In few years of Bt era – over Six hundred of Bt cotton hybrids are released – Just Handful of them are popular
Ultimately it’s the genetic potentiality for productivity that determines success of a Bt genotype
Breeding efforts of improving genetic potentiality of Bt cottons assumes greater importance
3. Dr.Yanal Ahmad Al-kuddsi
Biotechnology Researcher
Department of Biotechnology
General Commission for
Scientific Agricultural Research
Damascus – Syria
http://dr-yanal-alkuddsi.strikingly.com
6. 6 8/13/2016
Bacillus thuringiensis (Bt)
- Cry stalline proteins are classified according to structure &
have a specific nomenclature (e.g., Cry1Ac)
- Cotton has been transformed with Cry1Ac (narrow spectrum;
Lepidoptera only)
- Protein binds with receptors in the insect gut causing pores
which perforate the midgut & lead to cell leakage & insect death
Control Bt boll
Cotton Boll
Damage
Protected
Cotton Boll
8/13/20166
9. 9
The Transformation
The gene of interest is spliced
out of the bacterium using a
vector, like Agrobacterium
tumefasciens, & transferred to
cotton cells grown in tissue
culture
The cells are grown into a
plant & then, after testing,
plants are back-crossed into
commercial lines to make new
varieties
Recurrent
back-crossing 8/13/2016
9
11. Bt cotton…………….Why?
•To reduce use of chemical pesticides
•To increase production and productivity
• Ecofreindly
• Reduce risk to farmers
8/13/201611
12. Non Bt era
Bollworm
Square loss
Additional vegetative growth
No translation into sink
Reduction of harvest index
Reduction in yield
Bt Cotton
Reduce the cost of protection
Preventing the loss of Square
vegetative growth translated into matching
Increase in reproductive growth
Improvement of harvest index
Increasing in yield
Fig.1 Comparison between Bt cotton and non Bt cotton
8/13/201612
20. Most popular G. hirsutum hybrids
Until 2002, about 50 hybrids were released. In the past 5
years >500 G. hirsutum Bt hybrids have been released. The
area under hybrid cotton is about 90% in 2009.
8/13/201620
22. Improving Productivity of Hybrids
Bt gene is now part of the genetic background
•In few years of Bt era – over Six hundred of Bt
cotton hybrids are released – Just Handful of them
are popular
•Ultimately it’s the genetic potentiality for
productivity that determines success of a Bt
genotype
•Breeding efforts of improving genetic potentiality
of Bt cottons assumes greater importance
1
8/13/2016
22
23. Boll Number Boll Weight
SEED COTTON YIELD
Path of Productivity
8/13/201623
24. SEED COTTON YIELD
Boll Number
Bolls on
Monopodia
Monopodia length
No. of Monopodia
Bolls per monopodia
Bolls on
Sympodia
No. of Sympodia
Sympodia length
Bolls per
Sympodia
Other Traits
Rejuvination
Stay green nature
Subtending Leaf
Photosynthesis
Stay green nature
Leaf Thickness (SLW)
Boll Weight
Locule No.
Locule Weight
No. of Seeds
Seed Weight
GOT %
Boll Harvest Index
Kapas Wt/(KW + Rind Weight
8/13/201624
25. - Glabrousness
- Light green leaf
- Plant pigmentation long pedicel
- Cream petal
- Smaller bract
- High gossypol on gland
- High tannin
Genotype requirement – Bt cotton in India
8/13/201625
26. Developing diverse Heterotic Popuations
Exploiting them by Following Recurrent schemes of
Improving Combining ability (Increasing Genetic
distance) and Enhancing Heterosis
Maintaining such populations
Distributing them to breeders
Such Schemes are Integral part of Hybrid breeding in Cross pollinated crops
Can we Implement this concept in Cotton ?
8/13/201626
28. Understanding Combining Ability Pattern of Cotton
Genotypes and Forming Heterotic Groups
Evaluation of Crosses involving Diverse Cotton
genotypes at different centres is a Continuous
Process (over a decade)
Utilize this Data +Their Performance in national
trials
- Insight in to Combining ability pattern
(behaviour) of varietal lines
– HelpsTo Form Heterotic groups and Understand
basis of Heterosis
8/13/201628
29. Which Crosses are so far observed to be most Potential ?
Some crosses with diversity for plant types Robust vs
Compact are producing potential crosses
Complimenting Physiological Traits such as High
Photosynthetic Source genotypes (Stay green types) crossed to
genotypes having better Tanslocation efficiency (Harvest
Index ) , High RGR, Early maturing types
Though diverse types give potential hybrids at the same time
Some combinations involving apparently similar types may
also produce very potential crosses
8/13/201629
30. High RGR/HI
Group
Bushy/Robust Group
Stay Green Group
Compact Group
These Intergroup Crosses found to be equally productive
Potential
Additional heterotic groups without conspicuous
phenotypic differences are also formed 8/13/201630
33. Improving combining ability through reciprocal selection
Derived F4/F5 Lines Derived F4/F5 Lines
X X
cross II or its parents (C &D) cross I or its parents( A&B)
Cross I (A x B) Cross II (C x D)
Derived F1s compared with straight crosses and commercial checks
(Assessment of improved combining ability )
8/13/201633
34. Estimating combining ability effects and
forming heterotic pools for exploitation in
cotton (Gossypium hirsutum L.)
Y.A.Alkuddsi et al ,.2009
There is a constant need to develop more potential hybrids
and adopt noval approaches for improving hybrid
performance. In cross pollinated crops like maize heterotic
populations are developed and exploited through
population improvement schemes meant for improving
combining ability.
8/13/201634
35. Table1: List of cotton lines and testers in the study of combining
ability
Lines Testers
L1- RAH 318 T1- SC 14
L2- RAH 243 T2- SC 18
L3- RAH128 T3- SC 7
L4- RAH146 T4- SC 68
L5- RAH 97 T5- RGR 32
L6- RAH 124 T6- RGR 24
T7- RGR 58
Bushy/Robust Group Compact Group
8/13/201635
36. Table 2. Mean performance of experimental hybrids and and sca effects
and gca effects of parents for different characters.
Sl
No.
Crosses
Bolls per plant Mean boll Weight (g) Kapas yield (g/plant) Ginning per cent
Per se
performanc
e
Sca
effects
Per se
performan
ce
sca
effects
Per se
performan
ce
Sca
effects
Per se
performan
ce
sca
effects
1 YHH36 (L5T2) 16.1 1.16 3.52 0.04 139.96 62.53 ** 35.74 0.14
2 RCH2 Bt 31.6 3.32 107.58 32.25
3 YHH 134 (L4 T8) 29.5 0.83 2.89 -0.19 106.18 11.81 36.87 -3.91
4 YHH 133 (L4 T7) 33.8 3.05 2.98 -0.02 104.9 15.05 41.37 1.63
5 YHH 106 (L1 T6) 33.2 9.07 ** 2.98 -0.05 103.6 30.53 ** 35.06 0.56
6 YHH 129 (L4 T3) 31.1 2.56 2.87 -0.15 95.26 7.31 36.23 0.82
7 YHH 112 (L2 T 3) 31.4 4.35 2.91 0.17 91.61 19.69 * 31.6 0.33
8 YHH 123 (L3 T6) 31.1 3.49 2.97 0.01 91.16 11.89 31.38 -2.35
9 BUNNY Bt 26.4 3.3 91.14 34.88
10 YHH 124 (L3 T7) 30.1 -0.74 2.97 0.10 89.2 7.92 36.09 0.59
11 YHH 115 (L2 T 6) 28.5 2.47 3.05 0.23 89.09 17.27 33.44 0.45
12 YHH 131 (L4 T 5) 25.9 0.78 3.44 0.10 88.96 6.21 42.23 1.51
13 YHH 108 (L1 T8) 26.1 0.82 3.08 0.06 86.53 6.93 37.31 5.88 *
14 YHH 107 (L1 T7) 31.3 3.93 3.23 0.29 82.33 7.24 36.54 -0.10
15 RAHH 95 30.6 2.31 68.15 37.54
8/13/201636
37. Table 3.General combining ability effects of parents (lines and testers) in respect of
kapas yield and its attributing characters of cotton[G .hirsutum L.]
LINES Bolls per plant
Mean boll
weight(g) Kapas yield
(g / plant)
Ginning
Per cent
1 0.10 0.00 -0.76 -0.67
2 2.00 * -0.21 * -2.02 -2.20 *
3 3.57 ** -0.07 5.43 -1.61
4 3.49 ** 0.06 14.00 ** 2.19 *
5 -6.56 ** 0.40 ** -3.20 1.61
6 -2.60 ** -0.19 -13.45 ** 0.68
S.Em.± 0.86 0.09 3.06 0.86
CD ( ĝ i – ĝ j) at 5% 2.45 0.27 8.72 2.46
CD ( ĝ i – ĝ j ) at 1%
3.26 0.36 11.62 3.28
TESTERS
1 -0.53 -0.16 -7.27 * -1.80
2 -2.30 * 0.11 8.64 * -1.70
3 1.25 -0.02 1.96 -2.42 *
4 -1.33 -0.29 * -14.18 ** 0.24
5 -2.17 * 0.30 ** -3.24 2.64 *
6 0.23 0.06 1.85 -0.53
7 3.47 ** -0.04 3.86 1.62
8 1.38 0.04 8.38 * 1.95
S.Em.± 0.99 0.11 3.54 0.99
CD ( ĝ i – ĝ j ) at 5%
2.82 0.31 10.07 2.84 8/13/201637
38. Table 4. Identifying diverse pairs of crosses based on predicted
performance of double cross combinations
T 2 T 3
L4 78.48 95.26
L5 139.96 66.52
T 7 T 8
L1 82.33 86.53
L4 104.9 106.18
PREDICTED MEAN
(L4 T8) 106.18
(L4T7) 104.9
(L1 T8) 86.53
(L1 T7) 82.33
MEAN 94.985
PREDICTED MEAN
(L4 T2) 78.48
(L5T2) 139.96
(L4 T3) 95.26
(L4 T3) 66.52
MEAN 95.06
8/13/201638
39. Conclusion
- The performance of three top hybrids viz., hybrid YHH
133, YHH 134, YHH 136 in respect of kapas yield needs to
be verified for their performance on large scale basis.
- The results implies that the line 4 and the tester 5 studied
were high combiners across all the traits, indicating their
ability in transmitting additive genes in the desirable
direction to their progenies. Further, these line and tester
can be tested for the confirmation of their superiority as
good parents for hybridization
8/13/201639
41. India cottons are considered to possess inappropriate micronaire
value for aspecific staple owing to poorly developed cell wall in
fibres occasioned by immaturity resulting in weaker fibres.
The hybrids have been also found to be prone to immaturity with
subsequent pickings leading to immature and weaker fibres.
Concerted efforts and due to a very strong demonstrative
programme of integrated pest management, it has been possible to
improve significantly the micronaire status in most of the
popularly traded cottons .
8/13/201641
43. The tenacity of indian cottons , which continue to remain lower
even after sustained efforts in quality improvement.
Indian cottons are assessed for quality using the HVI in the ICC
mode unlike in the west where HVI mode is the normal
operating mode where the tenacity is invariably pushed up about
25 %.
The fibre mironaire has a predominant influence in deciding the
tenacity in the HVI mode.
8/13/201643
45. The fibre quality seems to be not a criteria under scrutiny before
the release of Bt hybrids in India, although Bt lint has been noted
to be cleaner and fluffy.
After handling for a few years, by now mills perspective about
processing of Bt cottons is that the micronaire value of the cotton
to begin with is considerably higher than what normally indian
cottons possessed prior to Bt release.
The deterioration in micronaire value in the case of Bt hybrid is
much steeper compared to the conventional hybrid.
The extra long staple erosion in the country has already been
known even before the Bt release.
8/13/201645
46. Comparative performance of Bt cotton with some elite
conventional cotton cultivars under arid to semi-arid
conditions
8/13/201646
Four cotton cultivars , CIM-496, BH-162,VH-144 and Bt-
121.
To compare yield potential, quality traits and disease
resistance of Bt and non-Bt cotton cultivars under arid
to semi-arid climatic conditions.
Ahsan Aziz et al., 2011
53. Conclusion
8/13/201653
Bt-121 with medium plant height gave better yield in arid climatic
conditions because of abundant sunshine and better response of
short statured cultivars to high doses of fertilizers.
The virus infection is a major hurdle for yield improvement in
cotton crop and the cultivars which showed resistance against virus
infection are preferred for general cultivation as compared to
susceptible varieties (Khan et al., 1993).
Variation in seed cotton weight per boll among cotton genotypes
may arise from variation in photosynthetic capacity, as well as
partitioning of these assimilates to various parts of the plant body.
54. 8/13/201654
All the four cotton cultivars differed in maturity percentage
and that the performance of Bt-121 was better than other
cultivars used for comparison.
LSD comparison showed significant differences among four
cotton varieties, depicting that BH-162 had maximum fiber
fineness as compared to other varieties.
Variation in fiber length was found within a cultivar or even
within a single boll. Uniformity in staple length improves
spinning performance.
High fiber strength is difficult to obtain without sacrificing
yield. CultivarVH-144 produced very strong fibers but its
seed cotton yield was the lowest indicating that, this quality
trait might have been influenced by the yield potential of a
cultivar.
56. 56
Is Bt gene affecting only boll worms ?
Bt gene produces poisonous protein to kill bollworm.
Is it causing any other effect on the plant?
YES
It induces even every early square to get converted
into a boll !
The capacity of sink is thus enhanced!
There is no evidence for it’s source being enhanced by
Bt gene!
Is the increased sink’s capacity realized?
What is the impact of this on the Source?
Is the plant phenology capable of sustaining
enhanced sink?
8/13/201656
57. 57
Impact of Bt gene on the relation between vegetative
and reproductive phase of growth in cotton”
Patil S. S et al., 2010
“Correlated Impact”
Introduced Bt Gene
Any impact on source?
Inducing every square to
bear a boll
Is it capable of bearing
this extra load?
Enhancement of sinks
capacity
Is this increased
sink’s capacity realized?
Is the plant phenology capable of sustaining enhanced
sink?
8/13/2016
57
58. 58
Objectives
- Evaluation of plant type, productivity and
physiological traits of Bt cotton hybrids
- Analyzing and interpreting causes for the differences
in productivity of these Bt cotton hybrids.
8/13/2016
58
61. 61
The impact of enhanced Sink
With same photosynthetic output (source) the quantum of sink has
increased
Some genotypes lack matching photosynthetic potential (source)
to meet extra demand of Sink
They cannot cope with this drain.
Leaves are weakened, subsequent reddening, disease incidence
and senescence
In some hybrids there is Mismatch between source and sink at a
later stage of growth (upper half of the plant)
8/13/2016
61
62. Physiological changes in Bt cotton
Leaf area Index Slightly reduced -
Photosynthetic rate Increased slightly -
Harvestable Bolls Increased
Boll weight Increased
Harvest index Increased
Boll growth /maturity Synchronous
Habit Determinate
Duration Reduced
Senescence Faster
Sink
8/13/201662
63. 63
Strata Yield
Harvest
Index
Upper half Low Low
Lower half High High
Because physiological impact of Bt background
was not visualized while developing them
8/13/2016
63
65. 65 STAY GREEN
Higher Photosynthetic ability, higher leaf
area, Stay green nature become very
important in overcoming this mismatch
8/13/2016
65
66. 66
Overcoming the demand of enhanced sink induced due to
Bt gene
Chemical intervention Genetic intervention
1.Ethrel spray Enhancing Source
to meet the demand of enhanced sink
Physiological Intervention
Ethylene spray is expected to induce initial
square dropping causing additional vegetative growth making the plant
competent to bear enhanced sink 8/13/2016
66
67. 67
- Often non Bt hybrid genotypes are selected based on
potentiality and the parents are subjected to transfer of Bt gene
through back cross breeding
- It is very important to know whether the genotype chosen
for Bt gene has ability to withstand the additional reproductive
load (sink).
If the enhanced sink is not matched by the source( mismatch
between source and sink ) this is reflected in the form of faster
senescence reduced boll load especially in the upper half of the
plant.
- These genotypes which lack matching photosynthetic
potential (source) wither under the pressure of enhanced sink.
Inference in Plant Breeding
8/13/2016
67
Results :
68. 68
- This impact is often noticed in the latter half of the
growth. As a result, the harvest index and seed cotton
yield are reduced in upper half of the plant.
- Some hybrids of non –Bt era , which on developing Bt
version are clearly showing weaker upper half since they
cannot cope with this drain.
- The symptoms of weakened leaves, more prone to
reddening, disease incidence and senescence are noticed
commonly.
This is leading to reduced boll load and lower harvest
index in the upper half as compared to lower half
8/13/2016
68
71. Armyworms on Cotton BractFeeding at Base of Boll
Damage to Inside of Boll
8/13/201671
72. Damaged Square: Bt CottonEconomically Damaged Square
Bollworm in Bloom Tag Color Phases of Beet Armyworms on
Cotton Flower
8/13/201672
73. While transfering Bt gene through Backcross
breeding
Grow progeny rows in backcross generation
Select for recurrent parents constitution and
retain or
Reject plants resembling donor parents
Grow recurrent and donor parents along with
backcross progenies
Develop such a donor as Bt source which has
distinct phenotypic marker traits helping in
rejecting plants in backcross generations
8/13/201673
74. Breeding for Sucking pest tolerance
Sucking pests cause maximum damage in Bt cottons
Conventional Mechanisms ofTolerance to Biotic Stresses
Increased tannin content, palisade layer features etc.,.
Antagonastic traits like hairiness of leaf can be freely used to
impart sucking pest tolerance
Introgression of traits from related species to cultivated
cottons
a) Desi cottons to hirsutums
b)Other related species to hirsutums
8/13/201674
75. Bollgard II Cotton
Benefits
Insect resistance
management
Increased control of targe
pests
Reduced insecticide
application
Control Bollgard
Cotton Boll Damage Cotton Boll Protected
Monsanto,USA
Bollgard I – event 531 – cry 1 Ac
Bollgard II- event 15985 cry 1 Ac + cry 2 Ab
8/13/201675
76. Safety - Resistance
.Given time & exposure, insects
have the capacity to overcome
most insecticides. Bt cotton may
be no different, however, there are
safeguards:
.Refugia
.High-Dose Strategy
.Development of additional protein
8/13/201676
77. Refugia
•Objective: provide harborage for susceptible moth production
to reduce the chance of resistant (R) moths mating with each
other
•U.S. growers are required to plant a proportion of their
acreage to non-Bt cotton
• 5% Refuge, if no lepidopteran-active insecticides are used
on it, or else 20% Refuge
RR RR RR SS
RR
RS SS
8/13/201677
80. Can other host species of cotton bollworm be non-Bt refuges to prolong the
effectiveness of Bt-cotton?
TAN Shengjiang et al.,2001
• In China Bt-cotton is inter-planted with many other host species of H. armigera, and the migration
ability of this insect specie is strong.
•An effective refuge strategy that maximizes the probability that susceptible individuals arising from
a structured refuge will find and mate with the resistant individuals that survive exposure to the
delta endotoxin produced in the Bt-plant.
8/13/2016
80
84. Result
Field investigation indicated that the third and fourth generations
of cotton bollworm can survive from the Bt-cotton, and chemical
insecticides are needed to reduce the damage.
The population on Bt-cotton differentiated genetically from those
on the other host species.
Both maize and castor-oil plant can be refuges as normal cotton to
produce susceptible individuals and to prolong the effectiveness of
Bt-cotton.
8/13/201684
85. Survival and reproduction of natural populations of
Helicoverpa armigera on Bt-cotton hybrids in Raichur, India
M.T. Ranjith,A. Prabhuraj andY. B. Srinivasa 2010
• Individuals of Helicoverpa armigera, the most prominent among bollworms in India.
• Commercial Bt-cotton hybrids containing single (Cry1Ac) and double (Cry1Ac and Cry2Ab) genes
in experimental plots of the University of Agricultural Sciences, Raichur campus, India.
• NCS-145 and MRC-7918 Cry1Ac and Cry2Ab genes
(BollgardTM-II,Event MON 15985) hybrids
•MRC-6918 Cry1Ac gene (BollgardTM-I, Event MON 531)
hybrid).
•MRC-7918 (non-Bt) is similar to MRC-7918.
8/13/201685
86. Figure 1. Desigen Bt expression strips® exhibiting double bands
indicating the presence of Cry toxins in different plant parts of Bt
hybrids.
8/13/201686
Figure 2. Larva of Helicoverpa
armigera feeding on boll of Bt
hybrid.
89. Conclusion
Results on the developmental biology from the F0 generation do
not clearly suggest any particular pattern. All the larvae collected
on MRC-7918 successfully pupated,whereas 75% of the larvae
collected on NCS-145 and 70% on non-Bt hybrid pupated.
The trends obtained cannot be attributed to either the presence or
absence of Cry genes in the hybrids.
It can be inferred that the performance of individuals of H.
armigera on the Bt hybrids is ‘comparable’ to those on the non-Bt
hybrid.
We further enquired whether the next generation (F1) was able to
successfully complete its development on particular hybrids.
8/13/201689
91. YES, by anyone's definition of a public good. Bt is a
public good because it is a natural resource, a part of
the biotic community, that has evolved over the
millennium shaped by the forces of nature. It is a
biological resource that serves uniquely valuable
functions within biotic communities, both in natural
systems and those managed by humans. It is a product
of organisms widely dispersed in nature, owned by no
one.
Is Bt a "public good"?
8/13/201691