**3. Related donor cord blood transplantation for thalassemia**

In 1995, the first CBT for thalassemia was reported, using a HLA-identical sibling donor cord blood for a two-and-half-year old [36]. Busulfan/cyclophosphamide conditioning regimen and cyclosporine/methotrexate GvHD prophylaxis were used. The TNC dose was 3.9 × 107 /kg, and the thawed product was not washed and was directly infused on June 12, 1993. Neutrophil and platelet engraftment were achieved by day +23 and day +27, respectively. The patient experienced no GvHD, and the patient was alive and transfusion independent 48 months after transplantation. The various studies using related donor CBT for thalassemia are summarized in **Table 1** [23–25, 28–30, 36, 56, 59, 63–68].

Various other approaches have been tried to improve the outcome of CBT for thalassemia, ranging from preference for superior HLA matches [4, 6, 26, 31, 34, 37, 51–53], usage of related HLA-identical donors [23–25], directed sibling cord blood bank efforts [30, 56], consideration of non-inherited maternal antigen (NIMA) matches [57], preference for IV busulfan over oral formulations [6, 58], the addition of thiotepa to the conditioning regimen [24, 25], reduced intensity conditioning regimens [3, 59, 60], the avoidance of methotrexate in the prophylaxis regimen [23–25], third-party MSC co-infusion [59], and intrabone direct injection of cord blood

In 1995, the first CBT for thalassemia was reported, using a HLA-identical sibling donor cord blood for a two-and-half-year old [36]. Busulfan/cyclophosphamide conditioning regimen and

the thawed product was not washed and was directly infused on June 12, 1993. Neutrophil and platelet engraftment were achieved by day +23 and day +27, respectively. The patient experienced no GvHD, and the patient was alive and transfusion independent 48 months after transplantation. The various studies using related donor CBT for thalassemia are summar-

**[36] [64] [28] [65] [23] [29] SDCB [30, 56] [24] [25]**

baby donor's PB

5.5/ 3.5–10

6 1 2

6.6/ 3.4–12.7 (1) 14 Thal 4/14 + PB (2) 7 Thal

(1) NA 5.9/ 2–11

(1) NA (2) 4.7/ 0.8–7.6

5/9 (1) Thal 12/14 (2) Thal 6/6 with

engraftment

1 33 9; 2 +

1–20

41 3

1.2–13 PF 4.0/ 1.2–10 PT

6.1 5.1/

1/1 ANC 89% D+23 (12–60)

0 1 /kg, and

27 66

5.9/ 2–20

100% 0/0 0/0

3.9 1.5–14

ANC 90 ± 4%; D +23 Plt

6 0.8–18

0 0

3.3 1.5–6

100% (27/27) 100% (27/27)

(1) 14 (2) 7 100%

cyclosporine/methotrexate GvHD prophylaxis were used. The TNC dose was 3.9 × 107

**3. Related donor cord blood transplantation for thalassemia**

186 Umbilical Cord Blood Banking for Clinical Application and Regenerative Medicine

ized in **Table 1** [23–25, 28–30, 36, 56, 59, 63–68].

5 CB + BM

2/1.2/2.5 Combined 3.2/2.8/3.7

5/5 CB + BM

2.5 2.2/3.8 4, 13, 15 3 5

128 0

2/2 1/3CB

3.9 6.2/11.4 CB

ANC 1/1; D+23 Plt


**5. Conclusions**

In this study, 2D nonlinear effective stress dynamic analyses have been carried out to inves‐ tigate the seismic behavior of gravity-type quay walls. A reference model has been constructed and then subjected to seismic loading. The Finn and Byrne model has been adopted with some slightly modifications which take into account the pore water pressure generation and liquefaction process under dynamic loading. The numerical model has been validated by simulating 1g shaking table test. It is shown that the obtained numerical results agree reason‐ ably with actual observation in the shaking table test. The seismic response of the gravity wall itself has well captured by the numerical analyses with satisfactory predictions of acceleration, displacement, total pressure and pore water pressure time histories. Additional computational parametric studies have been conducted by varying backfill soil relative density, shear modulus, friction angle and maximum amplitude and frequency of input excitation to study the extent of liquefaction and deformation mechanism of quay wall system. It is concluded that soil properties and input motion characteristics are among the most influential factors in dictating seismic performance of the quay wall system. The results show that the backfill in the soil-wall interaction zone and the foundation soils beneath the quay wall experiences less excess pore water pressure even liquefaction occurs in the far field during shaking. The alternative pumping and suction process in excess pore water pressure which are caused by wall's vibrations increase the level of damage because large amounts of backfill are forcedly leaked into the sea. The lack of backfill liquefaction near the wall is attributed to the lateral displacement of the wall. In the other words, excess pore water pressure does not attain 100% liquefaction behind the quay wall contrary to the far field. The current study states that the numerical simulation incorporated with the special numerical techniques is capable of modeling the seismic response of gravity-type quay walls.


*2.1. Materials*

All reagents were purchased from Sigma-Aldrich Co. (Merck KGaA, Darmstadt, Germany) and used without further purification. Analytical grade solvents were used for the crystallization experiments. 9-Ethyladenine (9ETADE) was obtained as previously described by some of us [19].


**5. The pollen cell wall**

#### **5.1. Exine**

The synthesis of the pollen cell wall starts during meiosis and depends on the activity of the tapetum. The composition of the pollen wall is unique compared to other plant cell walls and shows species-specific diversity. The biodiversity in pollen cell walls is functionally important for the plant to distinguish its own pollen from that of other plants [128–131]. The pollen cell wall can vary physically and chemically to match environmental aspects of pollination. Pollen wall diversity serves a taxonomical value, forming the basis of palynology [132]. The extremely resistant and elastic outer exine wall has evolved to protect pollen during dispersal. Exine is deposited first to provide pollen grains with their distinctive and characteristic features (Figure 3A). Pollen cell wall organisation starts just before meiosis when meiocytes become surround‐ ed by callose secreted by the tapetum [92, 93, 133]. The callose special cell wall (SCW) is formed during prophase and interphase and closes the cytomictic channels that synchronise the first meiotic division. Exine is patterned under the callose layer and the microspore plasma membrane (primexine or exine presursor) at the end of meiosis (late tetrad stage) and is completed after the release of the microspores from the SCW at the end of the tetrad stage. The tapetum then produces callase, a β-1,3-D-glucanase enzyme responsible for dissolution of the callose wall, as well as sporopollenin precursors, a complex polymer of fatty acids and phenolic compounds. These are released in the loculus and polimerise on the primexine of the micro‐ spore following its release from the tetrad [132, 134, 135]. Mutant screens for impaired pollen walls in Arabidopsis revealed several genes involved in sporopollenin biosynthesis and most of these mutants are male sterile [92, 136–140]. Sporopollenin precursors are deposited by ABC transporters that are expressed in the tapetum at the early vacuolated microspore stage [134, 141]. Sporopollenin biosynthetic enzymes form a complex ('metabolon') in the endoplasmatic reticulum of the tapetum [142]. Recent ultrastructural studies reveal the involvement of specialised tapetum organelles, elaioplasts or tapetosomes, in exine wall deposition [140, 143]. Exine deposition is reduced, interrupted and can even be absent altogether in aquatic plants or plants living and pollinating in extremely wet environments [144]. The absence of exine in species having underwater pollination (e.g., seagrasses) is correlated with the fact that in water pollen grains do not undergo desiccation and have to remain hydrophilic; there is no developmental arrest and changes in shape and volume do not occur [145].

The Trials and Tribulations of the Plant Male Gametophyte — Understanding Reproductive Stage Stress Tolerance http://dx.doi.org/10.5772/61671 715

**Figure 3.** Different stages of pollen development in angiosperms.

*Stages Stress type Targeted stage and/or*

714 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives

⋅ High/low relative

humidity

mechanisms of the male gametophyte.

**5. The pollen cell wall**

**5.1. Exine**

*compartment Defence mechanisms*

grains ⋅ Anthers can delay pollen dispersal

⋅ Number of viable, dispersed

The synthesis of the pollen cell wall starts during meiosis and depends on the activity of the tapetum. The composition of the pollen wall is unique compared to other plant cell walls and shows species-specific diversity. The biodiversity in pollen cell walls is functionally important for the plant to distinguish its own pollen from that of other plants [128–131]. The pollen cell wall can vary physically and chemically to match environmental aspects of pollination. Pollen wall diversity serves a taxonomical value, forming the basis of palynology [132]. The extremely resistant and elastic outer exine wall has evolved to protect pollen during dispersal. Exine is deposited first to provide pollen grains with their distinctive and characteristic features (Figure 3A). Pollen cell wall organisation starts just before meiosis when meiocytes become surround‐ ed by callose secreted by the tapetum [92, 93, 133]. The callose special cell wall (SCW) is formed during prophase and interphase and closes the cytomictic channels that synchronise the first meiotic division. Exine is patterned under the callose layer and the microspore plasma membrane (primexine or exine presursor) at the end of meiosis (late tetrad stage) and is completed after the release of the microspores from the SCW at the end of the tetrad stage. The tapetum then produces callase, a β-1,3-D-glucanase enzyme responsible for dissolution of the callose wall, as well as sporopollenin precursors, a complex polymer of fatty acids and phenolic compounds. These are released in the loculus and polimerise on the primexine of the micro‐ spore following its release from the tetrad [132, 134, 135]. Mutant screens for impaired pollen walls in Arabidopsis revealed several genes involved in sporopollenin biosynthesis and most of these mutants are male sterile [92, 136–140]. Sporopollenin precursors are deposited by ABC transporters that are expressed in the tapetum at the early vacuolated microspore stage [134, 141]. Sporopollenin biosynthetic enzymes form a complex ('metabolon') in the endoplasmatic reticulum of the tapetum [142]. Recent ultrastructural studies reveal the involvement of specialised tapetum organelles, elaioplasts or tapetosomes, in exine wall deposition [140, 143]. Exine deposition is reduced, interrupted and can even be absent altogether in aquatic plants or plants living and pollinating in extremely wet environments [144]. The absence of exine in species having underwater pollination (e.g., seagrasses) is correlated with the fact that in water pollen grains do not undergo desiccation and have to remain hydrophilic; there is no

developmental arrest and changes in shape and volume do not occur [145].

**Table 3.** Effect of abiotic stresses on different stages of pollen development, and possible evolutionary defence

A1-4: Scanning electron micrographs of mature desiccation-sensitive recalcitrant grains which are devoid of furrows.

A1: A rice pollen grain close to anthesis, showing the cell wall surface and the germination pore (arrow). A2: three pollen grains of *Lavatera arborea* (Malvaceae) kept together by pollenkitt, a viscous fluid covering the pores of the grains. A3-6: Pollen grains of different members of the Cucurbitaceae family with recalcitrant pollen grains. A3: *Cucurbita pepo*, with a germinating pollen grain (arrow) taken 10' after pollination. A4: *Cucumis sativus*, a species with recalcitrant pollen grains. A5 and A6: *Bryonia dioica* and *Cytrullus lanatus* orthodox pollen grains with furrows.

B-E: Asynchrony in vacuolisation and starch storage in olive (*Olea europaea*) pollen grains. B: A section of anther at mid-bi-cellular stage during the second vacuolisation, with degenerating tapetum (toluidine blue O staining). The asynchrony of development of pollen is evident: grains have vacuoles of different sizes and some grains are degenerating (arrows). C: A section of an anther at mid-microspore stage at the first starch engorgement (stained with Periodic Acid Schiff). Starch grains have different sizes because of the asynchrony of starch storage. D: Section of an anther at the early bi-cellular stage and second starch engorgement (PAS stained). Grains have an asynchronous development with respect to starch engorgement and in some grains the generative cell (arrow heads) can be discerned because of the thin polysaccharide wall. E: Pollen grains of *Cerinthe major* (Boraginaceae) displaced by flower visitors on the corolla (SEM). Only one has emitted a pollen tube because of precocious rehydration due to high humidity during the night – probably indicating asynchronic development of the grains.

#### **5.2. Intine**

The exine wall is completed by the mid-microspore stage before the internal intine layer is deposited. Intine is less elastic and consists of a pecto-cellulose mixture. Intine synthesis also starts before the first mitotic division and is always completed by the time the vegetative and generative cells are formed [132, 146]. Mutagenesis approaches have identified genes involved in the biosynthesis of pectins for the intine cell wall [147–152]. Some of these genes are expressed in the tapetum and ABC transporters transfer intine components to the pollen grains [153]. Mutations affect pollen shape and fertility, as well as growth of the pollen tube. Pectin is the main component of intine and is secreted by the tapetum into the locular fluid. Accu‐ mulation is highest at the vacuolated microspore stage [146]. The *Brassica campestris Male Fertility2* and *9* (*BcMF2, 9*) genes encode novel polygalacturonase enzymes that play a role in pectin metabolism, intine formation and tapetum degradation [151, 152]. At pollen germina‐ tion, the intine wall forms a continuum with the the pollen tube pectocellulose wall.

#### **5.3. The role of the cell wall in regulating pollen size and shape**

The pollen wall controls homeostasis of the cytoplasm and reduces fluctuations in pollen volume due to variations in water content. This is important during dispersal, when pollen is exposed to air. The characteristic exine wall furrows and surface pattern are crucial for the harmomegathic functions that regulate pollen shape during dehydration [154] (Figure 3 A5–6). After landing on the stigma, the pollen wall controls the rehydration process with water coming from the stigma in angiosperms or from the ovule in gymnosperms (pollina‐ tion drop) [155].

The exine layer has generally one or more pores through which the pollen tube is emitted (Figure 3 A1, 3). When pollen pores are absent, the pollen tube is emitted at the site where the pollen grain contacts the stigma surface. The pattern and distribution of the apertures are determined by the tetrad shape and callose deposition at the intersporal walls [156, 157]. The Arabidopsis *tam* mutant (*t*ardy *a*synchronous *m*eiosis) shows an altered cytoplasmic parti‐ tioning (cytokinesis) during tetrad formation and altered aperture patterning, suggesting that the last contact points between the cytoplasms of the future microspores during cytokinesis are the place where apertures are formed [158]. The number of pores per pollen grain can vary within one species and germination speed is positively correlated with pore number [159] and pollen water content at dispersal [160]. The intine wall is a continuous layer but is generally thicker and more elaborate at the pores and/or furrows to support the harmomegathic process [161]. Exine and intine have a similar thickness but in some cases intine, especially in the poral region, is much thicker and very pectin-rich, which may help in keeping pollen cytoplasm hydrated during dispersal [162–164].

### **5.4. Pollenkitt**

the Cucurbitaceae family with recalcitrant pollen grains. A3: *Cucurbita pepo*, with a germinating pollen grain (arrow) taken 10' after pollination. A4: *Cucumis sativus*, a species with recalcitrant pollen grains. A5 and A6: *Bryonia dioica* and *Cytrullus lanatus* orthodox pollen grains with

716 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives

B-E: Asynchrony in vacuolisation and starch storage in olive (*Olea europaea*) pollen grains. B: A section of anther at mid-bi-cellular stage during the second vacuolisation, with degenerating tapetum (toluidine blue O staining). The asynchrony of development of pollen is evident: grains have vacuoles of different sizes and some grains are degenerating (arrows). C: A section of an anther at mid-microspore stage at the first starch engorgement (stained with Periodic Acid Schiff). Starch grains have different sizes because of the asynchrony of starch storage. D: Section of an anther at the early bi-cellular stage and second starch engorgement (PAS stained). Grains have an asynchronous development with respect to starch engorgement and in some grains the generative cell (arrow heads) can be discerned because of the thin polysaccharide wall. E: Pollen grains of *Cerinthe major* (Boraginaceae) displaced by flower visitors on the corolla (SEM). Only one has emitted a pollen tube because of precocious rehydration due to high humidity during the night – probably indicating asynchronic development of the grains.

The exine wall is completed by the mid-microspore stage before the internal intine layer is deposited. Intine is less elastic and consists of a pecto-cellulose mixture. Intine synthesis also starts before the first mitotic division and is always completed by the time the vegetative and generative cells are formed [132, 146]. Mutagenesis approaches have identified genes involved in the biosynthesis of pectins for the intine cell wall [147–152]. Some of these genes are expressed in the tapetum and ABC transporters transfer intine components to the pollen grains [153]. Mutations affect pollen shape and fertility, as well as growth of the pollen tube. Pectin is the main component of intine and is secreted by the tapetum into the locular fluid. Accu‐ mulation is highest at the vacuolated microspore stage [146]. The *Brassica campestris Male Fertility2* and *9* (*BcMF2, 9*) genes encode novel polygalacturonase enzymes that play a role in pectin metabolism, intine formation and tapetum degradation [151, 152]. At pollen germina‐

tion, the intine wall forms a continuum with the the pollen tube pectocellulose wall.

The pollen wall controls homeostasis of the cytoplasm and reduces fluctuations in pollen volume due to variations in water content. This is important during dispersal, when pollen is exposed to air. The characteristic exine wall furrows and surface pattern are crucial for the harmomegathic functions that regulate pollen shape during dehydration [154] (Figure 3 A5–6). After landing on the stigma, the pollen wall controls the rehydration process with water coming from the stigma in angiosperms or from the ovule in gymnosperms (pollina‐

The exine layer has generally one or more pores through which the pollen tube is emitted (Figure 3 A1, 3). When pollen pores are absent, the pollen tube is emitted at the site where the

**5.3. The role of the cell wall in regulating pollen size and shape**

furrows.

**5.2. Intine**

tion drop) [155].

In some plant species, the surface of the pollen wall contains various amounts of pollenkitt, a viscous hydrophobic substance. The sticky nature of pollenkitt is thought to play a role in pollen adhesion to pollinators during dispersal [165, 166], but several other functions have been suggested [167]. Plants with zoophilous or entomophilous pollination, some of which having secondary anemophylous pollination, have exine cavities or ornamenations containing pollenkitt [168]. A simple and effective method was developed to reveal its presence or absence [169, 170]. The synthesis of pollenkitt is linked to tapetal degeneration [171] and plastids are implicated in its formation [104, 167]. In anemophilous plants, the plastids develop into elaioplasts which are resorbed by other tapetum cell components during degeneration. In entomophilous plants, the elaioplasts or tapetosomes (plastids accumulating lipids) are the more abundant organelles in the degenerating tapetum cytoplasm [167]. Tapetosomes are oil and flavonoid containing organelles in the tapetum that contribute to pollenkitt formation [172–174]. Pollenkitt is formed by the fusion of elaioplasts and spherosomes of tapetal cells during the late microspore stage [167]. After release in the locule, pollenkitt is deposited on the exine surface of the pollen grains, covering the exine ornamentations at the onset of anther dehydration [171]. In the entomophilous Brassicaceae family, elaioplasts are involved in forming tryphine, which plays a role in adhesion of pollen to the stigma [104] (Table 1). A conditionally male sterile mutant that affects tryphine production in Arabidopsis is affected in pollen-stigma recognition [69]. Pollenkitt consists mainly of saturated and unsaturated lipids, carotenoids, flavonoids, low molecular weight proteins and carbohydrates [167, 175]. An additional role of pollenkitt in biotic pollination could be in preventing water loss and other damage [167].