1. Genetic manipulation of stay-
green traits
for crop improvement
Shantanu Das
Regn.no. : 11-AMJ-65
Dept. of Plant breeding and Genetics

2. Contents
Introduction.
Strategy for development of genotype with
stay green traits.
Physiological basis of stay-green traits.
Importance of stay green traits.
Genetic manipulation of stay-green traits.
Case study on stay-green QTL introgression.
Ideotype of stay-green genotype.
The relationship between stay-green traits and grain yield.
Conclusion.

4. What is stay-green ?
 The extended foliar greenness
during grain filling under post
anthesis drought.
 Stay green is one such trait, and
genotypes possessing this trait
maintain more photosynthetically active leaves
than genotypes not possessing this trait .
 Genotype with stay green traits is called stay green
genotype.

5. Where, When and Why stay green traits
is required?
 It’s required specially in a drought
environmental condition.
 To keep greenness of leaves alive for
longer period of time, specially during
the grain filling stage.
 To maintain or increase higher grain
yield.
 Stay-green results when the plant’s
normal process of senescence is
disrupted.

6. Strategy for development of genotype
with stay green traits.
Keeping alive for longer is the fundamental
strategy for increase
 Crop yield
 Market value

8. Components of stay green traits.
 Key components determining stay green:
 Total plant leaf area (TPLA): (+ve) correlated
with green leaf area at maturity (GLAM) .
 Duration of leaf senescence: (+ve) correlated
with GLAM.
 Rate of leaf senescence (LS): (-ve) correlated with
GLAM
 NB: GLAM is an indicator of stay-green.

9. Classes of stay green
 There is a five way of stay green on the basis of time and
duration of occurrence of senescence.
 Type A stay greens (delayed initiation of yellowing)
 Type B stay greens (initiate senescence on schedule, but
comparatively slow)
 Type C stay greens (arise due to specific defects in
chlorophyll
degradation pathway.)
 Type D stay greens (cell are dead)
 Type E stay greens ( chlorophyll content is remain same but
enzyme activity is reduce)

10. Five ways to stay‐green.

11. Importance of chlorophyll in
stay green traits.
 Chlorophyll pigment is responsible for greenness of the leaves.
 Chlorophyll pigment involved in photosynthesis.
 The result of photosynthesis is biomass production.
 When there wii be degradation, during the reproductive stage ,
ultimate biomass production will be less.
 Chlorophyllase (CLH) is responsible for degradation of
chlorophyll a to chlorophyllide a (Chlide a)
 When a mutants disrupt the activity of chlorophyllase (CLH), it
leading to stay-green phenotypes.

12. Chlorophyll degradation initiation
×

13. Relation of N2 with stay green traits
Chemical structure of chlorophyll
 Chlorophyll pigment is consist of
N2, Mg+2 and others constituent.
 Stay-green can be viewed as a
consequence of the balance between
N demand by the grain and N supply
during grain filling stage.
 While N2 is the constituent of most of the
proteins (Rubisco involved in photosynthesis).)

15. 1. Importance of stay green traits in
agronomical crops.
 Delayed senescence is useful only when it contributes to
increase yield.
 N uptake during grain filling is higher in stay-green
genotype than senescent hybrids. (Borrell and Hammer,
2000).
 In some crop it leads to drought resistance.
 It leads to lodging resistance.

16. 2. Importance of stay green traits in
horticultural crops.
 In horticultural crop it increases market
value.
 Several approaches have been utilized to
manipulate hormone level or responses in
transgenic plants to reduce the incidence of
postharvest yellowing.
 It extends shelf-life and helps in long term
transportation.

17. Genetic manipulation of
stay green traits

18. A. Identification of QTL for stay green.
Requirement for QTL mapping:
1. A suitable mapping population generated from
phenotypically contrasting parents.
2. Reliable Phenotypic screening of mapping population.
3. A saturated linkage map based on molecular markers.
4.Appropriate statistical packages to analyze the
genotypic information in combination with phenotypic
information for QTL detection.

19. 1. and 2. Phenotypic screening of mapping population.
P1 x P2
Recipient Donor
F1
F2
F2:F3
Phenotypic screening
Trials plot Screening is going on

20. (1) LEAF TISSUE SAMPLING
(2) DNA EXTRACTION
(3) PCR
(4) GEL ELECTROPHORESIS
(5) MARKER ANALYSIS

21. Stay-green QTL have been identified – Few to
mention
Crop Identified QTL Reference
English blue Recessive nuclear
grass (Festuca allele sid Thomas(1997)
Pratensis )
Stg1, Stg2, Stg3, and
Sorghum Stg4. Wenwei Xu et al.
(2000)
recessive gene fiw
Arabidopsis maped on chromosome Nakamura et al.
4 ( 2000)

22. Stay-green QTL have been identified – Few to
mention
Crop Identified QTL Reference
Recessive mutant gene
Rice sgr(t) on Cha et al. (2002)
chromosome 9
46 main QTL for six Jiang et al. (2003)
Rice trait are localized in 25
chromosomal region
Wheat QSg.bhu-1A, QSg.bhu-3B Uttam Kumar et
(Triticum and QSg.bhu-7D al. (2010)
aestivum L.)

24. Marker assisted breeding
P1 x P2
Susceptible Resistant
F1
F2
MARKER-ASSISTED SELECTION (MAS)

25. Marker assisted breeding
P1 x P2
Susceptible Resistant
F1
F2

26. Marker assisted backcrossing

27. Marker-assisted backcrossing (MAB)
There are three levels of selection in which markers may be
applied in backcross breeding.
1 2 3 4 1 2 3 4 1 2 3 4
Target locus
FORGROUND RECOMBINANT BACKGROUND
SELECTION SELECTION SELECTION
 Foreground selection : to screen for the target trait.
 Recombinant selection: to minimize linkage drag.
 Background selection: to the recovery of the recurrent parent
genome.

28. CASE STUDY
INTROGRESSION OF STAY-GREEN
TRAITS INTO A KENYAN FARMER
PREFERED SORGHUM VARIETY
K. NGUGI, W. KIMANI1 and D. KIAMBI1

29.  A MAB breeding programme was conducted to
introgress stay green QTL from ICRISAT sorghum line
E36-1 well-characterised for stay-green QTL, into the
background of the Kenyan farmers’ preferred
sorghum variety, Ochuti.
 E36-1 donor parental line
 Ochuti recurrent parental line
 The parental lines E36-1 has 3 stay green
QTLs, SBI-01, SBI-07 and SBI-10 located at
various chromosomes.

30. Make the backcross and to select the
genotypes with Stay-green QTL
E36-1 Ochuti
Donor parent Recurrent parent
X
Collection of leaves at 15
Collection of leaves at days after sowing
15 days after sowing
for genotyping
for genotyping
`
Ochuti
F1 X
Collection of leaves
from 96 F1 progenies
BC1F1 Leaves collection from 128 individual
for genotyping
for identifying genotype with the Stay-green QTL

31. Contd.
 Seven forground marker were used to identify individuals
of F1 generation that had stay green QTLs transferred
into Ochuti.
 Out of 96 F1 genotype only five genotypes that had at
least one stay green QTL introgressed, 3 with two QTL
( SBI-07 and SBI-10) and 2 other F1 genotypes had only
one QTL (SBI-10) transferred into Ochuti.
 A total of 128 BC1F1 DNA samples together with the two
positive controls (the two parents) were genotyped with
the five foreground SSR markers.

32. Ideotype of genotype with stay green traits
 Plant should have spread and deep
root system.
 Genotype with stay green traits
should show slow rate of leaf
senescence(LS).
 Delay onset of leaf senescence(LS).
 Genotype should have more total plant leaf area
(TPLA).

33. The relation between
the stay green traits and
grain yield
 Grain yield was correlated positively with green leaf
area at maturity and negatively with rate of leaf
senescence (from the study of nine genotypes varying
in stay-green, Borrell et al.,1999).
GLAM ⍺ Grain yield
Rate of leaf senescence ⍺

34. CONCLUSION
 Several stay-green QTL have been identify in Several crops
like sorghum, wheat, rice etc.
 By hybridization programme, its is enable to transfer
identified stay-green QTL to the genotype having good
agronomic background.
 Incorporation of stay-green trait in a genotype will increase
the ultimate grain yield and market value which
will contribute to our national economy.