Ⅲ-4. Radiation Chemistry & Biology

RIKEN Accel. Prog. Rep. 49 (2016)

Sexualreproduction
reproduction observed
observed inSexualin thethe loss-of-apomixis
loss-of-apomixismutants
mutants ofof guineagrass
guineagrass
induced by heavy-ion beam
beam irradiationinducedirradiation
M. Takahara,＊1 R. Morita,＊2 Y. Kazama,＊2 T. Abe,＊2 T. Takamizo,＊1 and H. Nakagawa＊1, ＊3
Apomixis, an asexual mode of reproduction, provides amethod for reproducing clonal plants through the seed. By
using apomixis in the breeding program, we can develop a
new efficient method for fixing desirable genotypes of F1
hybrids and simplifying commercial seed production.
Therefore, we aimed to isolate the gene(s) controlling
apomixis from a tropical forage grass, guineagrass(Panicum maximum Jacq.). In a previous study, we found
two mutant lines (SM-1 and SM-2) that were suggested tohave lost the apomictic pathway of reproduction and to
propagate using the sexual mode of reproduction.1), 2) In thepresent study, we analyzed the mode of reproduction ofthese loss-of-apomixis mutants with two methods: theprogeny test using amplified fragment length polymorphism(AFLP) patterns and embryo sac analysis. The results
showed that they lost the apomictic mode of reproduction.
The M1 plants of SM-1 and SM-2 were generated fromthe dry seeds of an apomictic cultivar “Natsukaze”
irradiated with 20Ne10+ (63 keV/μm) ions at 200 Gy andwith 56Fe24+ (624 keV/μm) ions at 20 Gy, respectively.1)
Forty M2 plants of each line were grown in a field. DNA
from the leaves of each M2 plant was extracted andanalyzed using the AFLP method. For the embryo sacanalysis, inflorescences at anthesis were fixed in Farmer’s
fixative (100% ethanol:acetic acid = 3:1), and thendehydrated and cleared as described previously.3) The
cleared pistils were observed with a differential interference
contrast microscope (Nomarski).
The AFLP patterns of 40 M2 plants are shown in Fig.1. Incase of the apomictic cultivar “Natsukaze”, the AFLP
results show the same pattern among progenies. In contrast,
all the AFLP patterns of M2 plants of both SM-1 and SM-2were different from each other. This result suggested thatSM-1 and SM-2 propagated with the sexual mode ofreproduction.
To confirm the sexual mode of reproduction of SM-1 andSM-2, embryo sac analysis was performed. In the case ofguineagrass, an apomictic embryo sac has four nuclei (anegg, two synergids, and a polar nucleus; Fig. 2A), while asexual embryo sac has eight nuclei (an egg, two synergids,
two polar, and three antipodal cells’ nuclei; Fig. 2B). The
most clear differences between the sexual and apomictic
embryo sacs are whether antipodal cells are formed or not,and whether the number of polar nuclei is two or one. 3) In
National Agriculture and Food Research Organization (NARO)
Institute of Livestock and Grassland Science
RIKEN Nishina Center＊3
Hamamatsu Photonics K. K.

the embryo sac analysis of SM-1 and SM-2, we observed afew sexual embryo sacs (Fig. 2B), many aborted embryosacs (Fig. 2C), and no apomictic embryo sac in each line.
These results suggested that they lost the apomictic pathwayof reproduction.
Our previous results suggested that the deleted region inthe loss-of-apomixis mutants were relatively small.2) We are
currently searching for the genes located within thesedeletion regions.

Fig. 1. AFLP patterns of M2 plants in the sexual mutant line
SM-2. (A) The AFLP pattern of “Natsukaze” detected by theABI 3130xl capillary sequencer (as a reference). Red
arrowheads indicate the loci that are polymorphic among M2
plants. (B) Schematic presentation of AFLP patterns of M2
plants in SM-2. White box shows the presence of the peak, andgray box shows the absence of the peak. Similar results havebeen obtained for SM-1 (not shown).

Fig. 2. Typical embryo sacs in guineagrass. (A) Apomicticembryo sac observed in “Natsukaze” (apomictic cultivar). (Band C) Sexual embryo sac (B) and aborted ovule (C) observedin the loss-of-apomixis mutants. a, antipodal cell; e, egg cell;p, polar nuclei. Synergid cells are small and difficult toobserve in both types.3) Bar = 200 µm.

＊1＊2

References

1) M. Takahara et al.: RIKEN Accel. Prog. Rep. 46, 263 (2013).
2) M. Takahara et al.: RIKEN Accel. Prog. Rep. 47, 294 (2014).
3) H. Nakagawa: JARQ 24, 163-168 (1990).

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