Biotechnological approaches for crop improvement


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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

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  • This slide shows the actual biochemical pathway that we discussed in the previous slide. EPSP synthase synthesizes 3-enolpyruvly shikimic acid-5-phosphate. This is the essential precursor to aromatic amino acids. When plants are sprayed with a glyphosate-containing herbicide, such as Roundup, this important precursor is not synthesized, and consequently the plant is starved of aromatic amino acids. The result is plant death.
  • Roundup Resistant plants have a very simple solution. An engineered version of EPSP synthase, one that was discovered in a bacteria, is introduced into the plant. This enzyme can not be bound by glphosate. Therefore, if a field is sprayed with the herbicide, the introduced version of the gene produces a functional enzyme. The 3-enolpyruvl shikimic acid-5-phosphate precursor is synthesized normally, and the plant produces enough aromatic amino acids to survive.
  • What is needed is for the public to accept these crops. Examples such as these, were a corn crop is freed of weed pressure make a compelling case for acceptance of these new agricultural products. But, it should be noted that these traits are all producer orientated.
  • Biotechnological approaches for crop improvement

    1. 1. 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
    2. 2. 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
    3. 3. 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
    4. 4. 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
    5. 5. Broad leaf weeds
    6. 6. Weed grasses
    7. 7. 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
    8. 8. + 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
    9. 9. 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
    10. 10. Final Test of the Transgenic Plants RoundUp Ready GM Crop Before After
    11. 11. Round up Ready GM Crops Soybean Cotton Sorghum Corn Canola Wheat ( in development)
    12. 12. Classical example of biotechnology based variety breeding
    13. 13. 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
    14. 14. 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.)
    15. 15. 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
    16. 16. 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.
    17. 17. 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
    18. 18. 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.
    19. 19. 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
    20. 20. 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.
    21. 21. 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.
    22. 22. Fig. 8 Haploid bread wheat plants with n=3x=21, ABD chromosomes in (a) predominant univalent metaphase-1 meiosis (b)
    23. 23. 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%)