Developmental Morphogenesis Of Rice Seedling Biology Essay

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The developmental morphogenesis of a rice plant can be broadly classified into three phases: seedling, vegetative and reproductive. Seedling establishment is normally defined as the development of autotrophic seedling by utilizing the stored seed reserves. 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). The physiological processes involved in the transition from dry seed to the point of an established seedling involve seed germination, meristematic expression, cell division and organization leading to sequential and programmed tissue development. Organogenesis in the Gramineae is varied (ref) and particularly so amongst rice cultivars. Moreover the sequence of organ development is noticeably determined by gaseous concentrations typically by flooding regimes.

3.2. Seed and embryo size in relation to seedling establishment

Although seed weight provides an indication of carbohydrate reserves available, the rate at which these are mobilized is possibly influenced by the characteristics of the meristematic zone or embryo, by enzyme activity (Sung and Chen, 1988). Seed reserve mobilization might have substantial impact on early emergence. Differential rates of enzyme activity could be implicated in the differences in seed reserve mobilization rates. Other factors, too, may affect seedling growth.

3.3. Flooding effects on coleoptile growth and the developing seedling

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Young seedlings are vulnerable to flooding and are too small to escape this by driving the limited carbon resource involving in shoot elongation. Rice coleoptiles have a unique growing pattern which attracted many research studies on coleoptile expansion in response to environment. Rice coleoptiles elongate fast reaching a greater length under water (70 mm) than in air (25 mm) in 3.5 to 5 days (Wada 1961; Zarra and Masuda, 1979; Kamisaka et al., 1991). Root formation is delayed when seed germination occurs under water (Kutschera et al., 1990). 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). Coleoptile elongation in water (air bubbled) is slow compared to stagnant water but greater than the growth in air (Pjon and Furuya, 1974; Zarra and Masuda, 1979). Coleoptile growth under submerged conditions is due to cell elongation since the cell division ceases in 60 hr after sowing and further growth is resulting from the existing cell elongation (Wada, 1961a). 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). Coleoptile elongation is enhanced by low concentrations of oxygen (Ohwaki 1967; Alpi and Beevers, 1983). 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 meet the energy requirement necessary 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). Inhibition of leaf growth, suppression of root expression and coleoptile elongation are as a result of accumulation of carbon dioxide, ethylene and oxygen depletion under water and coleoptile expansion is due to water absorption (Raskin and Kende 1983). 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 when 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.

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Coleoptile elongation is initiates by two mechanisms; one that initiates the growth in all coleoptiles while the second initiates rapid growth at different times in different coleoptiles of barley (Liptay and Davidson, 1971). Coleoptile expansion above the water surface as a result of earlier submergence ensures that the seedling is exposed to aerobic conditions. This helps in adequate supply of oxygen and carbon assimilation supporting more energy producing aerobic respiration and photosynthesis rather than anaerobic fermentation under flooded conditions. Coleoptile extension under flooded conditions depends on photosynthesis which is limited due to reduced availability of light and gaseous. Differences in coleoptile lengths influence the seedling establishment. Though the biochemical mechanisms underlying the coleoptile responses to flooding have been studied for many years, it is still unclear about the factors involved in such responses. Gene expression studies may determine the role of genes that are determining the coleoptile responses to flooding. Irrespective of the flooding depths, it is observed that the coleoptile reach the maximum length that can be supported by the seedling which in turn depends on the seed reserves and is a genetic character. Once the seedling is independent (autotrophic) and established, the energy involved in coleoptile length can be diverted for seedling growth and there energy involved in coleoptile elongation can be saved for the survival of the seedlings. Existence of variation in growth rate and time of active growth in different coleoptiles of barley has been identified (Liptay and Davidson, 1971). Liptay and Davidson, (1971) concluded that coleoptiles of different seedlings are at different physiological states, coleoptile elongation initiated within hours after seeds begin water uptake but not always immediately followed by rapid growth. In addition to the processes resulting in initial coleoptile growth there are subsequent events promoting rapid growth. Physical factors are reasonably uniform in all experiments for all seeds and it appears that the observed variations in growth and development reflect variation in some internal factor(s). The growth pattern observed during germination may be induced in seeds during grain-filling or ripening. Initial rapid growth requires small volumes of water (10-12 ml) and large volumes (15-25 ml) inhibit growth in barley (Liptay and Davidson, 1971). The large variations in groups of germinating seeds are because of metabolism and hormones in the embryos of barley (Liptay and Davidson, 1971).

Rice coleoptile growth was rapid between day 2 and day 4 after sowing under flooding but decreased after day 4 whilst the aerobic coleoptile growth remained relatively steady and slows (Kamisaka et al., 1993). Changes in the cell wall properties like cell wall extensibility, amounts of diferulic acid suggest a role behind rapid coleoptile growth under water. Decrease in diferulic acids bound to cell wall leading to formation of diferulic acid bridges in hemicelluloses making the cell wall rigid mechanically thereby inhibiting cell elongation in coleoptile grown aerobically (Fry, 1979). Low concentrations of ethylene, oxygen and carbon dioxide increase growth rate of rice coleoptiles, promotion of rice coleoptile elongation is due to synergistic action of carbon dioxide and ethylene. Optimum oxygen concentration required for coleoptile growth is about 3-3.5 % (Ku et al., 1970). Rice coleoptile elongates proportionally with the depth of water under submerged conditions (Yamada, 1954 and Kefford, 1962). Ku et al., 1970 suggested submergence reduces diffusion of endogenous ethylene from the coleoptile resulting in increased efficiency of gas stimulated coleoptile elongation. Coleoptiles grow exclusively by cell elongation (Wada, 1961) and responds to environmental signals (Chaban et al., 2003). Auxin, plant hormone plays a major role in growth and elongation of coleoptile. It is produced mainly in the tip and moves basipetally. Auxins have crucial role in signal dependent growth regulation (Went, 1928). Auxin related growth mechanism is complex and are still far from being understood. Auxin induced cytochrome P450 gene CYP87A3 is only expressed in roots and coleoptiles, but not in leaves (Chaban et al., 2003).

Seedling establishment varies widely among genotypes at low temperatures (Sasaki and Yamasaki, 1971; Ikehashi, 1973; Jones and Peterson, 1976; Li and Rutgar, 1980; Kotaka and Abe, 1988; Kowata et al., 1992; Amano et al., 1993; Redona and Mackill, 1996; Inoue et al., 1997). Kotaka and Abe (1988) found poor seedling establishment in several low land and some upland rice cultivars inspite of rapid germination. Sasaki and Yamasaki (1971) and Kotaka and Abe (1988) reported faster geminating varieties exhibit better seedling establishment. Tanaka and Yamazaki (1989) identified the importance of coleoptile growth for seedling establishment, which is severely hampered if supplies with cold water during coleoptile elongation phase rather than during germination. Coleoptile is an essential organ to absorb oxygen from air or water enriched with dissolved oxygen (Kordan, 1977). Oxygen uptake is necessary for further seedling growth. It can be inferred that faster coleoptile elongation in a cultivar contributes towards its survival under submergence through faster transition from anaerobic to aerobic condition. First leaf and radicle appear after enough oxygen is supplied through the coleoptile (Kordan, 1972; Kordan, 1974; Alpi and Beevers, 1983; Alpi et al., 1985; Setter et al., 1994). Aerobic respiration in mitochondria resumes upon receiving enough oxygen (Shibasaka and Tsuji, 1988).

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Shoot elongation under flooding is controlled by interacting hormones such as ethylene, ABA, GA and auxin (Jackson, 2008). ABA decline is seen in submerged leaves (Ram et al., 2002). Kawano et al., 2008 observed negative correlation between the dry matter of leaves developed before submergence and during submergence.

Anaerobic treatment for 4 hours resulted in high fermentation, 40 % decline in carbohydrate in roots and shoots of rice. Shoots show 20 and four times more ethanol fermentation in rice and wheat respectively indicating more efficient fermentative metabolism in rice (Mustroph et al., 2006). Post- anoxic production of acetaldehyde is low suggesting more efficient detoxification of acetaldehyde. Faster rates of fermentation in shoots and suppression of toxic acetaldehyde formation is observed after the seedlings were re-aerated. Sugar and starch metabolism varies in rice compared to wheat and barley (Guglielminetti et al., 1995, 1997; Perata et al., 1996, 1998). Ethanol fermentation is comparable in these three cereals only during early anoxia because of the existence of the soluble sugars allowing fermentation to proceed. But rice continues fermentation actively for several days (Guglielminetti et al., 2001). High fermentation rates, high soluble carbohydrate and optimized ATP utilization under anoxia may explain the success of rice compared to intolerant cereal crops (Mustroph et al., 2006). Setter et al., (1994) showed a poor relationship between coleoptile growth and survival of seedlings during 7 day aeration after anoxia. Carbohydrate concentrations differed among the cultivars under anoxia treatment in 4 day old coleoptiles suggesting difference rates of metabolism existence among the coleoptiles (Setter et al., 1994).

3.4. Seed Germination

Rice exhibits hypogeal germination which is initiated under aerobic conditions. In hypogeal germination the cotyledons of the germinating seed remain non-photosynthetic and are retained inside the seed shell and below the ground. 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 seed inflation resulting in the breaking of the seed coat. Hydrolytic enzymes are activated that break down the stored seed reserves into metabolically useful products that allow the cells of the embryo to divide and grow, and seedling morphogenesis can take place. Germination can be broadly classified into three stages (Takane Matsuo et al., 1995). Phase I is characterized by a metabolically passive state, rapid water uptake and extremely low oxygen consumption. Phase II is an active stage with a plateau phase in water uptake, and where substantial metabolic activities are initiated and increased oxygen consumption occurs. This phase is characterized by hydrolysis of seed reserves, rapid carbohydrate metabolism and new cell materials are synthesized. In Phase III cell division occurs in both the plumule and radicle accelerating their development. Once the stored seed reserves are exhausted, further growth and development is supported by the growing seedling that requires continuous supply of nutrients, water and light for carbon assimilation.

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 radicle emergence is delayed until the first complete leaf emergence (Counce et al., 2000). Setter et al., 1994 found difference between cultivar seed lots and concluded that the relationship between coleoptile elongation and alcohol fermentation under anoxia 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. The ability to achieve the autotrophic state depends on the extent of exposure to anoxia which in turn critically depends on the depth and duration of flooding exposure. Coleoptile elongation under flooding drives its energy requirement from the fermentative pathway, but the seed reserves in the embryo is the limiting factor that allows the energy providing pathway to proceed for a limited period that in turn sets the limit on coleoptile expansion (Perata and Alpi, 1993). Reduced partial pressure of oxygen is the primary signal for enhanced elongation under submergence.

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. 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. 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 semi-dwarf cultivar seeds drilled into soil emerge slowly compared to tall stature plants, cultivars differ in establishment and that mesocotyl and coleoptile lengths contribute distinctly towards the seedling emergence in field under aerobic conditions.

Rice seeds undergo fermentation during the first 48 hr of sowing irrespective of aerobic and anaerobic environment, indicating the dry matter changes is not as a result of the environment (Tsuji 1968). Carbohydrate metabolism is seedlings under anoxia can be divided into two phases; (1) on the day of germination it is primarily the sugar degradation whereas after the α-amylase induction it is both starch degradation and sucrose synthesis (Guglielminetti et al., 1995).

The seed respiration is accelerated sharply with the start of water uptake in dark than in light indicating early initiation of seed reserve mobilization. Continuous supply of substrates such as carbohydrates and amino acids is essential for further growth of plumule and radicle. Protein synthesis is pre-requisite for normal growth at germination. Tissue differentiation and growth of the growing ends occur simultaneously along with protein and DNA synthesis that require the newly synthesized RNA. These processes occur irrespective of the environment in which the seed germination proceeds i.e. under water or in air.

In the early stage of germination, the plant hormone gibberellic acids (GAs) are synthesized in the embryo, penetrate through the scutellar epithelium, diffuse to the aleurone layers, and induce the de novo synthesis and secretion of α-amylase proteins in scutellum and aleurone cells. β-amylase is expected to be important in a synergistic role for starch degradation, and is synthesized de novo in aleurone cells for which GAs are not required.

Seed germination and early seedling growth are dependent upon the enzymatic hydrolysis of the starchy endosperm into metabolizable sugars. During the early stage of germination, α-amylase is actively synthesized and secreted into the endosperm. Although many enzymes are involved in the germination process, α-amylase is primarily responsible for the endoglycolytic cleavage of amylose and amylopectin. During germination, -amylase synthesis under anoxia allows rice to degrade the starchy reserves present in the endosperm (Atwell and Greenway, 1987: Perata et al., 1992), thus obtaining readily-fermentable carbohydrates providing ATP for the germinating embryo, and this contributes to rice tolerance to anaerobic conditions (Perata et al., 1992). In cereal seeds, the synthesis of -amylase is controlled, at the transcriptional level, by GA (Akazawa, Mitsui and Hayashi, 1988).

Little information is available concerning the anaerobic fate of the soluble carbohydrates either originally present in the dry seed cereals or resulting from starch degradation during imbibition and germination. Mayne and Kenede (1986) found that rice seedlings aerobically germinated and then subsequently transferred to anaerobic conditions were able to metabolize glucose at a rate similar to that of the tissue incubated under aerobic conditions, indicating that anoxia does not interfere with the potential for glucose metabolism in rice.

3.5. Aims

The main aim of this chapter was to scrutinize the processes and variability that occurs during the developmental morphogenesis during rice seed germination and seedling development. Secondly to investigate the variations in cultivars with respect to mesocotyl and coleoptile development.

3.6. Observations on germination and seedling development

3.6.1. Aerobic conditions

Bud scale

Ventral scaleTime Scale Dry seed Vertical section of the

developing embryo

Embryo before water absorption

0 DAS dry seed-edited.jpg img022.jpg img017.jpg

Imbibed seed

0 DAS Imbibed seed-edited.jpg img022.jpg

Pigeon breasted stage

0 DAS PB.jpg img024-1.JPGDSCN1255.JPG

Coleoptile Plumule emergence

Seminal root

1DAS Plumule emergence-edited.jpg Picture3.png

Radicle emergence

2 DAS Radicle emergence-edited.jpg

Figure 3.1. Process of the seed germination as observed (left corner), found in the literature adapted from Hoshikawa 1975 (middle column, not to be scaled) and cross section (right corner). Bar in the figures represent scale =1 mm. Note some cultivars vary in the expression of radicle or plumule first to emerge.

Seedling development following seed imbibition during the early 40 GDD. During this period cell division occurs in both plumule and radicle accelerating their development. The embryo's radicle and cotyledon are covered by coleorhiza and coleoptile respectively. The coleorhiza is the first part to grow out followed by radicle that later becomes seminal root.

3 DAS

3DAS(72)1-edited.jpg

4DAS

Leaf emergence out of coleoptile (not to be scaled)4DAS--(72)-edited 65% 2.jpg img028.jpg

5DAS

Structure of shoot (Hoshikawa 1975) (not to be scaled)5DAS--(72)-edited.jpg

Figure 3.2 Developmental morphogenesis during leaf emergence during seedling establishment. Observations taken as pictures towards the left column corresponding evidence from literature on right column. Bar in the figures represent scale =1 mm.

Morphogenesis of the developing seedling during 60-100 GDD during which seminal root establishes and leaf emergence from the coleoptile occurs. Coleoptile is cylindrical and sheath-shaped with a cone- shaped top. It consists of morphologically underdeveloped stomata and lacks photosynthetic tissue. Coleoptile is the pointed protective sheath covering the emerging shoot. The coleoptile is then pushed up through the soil until it reaches the surface. After germination, cells in coleoptile grow lengthwise. Coleoptile growth ceases at a length of 1-2 cm under aerobic conditions. However it grows longer in conditions short of oxygen. Upon cessation of coleoptile growth, the first leaf emerges from inside by breaking the coleoptile lengthwise near the ventral side.

2nd leaf stage

First and second leaves also start to become gradually bigger with the seminal roots growing downward (Not to be scaled)2nd leaf stage-edited.jpg

Mesocotyl

MesocotylMesocotyl-edited.jpg

Figure 3.3. Observations during the rice seed germination and seedling establishment. Mesocotyl expression in the seedling was observed under dark conditions. Bar in the figures represent scale =1 mm.

The region between the coleoptile and the basal part of seminal root called mesocotyl grows under dark conditions. In indica-type rice the mesocotyl length varies from 5-80 mm while in japonica-type usually 2-5 mm with a maximum of 50 mm in some.

3.6.2. Anoxic conditions

3 DAS Aerobic Flooded

First leaf Aero 3 DAS-X1.jpg Flood 3 DAS-X.jpg

4 DAS

Aero 4 DAS-X.jpg Flood 4 DAS-X.jpg

5 DAS

Leaf length varies in flooded conditions among the same seed lot.Aero 5 DAS-X.jpg Flood 5 DAS-X.jpg

Figure 3.4. Microscopic observations of IR-72 seedling response to flooding. Note the leaf development variations seen under aerobic and flooded conditions (indicated by arrows in the pictures). The leaf length varies in a given population exposed to similar environmental conditions in the same cultivar.

Coleoptile elongation under flooded conditions during 60-100 GDD. Coleoptile elongation is because of the expansion of the cells but not as a result of increased cell division (Section 3.3). Leaf growth in rendered until the coleoptile comes in contact with aerobic environment. Leaf length inside the coleoptile differs among the given seed sample within the same cultivar showing existence of variability.

3.6.3. Coleoptile response to different depths of flooding

The aim of this experiment was to understand the behavior of the coleoptile as a response to flooding. Rice cultivars Azucena, IR-72 and PSBRC09 were grown initially under aerobic conditions for 3 days and later flooded to 50 and 100 mm in two different treatments. Seedlings were allowed to grow for 8 days and then pictures were taken to determine the coleoptile lengths as mentioned previously (Section 2.3.4.).

Observations:

Azucena 50 mm flooding depth 100 mm flooding depth

DSCN0796-1.jpg DSCN0799-1.jpg

IR-72

DSCN0797-1.jpg DSCN0800-1.jpg

PSBRC09

DSCN0798-1.jpg DSCN0801-1.jpg

Figure 3.5. Seedling pictures of Azucena, IR-72 and PSBRC09 under 50 and 100 mm flooding depth. Bar in pictures indicate scale=1 mm.

The flooding depths were imposed at 3 DAS by which the seedling emergence is expected to complete. The above pictures (Fig 3.5) shows that the coleoptile length in response to differing depths of flooding does not vary significantly revealing that it is the best way possible under the given circumstances to survive and grow.

3.6.4. Coleoptile response to prolonged flooding

The objective behind this experiment was to determine the coleoptile response to prolonged flooding. Seeds of Azucena, IR-72 and PSBRC09 were imbibed and then immediately flooded to a depth of 40 mm till 8 days. After 8 days the seedling pictures were taken to measure the coleoptile length by using the earlier mentioned method (Section 2.3.4.).

Observations:

Azucena IR-72

DSCN0802-1.jpg DSCN0803-1.jpg

PSBRC09

DSCN0804-1.jpg

Figure 3.6. Seedlings of Azucena, IR-72 and PSBRC09 expressing coleoptile and no leaf and root expression.

The observations reveal that the cultivars vary in the maximum coleoptile lengths where IR-72 has minimum coleoptile expansion while Azucena and PSBRC09 coleoptiles expand rapidly under flooded conditions (Fig 3.6). There was no root expression and coleoptiles remained white with no leaf emergence in all the cultivars under these experimental conditions.

3.7. Mesocotyl expression in germinating seeds

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.).

Results

Table 3.1. Analysis of variance for mesocotyl length in rice cultivars under aerobic conditions in dark

Source

DF

SS

MS

F

P

Cultivar

4.000

254.122

63.531

76.220

0.000

Error

141.000

117.526

0.834

Total

145.000

371.649

Figure 3.7. Mean mesocotyl length (mm) (±SEM) of five rice cultivars (8 DAS) grown under aerobic condition in dark

The results (Fig 3.7.) 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). The mesocotyl length was < 1 mm in IR-64, PSBRC09 and Sabita while in Azucena it was 1.12 mm. All the seedlings were of same age (8 day old) where in IR-72 shows prominent mesocotyl expression and the rest of the cultivars show 25% of the mesocotyl length of IR-72.

Table 3.2. Summary of coleoptile experiments in response to flooding

MEASUREMENT OF COLEOPTILE RESPONSES TO FLOODING

IR72

IR64

PSBRC09

Azucena

Sabita

Experiment 3.8. immediate deep flooding

Immediate (1 day imbibed then flooding to 50 mm)

-

-

-

-

population responses and 'survivor' responses measured

Experiment 3.9. time of shallow

Time

Depth

flooding in 'survivors'

-

0 mm

-

-

-

Saturated soil and then flooded

1 DAS

5 mm

-

-

-

2 DAS

5 mm

-

-

-

3 DAS

5 mm

-

-

-

Experiment 3.10. depth of flooding  at 3 DAS in  'survivors'

5 mm

-

-

-

-

40 mm

-

-

-

-

3.8. 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.).

3.8.1.Results

% Mean ± S.E.M. of seed population

IR-72

Sabita

PSBRC09

Azucena

Figure 3.8. Population 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.

The results reveal that there is a stratified response in the seed population reveling that different metabolic events occur at varying rates in the seeds and these implicate that cultivars vary in response. There is considerable variation in coleoptile length that is evident at an early stage and persists throughout. Many observations were taken which show that there are highly significant differences (Table 3.4).There is a similar pattern of response in cultivars Azucena and IR-72 where more seeds express coleoptiles with roots while in PSBRC09 and Sabita more seed express coleoptiles with no roots (Fig. 3.8).

Figure 3.9. Showing the variation in dissolved oxygen (mg/l) among the cultivars all through the experiment.

Table 3.3. ANOVA for dissolved oxygen (mg/l) among cultivars.

Source of variation

d.f.

s.s

m.s

v.r.

F pr.

Cultivar(Cv)

3

5.1374

1.7125

8.27

<.001

Residual

20

4.1396

0.207

0.81

d.f. Correction factor 0.7513

Time

5

740.455

148.091

581.77

<.001

Time.Cv

15

4.0398

0.2693

1.06

0.407

Residual

100

25.4554

0.2546

The rate of depletion of dissolved oxygen (mg/l) varies significantly among the cultivars (Table 3.3). Azucena shows fast rate of depletion in comparison to other cultivars, while in IR-72 the rate of dissolved oxygen utilization is slow suggesting that there might be slow rate of metabolism (Fig 3.9).

Sabita

Azucena

IR-72

PSBRC09

Box plots of coleoptile length (mm)

Figure 3.10. Box plots of coleoptile lengths among the cultivars under aerobic and flooded treatments. Flooded treatments are towards the right of each pair.

The coleoptile length increases gradually and then a flat stand is seen suggesting that the coleoptile reached a maximum length in cultivars Azucena and PSBRC09 (Fig 3.10). The pattern of coleoptile expansion differs in IR-72 and Sabita showing a linear increase till 120 GDD. It cannot be concluded that the coleoptile lengths have reached their maximum length in these cultivars until the coleoptile response is studied beyond 120 GDD suggesting that as the coleoptile maximum length.

Table 3.4. ANOVA for coleoptile lengths among the rice cultivars under aerobic and flooded conditions. (Regime indicates either aerobic or flooded)

Source of variation

d.f.

s.s

m.s

v.r

F pr

Time

3

4912.26

1637.42

345.37

<.001

Cultivar (Cv)

3

778.978

259.659

54.77

<.001

Regime

1

17049.6

17049.6

3596.1

<.001

Time.Cv

9

353.543

39.283

8.29

<.001

Time. Regime

3

1410.83

470.277

99.19

<.001

Cv. Regime

3

1130.66

376.886

79.49

<.001

Time. Cv. Regime

9

331.807

36.867

7.78

<.001

Residual

864

4096.33

4.741

Total

895

30064

Mean response at 6 DAS SE=0.4115

Mean coleoptile lengths in cultivars

Aerobic

Flooded

Azucena

6.902

22.942

IR-72

7.54

16.064

PSBRC09

6.82

15.611

Sabita

6.282

21.184

Results (Table 3.4.) indicate there is a significant difference of coleoptile response to immediate flooding compared to aerobic condition. Coleoptile length varied significantly under flooded conditions when compared to aerobic conditions. Maximum coleoptile length (22.94 mm) was observed in Azucena under flooded conditions with minimum in PSBRC09 (15.61 mm). IR-72 had a maximum coleoptile length (7.54 mm) under aerobic conditions while Sabita the minimum coleoptile length (6.28 mm). Azucena and Sabita show a threefold increase in coleoptile length under flooded condition compared to the length under aerobic conditions while IR-72 and PSBRC09 have a two fold increase in coleoptile length.

3.9. 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.

Statistical analysis

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 selected 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

Where:

y = coleoptile length

x = GDD

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.

3.9.1. Results

The illustration of fit of equation is done using one data set (Azucena) at flooding 1 DAS.

Coleoptile length (mm)

GDD

Figure 3.11. Illustrates the fit of Equ 3.2 for the expression of coleoptiles length over time for cultivar Azucena where shallow flooding (5 mm) was imposed 1 DAS.

The coleoptile response has been measured beyond 120 GDD to estimate if the maximum length of coleoptile was obtained (Fig 3.11).

Table 3.5. 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

0.8015

Parameters

Value

Std Error

P>|t|

a

-0.102

1.235

0.934

b

16.133

1.206

0.000

c

77.694

2.865

0.000

d

12.927

5.865

0.029

e

0.962

1.127

0.394

Source

Sum of Squares

DF

Mean Square

F Statistic

P>F

Regr

3532.814

4.000

883.204

191.892

0.000

Error

874.495

190.000

4.603

Total

4407.309

194.000

Lack Fit

39.971

6.000

6.662

1.469

0.191

Pure Err

834.524

184.000

4.535

The equation fitting shows there is no significant difference in lack of fit supporting this model is a good fit for the data (Table 3.5).

Table 3.6. 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. Flooding time 0, 1, 2, and 3 DAS.

 

Flooding time DAS

b

b(s.e.)

c

c (s.e)

R2

P (Ho) overall fit

Model

IR-72

0

7.503

1.6758

50.621

7.7240

0.573

>0.000

S

 

1

15.092

5.8449

74.198

10.6917

0.606

>0.000

A

 

2

17.632

5.7758

74.413

13.9585

0.640

>0.000

A

 

3

13.459

2.2409

79.227

8.5686

0.683

>0.000

A

 

 

 

 

 

 

 

 

 

PSBRC09

0

7.319

0.4816

55.650

1.8714

0.787

>0.000

S

 

1

15.342

4.0821

61.742

12.1540

0.749

>0.000

A

 

2

18.614

2.4467

58.969

3.5086

0.797

>0.000

A

 

3

15.991

1.2684

65.296

2.0374

0.774

>0.000

S

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Azucena

0

9.201

1.1874

64.860

4.4189

0.624

>0.000

S

 

1

16.133

1.2065

77.694

2.8645

0.802

>0.000

A

 

2

18.565

3.1460

85.089

8.9465

0.775

>0.000

A

 

3

15.079

1.7494

74.202

5.0140

0.748

>0.000

A

Coleoptile length shows significant differences among the cultivars in response to shallow flooding (5 mm) imposed at 1, 2 and 3 DAS (Table 3.6.). Flooding imposed at 2 DAS shows the maximum coleoptile length (parameter b, Table 3.5) among all the cultivars compared to flooding imposed at 1 and 3 DAS. The minimum coleoptile length was observed under 0 mm flooding conditions in all the cultivars. Among the cultivars the maximum coleoptile length was in PSBRC09 (18.61 mm) followed by Azucena (18.56 mm) and minimum in IR-72 (17.63 mm) at 2 DAS flooding, under 0 mm flooded conditions PSBRC09 has minimum coleoptile length (7.31) followed by IR-72 (7.50) and Azucena the maximum coleoptile length (9.20). While the time taken (GDD) to reach half the maximum coleoptile length (parameter c, Table 3.6) was least in PSBRC09 (58.96) followed by IR-72 (74.4) and Azucena (85.08) under 2 DAS flooding. This suggests that Azucena takes long time for coleoptile expansion while PSBRC09 coleoptile expansion is rapid.

3.10. 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 was measured periodically through the experimental period.

3.10.1. Results

F

F

Figure 3.12. 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 3.12.). 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 3.7. Coleoptile lengths in response to flooding imposed 3 DAS in the cultivars and the parameters.

 

 

b

b (s.e.)

C

C (s.e)

R2

P (Ho) overall fit

Model

IR-72

0

7.781

1.2525

58.910

4.7679

0.6065

>0.000

S

 

5

11.822

1.8066

86.382

3.4062

0.6905

>0.000

S

 

40

15.562

1.2050

69.541

1.8580

0.8175

>0.000

S

 

 

 

 

 

 

 

 

 

PSBRC09

0

7.620

0.8614

55.381

2.9410

0.6228

>0.000

S

 

5

16.690

1.3736

70.258

2.0251

0.8000

>0.000

S

 

40

17.473

2.1937

70.956

3.1443

0.6977

>0.000

A

 

 

 

 

 

 

 

 

 

Azucena

0

6.796

0.7584

58.524

2.0398

0.5359

>0.000

S

 

5

19.883

2.1292

72.481

2.8197

0.7503

>0.000

S

 

40

22.838

1.2485

71.888

1.3220

0.9049

>0.000

S

 

 

 

 

 

 

 

 

 

Sabita

0

7.032

0.6278

56.874

1.8208

0.7275

>0.000

S

 

5

17.976

1.5928

65.398

2.0485

0.7755

>0.000

S

 

40

18.846

1.8127

67.414

2.2763

0.7637

>0.000

S

 

 

 

 

 

 

 

 

 

The maximum coleoptile length is obtained at 40 mm flooding depth in all the cultivars. Azucena shows maximum coleoptile length (parameter b, Table 3.7) (22.83 mm) followed by Sabita (18.84 mm), PSBRC09 (17.47 mm) and IR-72 (15.56 mm) at 40 mm flooding depth. Sabita requires less time (67.41) to reach maximum coleoptile length (parameter c, Table 3.7) followed by IR-72 (69.54), PSBRC09 (70.95) and Azucena takes long time to reach half the coleoptile length (71.88).

3.11. Discussion

Growth requires (1) suitable temperature, (2) adequate supply of hormones, metabolites, water and nutrients, and (3) absence of inhibitors. Under favorable environmental conditions the cells resume growth. During germination seeds show variation in the order of expressing plumule or radicle emergence first irrespective of the environment in which germination occurs. Liptay and Davidson (1971) suggested that the barley coleoptiles of uniform height do not have identical growth rates indicating that they are in different physiological states. In accordance with the literature the cultivars examined are inbred type despite there are variations among the given seed lot in terms of seed size reflecting the resource available for growth and development, suggesting differences during the developmental morphogenesis.

Germination under dark leads to mesocotyl expression among the cultivars ascertaining to embryo size variations (Hong et al., 1996). Genotypes vary in this expression with respect to the length of mesocotyl. From the above experiments it is seen that some cultivars express mesocotyl elongation prominently while others do not show mesocotyl elongation when grown under similar conditions. Among the cultivars studied, IR-72 shows significant mesocotyl elongation when grown in dark under aerobic conditions while the other genotypes represent existence of mesocotyl (Fig 3.7). Factors underlying mesocotyl elongation can be studied if the mechanisms involved in mesocotyl expression or the expansion of mesocotyl are studied.

Microscopic observations identified the leaf length variations inside the coleoptile. Leaf length varies under aerobic and anaerobic conditions, where coleoptile expands to a greater length under flooded conditions while the leaf bud growth is suppressed (Fig 3.4). Under the given seed lot, seeds vary in leaf length expression. Mechanisms behind such response can be answered with the help of physiological understanding and molecular studies identifying the role of different genes being expressed at varying intensities leading to such variations among the inbred lines.

Based on the test weight analysis, it has been observed that seed lot differ with in same cultivar in terms of seed weight. The cultivars investigates differ in size as well. The availability of seed reserves varies among seed and is a genotypic feature (Hong et al., 1996). This reflects the cultivar variations in rate of coleoptile extension. When flooding occurs before completion of germination, seeds do not express root but only coleoptile elongation and no leaf emergence. Leaf emergence is suppressed until the coleoptile reaches the aerobic surface (Fig 3.4). Cultivars vary in coleoptile length variation to understand the factors behind, the role of genes involved in coleoptile elongation has to be studied in detail. If the gene expression varies among the cultivars that can answer the coleoptile length variations.

Given seed population within the same cultivar differs in morphogenesis wherein some seeds express coleoptile with no root expression, while some show root expression (Fig 3.8). This shows that different metabolic events and molecular mechanisms occur at varying rates in the seeds responsible for such variations in seed lot. Factors underlying the suppression or induction of root expression remain unanswered challenge. Seeds also differ in leaf expression under submergence where some seeds showing greening of leaves whilst other lack leaf expression when grown under similar environment. The biochemical and molecular mechanisms involved in such response have to be studied in greater depth to understand such variations among genotypes. Few seeds germinate and then further growth and development is suppressed, may be because of lack of ability of seed to overcome the anoxia and grow. This might be because of competition among the seeds for limited resources available under submergence (oxygen, light and carbon dioxide). The vigorous seeds outgrow these seeds as they lack the potential strength. While some seeds remain dormant failing to germinate or show any developmental progress after imbibition, may be because of dormant or dead embryo. Tetrazolium test was done to investigate the seed viability and found dormant embryo in the seeds which did not germinate in flooded conditions. The driving force for such a varied seed response is unknown. Further molecular studies investigating the early gene expression may bring out the reason behind these cultivar variations.

Dissolved oxygen utilization is relatively slow in IR-72 indicating slow metabolism and hence a delayed development. While in Azucena there is rapid decline in dissolved oxygen indicating a faster rate to metabolism and seedling growth (Fig 3.9). This can be substantiated if the biomass accumulation is studied. The net gain in biomass reflects the rate of seed reserve mobilization indicating rate of metabolism and oxygen depletion. Leaves that develop during submergence derive their dry matter from the shoot developed before flooding. To understand the seedling survival under submergence a closer inspection of biomass allocation may be helpful. Slow oxygen depletion under flooding suggests slow metabolism in IR-72 and Sabita reflecting the slow coleoptile growth, indicating the presence of potential for coleoptile elongation while in Azucena and PSBRC09 the oxygen decline is quick indicating rapid coleoptile elongation (Fig 3.10). Flooding imposed at different times of germination i.e. 0, 1, 2, and 3 DAS shows variations among cultivars wherein the maximum coleoptile response is seen when flooded at 2 DAS among all the cultivars. IR-72 shows the least coleoptile length indicating slow growth rate (Table 3.6). IR-72 did not show much coleoptile elongation to reach oxygenated water level and failed to establish prolonged seedling growth under low land conditions (Biswas and Yamauchi, 1997).

Coleoptile length in response to flood depth varied with cultivars. Irrespective of flood depth in cultivars Sabita and PSBRC09 coleoptile growth is stimulated to the same potential while in Azucena and IR-72 genotypes the coleoptile length depends on depth of flooding (Fig 3.12) showing more elongation under greater depths of flooding. This may be because of differential expression of the genes involved in coleoptile expansion at molecular level. Since the rate of developmental morphogenesis varies among genotypes accounting to the differences in seed weight, embryo size and different rates of reserve mobilization.

3.12. Conclusions

Most cultivars show coleoptile elongation in response to flooding as a means to gain access to aerobic environment. However an alternative escape strategy lies in adaption to slow carbohydrate metabolism and minimum shoot elongation preserving the available limited resource to resume growth and development after the water level recedes. All these mechanisms are genetically controlled.

Research into rice adaptation to anoxic conditions has provided many insights however still some questions remain unanswered. Promising tools like gene expression, microarray studies may answer the differences behind the cultivar responses. Furthermore understanding molecular mechanisms that enable seed germination, shoot elongation and low oxygen sensing might bring new clues to understand the genotypes.

Genetic improvement of seedling establishment is possible by using several genotypes superior in seedling establishment and good seedling vigor as genetic resources. Identification of critical crop growth stage for better seedling establishment under submergence is not only important for breeding but also for improvement of direct sowing cultivation. Variations in seedling growth can be analyzed in detail using advanced technologies like genetic markers and QTL analysis. Introduction of rapid germinating trait into modern cultivars has produced promising pedigree lines for direct seeding cultivation (Sasaki and Yamazaki, 1971; Fukuoka et al., 1999). However this has a risk of vivipary. Rapid coleoptile growth is important for better establishment of seedlings rather rapid germination suggesting the genetic crop improvement can be achieved avoiding the risk of vivipary. Relationship between seed germination, seedling growth and establishment, coleoptile elongation, seedling survival, successful crop establishment and submergence tolerance requires further detailed study.