Designing an appropriate selection procedure is important for successfully identifying mutants.
The identification of appropriate selection conditions typically requires careful dose-response curves to determine the correct concentration of a toxin (for example, selections using herbicides, FuDR, etc.) that results in lethality or sufficient and distinctive inhibition of gametophyte growth or development. Several factors may need to be considered when choosing the appropriate selection regime, for example:
- The nature of the toxic response, e.g. whether abrupt or gradual.
- The pattern of gametophytic growth and development over time, e.g. whether gametophytes exhibit differential response dependent upon age.
- Identification of phenotypes other than growth that may identify mutants.
- Also for identification of putative morphological mutants (for example clumped chloroplast or callus-like growth habit), careful observation of gametophytes at a sufficiently young stage is important.
The standard selection procedure is outlined in this illustration.(Figure not yet available!)
A number of confirmed and putative mutants have been obtained using both X-ray and EMS mutagenesis.
Mutant Selections of Ceratopteris and References
|Selection Agent / Criteria||Strain / Mutant Codes||Description||Reference|
|paraquat tolerant||HaPQn, pq2, pq45, pqa||Gametophytes and sporophytes tolerant to paraquat (recessive in sporophytes).|
|acifluorfen||HaBn / blt1||and Hickok, unpublished|
|HaGn / glt1, glt2||Gametophytes and sporophytes tolerant to glyphosate-containing herbicides, e.g. RoundUp*, (incompletely dominant in sporophytes)|
|NaCl tolerant||HaNn / stl1, stl2||Gametophytes and sporophytes tolerant to Na+ and Mg2+ salts|
|FUDR (2′-deoxy-5-fluorouridine) tolerant||HaFn / fdr1||Gametophytes tolerant to the nucleotide analog 2′-deoxy-5-fluorouridine|
|Al2(SO4)3 at pH 4.4||HaAT37,29|
|antheridiogen (ACe) – maleless||HaCn / her1||Does not form male gametophytes – insensitive to the male-inducing pheromone|
|abscisic acid||HaAn/ abr48, abr104||Gametophytes tolerant to the plant growth regulator, abscisic acid|
|dark germinator||HaDn / dkg1||Does not require light to initiate spore germination|
|germination: altered red / blue responses||GERM1,2,3,4|
|day-night responder||dnr1||Gametophytes and sporophytes grow very slowly and accumulate massive amounts of starch in their plastids under constant light. A day/night cycle, or reduced light, accelerates growth and causes depletion of excess starch reserves|
|non-etiolated||det30||Gametophytes do not show elongation response when grown in the dark|
|self sterility: 230X (C.thalictroides) |
spermatozoid / flagella development
|sleepy sperm||zzz1||Sperm are released normally, but swimming is delayed. After a few minutes, some sperm ‘wake up’ and swim slowly to moderately while the rest show only slight movement and remain close to the gametophyte. Excellent for observation at high magnifications (greater than 50X). Self-fertile|
|slow-mo sperm||slo1||Sperm are released normally and swim immediately. All but a few show very slow movement. Excellent for observations at high magnifications (greater than 50X). Self-fertile.|
|clumped chloroplasts / polka dot||HaCPn /cp1||Chloroplasts form clumps that resemble polka dots in both gametophyte and sporophyte tissue. Recessive in sporophytes|
|F1 polka dot||cp/CP||F1 gametophytes segregate in a ratio of 1 wild type to 1 polka dot. F2 sporophytes segregate in a ratio of 3 wild type to 1 polka dot.|
|highly male||HaHIM / him1||Gametophytes show a ‘supermale’ morphology. Forms high numbers of males (even in isolate cultures) with increased numbers of antheridia on both males and hermaphrodites||Hickok, unpublished|
|F1 pale||pal1/PAL1||Spores from an F1 sporophyte produce gametophytes in a 1:1 ratio of pale mutant to green wild type. Pale gametophyte mutants grow nearly as well as wild type and are easily distinguished. The recessive mutation results in a 3:1 ratio in the F2 sporophyte generation.|
polka dot – chloroplasts appear aggregated into clumps – here shown adjacent to same age wild type
bulbous – intermittent large clear cells
dwarf – cell size appears smaller – here dwarf shown here adjacent to same age wild type
highly male – large male and hermaphroditic gametophytes both with prolific numbers of antheridia
pale – lighter gametophyte color – here shown adjacent to same age wild type
rumpled – irregular cell development giving a crumpled appearance
pie – hermaphroditic gametophytes develop a more circular appearance rather than cordate
All listed selections, with the exception of 230X, were derived from the Hn-n strain. Strain designations ending with ‘n’ indicate that a number of independent selections were available, which have not all been fully characterized genetically.
Morphological mutants were identified by the tedious screening of mutagenized spores without a selection agent.