**4. Micropropagation an alternative to develop plants tolerant to water stress "hyperhydricity"**

works that the treatment response depends on the type of explant and variety of sugarcane. Several studies have reported that the rate of shoot formation is higher in temporary immersion bioreactors than in semi-solid cultures. It is important to mention that none of the previous works reported any problem with the hyperhydricity in the obtained *in vitro* plants. Only, Snyman [44] reports this condition on the induction and germination of somatic sugarcane embryos. Tesfa and coworkers [45], didn't report problems of hyperhydricity or a decrease in field survival rate out of *in vitro* plants after using a liquid culture medium with agitation

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**Figure 1.** Average of shoots at different densities of inoculum. T5-T20: 5 inoculum plants were used in semi-solid and continuous immersion medium, 20 plants in temporary immersion; T10-T40: 10 inoculum plants were used in semi-solid and continuous immersion medium, and 40 plants in temporary immersion; T15-T60: 15 inoculum plants were used in semi-solid and continuous immersion medium, and 60 plants in temporary immersion; semisolid (red rectangle), continue immersion (orange rectangle) and temporary immersion (green rectangle). At the bottom of the figure, the calculated growth index factor is reported using the obtained fresh weight under the same inoculum density conditions; T5-T20 (red rectangle), T10-T40 (orange rectangle) and T15-T60 (green rectangle). Five replicates were carried out for

each treatment.

Various micropropagation systems such as liquid cultures and automation have proven the potential to resolve manual handling of *in vitro* cultures at various stages and decrease production cost. However, hyperhydricity is a major problem during *in vitro* culture of many crops in liquid culture systems. Hyperhydricity (also known as "vitrification") is a physiological disorder occurring in plant material of tissue culture, which causes a reduction of propagation and death of tissues when transferred to *ex vitro* conditions [39–41]. The environment inside culture vessels normally used for plant micropropagation is characterized by high humidity, limited gaseous exchange between the internal atmosphere of the culture vessel and its surrounding environment, and the accumulation of ethylene; conditions that may induce physiological disorders [42]. The development of hyperhydric deformities represents a disadvantage for plant micropropagation and a barrier for the exploitation of bioreactor technologies to scale-up its production [41]. The concept of stress in relation to hyperhydricity is not completely established. Therefore, it remains difficult to assume when hyperhydric tissues are stressed. Previous studies argued that abnormal morphology observed in hyperhydricity could be attributed to changes occurring at cellular level due to the modifications of membrane composition or DNA content [42]. However, Rojas-Martínez and coworkers [41] considered this disorder as the result of the stressful conditions brought out by waterlogging of the apoplast. This causes hypoxia and thereby leads to severe oxidative stress. They concluded that hyperhydric features like vitreous appearance and wrinkled leaves are secondary events resulting from waterlogging of the apoplast.

The temporary immersion system (TIS) consists on the use of bioreactors with automated devices that control features such as gas exchange, liquid medium culture and lighting, required for the growth, development and survival of plants. TIS mainly consist of three phases: multiplication, elongation and rooting phase. Plantlets propagated in TIS have better performance than those propagated by conventional methods of micropropagation. TIS provides a rapid and efficient plant propagation system for many agricultural and forestry species, it utilizes liquid media avoiding intensive manual handling [43].

With the objective of evaluating the stress caused by hyperhydricity in the *in vitro* culture of sugarcane var. MEX69290, three types of culture were analyzed: Semisolid (Magenta) was used as control; Continuous immersion (250 ml Flask); and Temporary Immersion (BioMINT II Bioreactor). Multiplication, maturation, and *ex vitro* adaptation phases of sugarcane under these three types of culture were evaluated.

The obtained results in the adaptation of *in vitro* plants of *S. officinarum* at three different types of culture in the multiplication phase were surprising, as it is observed in **Figure 1**, where a notorious formation of shoots occurs in continuous immersion medium. Plants of var. MEX69290 obtained a much higher average shoot formation at the temporary immersion bioreactors than those observed in semi-solid medium. It was observed that invariable of the inoculum density applied (5, 10, 15 plants per bottle) was higher in continuous immersion. Similarly, growth index factor was higher in this culture system than that obtained in semi-solid medium or temporary immersion bioreactors (**Figure 1**). We can observe comparing our results with other works that the treatment response depends on the type of explant and variety of sugarcane. Several studies have reported that the rate of shoot formation is higher in temporary immersion bioreactors than in semi-solid cultures. It is important to mention that none of the previous works reported any problem with the hyperhydricity in the obtained *in vitro* plants. Only, Snyman [44] reports this condition on the induction and germination of somatic sugarcane embryos. Tesfa and coworkers [45], didn't report problems of hyperhydricity or a decrease in field survival rate out of *in vitro* plants after using a liquid culture medium with agitation

**4. Micropropagation an alternative to develop plants tolerant to** 

wrinkled leaves are secondary events resulting from waterlogging of the apoplast.

species, it utilizes liquid media avoiding intensive manual handling [43].

these three types of culture were evaluated.

The temporary immersion system (TIS) consists on the use of bioreactors with automated devices that control features such as gas exchange, liquid medium culture and lighting, required for the growth, development and survival of plants. TIS mainly consist of three phases: multiplication, elongation and rooting phase. Plantlets propagated in TIS have better performance than those propagated by conventional methods of micropropagation. TIS provides a rapid and efficient plant propagation system for many agricultural and forestry

With the objective of evaluating the stress caused by hyperhydricity in the *in vitro* culture of sugarcane var. MEX69290, three types of culture were analyzed: Semisolid (Magenta) was used as control; Continuous immersion (250 ml Flask); and Temporary Immersion (BioMINT II Bioreactor). Multiplication, maturation, and *ex vitro* adaptation phases of sugarcane under

The obtained results in the adaptation of *in vitro* plants of *S. officinarum* at three different types of culture in the multiplication phase were surprising, as it is observed in **Figure 1**, where a notorious formation of shoots occurs in continuous immersion medium. Plants of var. MEX69290 obtained a much higher average shoot formation at the temporary immersion bioreactors than those observed in semi-solid medium. It was observed that invariable of the inoculum density applied (5, 10, 15 plants per bottle) was higher in continuous immersion. Similarly, growth index factor was higher in this culture system than that obtained in semi-solid medium or temporary immersion bioreactors (**Figure 1**). We can observe comparing our results with other

Various micropropagation systems such as liquid cultures and automation have proven the potential to resolve manual handling of *in vitro* cultures at various stages and decrease production cost. However, hyperhydricity is a major problem during *in vitro* culture of many crops in liquid culture systems. Hyperhydricity (also known as "vitrification") is a physiological disorder occurring in plant material of tissue culture, which causes a reduction of propagation and death of tissues when transferred to *ex vitro* conditions [39–41]. The environment inside culture vessels normally used for plant micropropagation is characterized by high humidity, limited gaseous exchange between the internal atmosphere of the culture vessel and its surrounding environment, and the accumulation of ethylene; conditions that may induce physiological disorders [42]. The development of hyperhydric deformities represents a disadvantage for plant micropropagation and a barrier for the exploitation of bioreactor technologies to scale-up its production [41]. The concept of stress in relation to hyperhydricity is not completely established. Therefore, it remains difficult to assume when hyperhydric tissues are stressed. Previous studies argued that abnormal morphology observed in hyperhydricity could be attributed to changes occurring at cellular level due to the modifications of membrane composition or DNA content [42]. However, Rojas-Martínez and coworkers [41] considered this disorder as the result of the stressful conditions brought out by waterlogging of the apoplast. This causes hypoxia and thereby leads to severe oxidative stress. They concluded that hyperhydric features like vitreous appearance and

**water stress "hyperhydricity"**

92 Plant, Abiotic Stress and Responses to Climate Change

**Figure 1.** Average of shoots at different densities of inoculum. T5-T20: 5 inoculum plants were used in semi-solid and continuous immersion medium, 20 plants in temporary immersion; T10-T40: 10 inoculum plants were used in semi-solid and continuous immersion medium, and 40 plants in temporary immersion; T15-T60: 15 inoculum plants were used in semi-solid and continuous immersion medium, and 60 plants in temporary immersion; semisolid (red rectangle), continue immersion (orange rectangle) and temporary immersion (green rectangle). At the bottom of the figure, the calculated growth index factor is reported using the obtained fresh weight under the same inoculum density conditions; T5-T20 (red rectangle), T10-T40 (orange rectangle) and T15-T60 (green rectangle). Five replicates were carried out for each treatment.

(80 rpm) in which they obtained an average shoot emission of 6.95 and 6.30 in the two cultivars used. The shoot emissions and growing index of the sugarcane variety MEX69290 was not affected when cultivated in a stationary liquid medium for 28 days (**Figure 1**).

After 28 days in maturation phase, 120 plants from semi-solid culture, 120 plants under continuous immersion, and 75 from BIOMINT were adapted. In **Figure 3**, we can observe the

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Plants underwent a 28 days preadaptation period, and afterward were planted and placed in greenhouse conditions. Once plants where transferred into the greenhouse, their survival rate was evaluated, being 100% in all cases (**Figure 4**). Plants from the temporary immersion bioreactors were taller and with longer leaves, but those from semi-solid medium and continuous immersion continued to emit shoots during the following 4 months evaluation at the greenhouse. The results obtained in this phase are very similar to those reported by Arencibia et al. [46], Bernal et al. [47], and Silva et al. [48], who reported survival rates higher than 96% in the different cultivars using a temporary immersion bioreactor, and our result is much higher than the studies reported by Snyman et al. [44], with only 34% of survival rate from sugarcane grown in the RITA system.

**Figure 3.** Phase adaptation of *in vitro* plants of *S. officinarum* var. MEX69290, seeded in a germination mixture BM2, previously autoclaved. 15 plants per container were adapted in growth culture room at 25°C with 16/8 hours photoperiod light/dark. (A) and (B) day zero and twenty-eight, of plants coming from semi-solid culture; (C) and (D) day zero and twenty-eight, of plants coming from liquid culture; (E) and (F) day zero and twenty-eight, of plants coming

from temporary immersion system (BioMINT).

quality of the plants from the same clone at the three different cultivation systems.

The variety MEX69290 clones' response at the maturation phase showed the same behavior as that observed at the multiplication phase, with the average shoot emission and the growth index being higher in the liquid culture than the one obtained in half semi-solid or in the temporary immersion bioreactor culture (**Figure 2**).

**Figure 2.** Mean of shoots using 10 in vitro plants in semi-solid and continuous immersion cultures and 60 plants in temporary immersion bioreactors. At the bottom of the figure the calculated growth index factor is reported using the obtained fresh weight under the same inoculum density conditions. Five replicates were carried out for each treatment. Semisolid (red rectangle), continue immersion (orange rectangle) and temporary immersion (green rectangle).

After 28 days in maturation phase, 120 plants from semi-solid culture, 120 plants under continuous immersion, and 75 from BIOMINT were adapted. In **Figure 3**, we can observe the quality of the plants from the same clone at the three different cultivation systems.

(80 rpm) in which they obtained an average shoot emission of 6.95 and 6.30 in the two cultivars used. The shoot emissions and growing index of the sugarcane variety MEX69290 was not

The variety MEX69290 clones' response at the maturation phase showed the same behavior as that observed at the multiplication phase, with the average shoot emission and the growth index being higher in the liquid culture than the one obtained in half semi-solid or in the tem-

**Figure 2.** Mean of shoots using 10 in vitro plants in semi-solid and continuous immersion cultures and 60 plants in temporary immersion bioreactors. At the bottom of the figure the calculated growth index factor is reported using the obtained fresh weight under the same inoculum density conditions. Five replicates were carried out for each treatment. Semisolid (red rectangle), continue immersion (orange rectangle) and temporary immersion (green rectangle).

affected when cultivated in a stationary liquid medium for 28 days (**Figure 1**).

porary immersion bioreactor culture (**Figure 2**).

94 Plant, Abiotic Stress and Responses to Climate Change

Plants underwent a 28 days preadaptation period, and afterward were planted and placed in greenhouse conditions. Once plants where transferred into the greenhouse, their survival rate was evaluated, being 100% in all cases (**Figure 4**). Plants from the temporary immersion bioreactors were taller and with longer leaves, but those from semi-solid medium and continuous immersion continued to emit shoots during the following 4 months evaluation at the greenhouse. The results obtained in this phase are very similar to those reported by Arencibia et al. [46], Bernal et al. [47], and Silva et al. [48], who reported survival rates higher than 96% in the different cultivars using a temporary immersion bioreactor, and our result is much higher than the studies reported by Snyman et al. [44], with only 34% of survival rate from sugarcane grown in the RITA system.

**Figure 3.** Phase adaptation of *in vitro* plants of *S. officinarum* var. MEX69290, seeded in a germination mixture BM2, previously autoclaved. 15 plants per container were adapted in growth culture room at 25°C with 16/8 hours photoperiod light/dark. (A) and (B) day zero and twenty-eight, of plants coming from semi-solid culture; (C) and (D) day zero and twenty-eight, of plants coming from liquid culture; (E) and (F) day zero and twenty-eight, of plants coming from temporary immersion system (BioMINT).

sugarcane. For example, some recent works have employed High-throughput sequencing to identify sugarcane genes involved in leaf abscission [49], biomass content and composition [50], and abiotic stress [51]. Li and cols. [49] performed a transcriptome analysis to identify genes associated with leaf abscission in sugarcane. They employed the Illumina HiSeq 2000 platform (2x90pb) to analyze six cDNA libraries from parents and their F1 offspring, which present different leaf abscission behaviors. After a total assembly, they found 275,018 transcripts corresponding to 164,803 genes. Then, to identify genes related to leaf abscission in sugarcane [49], analyzed a core set of 1, 202 transcripts which were up-regulated in leaf abscission sugarcane plants (LASP) in comparison to leaf packaging sugarcane plants (LPSP). They found that some of these genes were associated with plant-pathogen interaction, response to stress, and ABA-associated pathways. On the other hand [50], performed an extensive transcriptome analysis to identify genes associated with biomass content. They employed the Illumina HiSeq 4000 platform to analyze cDNA libraries from 20 internodal samples of 10 different sugarcane genotypes, which were divided in low and high fiber containing groups. They found 5601 and 4659 unique expressed transcripts in High and Low fiber containing genotypes; and 83,421 shared expressed transcripts between both groups. Furthermore, they found 555 differentially expressed transcripts between low and high fiber containing genotypes. Of these, 151 and 23 transcripts corresponded to sugar and fiber accumulation, respectively. Some of these genes were involved in Carbohydrate metabolism, Photosynthesis, Cell-wall metabolism and Lignin

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Regarding abiotic stress, Belesini and cols. [51] analyzed the transcriptomic profile of the drought-tolerant 'SP81-3250' and the drought-sensitive 'RB855453' sugarcane cultivars under drought stress conditions for 30, 60, and 90 days. They analyzed a total of 54 cDNA libraries by Illumina HiScanSQ System and HiSeq 2500 platforms. Among the genes that were induced in the drought-tolerant cultivar, they found an ascorbate peroxidase, a MYB TF, an E3 SUMOprotein ligase SIZ2, a coenzyme A ligase (a key enzyme for the biosynthesis of flavonoids), and an aquaporin, among others. These types of genes are well known to play a role in abiotic stress tolerance. In the drought-sensitive cultivar they found several kinases that were induced upon stress like Receptor like protein kinases (RLK), which might play a role in stress stimulus perception; bHLH transcription factors; ACC oxidase from the ethylene biosynthetic pathway; and many undescribed genes. More recently (2017), in our laboratory Pereira-Santana and cols. [52] analyzed the transcriptomic profile of the 2nd most important sugarcane cultivar in Mexico, 'Mex 69-290', in response to osmotic stress. In such study, authors employed the High-throughput sequencing system HiSeq-Illumina (2x100bp) to analyze 16 cDNA libraries representing leaves and roots of *in vitro*-grown plantlets exposed to PEG-8000 during 0, 24, 48, and 72 hours. After assembly of a total of 140, 339 unigenes, Pereira-Santana and cols. Found core sets of 536 and 750 up-regulated genes in response to osmotic stress in roots and leaves, respectively; and core sets of 1093 and 531 down-regulated genes in roots and leaves, respectively. After gene annotation, the authors found that sugarcane 'MEX69290' responds to osmotic stress by increasing the expression of genes involved in transcription regulation, oxide-reduction, carbohydrate catabolism, and flavonoid and other secondary metabolites biosynthesis. Genes responsive to ABA, water deprivation, and heat stress were also up-regulated. On the other hand, this sugarcane cultivar responds to osmotic stress by

Pathway; DIR proteins were also represented [50].

**Figure 4.** Greenhouse adaptation of *in vitro* plants of *S. officinarum* var. MEX69290, from culture: (A) semi-solid; (B) continuous immersion; (C) temporary immersion. Substrate consisted on a 3: 1 mixture of sunshine: soil. All plantlets survived 100% after 30 days in the greenhouse.

The best results out of the measured parameters were obtained from the continuous immersion propagation system. It was concluded the reason for this may reside in the elimination of gelling agent, which additionally lowers production costs in the process of delivering this sugarcane's variety to the field. Plants obtained under this system achieved normal development, they developed shoots and roots cyclically and no vitrification was detected in any of the evaluated micropropagation phases. This suggests that the clone obtained from the MEX69290 variety is tolerant to liquid culture conditions. Apparently this system does not generate an abiotic stress, stationing it as a prospective medium to perform genetic transformation processes and to study its gene expression pattern that could further make enhanced tolerant clones.
