Pollen viability

1. Rationale
• Effective pollination enables fruit and seed development,
and hybridization
• Pre-fertilization barrier and incompatibility hinders
successful pollination
• Rice bean (Vigna umbellata) is a warm-season annual
legume and is considered underutilized crop
• Exhibit high degree of resistance to pest and disease
(Pandiyan et al., 2020; Nair et al., 2017; Pratap et al., 2013)
• Has superior traits and nutritional content (Pattanayak et al., 2019;
Bepary et al., 2016; Katoch, 2013)
• Crossability and floral biology has yet been fully explored
among intraspecific crosses of rice bean
• Investigating the pollen biology and mechanism provides
essential knowledge in the development of inbreds and
hybrids with superior quality

2. Objectives
• Evaluate the pollen quality and incompatibility among native accessions of Vigna umbellata (Thunb.)
Ohwi & Ohashi)) to improve production

3. Materials and Methods
Plant materials, experimental design, and crop management
Pollen viability test
Pollen viability = (no. of viable pollen/total no. of pollen) x 100
In vitro pollen germination test
Germination rate = (no. of germinated pollen/total no. of pollen) x
100
In vivo pollen germination test
Germination rate = (no. of germinated pollen/total no. of
pollen) x 100
Data collection and statistical analysis

4. Results and Discussion
Figure 1. Acetocarmine calorimetric assay of pollen grains
in different rice bean (V. umbellata) showing the
germinal pore (arrow): (A) Viable and (B) nonviable
pollen grains using a compound light microscope at
400x total magnification.
1. Pollen viability assay
• V. umbellata pollen is tricolporate
• Viable pollen appears pink while nonviable
is colorless upon staining with
acetocarmine dye

5. Results and Discussion
Figure 2. Pollen viability rate in different rice bean (V.
umbellata) accessions (The same letters indicate
no significant difference among accessions using
Duncan’s multiple range test (DMRT) at 5% level of
significance; P = 0.023)
1. Pollen viability assay
• VU 004 and VU 007 has 10.74 – 48.80%
higher viability rate compare to the other
accessions
• 44.90 + 3.14 % mean viability rate

6. Results and Discussion
2. Optimization of pollen germination medium
(PGM) in rice bean (V. umbellata)
Table 3. Pollen germination rate among rice bean (V. umbellata) accessions in different medium compositions.
TREATMENT
ACCESSION (+ SEM)
Average
Germinati
on
Rate
(+
SEM)
VU 001 VU 002 VU 004 VU 005 VU 006 VU 007
T0 1.18 + 1.8 bc 1.55 + 0.69 c 8.17 + 2.60 a 1.48 + 1.03 c 5.19 + 1.40 bc 8.18 + 1.44 b 4.29 + 5.30 b
T1 9.84 + 1.30 a 7.18 + 1.12 ab 9.59 + 2.60 a 13.65 + 1.02 a 9.76 + 2.34 ab 11.63 + 1.99 ab 10.38 + 5.54 a
T2 9.27 + 1.90 a 11.59 + 1.31 a 12.18 + 1.76 a 8.66 + 0.61 b 11.71 + 1.45 a 15.96 + 2.72 a 11.56 + 5.53 a
T3 4.90 + 1.68 abc 6.94 + 1.52 ab 10.34 + 2.22 a 7.00 + 2.35 b 14.17 + 1.91 a 13.48 + 1.68 ab 9.47 + 6.50 a
T4 5.19 + 2.57 ab 7.27 + 3.13 ab 7.56 + 2.36 a 6.85 + 1.64 b 15.05 + 2.28 a 9.33 + 247 ab 8.54 + 7.68 a
T5 2.68 + 1.52 c 3.68 + 1.94 bc 0.00 b 0.00 c 4.79 + 2.67 bc 12.06 + 4.25 ab 3.87 + 7.70 b
T6 0.00 c 0.64 + 0.64 c 0.00 b 0.00 c 0.00 c 0.00 c 0.11 + 0.79 c
Means within the column having the same letters are not significantly different using Duncan’s multiple range test (DMRT) at 5.0% level of significance.

7. Results and Discussion
3. Effects of boron and myo-inositol on pollen
development in pollen germination media (PGM)
Figure 3. Normal (A) and burst/lysed (B-F) pollen tube and pollen
grains as a response to varying media formulation in rice
bean (V. umbelata) using a compound light microscope at
400x total magnification.
TREATMENT
Average
Germination
Rate (+ SEM)
T0 4.29 + 5.30 b
T1 10.38 + 5.54 a
T2 11.56 + 5.53 a
T3 9.47 + 6.50 a
T4 8.54 + 7.68 a
T5 3.87 + 7.70 b
T6 0.11 + 0.79 c
• 50% increase in B
increases germination
rate by 10.21% (T2)
• 80% (T4) increase in B
decreases germination
by 17.73%
• myo-inositol inhibits
germination by 54.68 to
98.71% (T5-T6)

8. Results and Discussion
4. Effects of stages in flowering in in vitro pollen
germination
a
ab
b
b
a
a
a
b
b
0
5
10
15
20
25
30
35
40
T0 T1 T2 T3 T4 T5 T6
Germination
rate
(%
+
SEM)
Treatments
Bud phase
Anthesis phase
Post-anthesis phase
Figure 5. Pollen germination rate among rice bean (V. umbellata)
accessions in different stages of flowering.
Figure 4. Germinated pollen (pollen tube length is greater than pollen
diameter) in different media during bud phase (A), anthesis (B),
and post-anthesis (C) using a compound light microscope at
400x total magnification.
• T2 has higher germination rate in bud and post-anthesis
pollens
• T4 and T5 have the highest germination rate during the
anthesis phase

9. Results and Discussion
5. Incompatibility in rice bean (V. umbellata)
PARENT COMBINATION
GERMINATION RATE (+
SEM) IN STIGMA
POLLEN
PENETRATION IN A
SINGLE STYLE
Male (Pollen
Source)
Female
(Pollen
Receptor)
Remarks
VU 001 VU 002 46.24 + 11.53 ab +
VU 001 VU 004 0.00 c -
VU 001 VU 005 6.30 + 6.30 c +
VU 001 VU 006 2.29 + 2.29 c -
VU 001 VU 007 3.99 + 3.99 c -
VU 002 VU 001 3.62 + 3.62 c -
VU 002 VU 004 55.12 + 11.35 a +
VU 002 VU 005 51.83 + 5.49 a ++
VU 002 VU 006 43.49 + 2.37 ab ++
VU 002 VU 007 41.75 + 3.27 ab ++
VU 004 VU 001 47.58 + 1.12 ab ++
VU 004 VU 002 42.63 + 9.68 ab +
VU 004 VU 005 42.56 + 6.41 ab +
VU 004 VU 006 34.39 + 6.20 ab +
VU 004 VU 007 54.53 + 6.79 a +++
VU 005 VU 001 48.39 + 7.97 ab ++
PARENT COMBINATION
GERMINATION RATE
(+ SEM) IN STIGMA
POLLEN
PENETRATION IN
A SINGLE STYLE
Male
(Pollen
Source)
Female
(Pollen
Receptor)
Remarks
VU 005 VU 002 0.00 c -
VU 005 VU 004 6.67 + 6.67 c +
VU 005 VU 006 8.23 + 8.23 c ++
VU 005 VU 007 49.60 + 5.45 ab +
VU 006 VU 001 34.18 + 18.77 ab +
VU 006 VU 002 3.24 + 3.24 c +
VU 006 VU 004 0.00 c -
VU 006 VU 005 0.00 c -
VU 006 VU 007 48.87 + 10.10 ab +
VU 007 VU 001 37.98 + 10.04 ab +
VU 007 VU 002 27.81 + 1.11 b +
VU 007 VU 004 50.00 + 1.31 ab +
VU 007 VU 005 5.65 + 5.65 c +
VU 007 VU 006 0.00 c -
Average 26.57 + 2.49 +
Table 4. Cross-compatible pollen-stigma interaction and style penetration in rice bean (V. umbellata)
accessions.
Means within the column having the same letters are not significantly different using Duncan’s multiple range test (DMRT) at 5.0% level of significance. Remark’s symbol:
(+++)10 and above pollen penetration, (++) 7-9 pollen penetration, (+) 4-6 pollen penetration, (+) very rarely 1-3 pollen penetration, and (-) no penetration observed.

10. Results and Discussion
5. Incompatibility in rice bean (V. umbellata)
Figure 6. Normal (A) and swollen (B-D) pollen tube (pointed by an arrow) in
incompatible inter-specific crosses (male x female) of rice bean (V.
umbellata) using a compound light microscope at 400x total magnification.

11. Results and Discussion
5. Incompatibility in rice bean (V. umbellata)
PARENT
COMBINATION GERMINATION
RATE (+ SEM)
IN STIGMA
POLLEN
PENETRATION IN A
SINGLE STYLE
Male
(Pollen
Source)
Female
(Pollen
Receptor)
Remarks
VU 001 VU 001 64.01 + 1.83 +
VU 002 VU 002 47.44 + 4.92 +
VU 004 VU 004 45.33 + 13.32 +
VU 005 VU 005 43.94 + 7.96 -
VU 006 VU 006 54.85 + 10.98 +
VU 007 VU 007 48.98 + 8.67 +
Average 50.76 + 3.45 +
Table 5. Self-compatible pollen-stigma interaction and style
penetration in rice bean (V. umbellata) accessions.
Means within the column having the same letters are not significantly different using
Duncan’s multiple range test (DMRT) at 5.0% level of significance. Remark’s symbol: (+++)
10 and above pollen penetration, (++) 7-9 pollen penetration, (+) 4-6 pollen penetration, (+)
very rarely 1-3 pollen penetration, and (-) no penetration observed.

12. Conclusion
• Accessions VU 004 and V U 007 have the highest viability rate under acetocarmine
• BK medium treated with 0.2 g.l-1 and 0.3 g.l-1 boric acid enhances the germination rate in vitro
• 0.2 g.l-1 boric acid supplementation resulted optimum pollen germination rate during Bud and post-
anthesis phase
• Myo-inositol supplemented media is suitable for pollens during anthesis
• Rice bean (V. umbellata) accessions have lower intraspecific compatibility but have higher self-
compatibility.

13. Materials and Methods
Accession
No.
Date of
Collection
Place of Collection
Indigenous
Crop
Local
Name
Genus Species
VU-001 16-Jul-20 Balao, Barili, Cebu, Philippines Rice bean Tahori Vigna umbellata
VU-002 23-Jul-20
Purok San Jose, Kangdampas, Barili,
Cebu, Philippines
Rice bean Tahori Vigna umbellata
VU-004 23-Jul-20
Sitio Sambag 2, Pangpang, Barili, Cebu,
Philippines
Rice bean Tahori Vigna umbellata
VU-005 18-Aug-20 Poblacion, Barili, Cebu, Philippines Rice bean Tahori Vigna umbellata
VU-006 24-Aug-20 Bubud, Barili, Cebu, Philippines Rice bean Tahori Vigna umbellata
VU-007 24-Aug-20
Kangdampas (Centro), Barili, Cebu,
Philippines
Rice bean Tahori Vigna umbellata
Table 1. Rice bean (V. umbellata) accessions used in the study.
Plant materials, experimental design, and crop management

14. Materials and Methods
Table 2. Pollen germination media (PGM) composition.
In vitro pollen germination test
Treatment Composition
T0
Distilled water (control)
T1
100 g.L-1 sucrose + 0.1 g.L-1 H3BO3 + 0.3 g.L-1 CaNO3.4H2O + 0.2 g.L-1 MgSO4.7H2O + 0.1 g.L-1 KNO3
(Standard BK (1963) medium)
T2 100 g.L-1 sucrose + 0.2 g.L-1 H3BO3 + 0.3 g.L-1 CaNO3.4H2O + 0.2 g.L-1 MgSO4.7H2O + 0.1 g.L-1 KNO3
T3 100 g.L-1 sucrose + 0.3 g.L-1 H3BO3 + 0.3 g.L-1 CaNO3.4H2O + 0.2 g.L-1 MgSO4.7H2O + 0.1 g.L-1 KNO3
T4 100 g.L-1 sucrose + 0.5 g.L-1 H3BO3 (modified Gaaliche et al., 2013 medium)
T5 100 g.L-1 sucrose + 0.5 g.L-1 H3BO3 + 0.1 g.L-1 myo-inositol
T6
100 g.L-1 sucrose + 0.5 g.L-1 H3BO3 + 0.2 g.L-1 myo-inositol

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