2.6　 Crosses of mutants defective in the sporophyte formation

Takako Tanahashi

Introduction

Double disruptants of PpLFY1^*1 and PpLFY2^*1 have drastically decreased rates of sporophyte formation (less than 1%), compared to that of the wild type (more than 90%). Most of the leaky sporophytes have aberrant morphology and spores in those rarely germinate. By contrast, gametophores of the double disruptants show no phenotypic difference from those of the wild type. This is a cross method between the wild type and the PpLFY double disruptants utilizing the self-sterile character of the latter. We obtained sporophytes originated from cross-fertilization by wild-type sperm of egg cells of PpLFY disruptants. These are distinguished from other sporophytes formed on the double disruptants by their morphology and the germination rate of the spores they contain.

Our method is especially effective in crosses of mutants defective in sporophyte formation for the following two reasons.

1. The maternal strain is easily identified when crossing two strains with no morphological difference in their gametophores.　

2. Jiffy-7 (medium used, see section 2.3) increases the efficiency of the sporophyte formation.

　(^*1 homologs of FLORICAULA/LEAFY genes in Physcomitrella patens.　 Tanahashi et al. Development 132 1727-1736 (2005))

Procedures

1) Inoculate the wild-type and the double disruptant mosses (protonemata or gamtophores) on a Jiffy-7 pad (3 cm in diameter before expansion).

2) Grow mosses under long day conditions (24L or 16L8D) at 25ºC for 1-1.5 months and then under short day conditions (8L16D) at 15ºC for 3 weeks in separate plastic boxes (Fig. 1A).　 Antheridia and archegonia differentiate on gametophore shoot apices.

3) Check there are archegonia fully open and having uncolored neck canals (Fig. 2).

4) Place the wild-type and the disruptant mosses into a same plastic box (Fig. 1B).

5) Submerge these mosses with distilled water (Fig. 1C).　 Place it with gentle shaking for 30 seconds, and then remove the water above the moss tissue by decanting (Fig. 1D).

6) Continue the culture under short day conditions at 15ºC for another 5 weeks.　 Collect sporophytes formed on the double disruptant gametophores and examine the genotypes of the progeny.

Result (all the values are for colonies of PpLFY double disruptants)

No. Jiffy-7 used.	71
No. gametophores with archegonia	18019
No. sporophytes* formed	141
No. sporophytes analyzed for their genotype	87
No. sporophytes formed by cross-fertilization	5 (normal morphology, 　 germination rate was good)
No. sporophytes formed by self-fertilization 　　　　　　or parthenogenesis	82 (largely abnormal morphology, 　　　germination rate was bad)

Fig. 1 Outline of crosses.

(wt; wild type, dis; PpLFY double disruptant)

Comments

For the present, the most effective way for the success of crossing is just;

1. Growing healthy mosses

2. Performing crosses with proper timing.

Therefore you had better prepare MANY mosses to ensure success!

The following might increase the efficiency of cross-fertilization, though we have never tried this.

1. Repeating submergence several additional times. (If a sporophyte is not formed, new archegonia will differentiate successively on a shoot apex.)

2. Concentrating sperm by centrifugation and applying this close to an archegonium.