs.70,000-85,000 net return/ha)
• Job opportunities for rural poor (60-80
  persons/ha)

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Use of Male Sterility System

• Basic Features of Male Sterility
• Prevents self pollination, permits cross
  pollination.
• Leads to heterozygosity
• Female gametes function normally
• Assayed through staining techniques
• In nature, occur due to spontaneous mutations
• Can be induced artificially

• Types of Male Sterility in rice
• Cytoplasmic male sterility (CMS)
• Environment-sensitive genic male sterility
• Chemically induced male sterility
• Transgenic male sterility Induced by the
  technique of genetic engineering

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Schematic description of hybrid breeding using
cytoplasmic genetic male sterility system
(Three-line System)
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Discovery of Cytoplasmic male sterility (CMS) in
rice

• Cytoplasmic male sterility (CMS) is a maternally
  inherited trait in which plants fail to produce
  normal fertile pollen.
• The role of cytoplasm in causing male sterility
  in rice was first reported by Sampath and Mohanty
  (1954).
• CMS-WA was discovered in the 1970s in a wild rice
  (Oryza rufipogon), and this cytoplasm was
  backcrossed into indica rice (Oryza sativa spp.
  indica) to produce CMS-WA lines
• The first CMS line used to develop commercial F1
  rice hybrids was developed in China in 1973 from
  (Oryza sativa f.sp. spontanea ) growing on Hainan
  Island in 1970 (Yuan 1977). This plant was
  designated wild rice with aborted pollen (WA).
• Presently, about 90 of commercially cultivated
  rice hybrids have been derived through a
  three-line breeding strategy which involves the
  use of WA-CMS lines.

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• More than 20 other CMS sources have been
  developed from cultivated rice and wild
  species.eg. HL, Boro, RT102, CW, LD, Kalinga-I
  etc.
• CMS is encoded by the mitochondrial genome and
  probably some defect in mitochondrial function
  arrests normal pollen development observed in
  male sterile genotypes
• Generally, the regions whose expression is
  associated with CMS contain unusual open reading
  frames (ORFs) that are often chimeric in
  structure and frequently cotranscribed with
  conventional mitochondrial genes

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Identified ORF associated with BT-CMS and WA-CMS
in rice

• atp6 (orf79) in CMS-BT (Boro II cytoplasm), Wang
  et al. 2006, The Plant Cell, 18676687
• orfB, in CMS-WA (Das et al. 2010. BMC Plant
  Biology, 1039)
• Orf126, in CMS-WA (Bentolila and Stefanov 2012.
  Plant Physiology,1589961017)
• WA-352, in CMS-WA (Luo et al. 2013. Nature
  Genetics, 45 5)

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• The Chimeric Gene orf79 Encodes a cytotoxic
  peptide and confers gametophytic CMS

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WA-CMS (6A)
Restored CMS (R) CMS x Restorer
rf/rf
Rf/rf (Rf1A/Rf1D)
S
S
(unedited)
orfB 1.1 kb (edited)
orfB 1.1 kb (unedited)
0.7 kb (edited)
0.7 kb (edited)
Inner mitochondrial membrane
Defective ATP synthase complex
Sterility
Normal ATP synthase complex
Restored fertility
Cytoplasmic factor- unedited orfB
Identified nuclear factor Rf-1A/Rf-1D
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Plant Physiology, February 2012, Vol. 158, pp.
9961017

• Identified and functionally validated of the
  new mitochondrial gene WA352 for CMS-WA
• It originated in the mitochondrial genome of
  wild rice by multiple recombination events

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Fertility restoration system

• Natural suppressors of CMS, called restorers of
  fertility, are found in the nucleus and have the
  ability to restore the production of pollen to
  plants carrying the deleterious mitochondrial
  CMS-associated genes.
• CMS/restorer of fertility systems have been
  widely used in hybrid seed production, because
  they eliminate the need to emasculate the
  parental line used as the female.
• Fertility restoration is often associated with
  genes thought to regulate the expression of genes
  encoded by organellar genome e.g.
  pentatricopeptide repeat (PPR) proteins involved
  in processing organellar RNAs.
• Rf2 (a restorer for L-CMS) does not encode a
  pentatricopeptide repeat protein, unlike a
  majority of previously identified Rf genes. It
  encodes a glycine-rich protein (GRP)

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(No Transcript)
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Fertility restorer genes for cytoplasmic male
sterility systems in rice

• The inheritance of fertility restoration in the
  CMS system has been extensively investigated
• Rf genes encode PPR proteins which constitute a
  large family, with more than 400 members each in
  Arabidopsis and rice.
• PPR proteins thought to be RNA binding proteins
  involved in posttranscriptional processes (RNA
  processing and translation) in mitochondria and
  chloroplasts

S. No. Rf genes Fertility restoration of CMS system Ref.
1 Rf1a, Rf1b CMS-BT Wang et al. 2006
2 Rf2 CMS-L (Lead Rice-type ) Itabashi et al . 2011
3 Rf3 CMS-WA Zhang et al. 1997
4 Rf4 CMS-WA Zhang et al. 1997
5 Rf5(t) CMS-HL Huang et al. 2000
6 Rf6 CMS-HL Huang et al. 2000
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A model for the mechanism of the CMS-WA systems
restoration (Luo et al. 2013.
Nature Genetics, 45 5)

• Rf4 and Rf3 suppress WA352 by different
  mechanisms, probably with Rf4 functioning
  post-transcriptionally and Rf3 functioning
  post-translationally.

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Strength and Limitations of 3-line system

• Strength
• Stable male sterility
• Limitations
• Limits germplasm source (CMS, Restorer)
• Predominant use of a single CMS system (WA)
• One additional step for parental seed production
• Time consuming CMS breeding

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TWO LINE BREEDING OF HYBRID RICE
USE OF EGMS (ENVIRONMENT-SENSITIVE GENIC MALE
STERILITY)
PGMS (Photoperiod- sensitive Genic Male
Sterility) (Becomes sterile -
beyond 14 hrs. day length)
TGMS (Thermo-sensitive Genic Male
Sterility) (Becomes sterile
between 22-28? C)



Variety-1 (Fertile)
(Environment-A)





(Selfing)
X
Pollen Parent (Fertile)
Variety-1 (Male Sterile)
(Environment---B)
(Environment- B)

F1 Hybrid (Fertile)
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Two-line method using gametocides

• Another two-line approach for hybrid rice seed
  production is spraying the chemical hybridizing
  agents (CHAs)
• 1. Ethrel, ethyl 4' fluoro oxanilate, or
• 2. 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.
• Because of the inefficient seed production
  related to nonsynchronous tillering and flowering
  as well as health hazards associated with the use
  of arsenic compounds, CHA use in China was
  discontinued.

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Advantage Disadvantage of 2-line hybrid rice
system

• Advantages
• Simplified procedure of hybrid seed production
• Multiple and diverse germplasm available as
  parents
• Any line could be bred as female
• 97 (2-line) vs 5 (3-line) of germplasm as male
• Increased chance of developing desirable
  heterotic hybrids
• Multiple cytoplasm courses as female parents
• Disadvantages
• Environmental effect on sterility could cause
  seed purity problem

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Strategies for improvement of hybrid and heterosis

• Diversification of male sterility
• To enhance heterosis
• Improvement of parental lines
• Exploitation of two line system

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Diversifying the CMS system

• gt90 of commercial hybrid production involves
  only a WA cytoplasmic source for male sterility
• The uniformity of the WA cytoplasm can result in
  genetic vulnerability to disease and insect
  pests.
• To overcome this, it is essential that the
  genetic source of CMS be diversified
• To broadening the genetic base
• Diversification to enhance the outcrossing
  feature.
• Indica/japonica diversification to harness the
  inter-subspecific hybridity.

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Approaches for identifying New CMS systems
Intraspecific hybridization

X
X
P1
P2
P2
P1
Female
Male
Female
X
X
P2
F1
X
X
P1
F1
P2
P1
Male
Male
(Partly/Fully sterile)
Male
Male
(Fertile)
BC1
BC1
BC1
BC1
(Sterile)
(Fertile)
Male
(Fertile)
(Fertile)
BC 6
(New male sterile line)
Identification of new source of male sterile
cytoplasm
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Induced Mutation
Interspecific hybridization
WILD SPECIES
CULTIVATED VARIETY (A)
SS
X
F
X
A
F1
MUTATION
(Partly/Fully sterile)
SS
A
X
BC1
Male
(Sterile)
S
M1 - Sterile
BC 6
(New male sterile line)

• CMS has been induced in rice through gamma
  irradiation of IR62829B
• a maintainer of WA cytoplasm (IRRI 1995).

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CMS lines with different CMS sources developed at
the Central Rice Research Institute (CRRI),
Cuttack, India.
CMS Source CMS lines developed
WA-CMS CRMS5A, 6A, 7A, 8A, 10A, 11A, 13A, 15A, 16A, 17A, 18A, 19A, 23A, 24A, 25A, 26A, 27A, 28A, 29A, 30A, 31A, and 34A, 51A
O. perennis CRMS22A, 35A, 36A
V20B CRMS20A
Kalinga I CRMS21A, 32A, 33A
Lalruma CRMS37A
Miz.21 CRMS 44A, CRMS45A
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Transgenic Approach

• Male sterility acquired through mitochondrial
  genes causing sterility i.e. orfB and WA-352 that
  can be expressed transgenically in nucleus
• Maintenance of GEMS plants To be done by
  hybridizing with normal plant and linking may be
  with herbicide resistance to sterile plant.
• The hybrid seeds will be generated by crossing
  parent carrying either the Cre-lox system
  (Hodges et al, 1999) or with antisense approach
  (Van Tunan et al, 1999) to down regulate the
  expression of the synthetic orfB or WA-352 genes
  or by the approach of TALEN system to knock out
  the expression of the orfB /WA-352 gene.

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Transgenic approaches

• Stability of the male sterility acquired through
  mitochondrial genes responsible for CMS e. i.
  orfB, WA-352 etc. made to express transgenically
  in nucleus in terms of Mendelian pattern of
  inheritance in filial generations.
• Maintenance of GEMS plants To be done by
  hybridizing with normal plant and linking with
  herbicide resistance to sterile plant.
• The hybrid seeds will be generated by crossing
  parent carrying either
• the Cre-lox system (Hodges et al, 1999) or
  with antisense approach (Van Tunan et al,
  1999) to down regulate the expression of the
  synthetic orfB/WA-352 gene or by the approach of
  TALEN system to knock out the expression of the
  orfB /WA-352 gene.

Male sterile line
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Heterosis in Rice

• Heterosis varies according to the level of
  parental diversity and or presence of heterotic
  gene blocks in parental lines.
• indica/japonica crosses show maximum heterosis
  vis-à-vis any other combination between other
  subspecies.
• Heterosis can be positive or negative. Both
  positive and negative heterosis can be useful
  depending on the trait, i.e. positive heterosis
  for yield and negative heterosis for growth
  duration

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Basis of Heterosis

• Dominance Hypothesis (Devenport 1908, Bruce 1910)
• Over dominance hypothesis (Hull 1945, Crow 1948)
• Epistasis hypothesis (Powel 1944, Williams 1959)
• Marginal over dominance (Wallace, 1968)
• Phytohormone Gibberellic acid ( Rood et al.,
  1988)
• DNA methylation (Tsaftaris et al. 1997)
• Metabolic balance (Hagemann and Lambert, 1999)
• Complementation for absence of genes (Fu and
  Dooner, 2002)
• MicroRNA (Ding et al. 2011)

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Exploration of rice intersubspecific heterosis

• Generally, rice is classified as two subspecies
  indica and japonica. Sometimes, rice geneticists
  and breeders define tropical japonica as javanica
  subspecies.
• It has been known that F1 hybrids between
  typical indica and tropical japonica produce
  incomplete fertility.
• Mainly javanica, with the wide compatibility (WC)
  gene(s), can produce complete fertility by
  crossing with either indica or japonica.

Exploration and exploitation of WC positive
parental lines to enhance the intersubspecific
heterosis
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Improvement of parental lines

• To breed improved heterotic rice hybrids, we need
  to adopt new strategies to enhance the frequency
  of occurrence of maintainers and restorers, and
  ensure a constant supply of genetically diverse
  parental lines
• Parental lines should be stacked with major
  biotic and abiotic stresses tolerance gene(s).
• The parental lines should have desired grain
  quality, and a high out crossing parameters
  (stigma excretion, long duration spikelets
  opening etc.)
• Introgression of yield and component traits in
  parental lines.
• Pyramiding of QTLs for yield heterosis

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Constraints to Popularization of Hybrids

• Quality of hybrids
• Availability of pure seed
• Seed cost
• Procurement constraints
• Maturity duration
• Insufficient/Inconsistent heterosis over
  environments
• Inadequate awareness of the farmers
• Lack of suitable hybrids for different
  ecosystems/different regions
• Restricted area for large scale hybrid seed
  production in the country

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Strategies for expansion of hybrid rice area

• Popularizing hybrids throughout the state by
  organizing minikits and identifying suitable
  hybrids for different regions.
• Better linkages among state departments, SAUs and
  Institutes to conduct extensive FLDs at multiple
  locations in each state.
• Identifying suitable areas for HR seed
  production.
• Encouraging farmers to take-up hybrid seed
  production.
• Imparting training in HR cultivation and seed
  production to farmers / extension personnel.
• Creating proper awareness among farmers and
  removing misconceptions about HR.

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Strategies for expansion of hybrid rice area

• Arranging buy back of HR seed produced by
  farmers.
• Govt. subsidy in the initial stages to encourage
  HR cultivation
• Hybrid rice should be extended to stress
  situations like shallow lowlands, saline/alkaline
  tracts and boro areas where the hybrids have
  proved to perform extremely well.
• Public-private partnership should be further
  strengthened to make the hybrid rice technology
  more popular with the farmers.
• Research front higher heterosis, better seed
  producibility, two line system, parental improved
  for higher stress tolerance, region specific
  grain and cooking quality

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Thank You