maize

1. Maize Crop Improvement
COURSE TEACHER
Dr. G. ANURADHA
PROFESSOR
(IBT)
STUDENT
PHULE AMOL S.

2.  Introduction
 Maize or corn (Zea mays) is the most widely grown cereal crop in
the world
 At global level, maize accounts for 15% of proteins and 20% of
calories in world food diet
 CIMMYT Genebank holds ~27,000 maize entries, including
~24,000 landraces and wild relatives (teosinte & tripsacum)

3. Maize Genetics
 Genus: Zea
 Species: Z. mays
 Subspecies: Z. mays subsp. mays
 Family: Poaceae
 Subfamily: Panicoideae
 Maize is a diploid with 20 chromosomes
(n=10)

4.  Many forms of maize are used for food, sometimes classified as
various subspecies related to the amount of starch each has:
 Flour corn — Zea mays var. amylacea
 Popcorn — Zea mays var. everta
 Dent corn — Zea mays var. indentata
 Flint corn — Zea mays var. indurata
 Sweet corn — Zea mays var. saccharata and
Zea mays var. rugosa
 Waxy corn — Zea mays var. ceratina
 Amylomaize — Zea mays
Cont…..

5. Biotic stresses in Maize
 Major Pests
 Stem borer - Busseola fusca (Fuller). Order/ Family: Lepidoptera
 Major Diseases
 Banded Leaf and Sheath Blight (BLSB)
 Rust - Puccinia sorghi (rust fungus)
 Stalk rots - Stenocarpella maydis (fungus)
 Turcicum leaf bligh (TLB ) – Exserohilu turcicum
 Gray Leaf Spot (GLS) - Cercospora zeae-maydis
 Maize Streak Virus

6. Some Major Diseases of Maize
Maize Streak Virus
Gray Leaf Spot (GLS )
Turcicum leaf bligh (TLB)

7. Major Abiotic Stresses in Maize

8. Genetic Engineering (Transgenic) Approach
I. Genetic Engineering for Herbicide Tolerance
II. Genetic Engineering for Insect Resistance
III. Genetic Engineering for QPM
IV. Genetic Engineering for Drought Tolerance

9. Fig. Genetic transformation via Agrobacterium or bombardment

10. Genetic Engineering for Herbicide Tolerance and Insect
Resistance
 The most widely adopted GM traits are herbicide tolerance and
insect resistance
 These traits provide growers with benefits of increased yield,
reduced insecticide use and simplified management of weed control
with fewer and more flexible herbicide applications
 Herbicide (glyphosate) tolerance and insect resistance is the most
widely used trait and has the largest acreage in corn

11. Fig. Transformation Procedure in Maize

12. GM Maize Traits
 Stacked Traits
 The stacked maize category includes three combinations of traits
such as:
I. a double stack with insect resistance (Bt) and herbicide tolerance
(HT), Bt/HT
II. a double stack with two traits for insect resistance, Bt/Bt
III. a triple stack with two types of insect resistance, plus herbicide
tolerance, Bt/Bt/HT

13.  The second most dominant biotech crop after soybean was maize
with stacked traits, planted in 39.9 million hectares, up 2.6 million
hectares from 2011
 Stacked traits maize was planted in ten countries: the USA, Brazil,
Argentina, South Africa, Canada, the Philippines, Uruguay,
Honduras, Chile and Paraguay
 Maize with stacked traits occupied a total of 39.9 million hectares in
2012 compared with 37.3 million hectares in 2011 and occupying
23% of global biotech crop hectarage
Cont…..

14. Table 1. Transgenic products with molecularly stacked trait genes currently on
the market

15. Table 1. Transgenic products with molecularly stacked trait genes currently on
the market

16. Molecular Marker-Assisted Breeding for Improving
Stress Tolerance and Nutritional Quality of Maize

17.  CIMMYT’s Abiotic Stress Tolerant Maize Breeding
Sites

18.  Marker-assisted selection to improve drought
adaptation in maize
 Drought resistance or tolerance in maize is clearly a quantitative
character (Zarco et al., 2005)
 Drought stress is controlled by many minor genes (polygenes) that
have additive effects in their expression
 53 Drought tolerant varieties released in Africa
 Occupy ~2 million hectares in Eastern and Southern Africa,
yielding at least 1-2 t/ha more than the local varieties under stress
 The most popular variety released is ZM521

19. Case Study -I

20.  Results
 A marker-assisted backcross (MABC) selection program involved the
crossing of drought resistant line Ac7643 with a drought susceptible
line CML247
 Marker-based selection was carried out stepwise on all four
generations (from BC1F1 to BC2F3)
 After the four consecutive MABC cycles, the 70 BC2F3 individuals
exhibiting the closest allelic composition at target and non-target loci
were bred with two CIMMYT testers (CML254 and CML274)

21.  Target regions for MABC were identified primarily from QTLs
 Significant QTLs detected in 1994 and 1996 from the segregating
population Ac76433 X Ac7729 (Ribaut et al., 1996, 1997a)
 In 1996 from Ac76433 X CML247, where, in both cases, Ac7643
was the drought-tolerant parent, were used
 Five QTLs, located on chromosomes 1, 2, 3, 8, and 10, were
selected across these two segregating populations
Cont…..

22. Fig. 1. General MABC scheme conducted at CIMMYT

23. Case Study -II

24.  Different tropical maize genotypes
to Agrobacterium- mediated
transformation with the NPK1 gene
for conference of drought tolerance
 To achieve this, immature embryos
from thirteen maize genotypes (IL1,
IL3, IL15, IL16, IL28, IL38, IL42,
IL43, Hudiba-1, Hudiba-2,
Mojtamma-45, A188 and KAT)
 Transformed by cocultivating with
Agrobacterium tumefaciens strain
EHA101 harboring the pSHX004
vector
Fig. Map of the pSHX004 vector used in
Agrobacterium-mediated transformation of
maize
Cont…..

25.  Results
 Successful stable transformation of Sudanese in bred lines (IL3, IL1,
IL15 and IL38) and OPVs (Hudiba-2 and Mojtamaa-45) using the
NPK1 gene was achieved
 A total of 28 transgenic plants were obtained from these genotypes

26.  Biofortification
 Biofortification is the development of nutrient-dense staple crops
using the best conventional breeding practices and modern
biotechnology
 Without sacrificing agronomic performance and important
consumer preferred traits

27. Breeding for Nutritional Quality
 QPM (Quality protein maize)
 Provitamin A
 Kernel-Zn
 Vitamin E
 High methionine

28. How Biofortified Crops Improve Food and Nutrition
Security

29. QPM (Quality protein maize)
 Nutritional profile of maize is poor as it is deficient in essential
amino acids such as lysine, tryptophan
 Breeding for improved protein quality in maize began in the mid-
1960s with the discovery of mutants, such as opaque-2, that
produce enhanced levels of lysine and tryptophan, the two amino
acids deficient in maize endosperm proteins
 Improved o2 maize was renamed as quality protein maize (QPM)
 QPM essentially has about twice the levels of lysine and
tryptophan than normal maize and also increased levels of histidine,
arginine, aspartic acid and glycine

30.  These include the floury-2
(fl-2), Mucronate (Mc) and
Defective endosperm B30
(DE B30)
 The opaque mutants are
recessive (o1, o2, o5, o9-11,
o13, o17), the floury mutation
is semidormant (fl-1, fl-2 and
fl-3) where as Mucronate and
defective endosperm are
dominant mutations
Fig. Comparative composition of normal
and o2 maize for lysine and tryptophan
Cont…..

31. Fig. Chromosomal locations of various zein genes (Gibbon and Larkin, 2005).

32. Table. CIMMYT QPM gene pools and their characteristics

33. Marker-assisted selection for QPM breeding
 Marker assisted selection is an appropriate technology for selecting
o2 locus in breeding populations
(Dreher et al., 2003)
 Babu et al. (2005) used MAS for development of QPM parental
lines of Vivek-9 hybrid and could developed QPM hybrid in less
than half the time required through conventional breeding
 Danson et al. (2006) used various markers to introgress o2 gene into
herbicide tolerant elite maize inbred lines

34. Table . QPM varieties released since 1996

35. Table 3. QPM hybrids released since 1996

36. MAS in QPM Breeding in India
 Utility of MAS in QPM
development has been
successfully demonstrated in
India
 Vivek QPM-9 and several
early maturing QPM lines
developed by VPKAS
(Almora) using MAS
 QPM versions of three hybrids
(PEHM2, PEEHM5 and
Parkash) developed by Maize
Genetics Unit, IARI.

37.  In India first QPM composite variety ‘Shakti–1’ released in 1997
for commercial cultivation across the country
 It is an improved variety of maize which contains higher amount of
lysine and tryptophan
 With lower amount of leucine and isoleucine in the endosperm than
those contained in normal maize
Cont…..

38. QPM varieties in India
 Devloped by Rajendra Agricultural University, Pusa
 Plant breeder Dr. P. B. Jha developed first hybrid variety of QPM in
India in the year 1999-2000 as Shaktiman 1
 Later on Shaktiman 2, Shaktiman 3 and Shaktiman 4 were
developed with higher productivity

39. Provitamin A
 White corn is the predominant food corn used in sub-Saharan Africa,
and the white color of the kernels is ample demonstration of the lack
of carotenoids
 Maize is a staple food in many parts of Africa
 Vitamin deficiency affects up to 50% of the world’s population
 Vitamin A deficiency (VAD) is a significant human health problem
in developing countries, especially in sub-Saharan Africa and
Southeast Asia (WHO, 2009)
 Development of maize varieties that are biofortified with
biologically-usefully-high concentrations of provitamin A
carotenoids

40. Source of genes biosynthesizing and accumulating
b-carotene (provitamin A)
Gene Source Function
Phytone synthase
(psy)gene
Maize Phytone synthesized from GGDP in
rice grain
Carotene
desaturase (crtI)
gene
Bacteria
(Erwinia uredovora)
Introduction of adding four double
bonds
lycopene b
cyclase (lcy) gene
Daffodil Form a ring in b-carotene
(provitamin A) and cyclized

41. Fig. Essentials of carotenoid biosynthesis in cereals

42. Transgenic Approach
 The synthesis of carotenes in plants is a branch of the isoprenoid
pathway, and the first committed step is the joining of 2 geranyl
geranyl diphosphate (GGPP) molecules to form the precursor
phytoene
 The conversion of phytoene into -carotene requires 3 additional
enzyme activities: Phytoene desaturase, -carotene desaturase, and
lycopene -cyclase.
 Rice and other cereal grains accumulate GGPP but lack the
subsequent enzymes in the pathway, so the genes for all 3 enzymes
are required

43. Case Study -III

44.  Results
 Transformed 10–14-day-old immature zygotic embryos of the South
African elite white corn variety M37W by bombarding them with
metal particles coated with 5 constructs
 The selectable marker bar and 4 genes/cDNAs encoding enzymes in
the metabolic pathways for the vitamins - carotene, ascorbate, and
folate
 To increase -carotene levels, we introduced corn (Zea mays)
phytoene synthase (psy1) cDNA under the control of the wheat
LMW glutenin promoter and the Pantoea ananatis (formerly
Erwinia uredovora) crtI gene (encoding carotene desaturase) under
the control of the barley D-hordein promoter

45.  To increase ascorbate levels we introduced rice dehydro ascorbate
reductase (dhar) cDNA, and
 To increase folate levels, introduced the E. coli folE gene encoding
GTP cyclohydrolase (GCH1), both under the control of the barley
D-hordein promoter
 Twenty-seven independent transgenic plants was regenerated lines
was contained and expressed all input transgenes (at varying levels)
Cont…..

46. Maize Genomics
 Maize Genome Sequencing Consortium (MGSC) to use a novel
clone-by-clone approach to sequence the genome of the maize
inbred B73, a process that was completed in 2009
 The present day maize genome is genetically diploid (n=10)
 Genome size (GS) of approximately 2300–2700 Mb
 85% of which is composed of transposable elements
 over 80% of repeated sequences of various types

47.  Simple sequence repeats (SSRs) have been widely used in maize
genetics and breeding
 whole-genome sequences from16 maize inbreds and 1 wild relative
to determine SSR abundance and to develop a set of high-density
polymorphic SSR markers
 Atotal of 264 658 SSRs were identified across the 17genomes,with
an average of 135,693 SSRs per genome
 Marker density was one SSR every of 15.48 kb.(C/G)n, (AT)n,
(CAG/CTG)n, and(AAAT/ATTT)n
 The most frequent motifs for mono, di-, tri-, and tetra-nucleotide
SSRs, respectively
Cont…..

48. THANK YOU……..