Fig 1.
Emasculation of Setaria viridis.
(A) Panicles that had just emerged completely out of the flag leaf sheath, such that the lowest spikelets of the panicles were between 0 and 2 cm from the collar of the flag leaf were selected. (B) The spikelets from tip and base of the panicle were trimmed; leaving approximately 50 florets and the bristles were also trimmed. (C) The panicles were completely immersed in the MH solution for 2 min, and excess liquid was gently wiped using tissue paper and (D) then the plant was left to stand until the panicles were dry. (E) After wiping off the solution and drying, the panicles were placed in glassine bags of appropriate sizes. (F) The glassine bag was labeled with the plant designation and emasculation date. Steps D and E were repeated on the same panicle for three consecutive mornings. (G) Panicle, 10 days after pollination.
Fig 2.
Manual emasculation of Setaria viridis.
Equipment needed for manual emasculation included (A) a magnifying glass, (B) glassine bag, scissors, forceps and ear pick. (C) A panicle of correct developmental stage, when the lowest spikelet of the panicle was between 0 to 2 cm from the collar of the flag leaf, was selected for emasculation. (D) The spikelets from the tip and base of the panicle were trimmed leaving approximately 10 to 20 spikelets, and then their bristles were trimmed. (E) One third of each spikelet was removed using sharp scissors and then all three anthers from each spikelet of the panicle were removed using an ear pick and/or forceps. (F) The emasculated panicles were enclosed in glassine bags. (G) On the following day, the spikelets were checked and were dusted with pollen. (H) The pollination was repeated for two days. (I) The emasculated and pollinated panicles were then bagged until seed set. (J) Seed set was checked 10 to 12 days after pollination, if immature seed germination was required. (K) The seeds were allowed to mature and were harvested with care to avoid shattering.
Fig 3.
(A, B) A dehuller was used to remove the seed coats of mature seeds. S. viridis seeds have two layers of seed coats, (C) outer seed coat and (D) inner seed coat. (E) Seeds after removal of both the seed coats look translucent white. (F) The dehulled seeds were washed with 70% ethanol and then with distilled water and were sterilized by incubating in a solution of 50% sodium hypochlorite containing 1 drop of Tween 20 (~2%) for 30 min. (G) The sterilized seeds were dried by blotting on filter paper. (H, I) The seeds were plated in MS medium with the embryo side facing upward. (J) All seeds germinated well in MS medium producing good root and shoot. (K) The seedlings after 10 days of plating were transplanted to the pots with soil and taken special care. (L) Immature panicles 10 to 12 days after pollination were selected for germination in MS medium. (M) Filled spikelets were chosen. (N) Selected spikelets were sterilized in 1% sodium hypochlorite solution with one drop of Tween 20 (~2%). (O) Immature embryos were dissected using sterilized scalpel and forceps, and (P) plated in MS medium. (Q) The immature embryos emerged into a seedling after 18 hrs of incubation in medium. (R) Small seedlings with well developed roots and shoots (10 to 14 days old after plating). (S) Seedlings were transplanted into pots with fine soil and covered with a translucent white cup for 2 days to allow the seedlings to adapt to the soil condition. (T) Seedlings from immature embryos could establish well in soil and (U) grew normally to maturity.
Fig 4.
The effect of different chemical treatments on emasculation of Setaria viridis.
The percentage of seed set in the panicles treated with different methods of emasculation. Ten plants with 50 spikelets per panicle were used for each treatment. Error bars show the standard deviation. All solutions were prepared in water. Room temperature water treatment and no treatment were used as controls.
Table 1.
The number of seed set in maleic hydrazide treated panicles with and without pollination.
Fig 5.
The effect of maleic hydrazide (MH) on the pre-anthesis anthers of Setaria.
(A) Untreated control spikelet and (B) spikelet treated with 500 μM MH. (C and D) Androecium and gynoecium of control and MH-treated spikelets, respectively. (E and F) Anthers of control and MH-treated spikelets, respectively. (G and H) A representative anther of control and MH-treated spikelets, respectively. (I) In control panicles, mature seeds were observed in the spikelets. (J) Brown specks were observed as remnants of anther dehiscence in the MH-treated panicles and no seeds were set although the florets remained green. The scale bar represents 5 mm in A and B, 0.5 mm in C, D, E and F, and 0.75 mm in G and H; I and J are not to scale.
Fig 6.
PCR verification of the crossed progenies using SSR markers.
(A) Products of PCR using an SSR marker on F1 plants and the two parents (A10.1 and P1 202407). (B) PCR using the same SSR primer pair on the F2 plants obtained from self pollination of the F1 (F1-3, shown in Fig 6A), and phenotypes of the representative plants of parents A10.1 (A) and PI 202407 (P). Plants from F2 generation were labeled A-type, P-type and H-type (heterozygous having both A-type and P-type alleles) plants.