INTRODUCTION TO PLANT BREEDING

AGRONOMY 815 / COURSE NOTES

P. STEPHEN BAENZIGER, 338 Keim Hall, 472-1538

DEPARTMENT OF AGRONOMY / UNIVERSITY OF NEBRASKA

CROP TISSUE CULTURE

1. Tissue culture can be divided into two main types of cell growth and differentiation systems:



1. Formation of unorganized tissues (callus) which are later induced to form organized tissues, and



2. Maintenance of organized tissues (for example, micropropagation of bud tissues). Maintenance of organized tissues have limited but very specific uses that will not be discussed extensively. Those uses are germplasm maintenance, germplasm exchange due to virus free materials, rapid increases of rare genotypes in clonally propagated materials (flowers and woody plants).

1. Organized tissues can be formed from unorganized tissues by organogenesis (the development of organs which then form the rest of the plant -- example is shoot regeneration that is then induced to form roots) or embryogenesis (the development of embryo-like tissues which on the proper medium germinate and form whole plants). In general, embryogenesis is the preferred method because it is believed to be more normal and induce less variation.
   
   
2. Successful tissue culture systems pay close attention to:



1. Explant source (tissue, age of tissue, health of donor plant), media (sugar type and concentrations, inorganic salts, vitamins, amino acids, and particularly hormones), pre- or post-treatments (heat or cold shocks; light wavelength, levels and duration; and atmospheric conditions (elevated CO₂). In general, most tissue cultures use Murashige-Skoog inorganic salts, with high levels of auxins (2-4D, NAA, IAA, or dicamba) to induce callus. Occasionally very undefined materials are added (coconut milk or potato extract). Regeneration usually occurs after the removal of the auxins and the additions of cytokinens (known as "auxin step down" procedure). It should be recognized that much of tissue culture is prudent trial and error research because relatively little is known about hormone action and the developmental genetics.

1. In addition to micropropagation, what are some of the uses of tissue culture?



1. Tissue for transformation;



2. Tissue for biochemical selection and mutation research



3. Source of genes (particularly from protoplasts which have many of the same properties as bacteria for gene identification);



4. Introduced gene expression assays;



5. Direct use in breeding programs (doubled haploidy);



6. Synthetic seeds; and



7. Physiological assays (particularly cellular assays in which whole plant assays are confusing).

1. Some of the concerns with using tissue culture:



1. Many of the media components are mutagenic -- somaclonal or gametoclonal variation is possible;



2. Tissue culture is not necessarily a quick or inexpensive process;



3. Need to very carefully define the objective of the research and then determine if the system is useful in meeting this objective;



4. Transformation via cell cultures will require careful evaluation for gene stability, gene expression, gene position effects, and for background effects (most lines are highly heterogeneous, the transformed line will be highly homogeneous if derived from one cell which may affect line performance). Example: synthetic seeds, wheat cultivars in Nebraska, chromosomal loss.