Hence, today, We will discussapplications of biotechnology in plant breeding with special reference to

Wide cross breeding and Genetic engineeringTissue culture is must here

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

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

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 proteiniii.Gene cloning

iv.Gene expressionv. Transformation of plant (gene gun/agro-bacterium)

using gene constructs

vi.Regeneration of plant through tissue culture

Broad leaf weeds

Weed grasses

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

+ 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

Aromaticamino acids

Without amino acids, plant dies

X

Bacterial/plantEPSP synthase

Shikimic acid + Phosphoenol pyruvate

3-enolpyruvyl shikimic acid-5-phosphate(EPSP)

Aromaticamino acids

Roundup Resistant Plants

+ Glyphosate

With amino acids, plant lives

RoundUp has no effect;enzyme is resistant to herbicide

Final Test of the Transgenic PlantsRoundUp Ready GM Crop

Before After

Round up Ready GM Crops

SoybeanCottonSorghumCornCanolaWheat ( in development)

Classical example of biotechnology based variety breeding

Doubled haploid production using wheat x maize crossing

Haploid production in bread wheat viacrosses 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

There are two major methods to making DH lines1. 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.)

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, and3. In-vitro application of 2,4-D have proved to be

an efficient and cost effective methods for DH production

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.

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 (430C) in a water bath for three minutes. The emasculated should immediately be covered with poly ethylene bags to maintain humidity.

Procedure continue

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.

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 22.5oC, 12 hours day length and 60-70% relative humidity. Haploid seeds would be developed on these spikes

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 25oC, 16 hrs day-length and 5000 lux light intensity.

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.

Fig. 8 Haploid bread wheat plants with n=3x=21, ABD chromosomes in (a) predominant univalent

metaphase-1 meiosis (b)

Table 2. Crossability of bread wheat F1s with maizeSr. 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%)