2.6　 Crosses of mutants defective in the sporophyte formation

Takako Tanahashi

Introduction

Double disruptants of PpLFY1*1 and PpLFY2*1 have drastically decreased rate of the 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.　 On the other hand, gametophores of the double disruptants have no phenotypic difference from those of the wild type.　 This is a cross method between the wild type and the PpLFY double disruptants utilizing such characters of the latter.　 We obtained some sporophytes originated from cross-fertilization of wild-type sperm and egg cells of PpLFY disruptants.　 They are distinguished from other sporophytes formed on the double disruptants by their morphology and the germination rate of containing spores.

 

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

 

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

 

2. Jiffy-7 (medium used) 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, 3 cm in diameter before expansion.

2) Grow mosses under long day condition (24L or 16L8D) at 25ºC for 1-1.5 months and then under short day condition (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 canal yet (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 gently shaking for 30 seconds, and then remove water above mosses by decantation (Fig. 1D).

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

 

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)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Fig. 2 Development of an archegonium.　 Stage C is proper for the manipulation.

 

Comments

 

We could not obtain higher rate of sporophyte formation of PpLFY double disruptants when performing cross-manipulation than when not performing it.

　　 % sporophyte formation (with cross-manipulation)　　　 0.78% (N = 18019)

　　 % sporophyte formation (without cross-manipulation)　 0.79% (N = 3152)

Apparently, this procedure remains much to be improved.

　

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

1. Growing healthy mosses

2. Performing cross in a proper timing.

Therefore you had better to prepare MANY mosses for your success.

 

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

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

2. Condensing sperms with centrifugation and apply those close to an archegonium.