Reproductive biology in cassava: stigma receptivity and pollen tube growth

Figures & data

Table 1. Material used in experiments and the experiments sizes. For experiments 4 and 5, the number of pollinations made with different male progenitors is specified within parenthesis.

Female parent	Male parent	Number of flowers	Trait assessed
Experiment 1
MNga 11	Trial 1a: open pollination	483	Number of fruits
SM1219–9		438
Mixture 1^a		417
MNga11	Trial 1b: open pollination	600	Number of fruits and seeds
SM1219–9		600
HMC1		600
Experiment 2
SM1219–9	SM1219-9	30	PTT in 90 ovules
SM1219-9	HMC1	30	PTT in 90 ovules
SM1219-9	Isolation (unpollinated)	10	PTT in 30 ovules
Experiment 3
MNga 11	SM1219-9	70	PTT in 210 ovules per cross
SM1219-9	MCol1505	70
MCol 1505	MNga 11	70
Experiment 4
GM1070-17 GM1423-1 GM1458-18 GM1626-20 GM4371-1 GM4377-42 GM4414-5 GM5202-2 HB60 SM3060-34 SM3112-70 SM2629-36 SM3158-26 SM3690-1	GM4414-5 (1), GM4419-16 (65), GM6182-12 (70), and Unknown (1)	16 2 5 29 11 3 7 14 3 11 6 8 4 18	A total of 137 female flowers from which 411 ovules were available for analysis. However, for a more balanced data from only 324 ovules was used for the analysis.
Experiment 5
GM 3692–76	GM 4385–7 (37) GM 4393–14 (26) GM 5270–34 (39) GM 5270–38 (37) GM 5270–7 (41) MCol 638 (52)	23	A total of 232 female flowers were harvested from which 696 ovules could be extracted. Few samples were lost so only 691 ovules were analyzed
GM 3761–29		6
GM 4385–7		21
GM 4527–2		17
GM 5270–6		24
GM 5270–34		23
GM 5270–37		28
GM 5270–38		24
MCol 1505		22
MNga11		23
SM 1219-9		21

^aMixture of flowers of all remainin genotypes available in the field on the pollination day.

Figure 1. Cassava (Manihot esculenta Cranz). Visualization of pollen tube pathway from stigma to embryo sac. a. Crossection of the cassava pistil (paraffin section 8 µm thick stained with safranin and fast green). b. The same parts of pistil as visible on A shown on preparation of ovules dissected from the pistil and stained with aniline blue, analyzed under UV light. Labels 1–6 show the main checkpoints for pollen tube growth analysis [1]. PTT on the stigma surface or its shallow ingrowth into the transmitting tissue (TT) (a, b1) [2]; PTT in the transmitting tissue of the style (a, b2) [3]; PTT at the bottom of the style (a, b2) [4]; PTT in the obturator (Ob) (a, b2) [5]; PTT in the nucellar beak (Nb), (a, b2); and [6] PTT at the embryo sac (ES), (a, b2). Outer (Oi) and inner (Ii) integuments of the ovules are also visible. Pictures were composed of several images ensemble together. Magnification A, B2 10x, B1 20x (scale bar = 200 μm).

Figure 2. Cassava (Manihot esculenta Cranz) stigmas stained with aniline blue and analyzed with UV, wideband filter. a. Stigma in freshly exposed flower. Stigma’s edges are dark blue. Vascular bundles visible on the right lower corner show pale fluorescence. b. Germinating pollen on the stigma. One pollen tube shallow ingrowth to the stigma is visible in the center. Stigmatic tissue gives bright callose reaction in the vicinity of pollen grains. c. Stigma fixed three days after pollination. Fluorescence of two grains and their tubes has faded. Stigma surface gives strong yellow fluorescence typical for a fading process (scale bar = 100 μm).

Figure 3. Cassava (Manihot esculenta Cranz). Series of pictures from dissected ovules stained at different time points to demonstrate pollen tube passage through the ovule. a. An ovule dissected from a flower at time of bracts opening. Integuments were removed. Nucellus forms large beak (Nb). b-d. Fragments of ovules dissected 18 h after pollination (HAP) showing variability of pollen tubes length. Several tubes are visible on c and d. The dominating pollen tube reaches vicinity of the embryo sac on d. Callose reaction is visible in beak after pollen tubes passage. e. Ovule fixed 42 HAP with pollen tube entering the egg apparatus. f. A pollen tube entered the egg apparatus giving a strong fluorescence in one of the cells. The second pollen tube stopped and swollen at the narrowest part of the beak, so-called pseudomicropyle. Oi – outer integument, Ii – inner integument (scale bar = 100 μm).

Figure 4. Average monthly rainfall in the 1980–2014 period and during the period in which Experiments 3–5 were conducted. Boxes indicate the duration of each experiment. Arrows indicate the range of variation (minimum to maximum) temperatures, respectively, for each experiment. Black circles indicate average temperature along each experiment.

Figure 5. Fruit (a) and seed (b) set observed from female cyathia that were subjected to different treatments: flowers not covered (0 days) or covered for 1, 2 or 3 days after anthesis (horizontal axis). The two independent trials were carried out to assess the duration of stigma receptivity. The vertical axis in the left plot presents the number of fruits per flower counted in the first trial. The vertical axis in the plot on the right presents the actual number of seeds in relation to the expected number based on the total number of flowers considered (three seeds per pistil). SM1219–9, Nga 11, HMC1 are cassava clones, Mixture – represent numerous other clones available in the field when Trial 1a began. AVERAGE represents the mean value of three types of genotype in each trial.

Table 2. Analysis of variance for data from Experiment 2 in which pistils from SM 1219-9 were self- or cross-pollinatinated with HMC1 (30 flowers for each type of cross yielding a total of 180 ovules). Pollinations were made one day before anthesis, on the anthesis day or 1, 2, or 3 days after anthesis (Pollination time). Flowers were collected 1, 2, or 3 days after pollination (Collection time). Response variables were: the number of pollen grains counted on the surface of the stigma and the position of the furthermost pollen tube tip within the pistil.

Source of variation	df	Mean squares^a
		No of pollen grains per stigma	Position of pollen tube tip in the pistil^b
Self pollination vs cross (T)	1	0.568	6.349
Pollination time (P)	4	2.285***	91.125***
Collection time (C)	2	7.340***	7.107**
T*P	4	5.275***	3.946
T*C	2	0.312	3.255
T*P*C	16	1.902***	10.318***
Error	146	0.482	2.313

^a***, **Significant at P < 0.01, and P < 0.05 probability level, respectively.

^bFurthermost position of the pollen tube tip (PTT) was assessed at different anatomical points of the pistil. Score was based on the pistil anatomy as follows: 1 – PTT on the stigma surface or a shallow ingrowth to the transmitting tissue; 2 – PTT in the transmitting tissue of the style; 3 – PTT at the bottom of the style; 4 – PTT in obturator; 5 – PTT in the nucellar beak; and 6 – PTT at the vicinity of the embryo sac.

Table 3. Results from the second experiment. Assesment of pollen grains number on stigma after clearing and staining with aniline blue and the furthermost pollen tube tip position in pistil in samples collected at a different time in relation to anthesis and on a different number of days after pollination. Pooled data of SM 1219–1 crossed with HMC-1 or self-pollinated. A total of 60 flowers (180 ovules) were analyzed^a.

Pollination of stigma in relation to anthesis (days)	Collection time after pollination (hours)	Pollen grains number on stigma after aniline blue staining^b	Position in pistil of the furthermost pollen tube tip^c
One day before anthesis (-1)	24	1.25e	2.33cde
	48	2.67ab	3.67bc
	72	3.00a	5.25a
Anthesis day (0)	24	2.58abc	4.50ab
	48	2.50abc	5.25a
	72	2.75ab	4.83ab
One day after anthesis (1)	24	1.58de	2.00def
	48	1.55de	1.64ef
	72	2.75ab	1.08efg
Two days after anthesis (2)	24	1.58de	0.92fg
	48	2.50abc	0.08g
	72	2.00cd	2.08def
Three days after anthesis (3)	24	2.25bc	3.08cd
	48	2.25bc	1.33efg
	72	2.27bc	2.00def
-1	Averaged across all collection times in relation to pollination day	2.26b	3.75b
0		2.61a	4.86a
1		1.97b	1.57cd
2		2.03b	1.03d
3		2.26b	2.14c
Averaged across all pollination times in relation to anthesis day	24	1.85b	2.57ab
	48	2.3a	2.40b
	72	2.5a	3.07a

^aFor each section of the Table, values with the same letter within each column were not statistically different at P < 0.05, based on the LSD test.

^bScoring for pollen grain number was as follows: 1 – No pollen grains; 2 – from 1 to 5 grains; 3 – from 6 to 24 grains; and 4 – 25 or more pollen grains.

^cAverage position of the furthermost pollen tube tip (PTT) was assessed at different anatomical points of the pistil. Score was based on the pistil anatomy as follows: 1-PTT at the stigma surface or its shallow growth into the transmitting tissue; 2-PTT in the transmitting tissue of the style; 3-PTT at the bottom of the style; 4-PTT in the obturator; 5-PTT in the nucellar beak; and 6-PTT at the vicinity of the embryo sac.

Table 4. Relevant results from Experiment 3. Analysis of variance and proportion for the presence of pollen tube growth ^(a) after controlled pollinations in three cassava female parents: SM 1219-9, MNga 11 and MCol 1505. A total of 210 pistils (630 ovules) were analyzed. The source of pollen was the same three genotypes. Pollinated pistils were harvested at seven different times ranging from 6 to 74 h after pollination (HAP).

		Nucellar beak		Micropylar	Embryo
Source of variation	df	Proportion^(a)	depth^(b)	region^(a)	Sac^(a)
Analysis of variance
Female parent (F)	2	2.752***	3.172***	2.232***	0.334***
Collection time (C)	6	2.470***	3.333***	7.856***	0.848***
F*C	12	0.624***	0.374***	0.475***	0.272***
Error	605	0.205	0.147	0.126	0.047
Average for each female parent^(c)
SM1219-9		0.707a	0.600a	0.428a	0.096a
MNga 11		0.569b	0.440b	0.254b	0.019b
MCol 1505		0.478c	0.357c	0.244b	0.077a
Average pollen tube growth for samples collected at different HAP^(d)
6 HAP		0.449b	0.171d	0.000f	0.000d
13 HAP		0.822a	0.535b	0.000f	0.000d
18 HAP		0.511b	0.378c	0.122e	0.000d
26 HAP		0.433b	0.362c	0.267d	0.022cd
42 HAP		0.433b	0.417c	0.400c	0.067bc
66 HAP		0.693a	0.668a	0.636b	0.091b
74 HAP		0.753a	0.734a	0.742a	0.270a
Least square means for stigma/style quality (only 74 HAP)^(e)
Fresh		0.768a	0.748a	0.753a	0.301a
Deteriorated		0.645a	0.634a	0.661a	0.066b

*** Significant at P < 0.01 probability level.

^(a)Position of the furthermost pollen tube tip (PTT) was assessed in the pistil and score was based on the pistil anatomy: nucellar beak; PTT the micropylar region of the beak; and PTT in vicinity of the embryo sac. These variables relate to the proportion of pollen tubes at each of these milestones for pollen tube growth.

^(b)Proportion of the total length of the nucellar beak covered, on average, by the growth of the pollen tubes.

^(c)(d)(e) For each section of the Table, values with the same letter within each column were not statistically different at P < 0.05 (based on the LSD test).

Table 5. Analysis of variance and relevant averages for pollen tube growth in controlled pollinations in 14 cassava female parents in Experiment 4. A total of 108 pistils (324 ovules) were analyzed in this study. Pollinations were made using three male progenitors. In one case the tag identifying the source of pollen was missing. Pistils were harvested at four different times ranging from 16 to 96 h after pollination (HAP).

		Nucellar beak
Source of variation	df	Proportion^(1)	Depth^(2)	Micropylar region^(1)	Embryo sac^(1)
Analysis of variance
Female parent (M)	13	0.672***	0.529***	0.428***	0.119
Hours (H)	3	0.475*	0.170	0.402**	0.160
M*H	26	0.486***	0.374***	0.305***	0.104
Error	280	0.186	0.148	0.145	0.083
Average pollen tube growth for samples collected at different HAP^(3)
16 HAP		0.413ab	0.263b	0.100c	0.025b
48 HAP		0.333 bc	0.307ab	0.247ab	0.148a
72 HAP		0.247c	0.225b	0.173bc	0.062b
96 HAP		0.469a	0.423a	0.358a	0.148a
Least square means for stigma/style quality (96 HAP)^(4)
Fresh		0.438a	0.388a	0.335a	0.143a
Deteriorated		0.631a	0.377a	0.000b	0.016a

***, **, * Significant at P < 0.01, P < 0.05 and P < 0.10 probability level, respectively

⁽¹⁾Position of the furthermost pollen tube tip (PTT) was assessed in the pistil and score was based on the pistil anatomy: nucellar beak; PTT the micropylar region of the beak; and PTT in vicinity of the embryo sac. These variables relate to the proportion of pollen tubes at each of these milestones for pollen tube growth.

⁽²⁾Proportion of the total length of the nucellar beak covered, on average, by the growth of the pollen tubes.

⁽³⁾⁽⁴⁾For each section of the Table values with the same letter within each column were not statistically different at P < 0.05 (based on the LSD test).

Table 6. Analysis of variance and relevant averages for pollen tube growth in controlled pollinations in crosses between 11 female and 6 male cassava progenitors in Experiment 5. A total of 210 pistils (630 ovules) were analyzed in this study. Pistils were harvested at 72 or 96 h after pollination (HAP).

		Nucellar beak
Source of variation	df	Proportion^(1)	depth^(2)	Micropylar region^(1)	Embryo sac^(1)
Analysis of variance
Female (F)	10	5.383***	5.406***	5.232***	2.117***
Male (M)	5	3.700***	2.583***	3.340***	0.476***
Hours (H)	1	0.020	0.062	0.032	0.163
F*H	10	0.251*	0.376***	0.322**	0.316***
M*F*H	75	0.359***	0.391***	0.371***	0.217***
Error	589	0.120	0.117	0.123	0.115
Average pollen tube growth in samples collected at different HAP^(3)
72 HAP		0.495a	0.477a	0.479a	0.226a
96 HAP		0.487a	0.452a	0.469a	0.181a
Least square means for stigma/style quality (96 HAP)
Fresh		0.500a	0.486a	0.486a	0.192a
Deteriorated		0.368b**	0.340b***	0.312b***	0.188b*

***, **, *Significant at P < 0.01, P < 0.05 and P < 0.10 probability level, respectively

⁽¹⁾Position of the furthermost pollen tube tip (PTT) was assessed in the pistil and score was based on the pistil anatomy: nucellar beak; PTT the micropylar region of the beak; and PTT in vicinity of the embryo sac. These variables relate to the proportion of pollen tubes at each of these milestones for pollen tube growth.

⁽²⁾Proportion of the total length of the nucellar beak covered, on average, by the growth of the pollen tubes.

⁽³⁾Values with the same letter within each column were not statistically different at P < 0.05 (based on the LSD test).

Table 7. Average depth of pollen tube growth in the pistils of 11 female cassava progenitors and 6 male progenitors (across two harvesting times: 72 and 96 h after pollination) from Experiment 6.

		Nucellar beak
Genotype	n	Proportion^(1)	Depth^(2)	Micropylar region^(1)	Embryo sac^(1)
Mother^(3)
GM 3692-76	69	0.884a	0.884a	0.884a	0.580a
SM 1219-9	62	0.839ab	0.830ab	0.806ab	0.452ab
GM 3761-29	18	0.778ab	0.778ab	0.778ab	0.333bc
MCol 1505	66	0.712bc	0.692bc	0.682bc	0.212cd
MNga11	69	0.594cd	0.572cd	0.580cd	0.188de
GM 5270-37	82	0.512de	0.390ef	0.512de	0.073ef
GM 4527-2	51	0.510de	0.466de	0.431ef	0.196de
GM 5270-34	69	0.406ef	0.396ef	0.391ef	0.159de
GM 5270-38	70	0.329f	0.288f	0.300f	0.157de
GM 5270-6	72	0.042g	0.042g	0.042g	0.000f
GM 4385-7	63	0.032g	0.032g	0.032g	0.000f
Father^(4)
MCol 638	154	0.636a	0.588ab	0.610a	0.162a
GM 4393-14	78	0.615a	0.515ab	0.590a	0.218a
GM 5270-38	111	0.477bc	0.474bcd	0.468bc	0.234a
GM 4385-7	110	0.427bc	0.423bcd	0.418bc	0.227a
GM 5270-7	125	0.416bc	0.39cd	0.384bc	0.176a
GM 5270-34	113	0.363c	0.363d	0.36c	0.212a

⁽¹⁾ Position of the furthermost pollen tube tip (PTT) was assessed in the pistil and score was based on the pistil anatomy: nucellar beak; PTT the micropylar region of the beak; and PTT in vicinity of the embryo sac. These variables relate to the proportion of pollen tubes at each of these milestones for pollen tube growth.

⁽²⁾ Proportion of the total length of the nucellar beak covered, on average, by the growth of the pollen tubes.

⁽³⁾⁽⁴⁾ For each section of the Table values with the same letter within each column were not statistically different at P < 0.05 (based on the LSD test).

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