Mutants and Strategies For Mutant Selection

Designing an appropriate selection procedure is important for successfully identifying mutants.

Figure 1. A successful selection using media supplemented with FuDR (5-flurodeoxy uridine). There are about 12,000 spores on this dish and all except one are severely inhibited or killed by this concentration of FuDR. The single individual to the right of center exhibits tolerance to FuDR and has nearly normal growth and development.

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 / CriteriaStrain / Mutant CodesDescriptionReference
paraquat tolerantHaPQn, pq2, pq45, pqaGametophytes and sporophytes tolerant to paraquat (recessive in sporophytes).
acifluorfenHaBn / blt1 and Hickok, unpublished
glyphosate
tolerant
HaGn / glt1, glt2Gametophytes and sporophytes tolerant to glyphosate-containing herbicides, e.g. RoundUp*, (incompletely dominant in sporophytes)
NaCl tolerantHaNn / stl1, stl2Gametophytes and sporophytes tolerant to Na+ and Mg2+ salts
hydroxy-L-prolineHaYn
azetidine-2-carboxylateHaAZn
FUDR (2′-deoxy-5-fluorouridine) tolerantHaFn / fdr1Gametophytes tolerant to the nucleotide analog 2′-deoxy-5-fluorouridine
2-aminoethyl-L-cysteineHaCYn
Al2(SO4)3 at pH 4.4HaAT37,29
antheridiogen (ACe) – malelessHaCn / her1Does not form male gametophytes – insensitive to the male-inducing pheromone
abscisic acidHaAn/ abr48, abr104Gametophytes tolerant to the plant growth regulator, abscisic acid
dark germinatorHaDn / dkg1Does not require light to initiate spore germination
germination: altered red / blue responsesGERM1,2,3,4
day-night responderdnr1Gametophytes 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-etiolateddet30Gametophytes do not show elongation response when grown in the dark
self sterility: 230X (C.thalictroides)
spermatozoid / flagella development
 
sleepy spermzzz1Sperm 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 spermslo1Sperm 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.
MORPHOLOGICAL MUTANTS
irregular meristem HaTUBE1
clumped chloroplasts / polka dot HaCPn /cp1Chloroplasts form clumps that resemble polka dots in both gametophyte and sporophyte tissue. Recessive in sporophytes
F1 polka dotcp/CPF1 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.
bulbousHaBUB Hickok, unpublished
dwarfHaDWF Hickok, unpublished
highly maleHaHIM / him1Gametophytes 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
paleHaPAL Hickok, unpublished
F1 palepal1/PAL1Spores 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.
rumpledHaRUM Hickok, unpublished
pieHaPIEHickok, unpublished

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.


References

Thomas Warne