Global climate change caused by natural processes results in major environmental issues that affect the world. Climate variability results in changes that cause water stress in plants. Sugarcane is a tropical grass C4, perennial and a multi-purpose industrial cash crop which serves as the main source of raw material for the production of sugar and biofuel. Farmers face the challenge to provide biotech alternatives with potential benefits and minimize potential adverse impacts on sugarcane’s production. In order to find biotechnology strategies to diminish the impact of climate change, our laboratory teamworks with micropropagation, transcriptome and genetic transformation of sugarcane using the var. MEX69290. In the transcriptome of sugarcane, a total of 536 and 750 genes were differentially regulated under normal and water stress treatment respectively, of which key genes were selected to be inserted into sugarcane for tolerance to abiotic stress. Regarding results of micropropagation, it was concluded that the continuous immersion propagation system was the best culture strategy. This may be as result of the elimination of gelling agent, which additionally helps reduce production costs.
- micropropagation systems
- genetic transformation
- abiotic stress
Climate change alters environmental conditions and therefore has direct and biophysical effects on agricultural production. The biophysical and direct effects of climate change induce alterations on the prices and production of agriculture. Such changes are reflected on the economic system as farmers and other market participants make adjustments autonomously. They are both compelled to modify their crop combinations, use of supplies, level of production, and food demand, consumption and trade. Climate change causes a changes in rainfall regimes which have direct effects on crop yields as well as indirect effects through changes in the availability of water irrigation .
2. Climate change, water stress and its effect in sugarcane
A large scale of plant production grown under different agricultural production systems is lost under the effects of abiotic stresses, which may result in a 70% reduction of the potential yields of crop plants . During growth and developmental periods, crops suffer seasonal floods and droughts, extreme temperatures or salinity all year round. Globally, about 22% of global agricultural land is saline, and the increased damage caused by drought has been reported to limit plant growth and development followed by a loss of productivity, especially in crop species [6, 7]. Thus, drought stresses are one of the most serious kind of abiotic stresses that implies a threat on crop productivity worldwide.
Sugarcane, an important source of sugar and ethanol, is a relatively high water-demanding crop and its growth is highly sensitive to water deficit . It is estimated that sugarcane produces 8–12 ton cane per ML of water irrigation , and water deficit can lead to productivity losses of up to 60% [10–13]. For this reason, production areas are concentrated in regions with favorable rain regime to sugarcane growth and development , while in other areas crop production requires supplemental or full irrigation .
According to various studies, water stress triggers many physiological, biochemical, and molecular responses that influence various cellular processes in plants and this impacts on its productivity [16, 17].
Severe water stress such as drought affects the entire plant. Morphological and physiological responses in sugarcane plants vary according to its genotype, duration (rapid or gradual) and intensity (severe or mild) of stress and also the type of affected tissue [18–21]. Water stress also affects both cane and sugar yield substantially. The most common water stress responses in sugarcane are leaf rolling, stomatal closure, inhibition of stalk and leaf growth, leaf senescence and reduced leaf area [12, 22]. Moreover, under water stress, both cell division and cell elongation are interrupted  and stem and leaf elongation are the most severely affected growth processes [24, 25]. Root development is also influenced by water deficit [19, 26] but its overall biomass is relatively less than the above-ground biomass. Sugarcane is a tropical crop with C4 photosynthetic metabolism. A moderate water stress causes a stomatal limitation, which triggers a decrease in stomatal conductance (gs), transpiration rate (E), internal CO2 concentration (Ci), and photosynthetic rate [26–30]. Under water stress, a decline in photosynthetic rate is mainly caused by a decrease in phosphoenolpyruvate carboxylase (PEPcase) and ribulose-1,5-biphosphate carboxylase (Rubisco) activity [26, 27, 31]. It is worth to note that photosynthesis rate is also impacted by sugar accumulation in leaves . Under non-stressed condition low leaf sugar content is conducive to photosynthesis, while high sugar content moderates carbon fixation . Interestingly, increased levels of some sugars, such as trehalose, can help plants to cope with water deficit, reducing the damage on cell membrane . The capacity to accumulate trehalose was demonstrated in sugarcane roots under drought conditions. Sales et al.  reported an increase in starch hydrolysis, leading to higher levels of soluble sugars that helped sustain carbon supply even in a reduced CO2 fixation condition, facilitating growth recovery after stress.
3. Sugarcane and biotechnology
Sugarcane crop productivity has progressively increased to remarkable levels worldwide in the last century . This increase in productivity has been ascribed to the development and widespread use of improved cultivars with increased resistance to diseases and pests, better management of water, nutrients and other resources, and the availability of relatively cheap chemical fertilizers and pesticides. Sustaining this pace of improvement in crop productivity by innovative and intensive agriculture, whilst ensuring minimal environmental impact, will be one of the major challenges to maintain a profitable sugar industry in the future.
Biotechnology offers excellent opportunities for sugarcane crop improvement. Commercial sugarcane, mainly the interspecific hybrids of
4. Micropropagation an alternative to develop plants tolerant to water stress “hyperhydricity”
Various micropropagation systems such as liquid cultures and automation have proven the potential to resolve manual handling of
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 .
With the objective of evaluating the stress caused by hyperhydricity in the
The obtained results in the adaptation of
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).
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.
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. , Bernal et al. , and Silva et al. , 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. , with only 34% of survival rate from sugarcane grown in the RITA system.
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.
5. Transcriptomic analysis of an elite Mexican sugarcane cultivar (‘Mex 69-290’) in response to osmotic stress. Identification of genes with biotechnological potential
Modern sugarcane cultivars have been obtained by inter-specific hybridizations between the high-sucrose-yielding of
Regarding abiotic stress, Belesini and cols.  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 SUMO-protein 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.  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 addition to the insights about the global gene expression dynamics of ‘Mex 69-290’ in response to osmotic stress and the identification of novel TFs, the work of Pereira-Santana and cols. Provides a useful benchmark for the study of other specific gene families of biotechnological significance for sugarcane engineering, for example the DIR protein family. Plant DIR proteins are believed to be involved in lignin biosynthesis, defense [56, 57], and abiotic stress responses such as dehydration , and salinity and oxidative stress . In a recent study, 5 available sequence databases for sugarcane were surveyed, a total of 120 DIR proteins were identified . Phylogenetic analysis showed that these DIR proteins are divided in 64 groups and 7 major clades: Dir-a, Dir-b/d, Dir-c, Dir-e, Dir-g, Dir-h, and Dir-i . In the sugarcane transcriptome assembly of ‘sugarcane Mex 69-290’ performed in our laboratory by Pereira-Santana and cols, a total of 48 predicted proteins with DIR-like domains were identified. These DIR proteins were clustered in 7 groups according to their expression patterns (Figure 6). DIR42 protein from cluster 1 was significantly up-regulated in all time points of osmotic stress in root tissues. Conversely, DIR40 protein from cluster 7 was significantly down-regulated in all time points of osmotic stress in leaf tissues. In general, DIR genes from cluster 4 seem to possess a relative high expression in roots under control conditions, and those from cluster 7 seem to possess a relative high expression in leaves under control conditions. DIR genes from both clusters are down-regulated in response to osmotic stress. On the other hand, we also recovered a homolog of the ScDir gene (GenBank: JQ622282.1) from the sugarcane variety FN39 (DIR38 in cluster 5). The expression of ScDir from FN39 has been reported to be up-regulated in response to H2O2, NaCl, and PEG treatment . Furthermore, its heterologous expression in
6. Genetic transformation of cane, a very powerful biotechnological tool to generate tolerant plants to water stress
According to the International Service for the Acquisition of Agri-biotech Applications (ISAAA), the worldwide distribution of genetically modified crops involves a total of 26 developing countries and 7 industrialized countries, headed by USA, Brazil, Argentina, Canada, India, China and South Africa. There is a current approval on the use of two commercial varieties of genetically modified cane in Brazil and Indonesia. On the former, plants containing the Cry1Ab gene, which produces an insecticidal toxin capable of killing the
Scientific research in genetic transformation have focused on resistance to biotic and abiotic factors such as weed control, production of renewable primary products, energy crops and production of pharmaceutically active substances.
Some of the methods in genetic transformation of plants are by Agrobacterium or biolistic which are time consuming, laborious and have low transformation efficiency. Thus we have attempted different options to optimize genetic transformation in sugar cane. An option for efficient transformation is by using different types of vectors, for example Anderson & Birch  used Binary super vectors in addition of different types of promoters (constitutive and inducible). Niu et al.  is other case who used the SoCINI inducible promoters and the ScMybRI constitutive promoters respectively [62, 63].
On the other hand, different
In contrast, a genetic transformation protocol using
It should be clarified that the functionality of the CpRap2.4b gene belonging to the (AP2/ERF) transcription factors family was tested in tobacco plants, which were segregated to obtain F2 plants and were then subjected to water stress (drought) conditions to evaluate their function.
Climate change affects farmers economically, causing drought floods, which affect the productivity of the plant. Biotechnology is an alternative to reduce the impact of climate change on plants. In recent years there has been a continuing need to provide commercial clones of resistance to pests and long-lasting diseases in combination with superior agronomic performance. This led to considerable research in different areas of biotechnology including: micropropagation, transcriptomics and genetic transformation.
These areas of biotechnology together are a key tool in the pursuit of genetically enhanced plants that resist climate change.
|BAC||Bacterial artificial chromosome|
|EST||Expressed sequence tag|
|NGS||Next generation sequencing|
|LEA||Late embryogenesis abundant|
|NAC||NAM, ATAF, and CUC|
|HSF||Heat shock factor|
|ORF||Open reading frame|
|DEG||Differentially expressed genes|
|HMM||Hidden Markov Model|
|TMM||Trimmed mean of M values|
|GFP||Green fluorescent protein|