The chemical substances used to disintegrate the cell walls and electrical field protoplasts lose their viability and ability to divide. In cotton, some attempts have been made to transform cells directly in the shoot apex either through the gene gun or Agrobacterium [24], but these methods suffer from extremely low transformation efficiencies. Others have used particle bombardment of suspension cultures of cotton. This method suffers from the fact that additional time is required for establishment of suspension cultures [25]. The most distinguishing limiting factor of using the gene gun is that it causes the presence of multiple copies of the target genes, which may lead to alteration or concealing of the expression. In addition, gene gun is a very expensive method. The usage of in vitro cultures for plant transformation may give rise to few somaclonal changes. Due to this, they cannot be used in further studies, and analysis of the transformants becomes quite complex. As a result of the limitations mentioned, many new transformation methods are being introduced as substitutes. Most commonly mentioned methods out of them are (a) silicon carbide fiber-mediated transformation; (b) microinjection; (c) infiltration; (d) electrophoresis of embryos; (e) transformation through the pollen tube pathway; (f) electroporation of cells and tissues; and (g) liposome-mediated trans-

In 1970 and 1971, two major advances were made, which proved the beneficial role of protoplasts in the enhancement of plants: (i) somatic hybrid cells and novel hybrid plants

kanamycin selection.

212 Cotton Research

5.1. Indirect transformation

5.2. Direct transformation

formation.

transformation (mostly in monocots).

5. Methods involved in transformation

transformations, which are detailed in below sections.

6. Overview of transformation techniques

6.1. Protoplasts and somatic hybridization

Desiccated plant tissues can uptake foreign DNA through the process of imbibition [34]. Numerous asserts and disproof can be found in literature regarding this method. During the process of desiccation, various physiological and substantial alterations take place such as bursting of the cell wall, leakiness and changes in structure of the cell membrane, quick expansion of the cell, and development of a huge water flow among the peripheral solution and the dehydrated tissue. Uptake and transitory expression, by the cereal and legume seed embryos, of the DNA plasmid bearing the NPTII gene through the process of imbibition were confirmed by Töpfer et al. [22].

Permeability of the membrane is a key factor in this process. This was proved by an increase in uptake and expression of the DNA as a result of using 20% DMSO during the process. Successive studies have led to 70% transitory GUS expression in dehydrated somatic embryos of alfalfa [35]. Yoo and Jung have also stated a stability of transformation in rice through the process of imbibition. From 30% to 50% incidence of transient expression of GUSA and NPT genes was observed using vectors, where insertions were driven by CaMV35S promoter. Meristematic regions of the roots and leaf vascular bundles are the major locations for the expression of GUSA gene. Different PCR techniques and southern blot analysis of genome showed the uptake, incorporation, and expression of the HPT gene in rice. Hygromycin phosphotransferase (HPT) DNA was not found as a plasmid but rather it was present in an incorporated form in the genome of rice [36]. This method is the easiest of all the direct gene transformation techniques because the formation of target plant tissues is quite uncomplicated and no professional equipment is needed to perform it. Few drawbacks of this transformation method are given below; this method cannot be applied to very specific organs or tissues such as new pollinated flowers or hydrating embryos.

#### 6.3. Agrobacterium-mediated transformation

A. tumefaciens causes the crown gal disease in plants. Pett et al. [37] in 1958 performed an experiment and proposed that tumor-inducing property of this soil bacterium is responsible for transformation in plants. Bacteria-free tumor cells contain large number of opines, which is a new class of metabolites.

Chilton et al. [39] introduced the idea of virulent strain of Agrobacterium, which leads to the discovery of mega plasmid. The plasmid were called tumor-inducing plasmid and only a small portion of that plasmid known as T-border is considered responsible for tumor induction [38] and was found to be present in the nuclear DNA of the tumor-inducing cells [39].

Ti plasmid is an integrated part of the plant genome during tumor formation (transformation), suggesting that the plasmid could be used as a vector to transfer other genes. It was reported that various methods were tested to insert genes into the Ti plasmid. Transformed crown-gall tumor tissues, which were grown on hormone-free media, only formed highly aberrant shoots in culture [40]. This was related to the presence of genes controlling the expression of auxin and cytokinin synthesis. Deletion of these genes resulted in the production of transformed tissues that required media supplemented with auxin and cytokinin for continued growth and regeneration of normal shoots and plants. Nadolska-Orczyk et al. [43] showed that efficient Agrobacterium-mediated transformation of Metrosideros polymorpha provide molecular techniques to facilitate comparative genomics. It was reported that above-discussed facts led to the use of Ti plasmid of Agrobacterium as a vector for plant transformation and kanamycin resistance genes as selection marker for transformed cells, and then transformed plants are regenerated [41] (Figures 1 and 2). Agrobacterium-mediated transformation followed by somatic embryogenesis remains the method of choice for most cotton transformation. However, regeneration aspect of the transformation process remains more difficult and choices are limited in case of cotton, one of the most difficult crop for transformation [42].

As Agrobacterium is highly attracted to phenolics, this method of transformation is not preferable for monocots due to production of phenolics, whereas it can be used for dicots. However, recently, Agrobacterium-mediated transformation has been optimized and becomes the best method for transformation of monocots [43].

#### 6.4. Biolistic transformation

In early 1980s, direct DNA delivery methods for protoplast were developed [44], especially for the economically important cereal crops, which were not subjected to the umbrella Agrobacterium-mediated transformation [45]. In this procedure, osmotic or electric shocks are applied to the protoplasts suspended in solutions containing DNA. After that planting on selection media is performed. Transformation based on embryogenic cell suspension cultures produced the first transgenic cereals [13]. The use of protoplasts for genetic transformation became less striking once it was shown that monocots could be transformed by co-cultivation of embryonic tissues and supervirulent strains of Agrobacterium in the presence of acetosyringone [15]. In case of Ricinus (castor oil plant), transformation through gene gun is dependent on many factors such as helium pressure, target distance, osmoticum, microcarrier type and size, DNA quantity, explant type, and number of bombardments had significant influence on transformation efficiency (Figure 2). Recommended distance is 6.0 cm, helium pressure of 1100 psi, 0.6-μm gold microcarriers, furthermore single bombardment has shown

Figure 1. Basic concept of gene transformation through Agrobacterium.

positive results [46]. Finer and McMullen [47] demonstrated the successful recovery of transgenic cotton plants by biolistic bombardment of embryogenic suspension cultures and reported a transient to stable conversion frequency of about 0.7%. Biolistic method is not recommended for the crops, which cannot be tissue cultured.

#### 6.5. Biolistic with Agrobacterium

for transformation in plants. Bacteria-free tumor cells contain large number of opines, which is

Chilton et al. [39] introduced the idea of virulent strain of Agrobacterium, which leads to the discovery of mega plasmid. The plasmid were called tumor-inducing plasmid and only a small portion of that plasmid known as T-border is considered responsible for tumor induction [38] and was found to be present in the nuclear DNA of the tumor-inducing

Ti plasmid is an integrated part of the plant genome during tumor formation (transformation), suggesting that the plasmid could be used as a vector to transfer other genes. It was reported that various methods were tested to insert genes into the Ti plasmid. Transformed crown-gall tumor tissues, which were grown on hormone-free media, only formed highly aberrant shoots in culture [40]. This was related to the presence of genes controlling the expression of auxin and cytokinin synthesis. Deletion of these genes resulted in the production of transformed tissues that required media supplemented with auxin and cytokinin for continued growth and regeneration of normal shoots and plants. Nadolska-Orczyk et al. [43] showed that efficient Agrobacterium-mediated transformation of Metrosideros polymorpha provide molecular techniques to facilitate comparative genomics. It was reported that above-discussed facts led to the use of Ti plasmid of Agrobacterium as a vector for plant transformation and kanamycin resistance genes as selection marker for transformed cells, and then transformed plants are regenerated [41] (Figures 1 and 2). Agrobacterium-mediated transformation followed by somatic embryogenesis remains the method of choice for most cotton transformation. However, regeneration aspect of the transformation process remains more difficult and choices are limited in

As Agrobacterium is highly attracted to phenolics, this method of transformation is not preferable for monocots due to production of phenolics, whereas it can be used for dicots. However, recently, Agrobacterium-mediated transformation has been optimized and becomes the best

In early 1980s, direct DNA delivery methods for protoplast were developed [44], especially for the economically important cereal crops, which were not subjected to the umbrella Agrobacterium-mediated transformation [45]. In this procedure, osmotic or electric shocks are applied to the protoplasts suspended in solutions containing DNA. After that planting on selection media is performed. Transformation based on embryogenic cell suspension cultures produced the first transgenic cereals [13]. The use of protoplasts for genetic transformation became less striking once it was shown that monocots could be transformed by co-cultivation of embryonic tissues and supervirulent strains of Agrobacterium in the presence of acetosyringone [15]. In case of Ricinus (castor oil plant), transformation through gene gun is dependent on many factors such as helium pressure, target distance, osmoticum, microcarrier type and size, DNA quantity, explant type, and number of bombardments had significant influence on transformation efficiency (Figure 2). Recommended distance is 6.0 cm, helium pressure of 1100 psi, 0.6-μm gold microcarriers, furthermore single bombardment has shown

case of cotton, one of the most difficult crop for transformation [42].

method for transformation of monocots [43].

6.4. Biolistic transformation

a new class of metabolites.

cells [39].

214 Cotton Research

Limitations of Agrobacterium-mediated transformation and difficulties involved in isolation and maintaining of embryogenic cultures have led to the discovery of universal transformation method redundant [48]. However, not a single transformation technique had proved satisfactory to be universal; therefore, a novel transformation technique for transformation was developed by Sanford et al. [49]. DNA-coated gold or tungsten particles are bombarded with high velocity to the intact cells or tissues. Combination of Agrobacterium and biolistics methods of transformation is most widely used method. However, choice depends on the individual researcher or plant tissue/ sample [50]. Figure 2 explains the schematic overview of biolistic gene transformation in plants.

#### 6.6. Chemical method

To enhance the uptake of DNA, a combination of polybrene-spermidine treatment is used to obtain non-chimeric transgenic cotton plants. Polybrene-spermidine combination treatment for plant genetic transformation has the advantage because it is less toxic than the other chemicals; furthermore, it also protects condensation effect, DNA shearing, and integration of the plasmid with host genome [35]. To deliver plasmid DNA into cotton suspension culture obtained from

Figure 2. Schematic overview of gene transformation through gene gun.

cotyledon-induced callus, polybrene and/or spermidine treatments were used. Researchers have also regenerated and analyzed the cotton plants containing HPT genes as a selectable marker [51]. Major limitation in chemical transformation technique is its low efficiency when compared to other transformation techniques.

#### 6.7. Microinjection

Microinjection technique is based on introducing DNA in the cells using injection pipette of microcapillary glass [51]. This operation requires a micromanipulator. In this case, cells are immobilized by holding glass and gentle suction. Both pipettes are filled with mineral oil.

Microinjection is mostly used for animal cells, while with plants, a cell wall causes hindrance for transformation by microinjection as it works as barrier for microglass tools. Using

Figure 3. Schematic overview of gene transformation through chemical [12].

microinjection technique for protoplast, there is a risk of toxic compounds to be released, which may cause sudden death of the protoplast. It is also possible to remove vacuoles before microinjection but regeneration and division may be decreased [52].

Protoplast microinjection involves different methods for immobilization, in which instead of using sucking poly-L-lysine is coated to the protoplasts. One of the major advantages of the microinjection is that it not only allows the transformation of the DNA plasmid but also the whole chromosome [53]. This technique is being used for the cellular mechanism and functions of the plant cells and to study the physiology of the plastids especially for tobacco [54] (Figure 3). Major limitation of microinjection method involves the use of expensive micromanipulator and it is a time-consuming procedure. Furthermore, frequency of transformation is very low and dependent on the species, i.e., proved to be successful in tobacco [55], Petunia [53], Rape [56], and Barley [57].

#### 6.8. The pollen tube pathway method

Transformation by pollen tube pathway has got great intention in molecular breeding [58]. After pollination, a foreign DNA/plasmid is applied to the styles. To reach ovule, DNA uses the pollen tube pathway. This method of transformation was first used by Luo et al. [60] in rice [59]. In case of rice, a high frequency of transgenic plants was obtained, and this method was then applied to the other commercially important crops, such as wheat [60], soybean [61], Petunia hybrid [62], and watermelon [63]. At the premeiotic stage, bacterial inoculum can be applied to the inflorescence without removing the stigma. Pena et al. [65] used this protocol for transformation in rye. Limitation of pollen tube pathway method reported by Shou et al. [66] collected pollens from the genetically engineered plants and reported 10-fold less efficiency in soybean. It was concluded that pollen tube pathway is not reproducible in case of cotton and soybean (Figure 4).

#### 6.9. Liposomes

cotyledon-induced callus, polybrene and/or spermidine treatments were used. Researchers have also regenerated and analyzed the cotton plants containing HPT genes as a selectable marker [51]. Major limitation in chemical transformation technique is its low efficiency when

Microinjection technique is based on introducing DNA in the cells using injection pipette of microcapillary glass [51]. This operation requires a micromanipulator. In this case, cells are immobilized by holding glass and gentle suction. Both pipettes are filled with mineral oil.

Microinjection is mostly used for animal cells, while with plants, a cell wall causes hindrance for transformation by microinjection as it works as barrier for microglass tools. Using

compared to other transformation techniques.

Figure 2. Schematic overview of gene transformation through gene gun.

6.7. Microinjection

216 Cotton Research

Direct transformation of the foreign DNA into the plant cells using liposomes was Employed in the 1980s. Liposomes are phospholipids with spherical shape, carry nucleic acid, and

Figure 4. Schematic overview of gene transformation through pollen tube [12].

internally aqueous. Liposomes were put in a nutshell with the DNA fragments to get attached to the cell membranes. Thus, in this way DNA enters the cell and then to the nucleus. For the transfer of the bacterial, plant, and animal genes, lipofection has been a very competent technique. Lipofection takes place by fusion through membrane, and it has improved transformation efficiency because the genetic material used for lipofection is not naked as used in conventional techniques [64, 65]. In spite of cheap and less equipment demanding technique, liposome transformation is not so common. Major limitations in this technique are its low efficiency and being so hectic. Therefore, success story for liposome-mediated transformation have been published so far only for tobacco [66] and wheat [67].

#### 6.10. Shoot apex method of transformation

Transformation by shoot apex method is a rapid method of transformation in cotton, and it is a genotype independent method. In this method, shoots are isolated from the plant and subjected to a virulent strain of A. tumefaciens, an antibiotic selection is also applied, and shoots are regenerated in vitro. In shoots, dedifferentiation does not take place and is less prone to mutations. Rooting occurs in 6–10 days depending on the cotton cultivar [68]. The shoot apex gives hairy transformed roots because it is most reactive to develop tumor. Shoot apex method is a substitute method for Agrobacterium-mediated transformation of dicotyledonous cultivars in which regeneration by protoplasts, leaf disks, or epidermal strips does not take place. This technique offers a rapid method for transformation with less risk of mutations [27]. Katageri et al. [59,71] transformed an Indian variety with shoot apex method and showed a great potential for transformation. Shoot apex transformation method is a time consuming and laborious method for transformation, and there are higher rates of somaclonal variations.

#### 6.11. Sonication-assisted Agrobacterium-mediated transformation (SAAT)

SAAT method is based on principle of causing thousands of wounds by ultrasound. These wounds allow the Agrobacterium to infect and cause gall formation. The chance of transformation of gene of interest is increased in sonication-assisted method due to infecting deeper plant cells. For the GUSA, transient expression protocol was optimized. This method has shown competitive advantage for being used as a routine method [10]. A sonication-assisted method of transformation was used to improve the transformation efficiency of the flax. These wounds created by the ultrasounds allow the uptake of the plasmid DNA into the flax hypocotyls and cotyledons, and its efficiency is dependent on duration and frequency of the sonication applied. SAAT could be a potential tool for increasing transformation efficiency in flax [11]. Due to low efficiency of Agrobacterium-mediated method in cotton, an alternative method was required to overcome the barrier of efficiency. SAAT should be a valuable and alternative method for demonstrating the stable cotton transformation [69].

#### 6.12. Infiltration

internally aqueous. Liposomes were put in a nutshell with the DNA fragments to get attached to the cell membranes. Thus, in this way DNA enters the cell and then to the nucleus. For the transfer of the bacterial, plant, and animal genes, lipofection has been a very competent technique. Lipofection takes place by fusion through membrane, and it has improved transformation efficiency because the genetic material used for lipofection is not naked as used in conventional techniques [64, 65]. In spite of cheap and less equipment demanding technique, liposome transformation is not so common. Major limitations in this technique are its low efficiency and being so hectic. Therefore, success story for liposome-mediated transformation

Transformation by shoot apex method is a rapid method of transformation in cotton, and it is a genotype independent method. In this method, shoots are isolated from the plant and subjected to a virulent strain of A. tumefaciens, an antibiotic selection is also applied, and shoots are regenerated in vitro. In shoots, dedifferentiation does not take place and is less prone to mutations. Rooting occurs in 6–10 days depending on the cotton cultivar [68]. The shoot apex

have been published so far only for tobacco [66] and wheat [67].

Figure 4. Schematic overview of gene transformation through pollen tube [12].

6.10. Shoot apex method of transformation

218 Cotton Research

In vitro culture is required for some transformation procedures. Some transformation procedures do not require in vitro culture. In the case of infiltration, plant parts at meiotic or mitotic stages are applied with bacterial inoculums. This method is mainly applied for Arabidopsis species for the past many years.

This simple procedure in which plant at the early stages is placed upside down in the beaker containing 5% sucrose solution with A. tumefaciens in such a way that only the inflorescences are submerged in the inoculum. Beakers are placed in vacuum chamber with usually 0.05 bar pressure. Seeds are collected and sown on selected media under sterile condition in order to protect from microbial contamination. Up to 95% plants with transgenic seeds can be obtained by keeping optimal conditions.

Vacuum infiltration was applied for the first time in 1993 for transformation of Arabidopsis [11]. Over the next 5 years, the optimal conditions were improved and efficiency increased about 2%. Another version of this protocol is to use optimum concentration of sucrose and bacterial inoculum by the surfactant, Silwet L-19 [70]. This modified protocol, which was optimized by Clough and Bent, gives the best result in transformation of Arabidopsis [71]. Modified infiltration method was replaced the vacuum infiltration, and the protocol was modified a bit by immersing flowering plant in bacterial culture or spraying bacterial culture on plant. In another study, which was performed by Chung et al. [72], there is a comparison of the classical vacuum infiltration and new version which include modification. The results indicated that plants with bacterial suspension produced much better results, i.e., 2.41% vs. 1.76% for vacuum infiltration and 2.09% for immersion.

Infiltration method is most suitable to plants which have smaller genome. Vacuum infiltration is excluded for plants having genome greater than Arabidopsis. Attempts were failed with Chinese cabbage so far. Hence this technique is species specific [12].

#### 6.13. Silicon carbide-mediated transformation (SCMT)

SCMT is less complicated method of plant transformation. Silicon carbide fibers are added to a suspension containing plant tissue (cell clusters, immature embryos, and/or callus) and plasmid DNA; it is mixed and then vortexed. Kaeppler et al. [73] demonstrated that DNA-coated fibers penetrate the cell wall in the presence of small holes created by collisions between the plant cells and fibers. The fibers mostly used in this procedure are single crystals of silica organic minerals like silicon carbide, elongated in shape having a length of 10–80 mm, and a diameter of 0.6 mm and show a high resistance to expandability.

The factors controlling the efficiency of SCMT are fiber size, vortexing parameters, shape of the vessels used, the plant material, and the characteristics of the plant cells, especially the thickness of the cell wall. The main advantages of this procedure are the low expenses and its usefulness for various plant materials. Disadvantages of this method are low transformation efficiency, damage to cells negatively influencing their further regeneration capability, and the necessity of obeying extraordinarily rigorous precaution protocols during laboratory work, as breathing the fibers in, especially asbestos ones, can lead to serious sicknesses [74].

Transgenic forms, cell colonies, or plants were derived from maize [75], and rice [76], from wheat [77], from tobacco [78], and from Lolium multiflorum, Lolium perenne, Festuca arundinacea, and Agrostis stolonifera [79]. Maize variety Black Mexican Sweet (BMS) cell suspension was transformed with the plasmid carrying the BAR gene [73]. They found that 3.4% transgenic cell lines were expressing both transgenes from a 300 ml of packed cell volume, which shows that the integration of transgenes occurred in one per one million cells. The efficiency was significantly lower as described earlier by the same team or other authors employing micro bombardment for transformation. Vortexing with silicon carbide fibers caused the damage to cells decreasing their viability about 29% and causing the decrease in the efficiency of transformation. However, SCMT is speedy and cost-effective with easy to perform. Therefore, this method may prove an alternative method for plant transformation where gene gun method is restricted (i.e., most of monocots). It was suggested that SCMT system of using commercial shakers, which has been reported for maize, seemed to be very good for commercial largescale transformation [80]. Frame et al. [81] obtained first fertile transgenics for maize in 1994. Out of 22 independent transgenic cell lines, 311 transgenic plants were derived, and 8 of those turned out to be stable transformants. The efficiency of SCMT was 5- to 10-fold lower than gene gun-mediated transformation. Petolino et al. [79] reported that efficiency is much lower in comparison with micro bombardment. The authors concluded that silicon carbide fibers cause damage to transformed cells that is why the results obtained were unsatisfactory [75].

#### 6.14. Electroporation of intact plant cells and tissues

plants with bacterial suspension produced much better results, i.e., 2.41% vs. 1.76% for vac-

Infiltration method is most suitable to plants which have smaller genome. Vacuum infiltration is excluded for plants having genome greater than Arabidopsis. Attempts were failed with

SCMT is less complicated method of plant transformation. Silicon carbide fibers are added to a suspension containing plant tissue (cell clusters, immature embryos, and/or callus) and plasmid DNA; it is mixed and then vortexed. Kaeppler et al. [73] demonstrated that DNA-coated fibers penetrate the cell wall in the presence of small holes created by collisions between the plant cells and fibers. The fibers mostly used in this procedure are single crystals of silica organic minerals like silicon carbide, elongated in shape having a length of 10–80 mm, and a

The factors controlling the efficiency of SCMT are fiber size, vortexing parameters, shape of the vessels used, the plant material, and the characteristics of the plant cells, especially the thickness of the cell wall. The main advantages of this procedure are the low expenses and its usefulness for various plant materials. Disadvantages of this method are low transformation efficiency, damage to cells negatively influencing their further regeneration capability, and the necessity of obeying extraordinarily rigorous precaution protocols during laboratory work, as

Transgenic forms, cell colonies, or plants were derived from maize [75], and rice [76], from wheat [77], from tobacco [78], and from Lolium multiflorum, Lolium perenne, Festuca arundinacea, and Agrostis stolonifera [79]. Maize variety Black Mexican Sweet (BMS) cell suspension was transformed with the plasmid carrying the BAR gene [73]. They found that 3.4% transgenic cell lines were expressing both transgenes from a 300 ml of packed cell volume, which shows that the integration of transgenes occurred in one per one million cells. The efficiency was significantly lower as described earlier by the same team or other authors employing micro bombardment for transformation. Vortexing with silicon carbide fibers caused the damage to cells decreasing their viability about 29% and causing the decrease in the efficiency of transformation. However, SCMT is speedy and cost-effective with easy to perform. Therefore, this method may prove an alternative method for plant transformation where gene gun method is restricted (i.e., most of monocots). It was suggested that SCMT system of using commercial shakers, which has been reported for maize, seemed to be very good for commercial largescale transformation [80]. Frame et al. [81] obtained first fertile transgenics for maize in 1994. Out of 22 independent transgenic cell lines, 311 transgenic plants were derived, and 8 of those turned out to be stable transformants. The efficiency of SCMT was 5- to 10-fold lower than gene gun-mediated transformation. Petolino et al. [79] reported that efficiency is much lower in comparison with micro bombardment. The authors concluded that silicon carbide fibers cause

breathing the fibers in, especially asbestos ones, can lead to serious sicknesses [74].

damage to transformed cells that is why the results obtained were unsatisfactory [75].

Chinese cabbage so far. Hence this technique is species specific [12].

diameter of 0.6 mm and show a high resistance to expandability.

6.13. Silicon carbide-mediated transformation (SCMT)

uum infiltration and 2.09% for immersion.

220 Cotton Research

The principles of electroporation are the same for plant cells and protoplasts. However, difference may exist in other plant tissues such as pollen, microspores, leaf fragments, embryos, callus, seeds, or buds. During electroporation, the material used can be in the form of plasmid DNA and Agrobacterium inoculums. To check the transient expression of transgenes, different efforts were performed in early 1990s by applying electroporation technology.

Protocols were established for successful electroporation in cell suspensions, e.g., in tobacco [82], rice [24], and in wheat [83]. In early 1990s, experiments were performed to obtain transgenic plants. It was reported that the best results were obtained for maize. Researchers transformed immature embryos and embryogenic callus type I, which were treated by a solution of pectolytic enzymes, and then transferred into electroporating cuvettes [66]. The electroporation efficiency was relatively high when compared with micro bombardment conducted for same species, and about 90 transgenic plants were regenerated from 1440 embryos (6.25%) and 31 plants from 55 callus clusters (54.6%).

Laursen et al. [86] obtained similar results for this species. The authors estimated that the integration of transgenes took place approximately in one per 10,000 cells. Sorokin et al. [87] reported that much lower efficiency, i.e., about three transgenic plants from 1080 immature embryos (0.28%), was observed in the case of wheat electroporation. The transformation efficiency could be increased by the post-pulse addition of ascorbic acid or another ascorbate without any negative influence on cell viability [69]. Tissues were electroporated in liquid media containing 8 mg/L benzyl adenine that showed maximal regeneration through secondary somatic embryogenesis. DaSilva et al. [88] reported that the secondary somatic embryos regenerated from electroporation were positive for GUS expression. PCR analysis was positive for the GUS and BAR genes at torpedo shape somatic embryos. For some species, electroporation is an effective method but it is not widely used for plant transformation.

#### 6.15. Electrophoresis

At the end of the 1980s, Songstad et al. [78] developed a method employing electrophoresis for the transformation of immature embryos, especially in monocotyledonous plants. This method was adopted as an alternative for transformation, but it is very expensive and yield poor results when compared with micro-projectile bombardment [53]. Transfected embryos were placed between the tip of two pipettes and connected to electrodes. The pipette connected to the anode was filled with agar / agarose followed by an EDTA containing electrophoresis buffer.

The pipette connected to the cathode contained agar that was mixed with DNA and an electrophoresis buffer. This pipette was in contact with the apical meristem of the embryo, whereas the second one was located near basal apical part of embryo. Electrophoresis-mediated transformation efficiency depends on various factors, such as electrical field parameters, duration of electrophoresis, contents of electrophoresis buffer, and physicochemical properties of the embryonic tissues. Voltage of 25 mV and an amperage of 0.5 mA for 15 minutes are mostly used for electrophoresis program [74]. In spite of its simplicity, electrophoresis is not considerable method in plant transformation, and the reason behind is less viability of treated embryos. Ahokas et al. [84] showed that none of the plants showed expression of transgene inserted. Griesbach et al. [85] obtained successfully transformed plants of Calnthe orchid L.

## 7. Conclusion

Plant transformation is an essential tool for incorporating new characteristics in crop plant like cotton. Cotton is recalcitrant crop hence a reproducible regeneration is not available in local cotton varieties. Among all strategies developed by different researchers, a little success in cotton (G. hirsutum) genetic modification has been reported using Agrobacterium-mediated shoot apex cut method of cotton or embryo and sonication-assisted Agrobacterium-mediated transformation, except Coker genotypes, where regeneration potential leads to the use of particle bombardment or even Agrobacterium-mediated gene transformation as the best procedure. Limitations of gene transformation in cotton are observed in almost all of other procedures. One the most listed limitations of their application is the decreased viability of cells. Infiltration is the main transformation method for Arabidopsis, whereas SCMT is for maize, which does not have much success in cotton. With optimizing conditions, these transformation procedures might soon be available for a broad spectrum of plant transformation. The investigations like RNAseq of cotton regeneration in comparison with Coker genotypes or 2D-gel electrophoresis of embryogenic callus of cotton in comparison with control can give better insight to further improve the regeneration potential of cotton cultivars for improvement of cotton regeneration and for better cotton genetic modification in future.

## Author details

Abdul Qayyum Rao<sup>1</sup> \*, Muhammad Azam Ali<sup>1</sup> , Muhammad Azmat Ullah Khan<sup>2</sup> , Kamran Shahzad Bajwa1 , Adnan Iqbal<sup>1</sup> , Tahir Iqbal<sup>1</sup> , Ahmad Ali Shahid<sup>1</sup> , Idrees Ahmad Nasir and Tayyab Husnain<sup>1</sup>

\*Address all correspondence to<EMAIL_ADDRESS>
1 National Center of Excellence in Molecular Biology, University of the Punjab, Lahore Pakistan

2 Faculty of Life Sciences, University of Central Punjab, Lahore, Pakistan

## References

[1] Fraley RT, Rogers SG, Horsch RB, Sanders PR, Flick JS, Adams SP Bittner ML, Brand LA, Fink CL, Fry JS. Expression of bacterial genes in plant cells. Proceedings of the National Academy of Sciences. 1983;80:4803-4807. DOI: 6308651

[2] Hoekema A, Hirsch P, Hooykaas P, Schilperoort R. A binary plant vector strategy based on separation of vir-and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature. 1983;303:179-180. DOI: 10.1038/303179a0

considerable method in plant transformation, and the reason behind is less viability of treated embryos. Ahokas et al. [84] showed that none of the plants showed expression of transgene inserted. Griesbach et al. [85] obtained successfully transformed plants of Calnthe orchid L.

Plant transformation is an essential tool for incorporating new characteristics in crop plant like cotton. Cotton is recalcitrant crop hence a reproducible regeneration is not available in local cotton varieties. Among all strategies developed by different researchers, a little success in cotton (G. hirsutum) genetic modification has been reported using Agrobacterium-mediated shoot apex cut method of cotton or embryo and sonication-assisted Agrobacterium-mediated transformation, except Coker genotypes, where regeneration potential leads to the use of particle bombardment or even Agrobacterium-mediated gene transformation as the best procedure. Limitations of gene transformation in cotton are observed in almost all of other procedures. One the most listed limitations of their application is the decreased viability of cells. Infiltration is the main transformation method for Arabidopsis, whereas SCMT is for maize, which does not have much success in cotton. With optimizing conditions, these transformation procedures might soon be available for a broad spectrum of plant transformation. The investigations like RNAseq of cotton regeneration in comparison with Coker genotypes or 2D-gel electrophoresis of embryogenic callus of cotton in comparison with control can give better insight to further improve the regeneration potential of cotton cultivars for improvement of

, Muhammad Azmat Ullah Khan<sup>2</sup>

, Ahmad Ali Shahid<sup>1</sup>

, Kamran

, Idrees Ahmad Nasir and

cotton regeneration and for better cotton genetic modification in future.

\*, Muhammad Azam Ali<sup>1</sup>

\*Address all correspondence to<EMAIL_ADDRESS>
, Tahir Iqbal<sup>1</sup>

2 Faculty of Life Sciences, University of Central Punjab, Lahore, Pakistan

Academy of Sciences. 1983;80:4803-4807. DOI: 6308651

1 National Center of Excellence in Molecular Biology, University of the Punjab, Lahore

[1] Fraley RT, Rogers SG, Horsch RB, Sanders PR, Flick JS, Adams SP Bittner ML, Brand LA, Fink CL, Fry JS. Expression of bacterial genes in plant cells. Proceedings of the National

, Adnan Iqbal<sup>1</sup>

7. Conclusion

222 Cotton Research

Author details

Shahzad Bajwa1

Tayyab Husnain<sup>1</sup>

Pakistan

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Abdul Qayyum Rao<sup>1</sup>

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**Cotton Based Products and Textile Research**

#### **Chapter 11 Provisional chapter**

#### **UV Treatments on Cotton Fibers UV Treatments on Cotton Fibers**

Franco Ferrero, Gianluca Migliavacca and Monica Periolatto Franco Ferrero, Gianluca Migliavacca and Monica Periolatto

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/63796

#### **Abstract**

Ultraviolet (UV) radiations can act in different ways on the functionalization of textiles, through pre- or posttreatments, in order to modify their behavior in dyeing and finishing processes. In cotton fiber, unlike the wool, the UV absorption is not due to any of the structural groups of the normal cellulosic chains and can only be attributed to "impurities" or "faults" bearing carbonyl and/or carboxyl groups. In fact, UV irradiation coupled with mild oxidation can improve some properties of the cotton fibers such as pilling resistance, water swelling, and dyeability. Therefore, the process of differential dyeing with direct and reactive dyes assisted by UV irradiation was studied and interesting differential chromatic effects were obtained by a UV posttreatment capable to fade dyeing. On the other hand, the surface modification of cotton fabrics by UV curing and UV grafting with suitable chemicals was pursued to obtain finishing treatments able to confer oil and/or water repellency. Finally, antimicrobial finishing by chitosan UV grafting was proposed as valid environmental friendly method to confer a satisfactory washing-resistant antimicrobial activity to cotton fabrics even with low polymer add-on.

**Keywords:** cotton, dyeing, finishing, UV curing, UV grafting, UV radiation

#### **1. Introduction**

Cotton fiber differs markedly from other cellulose fibers in morphological traits. Due to its many features, cotton, even though it was discovered later than other fibers, gained a high position and strongly stimulated the development of textile industry. Cotton today is the most used textile fiber in the world. Its current market share is 56% for all fibers used for apparel and home furnishings; another contribution is attributed to nonwoven textiles and personal

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

care items. It is generally recognized that most consumers prefer cotton fiber for personal care items to those containing synthetic fibers.

Soft hand, good absorbency, and color retaining are among the advantages of cotton fabric; it is easy to print, handle, and sew; it is machine-washable and dry-cleanable; and it has good strength and hangs well. These are all the desirable properties for a textile but, for some applications, they can show drawbacks, which suggested modifications of the fibers achievable with chemical or physical treatments. Among the latter, ultraviolet (UV) irradiation was experimented.

UV radiations can act in different ways on the functionalization of textiles, through pre- or post-treatments, in order to modify their behavior in dyeing and finishing processes.

The UV treatments applied in textile field, in addition to required specific effects, should also have the following advantages: (1) easy application, not needing expensive and sophisticated equipment; (2) durability and fastness of the degree of treatment looking at the future uses; (3) limited treatment costs, without precluding any market segment; and (4) environmentally friendly requirements, for sustainable textile processes. Many research works were developed with the aim to utilize the effects of UV radiations on various natural as well as synthetic fibers [1–6]. Most of these studies were devoted to the surface modification of wool, but even cotton fiber was considered.

In cotton, unlike the wool, the true origin of the UV absorption, which is not due to any of the structural groups that make up the normal cellulosic chains, is uncertain and can only be attributed to "impurities" or "faults" bearing carbonyl and/or carboxyl groups.

Naturally pigmented green cotton fiber derives its color from caffeic acid that is deposited in alternating layers with cellulose around the outside of the fiber. The isolated compound is fluorescent (287 and 310 nm absorption wavelengths), and it has been theorized that its purpose is to absorb UV radiation in order to protect the seed. Brown and tan cottons derive their color from tannin vacuoles in the lumen of the fiber cells rather than in the wax layer as in green cotton. The brown color appears after the fibers are exposed to oxygen and sunlight, which happens when the seed pod opens.

The gray cotton fiber contains the above-cited impurities, so one or more pretreatment processes before dyeing are applied to attain its full textile exploitation. The pretreatment processes of cotton fabrics include scouring to get off noncellulosic impurities, pigments, and waxes. An oxidative bleaching is carried out to wipe out natural coloring matter for white or dyeing, while singing is applied to eliminate the protruding loose fibers [7].

Mansuri et al. [8] claimed that cotton-knitted fabric padded in a solution of hydrogen peroxide and then irradiated with UV lamp showed a significant pilling reduction, comparable to that obtained with industrial system of biopolishing. Moreover, Millington said that Siroflash® treatment (UV irradiation coupled with mild wet oxidation with hydrogen peroxide) is highly effective against pilling formation of wool as well as of cotton fiber [9]. In the latter case, the application of Siroflash® process would require the presence of a sensitizer or a photoinitiator to absorb UV radiation in the primary photochemical step. Such a photoinitiator must be odorfree, nontoxic, low expensive, and easily removable by aqueous washing. This rules out many of the commercial UV photoinitiators, but hydrogen peroxide is able to absorb UV radiations below 300 nm to produce highly reactive hydroxyl radicals. One would expect these radicals to reduce the strength of knitted cotton fabric by breaking the cellulose chains. In fact, significant dry-burst strength losses were observed in dependence on hydrogen peroxide concentration, but some fabric strength loss would be acceptable if the limiting factor is an unacceptable level of pills.

care items. It is generally recognized that most consumers prefer cotton fiber for personal

Soft hand, good absorbency, and color retaining are among the advantages of cotton fabric; it is easy to print, handle, and sew; it is machine-washable and dry-cleanable; and it has good strength and hangs well. These are all the desirable properties for a textile but, for some applications, they can show drawbacks, which suggested modifications of the fibers achievable with chemical or physical treatments. Among the latter, ultraviolet (UV) irradiation was

UV radiations can act in different ways on the functionalization of textiles, through pre- or

The UV treatments applied in textile field, in addition to required specific effects, should also have the following advantages: (1) easy application, not needing expensive and sophisticated equipment; (2) durability and fastness of the degree of treatment looking at the future uses; (3) limited treatment costs, without precluding any market segment; and (4) environmentally friendly requirements, for sustainable textile processes. Many research works were developed with the aim to utilize the effects of UV radiations on various natural as well as synthetic fibers [1–6]. Most of these studies were devoted to the surface modification of wool, but even cotton

In cotton, unlike the wool, the true origin of the UV absorption, which is not due to any of the structural groups that make up the normal cellulosic chains, is uncertain and can only be

Naturally pigmented green cotton fiber derives its color from caffeic acid that is deposited in alternating layers with cellulose around the outside of the fiber. The isolated compound is fluorescent (287 and 310 nm absorption wavelengths), and it has been theorized that its purpose is to absorb UV radiation in order to protect the seed. Brown and tan cottons derive their color from tannin vacuoles in the lumen of the fiber cells rather than in the wax layer as in green cotton. The brown color appears after the fibers are exposed to oxygen and sunlight, which

The gray cotton fiber contains the above-cited impurities, so one or more pretreatment processes before dyeing are applied to attain its full textile exploitation. The pretreatment processes of cotton fabrics include scouring to get off noncellulosic impurities, pigments, and waxes. An oxidative bleaching is carried out to wipe out natural coloring matter for white or

Mansuri et al. [8] claimed that cotton-knitted fabric padded in a solution of hydrogen peroxide and then irradiated with UV lamp showed a significant pilling reduction, comparable to that obtained with industrial system of biopolishing. Moreover, Millington said that Siroflash® treatment (UV irradiation coupled with mild wet oxidation with hydrogen peroxide) is highly effective against pilling formation of wool as well as of cotton fiber [9]. In the latter case, the application of Siroflash® process would require the presence of a sensitizer or a photoinitiator to absorb UV radiation in the primary photochemical step. Such a photoinitiator must be odor-

attributed to "impurities" or "faults" bearing carbonyl and/or carboxyl groups.

dyeing, while singing is applied to eliminate the protruding loose fibers [7].

post-treatments, in order to modify their behavior in dyeing and finishing processes.

care items to those containing synthetic fibers.

experimented.

234 Cotton Research

fiber was considered.

happens when the seed pod opens.

Mercerization is the treatment of cotton yarns, fabrics, and knit goods with cold, strong caustic soda bath under tension to give increased luster, smoothness, dye and finishing chemical uptake, dimensional constancy, and better mechanical properties. Zuber et al. [10] demonstrated that UV rays have a similar effect, on cellulosic fibers, as the alkaline treatment; irradiated cellulosic fibers showed higher swelling in comparison with any concentration of soda treatment, but tear and tensile strength were worse compared to the untreated and to the alkali-treated ones. They emphasized that UV rays do not produce loss in weight after exposure of cotton fabrics, and affinity for direct dyes is slightly enhanced in comparison with that found with mercerization. However, with a reactive dye it is found that UV irradiation not only enhanced the strength of dye on irradiated fabric but also improved the dyeing properties [11]. The reason for this might be that the exposure of cotton fiber to UV radiation in the presence of oxygen causes cellulose oxidation to carboxylic acids and opens spaces between the fibers causing imbibition of more dye, hence the interaction for dyeing becomes more significant [12].

Iqbal et al. [13] and more recently Adeel et al. [14] observed that UV radiation not only enhances the uptake and fastness of natural dyes on pre-irradiated cotton fabric using low concentrations of dye but also with a low concentration of mordant.

Cotton fiber immersed in water develops a negative surface potential, which consequently gives rise to electrostatic repulsion of anionic dyes. Therefore, a large amount of electrolyte, such as sodium sulfate or chloride, is required in order to reduce the repulsion between the negatively charged fibers and the anionic dyes. Moreover, the complete dye exhaustion is not normally reached, causing environmental problems due to the discharge of colored effluent having a high salt concentration. To overcome these problems, the cationization of cotton fiber was applied to increase the substantivity of anionic dyes by introducing positively charged sites on the fibers. Pretreatments with glycidyltrimethylammonium chloride and other cationization reagents were reported. However, cationic polymerizable monomers, such as methacryloyl quaternary ammonium compounds, have also been fixed onto cotton fabric using UV radiation with a view to producing print-patterned dyeings [15].

Instead, Jang et al. [16] have examined the potential of photografting cationic monomers onto crease-resistant cross-linked cotton fabric by incorporation of a water-soluble photoinitiator to improve grafting. In fact, whereas untreated cotton is readily dyeable with anionic dyes in the presence of salt, the cross-linked fiber is undyeable to a comparable deep shade, since the diffusion of relatively large dye molecules into the cross-linked substrate is hindered by a low swelling under aqueous conditions. The dyeability of cationized cotton fiber by three classes of dyes (direct, reactive, and sulfur dyes) has been significantly improved owing to the increased ionic attraction between dyes and cationized cellulose, even in the absence of salts [17].

On the other hand, Dong and Jang [18] proposed the direct photografting of wool-reactive dyes themselves onto cotton fabric. This coloration utilizes the photoreactivity of certain dyes under UV irradiation, through photopolymerization, photografting, and photocross-linking processes, to form a polymerized and cross-linked dye network. Thus, a single class of dye can color almost any textile substrate since no specific affinity of particular dyes to individual fibers is required. Reactive dyes containing an α-bromoacrylamido reactive group are among the most successful metal-free dyes for protein fibers because of their brilliant color and high wetfastness properties. However, these reactive dyes hardly react with cotton fiber under similar conditions owing to the rather low nucleophilicity of the hydroxyl groups in cellulose compared with the thiol and amino groups in proteins. Therefore, dyes containing α-bromoacrylamido groups were employed as grafting monomers to be photografted onto cellulose under continuous UV irradiation. This novel approach may realize the photoactive coloration of cotton fiber even with dyes of low affinity for conventional reactive dyeing. Furthermore, the coloration does not require large amounts of salt, time, and energy, which makes it an alternative process of excellent environmental friendliness.

## **2. Differential dyeing on cotton fabric by UV irradiation**

#### **2.1. Differential dyeing**

Textile world is always looking for new color effects, both for artistic questions and in order to meet the current fashion demands; for these reasons, differential dyeing effects mainly on wool fibers were studied. These can be achieved by surface modification on selected areas of fabric by a physical surface treatment such as plasma [19] or UV irradiation on one side followed by a conventional dyeing [20]. Moreover, patterning effects can be obtained if a suitable mask is interposed between energy source and fabric for selective surface modification. This represents an alternative and less expensive method than the usual production of patterned fabrics by printing or color weaving.

In the Sun-Wash method patented by Nearchimica with Stamperia Emiliana (Italy) [21], the continuous UV pretreatment of wool fabric on one face before dyeing has been proposed to obtain different shades on the two faces or patterning effects. A careful selection of dyes is needed to obtain satisfactory tone-on-tone effects and even more to produce double-face effects with different colors. These suggestions inspired the experiments on differential dyeing of wool by UV irradiation carried out by Migliavacca et al. [22]. A good final shade uniformity was obtained, with an acceptable color difference (Δ*E* ≥5.0) between UV-treated and untreated fabric area (double-face effect), due to an increased dye-fiber affinity of the side previously treated with UV radiation.

The same type of chromatic effect was also wanted for cellulosic fibers, in particular for cotton [23]. An unscoured 100% cotton yarn was chosen (*Nm* = 2/34), and from this material many knitted fabrics were prepared in order to do dyeing tests with direct dyes. At first, these fabrics were subjected to a dynamic UV irradiation under a Hg-medium pressure vapor lamp, with the following parameters:

Colorimetric measurements were made comparing the UV-treated fabric with the untreated one, using a reflectance spectrophotometer Datacolor Check II Plus, with the results reported in **Table 1**.

**Table 1.** CIE L a b difference parameters for unscoured cotton fabric after UV treatment.

In these conditions of treatment, a global fiber-yellowing effect is observed, mainly due to a process that involves both photodegradation and photochromic reactions; chemical species probably involved in this phenomenon are quinol and phenylcoumaran residues (belonging to lignin impurity).

#### **2.2. Dyeings performed with direct dyes**

increased ionic attraction between dyes and cationized cellulose, even in the absence of salts

On the other hand, Dong and Jang [18] proposed the direct photografting of wool-reactive dyes themselves onto cotton fabric. This coloration utilizes the photoreactivity of certain dyes under UV irradiation, through photopolymerization, photografting, and photocross-linking processes, to form a polymerized and cross-linked dye network. Thus, a single class of dye can color almost any textile substrate since no specific affinity of particular dyes to individual fibers is required. Reactive dyes containing an α-bromoacrylamido reactive group are among the most successful metal-free dyes for protein fibers because of their brilliant color and high wetfastness properties. However, these reactive dyes hardly react with cotton fiber under similar conditions owing to the rather low nucleophilicity of the hydroxyl groups in cellulose compared with the thiol and amino groups in proteins. Therefore, dyes containing α-bromoacrylamido groups were employed as grafting monomers to be photografted onto cellulose under continuous UV irradiation. This novel approach may realize the photoactive coloration of cotton fiber even with dyes of low affinity for conventional reactive dyeing. Furthermore, the coloration does not require large amounts of salt, time, and energy, which makes it an

Textile world is always looking for new color effects, both for artistic questions and in order to meet the current fashion demands; for these reasons, differential dyeing effects mainly on wool fibers were studied. These can be achieved by surface modification on selected areas of fabric by a physical surface treatment such as plasma [19] or UV irradiation on one side followed by a conventional dyeing [20]. Moreover, patterning effects can be obtained if a suitable mask is interposed between energy source and fabric for selective surface modification. This represents an alternative and less expensive method than the usual production of

In the Sun-Wash method patented by Nearchimica with Stamperia Emiliana (Italy) [21], the continuous UV pretreatment of wool fabric on one face before dyeing has been proposed to obtain different shades on the two faces or patterning effects. A careful selection of dyes is needed to obtain satisfactory tone-on-tone effects and even more to produce double-face effects with different colors. These suggestions inspired the experiments on differential dyeing of wool by UV irradiation carried out by Migliavacca et al. [22]. A good final shade uniformity was obtained, with an acceptable color difference (Δ*E* ≥5.0) between UV-treated and untreated fabric area (double-face effect), due to an increased dye-fiber affinity of the side previously

The same type of chromatic effect was also wanted for cellulosic fibers, in particular for cotton [23]. An unscoured 100% cotton yarn was chosen (*Nm* = 2/34), and from this material many

alternative process of excellent environmental friendliness.

patterned fabrics by printing or color weaving.

**2.1. Differential dyeing**

treated with UV radiation.

**2. Differential dyeing on cotton fabric by UV irradiation**

[17].

236 Cotton Research

Dyeing tests were carried out on cotton fabrics (UV irradiated and not) using Solophenyl® (Huntsman) direct dyes for cellulosic fibers applied on 0.1% o.w.f. (on the weight of fabric), 1:40 material to liquor ratio, with 2.0 g/L Albatex UNI as leveling agent, and 1.0 g/L NaCl. The dyeing was performed at 100°C for 30 min, then the samples were cooled at 80°C, and maintained at 80°C for 15 min. The irradiation conditions were the same as reported in Section 2.1.

The final bath exhaustion was about 70% for all the dyes, and the resulted dyeings showed good color level and chromatic homogeneity. However, the color differences between UVtreated and untreated fabrics were minimal, as shown in **Table 2**, where a color difference greater than 5.0 Δ*E* units was evidenced only with Navy Solophenyl BLE, evidenced in bold.

Increasing the dyeing depth over 0.1%, the chromatic differences between UV-treated and untreated fabrics would increase; however, the wet fastness of dyed cotton fabric would be less, then not acceptable. Therefore, this dye selection seems not to be suitable for the differential dyeing.

**Table 2.** Color difference parameters for 0.1% dyeings with Solophenyl dyes.

On the other hand, differential dyeing effects can be pursued applying UV irradiation on dyed samples, just using the discoloration induced by radiation. In this case, the fading ability of the dyes on cotton yarn was exploited and in many cases significant color differences (Δ*E* higher than 5.0) were found between UV-irradiated and untreated samples.

#### **2.3. Differential dyeing performed with UV fading of reactive dyes**

Fading of dyed cotton yarns was carried out on dyed yarn samples wrapped on cardboard where one side was exposed for half area to UV rays. The irradiation condition and color evaluation were the same as reported in Section 2.1.

Two series of reactive dyes for cellulosic fibers were investigated, at first reactive dyes namely Kayacelon React® (Nippon Kayaku) and Kemacelon® (Kem Color) having reactive groups belonging to triazinyl betaine (**Figure 1**).

**Figure 1.** Triazinyl reactive group.

These dyes were applied at 1.0% o.w.f. with liquor ratio of 1:20, in the presence of an alkali donor (1.0 g/L of Buslid 509, which slowly releases hexamethylenetetramine increasing pH) and 40 g/L Na2SO4 working at 90°C for 60 min. The results are reported in **Table 3**.

Even this series of reactive dyes presents only a term (Kayacelon React Red CN-3B, evidenced in bold) with acceptable color difference for a differential dyeing between UV-treated and untreated samples.

The chromatic investigation was continued with the reactive dyes Avitera SE® (Huntsman), constituted by a chromogen with three reactive groups (chemical structure still under patent), applied at 0.5% o.w.f., 1:20 liquor ratio, and 50 g/L NaCl working at 60°C for 30 min adding 9

g/L of Na2CO3 divided into three aliquots (1/6, 2/6, 3/6), always working at 60°C for another 30 min. The results are reported in **Table 4**.

**Table 3.** Color difference parameters for 1.0% dyeings with Kayacelon React dyes.

**Dye Δ***L* **Δ***a* **Δ***b* **Δ***E* Orange Solophenyl TGL 182% 0.58 −1.0 −1.20 1.68 Bordeaux Solophenyl 3BLE 1.20 −0.75 0.52 1.51 Blue Solophenyl FGLE 220% 1.30 0.75 2.30 2.75 Navy Solophenyl BLE 250% 4.94 0.06 3.58 **6.10**

On the other hand, differential dyeing effects can be pursued applying UV irradiation on dyed samples, just using the discoloration induced by radiation. In this case, the fading ability of the dyes on cotton yarn was exploited and in many cases significant color differences (Δ*E*

Fading of dyed cotton yarns was carried out on dyed yarn samples wrapped on cardboard where one side was exposed for half area to UV rays. The irradiation condition and color

Two series of reactive dyes for cellulosic fibers were investigated, at first reactive dyes namely Kayacelon React® (Nippon Kayaku) and Kemacelon® (Kem Color) having reactive groups

These dyes were applied at 1.0% o.w.f. with liquor ratio of 1:20, in the presence of an alkali donor (1.0 g/L of Buslid 509, which slowly releases hexamethylenetetramine increasing pH)

Even this series of reactive dyes presents only a term (Kayacelon React Red CN-3B, evidenced in bold) with acceptable color difference for a differential dyeing between UV-treated and

The chromatic investigation was continued with the reactive dyes Avitera SE® (Huntsman), constituted by a chromogen with three reactive groups (chemical structure still under patent), applied at 0.5% o.w.f., 1:20 liquor ratio, and 50 g/L NaCl working at 60°C for 30 min adding 9

and 40 g/L Na2SO4 working at 90°C for 60 min. The results are reported in **Table 3**.

higher than 5.0) were found between UV-irradiated and untreated samples.

**2.3. Differential dyeing performed with UV fading of reactive dyes**

**Table 2.** Color difference parameters for 0.1% dyeings with Solophenyl dyes.

evaluation were the same as reported in Section 2.1.

belonging to triazinyl betaine (**Figure 1**).

**Figure 1.** Triazinyl reactive group.

238 Cotton Research

untreated samples.

**Table 4.** Color difference parameters for 0.5% dyeings with Avitera dyes.

This series of reactive dyes presents only two terms, evidenced in bold, with acceptable values for differential dyeing effects. It was then decided to consider other series of DyStar reactive dyes, based on the monochloro-difluoro-pyrimidine reactive group, Levafix type (**Figure 2a**), and β-sulfatoethylsulfonic reactive group, Remazol type (**Figure 2b**).

**Figure 2.** Molecular structure of dyes with monochloro-difluoro-pyrimidine reactive group (a) and β-sulfatoethylsulfonic reactive group (b).

In accordance with the works carried out by Batchelor et al. [24] about photofading, it was observed that light fastness (and therefore also the fading ability toward UV radiations) is instead related to the chemical structure of the chromogen; they showed that fading is caused by both visible and UV light, with visible light responsible for azo dyes and UV light responsible for phthalocyanines.

Generally speaking, the photofading is mainly due to singlet oxygen development, 1 O2, which can be formed by the quenching of excited states of dyes by the triplet ground state of oxygen, 3 O2:

$$\text{dye} \stackrel{\text{light}}{\rightarrow} \text{dye}^\* + ^3 \text{O}\_z \stackrel{\text{quench}}{\rightarrow} \text{dye} + ^1 \text{O}\_z$$

**Table 5.** Light fastness, structure, and groups of commercial dyes experimented.

**Table 5** shows light fastness and structure types of the investigated reactive dyes (data derived from color charts of the producers).

**Table 6** shows the measured color differences between UV-irradiated and untreated cotton yarns of the dyeings carried out with 0.5% Levafix dyes, while **Table 7** reports those obtained with 0.5% Remazol dyes (in both tables Δ*E* > 5 are evidenced in bold).

**Table 6.** Color difference parameters for 0.5% dyeings with Levafix dyes.

by both visible and UV light, with visible light responsible for azo dyes and UV light respon-

can be formed by the quenching of excited states of dyes by the triplet ground state of oxygen,

light quench \* 3 <sup>1</sup> <sup>2</sup> <sup>2</sup> dye ® ® dye + O dye+ O O2, which

Generally speaking, the photofading is mainly due to singlet oxygen development, 1

**Color index Commercial name Light fastness (1/6 SD) Structure Reactive group** R.Y. 160 Yellow br. Remazol 4GL 5 Azo Vinylsulfone R.Y. 176 Yellow Remazol 3RS 5 Unknown Unknown R.O. 107 G. yellow Remazol RNL 150% 4/5 Azo Vinylsulfone R.O.16 Orange br. Remazol 3R spec. 4/5 Azo Vinylsulfone

Unknown Scarlet Levafix CA 4 Azo Triazinyl + vinylsulfone Unknown Red Levafix CA 4 Azo Triazinyl + vinylsulfone

Unknown Navy Levafix CA 3/4 Azo Triazinyl + vinylsulfone

R.R. 180 Red br. Remazol F-3B 3/4 Azo Vinylsulfone R.Bl. 21 Turquoise Remazol G 133% 5 Phthal. Vinylsulfone

R.Bk. 5 Black Remazol B 133% 3/4 Disazo 2 vinylsulfone Unknown Yellow Remazol RR 4 Unknown Unknown Unknown Orange Remazol RR 4 Unknown Unknown Unknown Red Remazol RR 4 Unknown Unknown Unknown Blue Remazol RR 4 Unknown Unknown

R.Y. 217 Yellow Avitera SE 5/6 Unknown 3 reactive groups R.R. 286 Red Avitera SE 4 Unknown 3 reactive groups R.Bl. 281 Deep blue Avitera SE 4 Unknown 3 reactive groups

**Table 5** shows light fastness and structure types of the investigated reactive dyes (data derived

Unknown Blue Farbofix SP-BRF 150% 4/5 Unknown Unknown Unknown Navy Farbofix SP-BRK/N 3/4 Unknown Unknown R.Bl. 225 Navy Levafix E-BNA 2/3 Unknown Unknown Unknown Blue br. Neareafix AC-BRF 150% 5 Unknown Unknown

**Table 5.** Light fastness, structure, and groups of commercial dyes experimented.

from color charts of the producers).

sible for phthalocyanines.

3 O2:

240 Cotton Research

**Table 7.** Color difference parameters for 0.5% dyeings with Remazol dyes.

Specifically, it was noted that the chromogens more difficult to fade under UV radiations are those providing yellow shades, typically made up of pyrazolone. Only the amber tone (Levafix) is interesting as a yellow, but it is an orange yellow; the fading ability increases going from orange shades (azo) toward red (azo), while in the case of shades provided by diazo dyes the fading ability increases going from navy until the maximum value in the case of Remazol Blue RR, as demonstrated in **Table 7** (Remazol dyeings).

A selection of dyes sensitive toward UV-generated fade was thus performed offering a combination of the six terms reported in **Table 8**.

**Table 8.** Total color difference (Δ*E*) for 0.5% selected dyeings.

It is possible to observe, as reported in **Tables 6** and **7**, that the total color difference (Δ*E*) is due not only by an achromatic fading (ΔL) but also results from a variation of chromatic parameters (Δ*a* and Δ*b*), because a tone change after UV exposure occurs.

The selection shown in **Figure 3** has characteristics of good mutual combinability and synchronism in dye exhaustion, together with a color difference higher than 5.0 units of Δ*E*.

**Figure 3.** Dyeings with 0.5% selected dyes: (a) Levafix Amber CA, (b) Remazol Orange RR, (c) Remazol Red RB 133%, (d) Remazol Navy GG 133%, (e) Remazol Black B 133%, (f) Remazol Blue RR (for each shade: untreated yarn on the left side, UV faded on the right side).

In conclusion, on cotton fibers, unlike wool, a UV pretreatment does not substantially change the dyeing affinity, but a UV posttreatment is capable to fade dyeing, allowing to obtain interesting differential chromatic effects.

## **3. Surface modification of cotton fabrics by UV curing and UV grafting**

**Dye Δ***E* Levafix Amber CA **6.31** Remazol Orange RR **8.93** Remazol Red RB 133% **12.90** Remazol Navy GG 133% **5.56** Remazol Black B 133% **7.16** Remazol Blue RR **8.67**

It is possible to observe, as reported in **Tables 6** and **7**, that the total color difference (Δ*E*) is due not only by an achromatic fading (ΔL) but also results from a variation of chromatic

The selection shown in **Figure 3** has characteristics of good mutual combinability and synchronism in dye exhaustion, together with a color difference higher than 5.0 units of Δ*E*.

**Figure 3.** Dyeings with 0.5% selected dyes: (a) Levafix Amber CA, (b) Remazol Orange RR, (c) Remazol Red RB 133%, (d) Remazol Navy GG 133%, (e) Remazol Black B 133%, (f) Remazol Blue RR (for each shade: untreated yarn on the left

In conclusion, on cotton fibers, unlike wool, a UV pretreatment does not substantially change the dyeing affinity, but a UV posttreatment is capable to fade dyeing, allowing to obtain

parameters (Δ*a* and Δ*b*), because a tone change after UV exposure occurs.

**Table 8.** Total color difference (Δ*E*) for 0.5% selected dyeings.

242 Cotton Research

side, UV faded on the right side).

interesting differential chromatic effects.

Cotton fabrics often find its application in producing home furnishing, such as table linen, clothing, or work wear with high hygienic requirements. The high concentration of hydroxyl groups on cotton fabric surface makes the fabrics water adsorbent and easily stained by liquids. Moreover, the chemical composition and morphological properties of cellulosic fibers provide an excellent medium for bacterial growth. To overcome these limits, a finishing treatment is required on cotton fabrics to confer oil and/or water repellency and antibacterial activity.

Usually, the desired properties are achieved by thermal polymerization, regardless of energy consumption and costs of the process [25]. Fluorinated or silicone monomers, applied to the fabrics by padding, can confer oil and water repellency, while Triclosan, quaternary ammonium salts, and silver ions [26] are commonly used as antibacterial agents [27].

Ferrero et al. [3] proposed the UV curing as eco-friendly and cheap alternative to thermal curing of functional monomers onto cotton fibers. In UV-curing processes, a suitable photoinitiator is able to interact with UV radiations yielding radical or cationic species, which induce a rapid curing of reactive monomers and oligomers at low temperature, with lower environmental impact and lower cost than thermal process.

For textile applications on cotton fabrics, if a mixture of monomer and initiator is absorbed by the fibers and subsequently UV-irradiated, the polymeric chains can form inside the textile structure, establishing also graft bonds (UV grafting) with the cellulose macromolecules and making the treatment solid and water resistant. Moreover, the interpenetration of components and homogeneous distribution of monomers, even at a low concentration, contribute to obtain textile materials with modified surface properties without high add-on of polymer. In this way, the bulk properties of the fibers, such as mechanical and thermal resistance, are not affected and also the fabric breathability can be kept unvaried.

#### **3.1. Water- and oil-repellent finishing of cotton fabrics by UV radiation**

Water and oil repellency are among the most common functional properties that need to be assessed for protective clothing. This property can be conferred by the modification of the surface energy of textile fibers, possibly confined to a thin surface layer, so that the bulk properties of the textile fabric such as mechanical strength, flexibility, breathability, and softness should remain uncompromised.

Hydrophobic or oleophobic surfaces are difficult to wet by water or apolar liquids, respectively, and are called low-energy surfaces. Wetting primarily comes from the non-ideality of solid substrates that are both rough and chemically heterogeneous. The surface modification of textile fibers to confer these properties can be achieved by physical or chemical methods or by the combination of both. Plasma treatments and exposure to radiations, often accomplished in the presence of reactive gases or after impregnation with suitable chemicals, are mainly representative of physical methods, while chemical treatments can generally be carried out with oxidants or other finishing agents, followed by thermal treatment or by sol-gel techniques. Polysiloxanes are widely used for textile finishing to impart desirable properties such as softness, crease resistance, and water repellency, depending on the nature of organic functional groups incorporated in the polymer structure. The application of a polymeric coating to a cotton fabric in the form of a thin film ensures waterproof properties, but the fabric could lose comfort characteristics, such as handling and breathability. Therefore, hydrorepellency obtained by homogeneous adsorption of monomers onto each fiber and followed by a radiation curing method should be preferred. This was proposed by Ferrero et al. [3], which obtained water-repellent cotton fabrics by radical UV curing of silicone and urethane acrylates. The values of contact angle (**Table 9**), wettability, and moisture adsorption showed that a low resin add-on on the fabric is enough to confer water repellency, while scanning electron microscopy (SEM) analysis confirmed that UV curing yields a coating layer onto each single fiber than a film on the fabric surface (**Figure 4**) without damage of breathability.

**Table 9.** Hydrorepellent finishing of cotton fabrics by radical UV curing of silicone and urethane acrylates.

**Figure 4.** SEM images of cotton fibers untreated (a) and treated by radical UV curing of a silicone acrylate resin 3% add-on (b).

Periolatto et al. [28] recently investigated the application by UV grafting of polyhedral oligomeric silsesquioxanes (POSSs) and a polysilazane (KION 20) to cotton fabrics to confer hydrorepellency.

Polysiloxanes are widely used for textile finishing to impart desirable properties such as softness, crease resistance, and water repellency, depending on the nature of organic functional groups incorporated in the polymer structure. The application of a polymeric coating to a cotton fabric in the form of a thin film ensures waterproof properties, but the fabric could lose comfort characteristics, such as handling and breathability. Therefore, hydrorepellency obtained by homogeneous adsorption of monomers onto each fiber and followed by a radiation curing method should be preferred. This was proposed by Ferrero et al. [3], which obtained water-repellent cotton fabrics by radical UV curing of silicone and urethane acrylates. The values of contact angle (**Table 9**), wettability, and moisture adsorption showed that a low resin add-on on the fabric is enough to confer water repellency, while scanning electron microscopy (SEM) analysis confirmed that UV curing yields a coating layer onto each single fiber than a

**Resin Chemical nature Weight on cotton (%) Contact angle (°)**

Coatosil 3503 Silicone (medium) acrylate 1.3 143 Coatosil 3509 Silicone (high) acrylate 2.9 114 Tego Rad 2600 Polysiloxane epoxy 2.6 100 Ucecoat 7849 Urethane acrylate (MW = 10,000) 2 100

**Table 9.** Hydrorepellent finishing of cotton fabrics by radical UV curing of silicone and urethane acrylates.

**Figure 4.** SEM images of cotton fibers untreated (a) and treated by radical UV curing of a silicone acrylate resin 3%

add-on (b).

244 Cotton Research

film on the fabric surface (**Figure 4**) without damage of breathability.

POSSs are polyhedral clusters yielded by hydrolytic condensation of trifunctional silanes. The generic formula is (RSiO1.5)n, where each silicon atom is bound on average to one and a half oxygen atoms and to one hydrocarbon group. The single nanoparticle may be represented as a silica cage core (diameters in the range of 1–3 nm) bearing organic functional groups attached to the corners of the cage (**Figure 5a**).

**Figure 5.** Molecular structures of polyhedral oligomeric silsesquioxanes (POSSs), where R = i-butyl, Y = methacryloisobutyl functional groups (a) and KION 20 structural units (b).

KION polysilazane contains repeating units in which silicon and nitrogen atoms are bonded in an alternating sequence. Both of these units contain cyclic and linear features. In addition, KION 20 contains fewer low-molecular-weight polysilazane components (**Figure 5b**).

For what concerns the hydrorepellency, it was clearly conferred by the treatment, as confirmed by water contact angles measured on the as-prepared samples higher than 90°, for both POSSand KION-treated samples. Measurements on aged samples revealed that KION-treated samples could maintain better properties during time, with respect to POSS treated. Moreover, for these two oligomers, higher contact angles were measured on samples treated with solutions at higher concentration. In particular, samples treated with 1- and 5-g/L POSS solutions, although showing an initial water contact angle higher than 100°, after aging immediately absorb the water drop, denouncing the total loss of hydrorepellency.

Many research papers have been published on the production and application of different types of fluorochemicals to textile finishing [29]. Fluorochemicals are organic compounds consisting of perfluorinated carbon chains, which impart water and oil repellency to the fiber surface when incorporated into polymer backbone with perfluoro groups as side chains. The old fluorochemicals used were based on C8 carbon chains, which release highly hazardous and toxic substances, such as perfluoro‐octanoic acid and perfluoro‐octanesulfonates. Nowa‐ days, C6 fluorochemicals are still in use although the rules on this topic became more stringent, banding also C6 fluorochemicals, so studies about the performance conferred by C2–C3 products are of great interest [30].

Fluorochemical finishings are commercially available as water emulsions and are applied to fabrics by the pad‐dry‐cure method, with a thermal‐curing step at 150–175°C in hot flue for some minutes. As alternative, Ferrero et al. [2, 6] proposed the UV curing of perfluoro‐alkyl‐ polyacrylate resins (Repellan EPF and NFC by Pulcra Chemicals and Oleophobol CP‐C by Huntsman), in water emulsions, able to impart water as well as oil repellency to cotton fabrics, and the results were compared with those obtained by thermal polymerization. A radical photoinitiator was added in the proper amount, then the solution was diluted with water, mixed, and applied by dipping or spraying onto strips of fabric that were dried in an oven. Final weight add‐ons of 3 and 5% o.w.f. were usually applied in order to obtain the desired properties without loss of fabric handling. Then, the samples were UV radiated on both sides by a medium pressure mercury lamp (about 60 mW/cm2 ) under inert atmosphere for 30–60 s. Thermally cured fabrics were considered as reference, treated for 2–3 min at 140 or 150°C according to the indications of the producer.

**Table 10.** Dynamic water contact angles (average of repeated measurements, standard deviation about 4°, θA = advancing, θR = receding, Δθ = hysteresis) before and after washing on cotton fabrics cured with Repellan EPF and NFC.

The effectiveness of the UV treatment was evaluated by the determination of weight loss in chloroform. Repellan EPF showed the highest yields after UV curing, quite similar to those reached with the thermal treatment (93–96%), whereas Repellan NFC showed lower, although acceptable, yields in UV curing (80–81%) than in the thermal one (98%), without the influence of irradiation time and polymer add‐on. However, with Oleophobol CP‐C lower yields were obtained in both finishing treatments, and longer UV irradiation time, at least 60 s, is necessary to achieve a yield of 91% with a 3% add‐on. Measurements of dynamic contact angles of water and oil drops allowed comparing the repellent behavior of the cotton fabrics finished with both curing methods before and after five domestic washing cycles (**Table 10**). A hysteresis Δθ > 0 is typical of most real surfaces, as confirmed by all the results obtained. The good wash fastness of water repellency was proved by the slight reduction of the advancing contact angles after washing, regardless of the curing type and polymer add-on. Oleophobol (data not reported) gave slightly lower contact angles but was practically unaffected by repeated washings.

and toxic substances, such as perfluoro‐octanoic acid and perfluoro‐octanesulfonates. Nowa‐ days, C6 fluorochemicals are still in use although the rules on this topic became more stringent, banding also C6 fluorochemicals, so studies about the performance conferred by C2–C3

Fluorochemical finishings are commercially available as water emulsions and are applied to fabrics by the pad‐dry‐cure method, with a thermal‐curing step at 150–175°C in hot flue for some minutes. As alternative, Ferrero et al. [2, 6] proposed the UV curing of perfluoro‐alkyl‐ polyacrylate resins (Repellan EPF and NFC by Pulcra Chemicals and Oleophobol CP‐C by Huntsman), in water emulsions, able to impart water as well as oil repellency to cotton fabrics, and the results were compared with those obtained by thermal polymerization. A radical photoinitiator was added in the proper amount, then the solution was diluted with water, mixed, and applied by dipping or spraying onto strips of fabric that were dried in an oven. Final weight add‐ons of 3 and 5% o.w.f. were usually applied in order to obtain the desired properties without loss of fabric handling. Then, the samples were UV radiated on both sides by a medium pressure mercury lamp (about 60 mW/cm2 ) under inert atmosphere for 30–60 s. Thermally cured fabrics were considered as reference, treated for 2–3 min at 140 or 150°C

Resin Add‐on (%) Curing type θA θR Δθ θA θR Δθ Repellan EPF 3 Thermal 134 126 8 142 136 6

Repellan NFC 3 Thermal 140 130 10 139 122 17

**Table 10.** Dynamic water contact angles (average of repeated measurements, standard deviation about 4°, θA = advancing, θR = receding, Δθ = hysteresis) before and after washing on cotton fabrics cured with Repellan EPF and

The effectiveness of the UV treatment was evaluated by the determination of weight loss in chloroform. Repellan EPF showed the highest yields after UV curing, quite similar to those reached with the thermal treatment (93–96%), whereas Repellan NFC showed lower, although acceptable, yields in UV curing (80–81%) than in the thermal one (98%), without the influence of irradiation time and polymer add‐on. However, with Oleophobol CP‐C lower yields were obtained in both finishing treatments, and longer UV irradiation time, at least 60 s, is necessary to achieve a yield of 91% with a 3% add‐on. Measurements of dynamic contact angles of water

3 UV 138 129 9 134 131 3 5 Thermal 145 133 12 143 140 3 5 UV 142 134 8 138 134 4

3 UV 140 132 8 137 123 14 5 Thermal 136 125 9 134 124 10 5 UV 133 126 7 131 119 12

**Contact angle (°) Before washing After washing**

products are of great interest [30].

246 Cotton Research

according to the indications of the producer.

NFC.

In conclusion, the laboratory-scale application of the hydro- and oil-repellent finishing on textile fabrics by UV curing of silica based or fluorocarbon resins, with the optimization of process parameters, followed by a deep characterization of treated samples, confirmed the effectiveness of the treatment.

The study of Ferrero and Periolatto went on with the semi-industrial scale-up of the process: a great number (about 80) of larger fabric samples were padded by foulard with Oleophobol and then were irradiated in air by Sun-Wash®, an apparatus for continuous treatment of fabrics by UV light providing an irradiance of 913 mW/cm2 on an exposed area of about 120 × 60 cm2 . Samples were exposed to the radiation with a carpet speed of 5 m/min, by five passes, corresponding to an irradiation time of 35 s, on both sides [31]. Fabric add-on was significantly reduced in order to hold down the finishing cost. White and dyed samples of different textile composition were treated and evaluated in terms of conferred repellency, yellowing, or color changes. Most relevant process parameters were investigated, considering the thermal process normally adopted at industrial level as reference. Results were so statistically evaluated by Six Sigma method with Minitab 16 software, to point out the most influencing parameters and the real possibility to replace the thermal treatment with UV. Water and oil drop sorption times higher than 2 h were found on all treated samples, showing that Oleophobol works very well as oil- and water-repellent agent for textiles.

UV process was revealed to work better than thermal one, in fact higher water and oil contact angles were obtained with a lower amount (1% o.w.f.) of finishing agent. Considering the UV process, best results were related to white fabrics, rather than dyed, and medium values of both radiation dose and product concentration, taking into account both contact angles and color reflectance evaluations. Finally, contact angle measurements carried out on aged samples (2 years) showed no variations with respect to fresh samples, meaning that the finishing is not affected by aging. Obtained results were considered encouraging and can open the way for a real application of the UV process to industrial field.

#### **3.2. Antimicrobial finishing by chitosan UV grafting**

The textile manufacturing industry is going through a period of severe crisis due to the globalization of the world market. A highly competitive context and stringent ecological regulations make quality and eco-friendly processes the major demands for a company.

A first objective aims to lower water and energy consumption; another relevant factor is the possibility of replacing high-polluting or toxic chemicals with others characterized by lower or zero environmental impact. From this point of view, the products of green chemistry, of natural origin, are particularly interesting for applications in the field of textile finishing.

Increased attention toward health and hygiene, due to frequent diseases and invasive infections, brought the attention of the research in textile field on antibacterial materials, which can not only prevent degradation and discoloration of the fabrics by microorganisms but also effectively prevent the spread of pathogenic bacteria. An antibacterial finishing, by means of a suitable surface chemical modification of fibers, is mainly required on natural fibers for furnishing, technical textiles, medical devices, hygienic textiles, food industry, and packaging. Chemicals bearing functional biocide groups are usually applied by padding, followed by a thermal treatment. Unfortunately, most of these products are toxic or carcinogenic, so the application to textiles is not advisable, also considering a possible release of the antibacterial agent during the use, in skin contact.

For these reasons, a strong chemical grafting to treated fibers is mandatory for a fast, stable, and resistant treatment. However, the finishing should not compromise the hand, appearance, and color of the fabric, considering that finishing processes are normally carried out after dyeing. In this view, the application of natural biopolymers by an eco-friendly and cheap process can be the winning choice to develop bioactive eco-sustainable textiles from renewable sources [32].

Cellulose and chitin, the main components of cotton fibers and crustacean shells, respectively, are the two most abundant polysaccharides in nature. They are mutually compatible, due to their similar structure (**Figure 6**), and biodegradable; it makes them good candidates as ecofriendly and eco-sustainable substrates for textile applications. Chitosan (2-amino-2-deoxy-(1– 4)-β-d-glucopyranan) is a carbohydrate biopolymer derived from the deacetylation of the chitin with unique biological, physiological, and pharmacological properties, such as biodegradability, no toxicity, and high antibacterial activity toward both Gram-positive and Gramnegative microorganisms, due to the combined bacteriostatic and bactericide action.

**Figure 6.** Molecular structures of cellulose (a) and chitosan (b).

In textile field, chitosan is mainly used as a dyeing auxiliary or a finishing agent, but the finishing fastness is limited by the weak interactions between chitosan macromolecules and fibers. To obtain a stable treatment, a thermal wet process is required, with high energy, water consumption, and possible degradation of the treated substrate. Moreover, the addition of cross-linking agents is required. Usually, toxic chemicals bearing aldehyde groups are used in thermal processes; recently, genipin was proposed as natural, nontoxic alternative: encouraging results in terms of fastness improvement were obtained, opening the way for biomedical and pharmaceutical applications [33], but the prohibitive cost of genipin makes it not applicable in textile field.

Increased attention toward health and hygiene, due to frequent diseases and invasive infections, brought the attention of the research in textile field on antibacterial materials, which can not only prevent degradation and discoloration of the fabrics by microorganisms but also effectively prevent the spread of pathogenic bacteria. An antibacterial finishing, by means of a suitable surface chemical modification of fibers, is mainly required on natural fibers for furnishing, technical textiles, medical devices, hygienic textiles, food industry, and packaging. Chemicals bearing functional biocide groups are usually applied by padding, followed by a thermal treatment. Unfortunately, most of these products are toxic or carcinogenic, so the application to textiles is not advisable, also considering a possible release of the antibacterial

For these reasons, a strong chemical grafting to treated fibers is mandatory for a fast, stable, and resistant treatment. However, the finishing should not compromise the hand, appearance, and color of the fabric, considering that finishing processes are normally carried out after dyeing. In this view, the application of natural biopolymers by an eco-friendly and cheap process can be the winning choice to develop bioactive eco-sustainable textiles from renewable

Cellulose and chitin, the main components of cotton fibers and crustacean shells, respectively, are the two most abundant polysaccharides in nature. They are mutually compatible, due to their similar structure (**Figure 6**), and biodegradable; it makes them good candidates as ecofriendly and eco-sustainable substrates for textile applications. Chitosan (2-amino-2-deoxy-(1– 4)-β-d-glucopyranan) is a carbohydrate biopolymer derived from the deacetylation of the chitin with unique biological, physiological, and pharmacological properties, such as biodegradability, no toxicity, and high antibacterial activity toward both Gram-positive and Gram-

In textile field, chitosan is mainly used as a dyeing auxiliary or a finishing agent, but the finishing fastness is limited by the weak interactions between chitosan macromolecules and fibers. To obtain a stable treatment, a thermal wet process is required, with high energy, water consumption, and possible degradation of the treated substrate. Moreover, the addition of cross-linking agents is required. Usually, toxic chemicals bearing aldehyde groups are used in thermal processes; recently, genipin was proposed as natural, nontoxic alternative: encouraging results in terms of fastness improvement were obtained, opening the way for biomedical

negative microorganisms, due to the combined bacteriostatic and bactericide action.

agent during the use, in skin contact.

**Figure 6.** Molecular structures of cellulose (a) and chitosan (b).

sources [32].

248 Cotton Research

Ferrero and Periolatto [34] proposed photocuring (UV curing and/or UV grafting) as cheap and eco-friendly process to bind chitosan to textiles by means of radical reactions. Studies about the photodegradation of chitosan macromolecules due to UV exposure confirmed the formation of macroradicals on the polymer. Same radicals can be involved in cross-linking process, promoted by the presence of a suitable radical photoinitiator. Among the substrates considered, there are wool [35], silk [36], polyester, polyamide, and cotton fiber [37], in the form of weft-warp and knitted piquet fabrics [38] but also filter substrates and gauzes with more open structures [39–41].

Focusing on cotton, the research work started with laboratory test on samples of small dimensions, aimed to optimize the process parameters and confirm the treatment efficiency and fastness [36]. A low-viscosity chitosan with deacetylation degree of 75–85% was dissolved in acetic acid solution at pH 4. The solution was added of 2-hydroxy-2-methyl-phenylpropane-1-one, 4% wt on chitosan, as radical photoinitiator, properly diluted and applied to the fabric surface by dipping, to reach a chitosan add-on ranging from 1 to 3% wt. An impregnation time of 12 h at 25°C or 1 h at 50°C was necessary to obtain 100% process yield.

Chitosan UV curing yielded high antimicrobial properties, against *Escherichia coli* and *Staphylococcus aureus*, on cotton fabrics. The test was carried out according to ASTM E 2149-01 standard test. A microorganism reduction higher than 97% was found on all treated samples, regardless of the application method for chitosan. Moreover, about 2% polymer add-on was enough to confer a strong antibacterial activity to the fabrics without endangerment of hand and breathability. An impregnation time of 12 h at ambient temperature was necessary so that treated fabrics maintained about one half of their own antimicrobial activity after washing with standard ECE detergent, according to UNI-EN ISO 105 C01 standard test, while at lower impregnation time, the antimicrobial activity was more reduced. This was attributed to a notenough penetration of chitosan inside the fiber structure, due even to the high viscosity of the solution. A prolonged contact time between chitosan solution and fabrics improved this penetration and chitosan could graft to the fibers, showing increased washing fastness. However, this was strongly dependent on the ionic nature of the detergents, and a nonionic surfactant could assure that an antimicrobial activity completely retained after repeated washings.

Semi-industrial scaled tests were carried out on samples of higher dimension, using commercial chitosan powder (Peripret or Chitoclear) dissolved in acetic acid solution [38]. In some cases, a softener (Nearfinish SM/40) or an antioxidant agent (Nearcand) was added to the recipe, while the radical photoinitiator was kept in the same amount. The chitosan add-on was drastically lowered till 0.3% o.w.f, diluting the solution to 0.25% before the impregnation by padding. The as-impregnated samples were dried in a rameuse and finally radiated by Sun-Wash®, as reported in Section 3.1. Results of antibacterial activity tests are reported in **Table 11** and confirmed those previously obtained with samples prepared at laboratory scale.

**Table 11.** Antibacterial activity of chitosan-treated samples as prepared and after 10 and 30 washing cycles. Quantitative assessment of activity according to ISO 20743: 2007 (E) against *Staphylococcus aureus*.

"*Chitosan conferred a strong antibacterial activity, with the total reduction of the microorganism colonies on all the tested samples. Moreover, chitosan treated samples showed optimum washing fastness, maintaining their antibacterial activity unvaried even after 30 washes*" [38]. Comparing the results with those related to Ultra-Fresh, a commercial sanitizing agent considered as reference, the performances are similar or even better for chitosan-treated samples after 30 washes. Obtained results are of particular interest considering that the UV exposure was carried out in the presence of oxygen, even on wet samples. "*These are generally considered unfavorable conditions for radical reactions occurring or for photoinitiator effectiveness. Moreover, the strong antibacterial activity and washing fastness were obtained on all treated samples regardless the add-on or the presence of additives* [38]."

"*Results obtained with the lowest amount of chitosan on fibers is particularly interesting because makes chitosan competitive with other antibacterial agents commonly used, such as Triclosan, silver ions or quaternary ammonium salts, even from an economical point of view*" [38]. The homogeneous distribution of chitosan on fabrics, in particular in correspondence to the sample that presented the best fastness, was confirmed by dyeing tests with an acid dye (**Figure 7**) and by SEM analysis (**Figure 8**) that showed the optimal distribution of the finish on a single fiber surface.

**Figure 7.** Dyeing test with Direct Red 81: untreated cotton fabric (a), treated with 0.6% wt. chitosan (Peripret CTS) (b).

**Figure 8.** SEM analysis on cotton fabric treated with 3% wt. chitosan (4500×).

## **4. Conclusion**

**Sample description Condition Microorganism reduction (%)**

10 washes >99.9 30 washes 98.4

30 washes >99.9

30 washes >99.9

30 washes >99.9

30 washes >99.9

**Table 11.** Antibacterial activity of chitosan-treated samples as prepared and after 10 and 30 washing cycles. Quantitative assessment of activity according to ISO 20743: 2007 (E) against *Staphylococcus aureus*.

"*Chitosan conferred a strong antibacterial activity, with the total reduction of the microorganism colonies on all the tested samples. Moreover, chitosan treated samples showed optimum washing fastness, maintaining their antibacterial activity unvaried even after 30 washes*" [38]. Comparing the results with those related to Ultra-Fresh, a commercial sanitizing agent considered as reference, the performances are similar or even better for chitosan-treated samples after 30 washes. Obtained results are of particular interest considering that the UV exposure was carried out in the presence of oxygen, even on wet samples. "*These are generally considered unfavorable conditions for radical reactions occurring or for photoinitiator effectiveness. Moreover, the strong antibacterial activity and washing fastness were obtained on all treated samples regardless the add-on or the presence*

Untreated – Ultrafresh GH-20 2% Initial >99.9

250 Cotton Research

Chitosan finishing: 3% Peripret Initial >99.9 Wet irradiation 10 washes >99.9

Chitosan finishing 3% Peripret Initial >99.9 Dry irradiation 10 washes >99.9

Chitosan finishing: 3% Peripret Initial >99.9 With Nearfinish SM/40 10 washes >99.9 Dry irradiation 30 washes >99.9 Chitosan finishing: 0.3% Chitoclear Initial 98.6 Wet irradiation 10 washes >99.9

Chitosan finishing: 0.6% Chitoclear Initial 99 Wet irradiation 10 washes >99.9

Chitosan finishing: 0.6% Chitoclear Initial 99.2 With Nearcand 10 washes >99.9 Wet irradiation 30 washes >99.9

*of additives* [38]."

The UV treatments on cotton fibers can be utilized in dyeing and finishing processes. UV irradiation coupled with mild oxidation can improve some properties of the cotton fibers such as pilling resistance, water swelling, and dyeability. However, significant effects of differential dyeing were obtained by a UV posttreatment capable to fade dyeings with reactive dyes. UV curing and UV grafting with suitable chemical enabled to modify the surface of cotton fibers in order to confer oil and/or water repellency with an eco-friendly and cheap alternative to chemical and thermal finishing treatments. Moreover, the chitosan UV grafting was proposed as valid environmental friendly method to obtain cotton fabrics with a satisfactory washingresistant antimicrobial activity even with low polymer add-on.

## **Author details**

Franco Ferrero1\*, Gianluca Migliavacca1 and Monica Periolatto2

\*Address all correspondence to<EMAIL_ADDRESS>

## **References**

[8] Mansuri AH., Khoddami A, Rezaei Do A. Using ultraviolet radiation for the bleaching and pilling reduction of knitted cotton fabric. Fibres & Textiles in Eastern Europe 2011; 19: 74–77.

as valid environmental friendly method to obtain cotton fabrics with a satisfactory washing-

1 Department of Applied Science and Technology, Polytechnic of Turin, Torino, Italy

2 Faculty of Science and Technology, Free University of Bozen, Bolzano, Italy

and Monica Periolatto2

[1] Bhatti IA, Adeel S, Abbas M. Effect of radiation in textile dyeing. In: Hauser P (ed.) Textile Dyeing. Rijeka: InTech; 2011. p. 1–17. Available from: http://www.intechopen.com/books/textile-dyeing/effect-of-radiation-on-textile-dyeing [Accessed:

[2] Ferrero F, Periolatto M. Ultraviolet curing for surface modification of textile fabrics. Journal of Nanoscience and Nanotechnology 2011; 11:8663–8669. DOI: 10.1166/jnn.

[3] Ferrero F, Periolatto M, Sangermano M, Bianchetto Songia M. Water-repellent finishing of cotton fabrics by ultraviolet curing. Journal of Applied Polymer Science 2008;

[4] Ferrero F, Periolatto M, Bianchetto Songia M. Silk grafting with methacrylic and epoxy monomers: thermal process in comparison with ultraviolet curing. Journal of Applied

[5] Chen W-H, Chen P-C, Wang S-C, Yeh J-T, Huang C-Y, Chen K-N. UV-curable PDMScontaining PU system for hydrophobic textile surface treatment. Journal of Polymer

[6] Ferrero F, Periolatto M, Udrescu C. Water and oil-repellent coatings of perfluoropolyacrylate resins on cotton fibers: UV curing in comparison with thermal polymeri-

[7] Bhatti IA, Zia KM, Ali Z, Zuber M, Rehman F-u. Modification of cellulosic fibers to enhance their dyeability using UV-irradiation. Carbohydrate Polymers 2012; 89:783–

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resistant antimicrobial activity even with low polymer add-on.

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**Author details**

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[22] Migliavacca G, Ferrero F, Periolatto M. Differential dyeing of wool fabric with metalcomplex dyes after ultraviolet irradiation. Coloration Technology 2014;130:327–333.

[23] Migliavacca G. Applications of ultraviolet radiations in dyeing processes of yarn and

[24] Batchelor SN, Carr D, Coleman CE, Fairclough L, Jarvis A. The photofading mechanism of commercial reactive dyes on cotton. Dyes and Pigments 2003;59:269–275. DOI:

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[27] Shahid-ul-Islam, Faqeer M. High-energy radiation induced sustainable coloration and functional finishing of textile materials. Industrial Engineering Chemistry Research

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254 Cotton Research

#### **Glandless Cottonseed Protein for Environmentally Friendly Bioplastics Glandless Cottonseed Protein for Environmentally Friendly Bioplastics**

Hangbo Yue, Guoqiang Yin and Yingde Cui Hangbo Yue, Guoqiang Yin and Yingde Cui

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64045

#### **Abstract**

Environmentally friendly bioplastics have attracted renewed attention over the last few decades due to the ever‐growing awareness of the environmental impact of petroleum‐ based polymers and the rising costs of raw materials. Cottonseed protein (CP) extracted from cottonseed meal has abundant amino acid components and nutrition value, but are not carefully considered in non‐feed industries. For the purpose of being explored, glandless cottonseed flour is utilized in this work, as a raw material, to prepare cottonseed protein bioplastics (CPBs) as environmentally friendly products. The optimum synthesis conditions of CPBs were firstly investigated, followed by the analysis of protein modification and cross‐linking mechanism, with a close view on changes of their micro‐ or chemical structures. Detailed morphologies, element composition and biodegradabilities of CPBs were characterized via scanning electron microscopy, energy dispersive spectroscopy (EDS) and soil‐burial test, respectively, pointing out its structural heterogeneity as well as nature of biodegradability. Charac‐ terizations of the thermal stability and thermomechanical relaxation of thermal‐treated CPBs and its interaction with molecule revealed the presence of different thermal relaxation behaviours and different water states. Concluding remarks shortly summa‐ rize the importance of the work and point out possible solutions to addressing future potential challenges.

**Keywords:** biodegradability, bioplastics, cottonseed protein, synthesis, water states

## **1. Introduction**

The soaring consumption of organically synthesized polymers in agriculture and a wide variety of industries around the globe poses a great threat to our living environment, mainly

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

because of the non‐biodegradability of such polymers. The increased awareness of environ‐ mental impact of using petroleum‐derived polymers and the rising costs of chemical raw materials shift people's attention to seeking out environmentally friendly polymers for sustainable development. In the course of this searching over the last few decades across the world, protein‐based bioplastics (PBBs) have attracted renewed attention due to the following reasons: they can be completely degraded through regular composting, readily obtained from abundant natural resources (i.e. plant and animal proteins such as soy, corn zein, wheat gluten, sunflower, peanut, cottonseed, milk casein, fish gelatin, feather quill and serum albumin) [1– 4], as well as easily modified and manufactured. All these advantages together make PBBs suitable alternatives to petroleum‐based plastics, which are being considered or have already fabricated in many environmentally sensitive industries, including agriculture (e.g. mulch films, greenhouse films, flower pots, planting pots, etc.), green packaging (one‐time or short‐ term use before disposal) and possible biomedical industries (e.g. soft‐tissue scaffold, superabsorbent) [2, 5–7].

In addition, tons of wasted protein from cottonseed, feather quill, soya residue and animal keratin are being utilized to fabricate bioplastics, which have even emerged as a new family of renewable and sustainable plastics, known as 'Second‐generation bioplastics' [1, 8, 9] owing again mostly to their low cost, availability and biodegradability.

Cottonseed proteins (CPs) extracted from cottonseed meal after degreasing and peeling process contain rich amino acid components enjoying nutrition value, thereby they were used mostly as dairy‐cattle feeding products. However, this type of protein has not been considered as non‐feed industries, especially being used as a raw material for producing biodegradable plastics. The main component of cottonseed protein is globulin, approximately 90%, which contains 60% of globulins with gossypin (11S) and congossypin (7S) and 30% of albumins (2S) [10], resembling soy protein, the most studied material for bioplastic synthesis. In addition, the processability of CP with respect to plasticizer efficiency (PE) calculated from the relative contents of amino acids was similar to that of other proteins that have already been synthesized into various bioplastics [9]. Good amino acid composition together with a relatively high PE value (>5) makes cottonseed protein a good candidate as a renewable raw material for bioplastics production. However, little research to date has been carried out on cottonseed protein bioplastics (CPBs) while most studies focus on soy protein and wheat gluten bioplas‐ tics. Casting of CP films has been achieved by Marquié [11], but this process is time consuming and film formation is very limited. On the contrary, hot compression moulding is a more efficient strategy in achieving protein films.

In this chapter, a series of CPBs were synthesized starting from glandless cottonseed flour purified from cottonseed meal that was subjected to the processes of protein denaturation, plasticizing, cross‐linking and hot compression; they showed good biodegradability, thermal stability and a low degree of water absorption (WA), but they are structurally heterogeneous even after thorough mixing and dispersion. The results of this study should provide important theoretical guidance on the development of environmentally friendly protein‐derived bioplastics with improved properties, and necessary steps to pave the way towards expanding non‐feed industry of cottonseed protein after oil extraction.

## **2. Synthesis of glandless CPBs**

### **2.1. Optimum synthesis conditions**

because of the non‐biodegradability of such polymers. The increased awareness of environ‐ mental impact of using petroleum‐derived polymers and the rising costs of chemical raw materials shift people's attention to seeking out environmentally friendly polymers for sustainable development. In the course of this searching over the last few decades across the world, protein‐based bioplastics (PBBs) have attracted renewed attention due to the following reasons: they can be completely degraded through regular composting, readily obtained from abundant natural resources (i.e. plant and animal proteins such as soy, corn zein, wheat gluten, sunflower, peanut, cottonseed, milk casein, fish gelatin, feather quill and serum albumin) [1– 4], as well as easily modified and manufactured. All these advantages together make PBBs suitable alternatives to petroleum‐based plastics, which are being considered or have already fabricated in many environmentally sensitive industries, including agriculture (e.g. mulch films, greenhouse films, flower pots, planting pots, etc.), green packaging (one‐time or short‐ term use before disposal) and possible biomedical industries (e.g. soft‐tissue scaffold,

In addition, tons of wasted protein from cottonseed, feather quill, soya residue and animal keratin are being utilized to fabricate bioplastics, which have even emerged as a new family of renewable and sustainable plastics, known as 'Second‐generation bioplastics' [1, 8, 9] owing

Cottonseed proteins (CPs) extracted from cottonseed meal after degreasing and peeling process contain rich amino acid components enjoying nutrition value, thereby they were used mostly as dairy‐cattle feeding products. However, this type of protein has not been considered as non‐feed industries, especially being used as a raw material for producing biodegradable plastics. The main component of cottonseed protein is globulin, approximately 90%, which contains 60% of globulins with gossypin (11S) and congossypin (7S) and 30% of albumins (2S) [10], resembling soy protein, the most studied material for bioplastic synthesis. In addition, the processability of CP with respect to plasticizer efficiency (PE) calculated from the relative contents of amino acids was similar to that of other proteins that have already been synthesized into various bioplastics [9]. Good amino acid composition together with a relatively high PE value (>5) makes cottonseed protein a good candidate as a renewable raw material for bioplastics production. However, little research to date has been carried out on cottonseed protein bioplastics (CPBs) while most studies focus on soy protein and wheat gluten bioplas‐ tics. Casting of CP films has been achieved by Marquié [11], but this process is time consuming and film formation is very limited. On the contrary, hot compression moulding is a more

In this chapter, a series of CPBs were synthesized starting from glandless cottonseed flour purified from cottonseed meal that was subjected to the processes of protein denaturation, plasticizing, cross‐linking and hot compression; they showed good biodegradability, thermal stability and a low degree of water absorption (WA), but they are structurally heterogeneous even after thorough mixing and dispersion. The results of this study should provide important theoretical guidance on the development of environmentally friendly protein‐derived bioplastics with improved properties, and necessary steps to pave the way towards expanding

again mostly to their low cost, availability and biodegradability.

efficient strategy in achieving protein films.

non‐feed industry of cottonseed protein after oil extraction.

superabsorbent) [2, 5–7].

258 Cotton Research