Screening for apomixis—the process by which plants reproduce asexually through seeds without fertilization—involves several techniques to identify and characterize apomictic behavior. Apomixis can be complex, involving various forms of asexual reproduction, and detecting it requires a range of cytological, genetic, and molecular methods. Here’s a detailed explanation of the techniques used to screen for apomixis:

1. Cytological Analysis

Cytological analysis involves examining the development of the embryo sac to identify apomictic modes of reproduction.

  • Aposporous Embryo Sac: This is a form of apomixis where the embryo sac is formed directly from somatic cells. Aposporous embryo sacs are typically four-celled and lack antipodals, which are usually present in normal embryo sacs.
    • Technique: Microscopic examination of ovules and embryo sacs at various developmental stages can reveal the presence of aposporous sacs.
    • Example: Aposporous embryo sacs can be identified by their distinctive four-celled structure and the absence of the normal antipodal cells.

2. Callose Fluorescence and Clearing of Ovules

Callose fluorescence combined with clearing of ovules is used to detect diplospory, another type of apomixis.

  • Diplospory: This involves the formation of an embryo directly from a megaspore without fertilization.
    • Technique: Ovules are stained with callose-specific dyes and then cleared to observe the presence of callose, a polysaccharide associated with diplospory.
    • Example: In species showing diplospory, the ovules may exhibit distinct fluorescence patterns indicating the presence of callose.

3. Uniformity of Progenies

Uniformity of progenies from cross-pollinated parents can be an indirect indicator of apomixis.

  • Technique: If progenies from cross-pollinated parents are uniformly similar, it suggests the possibility of apomixis, as apomictic seeds produce genetically identical offspring to the maternal plant.
    • Example: High uniformity in traits among progeny from a cross-pollinated parent could indicate apomixis.

4. Maternal Phenotype Consistency

Maternal phenotype among progenies from F1 crosses is another indicator.

  • Technique: If the F1 progenies consistently show the phenotype of the maternal parent, it may indicate apomictic reproduction, as the progeny are clones of the maternal plant.
    • Example: In crops where the maternal phenotype is consistently observed in the offspring, it suggests apomictic seed production.

5. Genetic Variance in Progeny

Limited or no genetic variance in the F2 progeny of two distinct parents is a sign of apomixis.

  • Technique: By analyzing genetic variance among progeny, researchers can infer whether apomixis is occurring, as apomictic progeny should have minimal genetic diversity.
    • Example: A low level of genetic variation in F2 progeny compared to expected levels of genetic diversity from cross-pollination suggests apomixis.

6. High Seed Frequency in Aneuploid, Triploid, and Wide Crosses

High seed frequency in aneuploid, triploid, and wide crosses may indicate apomixis.

  • Technique: Observing the seed set in these crosses can provide clues about apomictic behavior, as apomictic plants often have high seed set in such crosses.
    • Example: An unusually high seed frequency in triploid or wide crosses may indicate the presence of apomixis.

7. Multiple Seedlings per Seed

Multiple seedlings per seed can be an indicator of apomixis.

  • Technique: Monitoring seed development and germination can reveal the presence of multiple seedlings emerging from a single seed, suggesting apomictic reproduction.
    • Example: The occurrence of multiple seedlings from a single seed is often a characteristic of apomictic species.

8. Molecular Markers

Molecular markers such as UGT 197 and OPC-04 are used to detect apomictic modes of reproduction in specific plant species.

  • Technique: These markers are used in genetic analysis to identify the presence of apomixis-related genes. Co-segregation of these markers with apomictic traits can confirm apomictic reproduction.
    • Example: In Pennisetum, molecular markers like UGT 197 and OPC-04 are linked with apomictic reproduction and can be used for screening.

9. Progeny Test

Progeny tests are conducted to determine the degree of apomixis.

  • Technique: By analyzing the offspring from plants known or suspected to be apomictic, researchers can assess the extent and nature of apomictic reproduction.
    • Example: Progeny tests can reveal the proportion of apomictic vs. sexual seeds and help in quantifying apomictic behavior.

Conclusion

Screening for apomixis involves a combination of cytological, genetic, and molecular techniques. Each technique provides different insights into the mechanisms of apomictic reproduction and helps in characterizing and utilizing apomictic traits in plant breeding and research. These methods collectively enhance our understanding of apomixis and its applications in agriculture.