What is crop breeding? Modifying, tailoring, and/or engineering plants making them more suitable for humans Modification means converting (e.g.): a. Tall height to short height, b. Late maturing to early maturing, c. Disease susceptible to disease resistant, d. Low yielding to high yielding, e. Stress susceptible to stress tolerant f. Low food quality to high food quality

6. Hence, today,
We will discuss
applications of biotechnology in plant breeding with
special reference to
Wide cross breeding and
Genetic engineering
Tissue culture is must here

7. Remember:
Biotechnology is utilization of
information available in
bacteria and fungi and cells and
tissues of plant and animals
Therefore, the gene(s) of
interest are either being
obtained from micro-
organisms, or from plants

8. Some of the hard core examples
of genetic engineering and tissue
culture in plant breeding
Bt. Cotton (GE+TC)
Herbicide resistance (GE+TC)
Golden rice and (GE+TC)
Doubled haploids (TC only)
production in wheat

9. Bt Cotton: insecticidal products from
Bacillus thuringiensis (Bt) engineered in
Cotton, rice, canola, soybean, maize etc.
The steps involved:
i.Identification of Bt gene,
ii.Isolation of insecticidal protein
iii.Gene cloning
iv.Gene expression
v. Transformation of plant (gene gun/agro-bacterium)
using gene constructs
vi.Regeneration of plant through
tissue culture

11. Broad leaf weeds

12. Weed grasses

14. Herbicide GE crop Mechanism that results in plant
death
Glyphosate Corn,
soybeans,
cotton.
Canola,
sugar-beets
Block the metabolic pathway of enzyme EPSPS. Trade
name is “Roundup Ready” by MONSANTO. Resistance
to glyphosate is engineered in plants from
Agrobacterium sp.
Glufosinate Corn,
soybeans,
cotton,
canola, rice,
sugar-beets
Active ingredient is “phosphino-thricin”similar to
“Glutamine” block the synthesis of “glutamine
synthase” (GS) required for N metabolism, commonly
knows as “Liberty” by Aventis (AgrEvo). Resistance is
engineered by PAT gene from Streptromyces bacteria
Bromoxynil Cotton Kill broad spectrum weed by inhibiting
Phosphosynthesis. Bromoxynil nitrilase (BXN) gene from
Klebsiella pneumoniae detoxify the effect in plants.
Known as “Buctril” by MOSANTO is largely for cotton
Sulfonylurea Cotton, Flax Sulphonylurea block acetolactate synnthase (ALS) used
for synthesis of leucine, isolucine and valine). ALS gene
of tobacco resists Sulphonyleurea. CDS , MONSANTO

15. + Glyphosate
X
Roundup Sensitive Plants
X
X
Shikimic acid + Phosphoenol pyruvate
3-Enolpyruvyl shikimic acid-5-phosphate
(EPSP: an enzyme of metabolic pathway
Plant
EPSP synthase
Aromatic
amino acids
Without amino acids,
plant dies
X

16. Bacterial/plant
EPSP synthase
Shikimic acid + Phosphoenol pyruvate
3-enolpyruvyl shikimic acid-5-phosphate
(EPSP)
Aromatic
amino acids
Roundup Resistant Plants
+ Glyphosate
With amino acids,
plant lives
RoundUp has no effect;
enzyme is resistant to herbicide

17. Final Test of the Transgenic Plants
RoundUp Ready GM Crop
Before After

18. Round up Ready GM Crops
Soybean
Cotton
Sorghum
Corn
Canola
Wheat ( in development)

19. Classical example of biotechnology based variety breeding

20. Doubled haploid production
using wheat x maize
crossing
Haploid production in bread wheat via
crosses with Zea mays has developed
into an efficient tool for addressing several
research areas. The most significant and
practical use is in wheat breeding especially
to get instant homozygosity at any stage of
segregation in hybridization program.
Plant breeding entirely based on
tissue culture

21. There are two major methods to making DH lines
1. Using microspores (pollen) beset with differential
responses with different wheat genotypes,
2. Using megaspores (egg cells) in ultra wide-crosses
like wheat x maize proved an efficient method as the
maize pollen can successfully hybridize with wheat
egg cell and produce hybrid zygote. The immature
hybrid embryos are reared on artificial nutrient
medium under controlled conditions. The plantlet
thus obtained are finally treated with colchicine to
get DH lines
Doubled Haploid (DH) production
using wheat x maize crossing (cont.)

22. The method however, has several crucial stages
that translate into significant variability in
outputs among various working groups
Modifications induced for
1. Using detached tillers instead of fixed tiller as
opposed to the conventional method,
2. Hot water emasculation instead of clipping
the florets for hand emasculation, and
3. In-vitro application of 2,4-D have proved to be
an efficient and cost effective methods for DH
production

23. The details of this includes:
1.Selection of material.
All hexaploid wheats,
F1 and BC1 crosses
monosomic and di-somic addition lines
can be used for crossing with maize.

27. 2.Emasculation:
Spikes can be selected for emasculation on
the basis of their morphological development
(2-3 days before anthesis). The selected spike
can be cut as a whole tiller (2-3 cm above the
ground) and can be kept in container having
tap water by keeping the base of the tiller
the water. Spike can be emasculated by
dipping in hot water (430
C) in a water bath
for three minutes. The emasculated should
immediately be covered with poly ethylene
bags to maintain humidity.
Procedure continue

29. Procedure continue
3. Pollination:
One day before predicted anthesis,
emasculated wheat spikes can be
pollinated by freshly collected
maize pollens. Maize pollens can be
simply dusted on the spikes. The
spikes can than be covered with
glassine bags.

30. 4. Spike culture:
Pollinated spikes can be cultured in
liquid culture medium containing 40g/L
Sucrose, 100 mg/L 2,4-D and 8 ml/L
sulfurous acid. The cultures can be
maintained in a growth chamber preset
at 22.5o
C, 12 hours day length and 60-
70% relative humidity. Haploid seeds
would be developed on these spikes

31. Procedure continue
5. Embryo culture:
After 12-16 days of pollination, immature seeds can excised from the spikes.
Seed set in wheat crossed with maize would be smaller than the normal
selfed seeds and would be filled with an aqueous solution instead of solid
endosperm.
Surface sterilize these seeds with sodium hypo-chlorite (1-2%)
supplemented with few drops of Tween-20 for ten minutes and rinse them
with sterilized water.
Dissect the seed for excision of immature embryo under aseptic conditions
in a laminar air flow and platted on artificial nutrient medium containing ½
MA (Murashige & Skoog) solution supplemented with 10 g/L sucrose and
1.5 g/L gel grow (Gelling agent by ICN).
The culture tubes can be maintained in an incubator preset at 25o
C, 16 hrs
day-length and 5000 lux light intensity.

35. Procedure continue
7: Chromosome doubling:
At tillering stage, roots can be cut to about 2 cm below the crown. The plants
can then be immersed in 1% Colchicine solution supplemented with 2%
DMSO and 15 drops/L Tween-20, for about 5 hrs. in a beaker at room
temperature.
Roots of the treated plants can then be washed under running tap water and
the plants can again be planted in potted soil to let them grow till maturity.
Chromosome number can again be determined to ensure proper doubling.
At maturity, seeds of the doubled haploid (DH) plants can be collected,
which can be evaluated in the following crop season for agronomic, morpho-
physiological, and pathological characteristics.

37. Fig. 8 Haploid bread wheat plants with n=3x=21,
ABD chromosomes in (a) predominant univalent
metaphase-1 meiosis (b)

38. Table 2. Crossability of bread wheat F1s with maize
Sr.
No
Female
parent
Male
parent
Florets
pollinated
% seed
set
% Emb.
formed
Haploid
plants
DH lines
obtained
1 Inqlab-91 Parent 441 64.63 9.75 33 9
2 -do- V-87094 199 77.89 18.59 17 8
3 -do- Pak-81 158 88.61 21.52 12 3
4 -so- Punjab 508 73.62 16.14 44 16
5 Kohistan-97 Parent 342 76.02 8.77 15 1
6 -do- Inqlab 322 74.53 9.01 14 1
7 -do- V-87094 86 63.95 13.95 6 4
8 Pak-81 Parent 260 70.38 8.85 16 6
9 -do- Inqlab-91 103 82.52 21.36 9 3
10 -do- Punjab-85 632 60.76 8.23 10 6
11 Uqab-200 Parent 248 55.24 11.29 12 6
12 -do- Inqlab-91 184 73.91 10.87 5 3
13 -do- Punjab-85 228 75.0 17.54 14 3
14 Punjab-85 Parent 264 94.70 17.42 20 5
15 -do- Inqlab-91 344 93.31 15.41 9 3
Total/Average 288 75.0 13.91 236 77 (36.6%)