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Cereal seeds are characterized by the presence of large persistant endosperm which is thought to affect the embryo size (Hong et al., 1996). During embryogenesis, the differentiation of the root and shoot apical meristems occurs. The size of root and shoot apical meristems influence the postembryonic development. Therefore, embryo size would affect the development of the plant. Rice cultivars vary slightly with the size of the mature embryo with an average 2 mm in length (Hong et al., 1996). Germination and the seedling growth require large amounts of energy that can be provided only by the seed since the germinating seed lack photosynthetic apparatus and mineral uptake system. Hence the seed reserve mobilization is critical for germination. Based on the seed reserves availability and environment different metabolic events occur at different rates in a population of rice seed germinating. Some cultivars express mesocotyl while others do not and also aerobic or anaerobic conditions in which the germination occurs have a great impact on the pace of the development processes.
Rice seeds can germinate under varied environments such as under water or in air. Young seedlings are vulnerable to flooding and are too small to escape this by driving the limited carbon resource involving in shoot elongation. Germination under water influences the growth wherein the coleoptile grows more rapidly expanding to a greater length than in air (Sircar et al., 1955). If the seedling develops in the dark such as seeds sown beneath the soil surface at a greater depth, a short stem called mesocotyl develops. Mesocotyl growth is suppressed under submerged conditions in contrast to the elongated mesocotyl growth in air (Sircar et al., 1955). Turner et al., 1982 has illustrated that cultivars differ in establishment and that mesocotyl and coleoptile lengths contribute distinctly towards the seedling emergence in field under aerobic conditions.
Rice morphological development can be broadly classified into three phases: seedling, vegetative and reproductive. The most common system to express the various growth stages of rice seedling development consists of mainly four stages: Dry seed (S0), radicle and coleoptile emergence (S1, S2) and prophyll (rudimentary leaf) emergence from the coleoptile (S3) (Counce et al., 2000).
3.1.1. Flooding effects on seed germination and developing seedling
Rice coleoptiles show increased growth rates in low concentrations of ethylene which is enhanced by low concentrations of oxygen and carbon dioxide (Ku et al., 1969). Rapid coleoptile elongation occurs in stagnant water saturated soils. Low oxygen concentrations inhibit seed germination and coleoptile growth in submergence-intolerant cultivar while the later growth stages were unaffected, suggesting these processes require high oxygen requirements possibly the cell division than cell extension (Atwell et al., 1982). Cultivars vary in terms of sensitivity to oxygen deficiency (Yamauchi et al., 1993; Turner et al., 1981). Tolerance to low oxygen concentrations is probably due to efficient production of ATP rather than the supply of reserves for growth and respiration, supporting that cultivars vary with metabolic efficiency. Anoxia prolongs the period between seed imbibition and germination at low oxygen levels due to low rates of ATP regeneration, since fermentation does not supply with the energy requirement needs for germination.
Anoxic rice coleoptile growth has been explained by several hypotheses (Masuda et al., 1998) and rice genotypes show variation in anoxic coleoptile extension (Setter et al., 1994). Differences in the coleoptile extension under submergence strongly influences the crop establishment since coleoptiles play a key role in enabling the seedling to come in contact with better aerated environment (Huang et al., 2003). Shoot elongation under submergence is an escape strategy exposing the rice plants to aerobic environment and shift to aerobic metabolism and raise the shoots above water for photosynthetic carbon fixation (Ram et al., 2002; Jackson and Ram, 2003). The rate of shoot elongation under flooding is a genetic feature depending on the genotype and influenced by the submergence environment or the seedling stage before submergence (Kawano et al., 2008). Rapid shoot elongation under submergence has a disadvantage of lodging after de-submergence at the cost of carbohydrate consumption (Voesenek et al., 2006). Carbon assimilation is restricted under flooded conditions by limiting gaseous exchange and irradiation. The gaseous diffusion is very slow because of the unstirred boundary layer around the leaves (Jackson and Ram, 2003). Shoot elongation under submergence is at the expense of limited photosynthetic carbon assimilated under water, raising the question of survival under limited carbon assimilation or recover growth after the post de-submergence and the seedling is brought into aerobic environment. In short term submergence caused by flash floods, rapid shoot elongation affects adversely the submergence tolerance (Jackson and Ram, 2003). Namuco et al., 2009 found genetic variation in early seedling vigor among cultivars. Rice coleoptile length of 25 mm in aerobic and 70 mm under submergence occurs in 3.5 to 5 days has been reported (Wada 1961; Zarra and Masuda, 1979).
3.1.1. Seed Germination
Seed germination is a complicated physiological process wherein many biochemical processes are involved. Germination begins with uptake of significant amounts of water, relative to the seeds dry weight, before cellular metabolism and growth can resume. Imbibition leads to the swelling and the breaking of the seed coat. Hydrolytic enzymes are activated that break down these stored food resources in to metabolically useful chemicals, allowing the cells of the embryo to divide and grow, so the seedling can emerge from the seed. The seed begins to germinate, and the embryonic tissues resume growth, developing towards a seedling provided with favorable environmental conditions such as appropriate water, atmosphere and suitable temperature. Depending on the water uptake, germination can be broadly classified into three stages: Phase I characterized by rapid water uptake, Phase II which is a plateau phase of water uptake and Phase III where growth initiation occurs. Once the seedling starts growing and the food reserves are exhausted, it requires a continuous supply of water, nutrients and light for photosynthesis, which now provides the energy needed for continued growth. While germination either coleoptile or the radicle may be first to emerge. Under dry-seeding, radicle emerges first whilst in water-seeding it is the coleoptile that emerges first, however cultivars vary in this expression where in some show coleoptile emergence before radicle under dry-seeding. Under hypoxia (low oxygen condition) radicle emergence is delayed until the first complete leaf emergence (Counce et al., 2000). Setter et al., 1994 found difference between seed lots and concluded that these growth responses are associated with environment during which the seed set and grain filling occurred and the variations in cultivar behavior under anoxic conditions can be attributed to the differences in their seedling vigor.
Table 3.1.1. Summary of stages during morphological development of the rice seedling
Uptake of significant amount of water resulting in swelling of seed, activation of hydrolytic enzymes allowing the embryo cells to divide and grow resulting in emergence of seedling from seed by breaking the seed coat (pigeon breast stage).
Radicle is covered by a coleorhiza, the first part to grow out of the seed followed by the radicle.
Coleoptile is protective sheath covering the emerging shoot. The coleoptile is pushed through the ground until it reaches the surface where it stops elongation and the first leaves emerge through an opening.
Leaf emergence from coleoptile
After the coleoptile emerges it splits and the primary leaf develops.
3.1.2. Seedling establishment
The appearance of the radicle marks the end of germination and the beginning of establishment, a period that ends when the seedling has exhausted the food reserves stored in the seed. Seedling establishment in the Gramineae involves the process of mobilization of stored reserves in the grain in the production of coleoptile, root and shoot. This process results in an initial loss in total plant biomass due to developmental costs in morphogenesis, principally respiration, until such time when photosynthesis results in a net gain in biomass.
3.1.3. Radicle emergence
In rice the embryo's radicle and cotyledon are covered by a coleorhiza and coleoptile, respectively. The coleorhiza is the first part to grow out of the seed, followed by the radicle.
3.1.4. Coleoptile emergence
Coleoptile is the pointed protective sheath covering the emerging shoot. The coleoptile is then pushed up through the ground until it reaches the surface. There, it stops elongating and the first leaves emerge through an opening as it is.
3.1.5. Leaf emergence
After the coleoptile emerges it splits and the primary leaf develops.
3.1.6. Seedling growth
Germination and seedling development results from mobilization of endosperm and seed reserves representing a cost reflected in loss of total biomass, until subsequent net gain in biomass result from photosynthesis - the onset of autotrophy. This period of transition from seed to seedling will be governed by the environmental conditions affecting seedling growth, particularly anoxia in relation to flooding.
Morphogenesis results in the development of photosynthetically capable leaves once leaf emergence has occurred from the coleoptile and is exposed to photosynthetically active radiation (PAR). The rate of accumulation of seedling biomass will depend upon temperature, the degree of hypoxia and PAR. In light, photosynthesis will accelerate the accumulation of biomass once photosynthetic tissue is established. If the seed develops in the dark such as sown beneath the soil surface, a short stem called mesocotyl develops.
bFigure 3.1. Process of rice seed germination following imbibition (Adapted from Hoshikawa 1975b)
In flooded conditions, coleorhiza hairs as well seminal root hairs appear rarely
Figure 3.2. Development of embryo (Adapted from Hoshikawa 1975b)
Figure 3.3. Process of the growth of rice seedling
Dry seed Imbibed seed
dry seed-edited.jpg Imbibed seed-edited.jpg
Pigeon breasted stage
Plumule emergence Radicle emergencePlumule emergence-edited.jpg Radicle emergence-edited.jpg
3 DAS 4DAS
3DAS(72)1-edited.jpg 4DAS--(72)-edited 65% 2.jpg
5DAS 2nd leaf stage
5DAS--(72)-edited.jpg 2nd leaf stage-edited.jpg
Mesocotyl expression in rice seedling (from literature)Mesocotyl-edited.jpg rice mesocotyl.png
Figure 3.4. Observations during the rice seed germination and seedling establishment. Bar in the figures represent scale =1 mm.
3.1.7. Seed germination under aerobic conditions in dark
Seeds of five cultivars were sown in pots as per mentioned above after 24 h imbibition (Section 2.2, 2.3.1). JI (sieved through 10 mm) was used as the potting material. The pots were wrapped in aluminum jacket and placed in external tanks. Aerobic soil conditions were maintained by retaining the water level half way up in external tanks. The mesocotyl lengths were measured by taking the pictures of the 8 day old seedlings and measuring them as mentioned above (Section 2.3.4.).
Table 3.1.2. Analysis of variance for mesocotyl length in rice cultivars under aerobic conditions in dark
S = 0.9130 R-Sq = 68.38% R-Sq(adj) = 67.48%
Pooled StDev = 0.9130
Figure 3.5. Mean mesocotyl length (mm) (±SEM) of five rice cultivars (8 DAS) grown under aerobic condition in dark
The results (Fig 3.5.) indicate that the rice cultivar IR-72 shows significant mesocotyl expression under aerobic conditions when grown in dark reaching a maximum of 4.2 mm (Table 3.1.7). The mesocotyl length was < 1 mm in IR-64, PSBRC09 and Sabita while in Azucena it was 1.12 mm.
Table 3.1.3. Summary of coleoptile experiments in response to flooding
MEASUREMENT OF COLEOPTILE RESPONSES TO FLOODING
Experiment 3.1.8. immediate deep flooding
Immediate (1 day imbibed then flooding to 50 mm)
population responses and 'survivor' responses measured
Experiment 3.1.9. time of shallow
flooding in 'survivors'
Saturated soil and then flooded
Experiment 3.1.10. depth of flooding at 3 DAS in 'survivors'
3.1.8. Coleoptile response to immediate flooding
Seeds of the cultivars Azucena, IR-72, Sabita and PSBRC09 were sown in glass beakers as per mentioned above after 24 h imbibition (Section 2.2, 2.3.1). Sand was used as the potting material. Seeds were subjected to aerobic and flooding depth of 50 mm immediately after imbibition. Daily destructive sampling of the population responses and survivor count were taken till 6 DAS (in terms of expression of root, leaves and greening of leaves and coleoptile expansion).The coleoptile lengths were measured by taking the pictures of the coleoptiles and measuring them as mentioned above (Section 2.3.4.).
% Mean ± S.E. of seed population
Figure 3.6. Coleoptile responses to aerobic and flooded conditions in rice cultivars representing seeds at different morphological development 1 coleoptile white with no roots, 2 coleoptile with roots, 3 coleoptile greening (leaf out of coleoptile), 4 Seeds germinated and no further development and 5 No germination.
Table 3.1.4. ANOVA for coleoptile lengths among the rice cultivars under aerobic and flooded conditions
Source of variation
Mean response at 6 DAS SE=0.4115
Mean coleoptile lengths in cultivars
Results (Fig 3.6.) indicate there is a significant difference of coleoptile response to immediate flooding. Coleoptile length varies significantly under flooded conditions when compared to aerobic conditions. Maximum coleoptile length was observed in Azucena under flooded conditions with minimum in IR-72. PSBRC09 had a maximum coleoptile length under aerobic conditions while Azucena the minimum coleoptile length.
Figure 3.7. Showing the variation in dissolved oxygen (mg/l) among the cultivars all through the experiment.
% Mean ± S.E. of coleoptile length (mm)
Figure 3.8. Mean coleoptile lengths (mm) (± SEM) among the cultivars under aerobic and flooded treatments. Flooded treatments are towards the right of each pair.
3.1.9. Coleoptile response to time of shallow flooding
Seeds of the cultivars Azucena, IR-72, Sabita and PSBRC09 were sown in petriplates as per mentioned above after 24 h imbibition (Section 2.2, 2.3.1). The seeds were flooded to a depth of 5 mm at different times such as 1, 2, 3 DAS with no flooding (0 mm) as a control treatment. Daily destructive sampling was done by taking the pictures of coleoptiles and measuring them as mentioned above.
The maximum coleoptile length expressed (b) and the time (GDD) required to achieve half of this maximum (c) were estimated by fitting either a symmetric sigmoid (equ 3.1) or asymmetric sigmoid (equ 3.2) response to the observed results. The choice of final response relationship was chosen on goodness of fit and coefficient of determination (R2).
y=a+b/ (1+exp (-(x-c)/d)) Equ 3.1
y=a+b (1-(1+exp ((x+dln (21/e-1)-c)/d))-e) Equ 3.2
Though they are different equations parameters b and c have the same interpretation. An asymmetric sigmoid response is likely to occur under flooded conditions where the coleoptile expansion is delayed.
Figure 3.9. Illustrates the fit of Equ 3.2 for the expression of coleoptiles length over time for cultivar Azucena where flooding was imposed 1 DAS.
Table 3.1.5. Curve fitting for coleoptile responses in Azucena cultivar flooded to 5 mm imposed 1 DAS. Using the following equation:
y=a+b (1-(1+exp ((x+dln (21/e-1)-c)/d))-e)
r2 Coef Det
Sum of Squares
Table 3.1.6. Parameters estimating maximum coleoptile length expressed (b) and the time (GDD) required to achieve half the maximum coleoptile length(c) in the cultivars with respect to time of flooding. S- symmetric and A-asymmetric model fitting.
P (Ho) overall fit
Coleoptile length shows significant differences among the cultivars in response to shallow flooding (5 mm) imposed at 1, 2 and 3 DAS (Fig 3.7.). In Azucena the coleoptile length does not vary significantly with the time of flooding. Flooding imposed at 2 DAS in IR-72 cultivar shows maximum coleoptile length, while coleoptile length did not vary much with flooding imposed at 1 and 3 DAS. Coleoptile length was maximum when flooded was imposed at 2 DAS followed by 3 DAS and 1 DAS in PSBRC09.
3.1.10. Coleoptile response to depth of flooding at 3 DAS
Aerobic germination of four rice cultivar seeds (Azucena, IR-72, Sabita and PSBRC09) was allowed for 3days. Aerobic conditions were maintained by sprinkle irrigation on daily visual basis. 3DAS flooding depths of 5 mm, 40 mm and 0 mm was given to the seedlings. Daily destructive sampling was done to obtain the coleoptile lengths by taking the pictures of the coleoptiles and measuring them as mentioned above. Daily mean temperature were measured periodically through the experimental period.
Figure 3.7. Mean coleoptile length (± SEM) to flooding depths of 0, 5 and 40 mm imposed at 3 DAS in (A) Azucena, (B) IR-72, (C) Sabita and (D) PSBRC09 cultivars.
Different flooding depths imposed at 3 DAS indicate significant coleoptile responses (Fig 3.7.). The coleoptile grows uniformly till the flooding is imposed and thereafter the coleoptile length varies depending on the depth of flooding imposed. Maximum coleoptile length was observed at 40 mm of flooding depth in Azucena cultivar. In cultivars Sabita and PSBRC09 the coleoptile length does not vary significantly at 5 and 40 mm flooding depths, indicating that the coleoptile length does not vary with the depths of flooding imposed.
Table 3.1.7. Coleoptile lengths in response to flooding imposed 3 DAS in the cultivars and the parameters.