Sugarcane propagation utilizing TIB
Sugarcane was propagated by initiating and regenerating callus into plantlets. Callus was successfully initiated (Fig. 1f) from spindle leaves explants of the four sugarcane varieties. The percentage of callusing explants was below 40%. The highest was 33.4% for PS 094, then 28.5% for PS 091, 24.8% for PSKA 942, and 11.1% for PS 881 (Fig. 1a). Therefore, the quantity of calli as propagation materials was sufficed by proliferating the calli in TIB (Fig. 1h). Interestingly, the proliferation rates of calli were high, ranged 4.5 – 33.8 multiplies of the calli fresh biomass (Fig. 1b). Calli of the sugarcane PSKA 942 achieved the highest proliferation rate while the lowest was PS 091 (Fig. 1b).
Callus proliferation or multiplication was affected by external stimuli such as nutrient and hormone in the media (Solangi et al. 2016; Abdelsalam et al. 2021; Saleem et al. 2022), temperature (Mostafiz et al. 2018), or light intensity (Notoya and Kim 1996). Besides, it was also affected by the plant genetic background or genotypes (Mostafa et al. 2020; Nguyen et al. 2020; Abdelsalam et al. 2021; Saptari et al. 2022), resulted in different cells regenerating capacity and response against treatments. Nguyen et al. (2020) revealed some single nucleotide polymorphisms (SNPs) associated with callus size on the micropropagation of 96 rose genotypes. Wu et al. (2022) also identified three quantitative trait loci (QTLs) associated with rice callus regeneration ability. Although, cells response still can be enhanced by external stimuli such as hormones.
Consistently, results of our study also showed the influence of plant genotypes in the callus proliferation and growth (Fig. 1). Moreover, different plant genotypes also showed different responses in the further development on the propagation, such as callus regeneration into shoots (Fig. 1c), and shoots maturation into plantlets (Fig. 1d). The sugarcane PSKA 942 and PS 094, having relatively higher callus proliferation rate than PS 091 and PS 881, the varieties also produced more shoots in the callus regeneration phase (Fig. 1c). PSKA 942 and PS 094 produced on average of 333.8 and 257.8 shoots per TIB flask, respectively. Meanwhile, PS 091 only produced on average of 9.4 shoots per TIB flask, and PS 881 could not regenerate shoots (Fig. 1c).
The lack of shoots regeneration in the sugarcane PS 881 might not necessarily due to low regeneration capacity of the variety. However, it might still need further optimization to find the suitable media or other treatments. In this study, media to induce callus regeneration into shoots was supplemented with polyamine (10 mg L-1 putrescine), and cytokinin (0.2 – 0.5 mg L-1 BAP). Cytokinin, including BAP was a shoot inducing hormone (Chickarmane et al. 2012). It plays a major role in maintenance and controlling meristematic properties of the shoot apical meristem (Chickarmane et al. 2012). Meanwhile, putrescine is among a group of polyamines which has a major role in plant environmental stress response (Chen et al. 2019). Some studies used putrescine supplemented media to induce sugarcane in vitro regeneration (Shankar et al. 2011; Reis et al. 2016; Sathish et al. 2020). Nevertheless, other hormones might also be used or tested for better inducing sugarcane in vitro regeneration. Chengalrayan and Gallo-Meagher (2001) experimented five types of cytokinins: BAP, kinetin, 6-γ, γ- (dimethylallylamino) purine (2iP), zeatin, thidiazuron, resulted in the best shoot induction by thidiazuron. Saleem et al. (2022) used kinetin, indole-3-butyric acid (IBA), BAP, and α-naphtaleneacetic acid (NAA), resulted in the best shoot induction by kinetin, followed by BAP. Therefore, thidiazuron and kinetin might be recommended for further optimization on the sugarcane shoots induction and regeneration.
Fig. 2 showed detailed regeneration process of the sugarcane callus into shoots. The early sugarcane callus (Fig. 2a) was translucent and watery. The histological figure of the early callus showed unregular structure with relatively big vacuoles and dispersed cytoplasm (Fig. 2b). Meanwhile, the embryogenic callus (Fig. 2c) obtained after some callus proliferations in TIB, showed nodular structures with relatively smaller vacuoles and dense cytoplasm, also reflected by an intense staining in the histological figure (Fig. 2d). This type of callus has regenerative capacity, and after cultured in TIB in 2 – 3 cycles, it developed greenish structures (Fig. 2e) indicating shoot primordia initiation. Histological figure on Fig. 2f showed the early formation of vascular tissues (v) on the greenish calli. The greenish callus further developed into shoot primordia (Fig. 2g), was also indicated by the elongation of the vascular tissue (Fig. 2h). Accordingly, the histological evidence (Fig. 2b,d,f,h) showed that the in vitro regeneration of the sugarcane in this study was via organogenesis route as indicated by the unipolar growth of the shoot meristem.
The sugarcane in vitro shoots were then successfully developed into plantlets (Fig. 1g, h, i). There were on average of 181, 146, and 6 sugarcane plantlets of PSKA 942, PS 094, and PS 091 respectively obtained per TIB flask (Fig. 1d). The quantity of plantlets obtained from each variety were in correlated with the number of regenerating shoots in the previous step, which was dominated by the PSKA 942. However, there was no morphological different or abnormality observed between plantlets from each sugarcane variety (Fig. 1i), indicating the true-to-type micropropagation products. Although, the occurrent of SV still needs further confirmation from the genetic analysis.
The sugarcane plantlets grew in clusters (Fig. 1i). The plantlets clusters were then transferred into test tubes containing liquid media (Fig. 1i) to maintain plantlets vigor and induce better root system. A well-developed root system is essential to plantlets survival during acclimatization. In this study, all plantlets successfully developed roots with the trigger of auxin (IAA) in the media. However, despite produced more plantlets, the survival rate of PSKA 942 (42.5 %) plantlets was lower than PS 094 and PS 091 plantlets during acclimatization process (Fig. 1e). Meanwhile, PS 094 and PS 091 plantlets were 100% survived after 6 weeks of acclimatization (Fig. 1e).
The survival rate was considered high compared to other sugarcane micropropagations. Meyer et al. (2009) reported the best survival rate at 52% of the temporary immersion-derived sugarcane seedlings acclimatization. The study also reported lack of roots formation and occurrence of hyperhidricity such as small and glassy leaves on plantlets, which contributed to mortality in acclimatization. Whereas in our study, there was no hyperhidricity observed, and plantlets mortality was due to lack chlorophyl synthesis resulting in downy plantlets of PSKA 942. High multiplication and regeneration rates of this variety (Fig. 1b,c,d) might contribute to the phenomenon. The cells energy might deplete due to rapid proliferation. Analysis on the media physicochemical during callus proliferation and regeneration in TIB revealed that in the rapid growth of PSKA 942, a considerably depleted of the total soluble sugar, total dissolved solid, and electrical conductivity were observed compared to those in other varieties (Supplementary Table 1). Sugar is source of energy and fixed carbon for synthesis of biological compound, including pigment (Nevins 1995). Sáez et al. (2016) reported chlorophyll content increase with the high sucrose supplemented media compared to those with low sucrose. Still, other factors also influence chlorophyll synthesis in the in vitro plant, and further analysis is necessary.
Acclimatization is an important step in the plant micropropagation, to adapt the plantlets into the ex-vitro environment, the plant natural environment. The in vitro plants were cultivated under controlled heterotrophic or photomixotrophic conditions. During acclimatization, plants must convert rapidly to an autotrophic growth in the uncontrolled environment. Therefore, to maintain the relative humidity during acclimatization, a UV plastic cover was installed in the acclimatization block (Fig. 1j), with gradual opening every week, supporting seedlings adaptation. Survive seedlings were then successfully transplanted into the field after 6 months (Fig. 1k).
Genetic stability of the TIB-derived sugarcane seedlings
Fifteen SSR markers were used to analyze the genetic stability of TIB-derived sugarcane seedlings. PCR amplification using the fifteen primer pairs resulted in the total of 204 bands, ranging from 150 – 4,000 bp in size, with over 170 bands were polymorphic. Among the 15 markers, 12 of them were highly polymorphic, as shown by the high polymorphism information content (PIC) value (Table 1). PIC value for co-dominant markers such as SSR ranged from 0 – 1, with higher value represents higher polymorphism. The PIC value of the SSR markers in this study ranged from 0.43 – 0.88. Markers with PIC value greater than 0.50 was considered ideal for this study because they have higher possibility to detect genetic changes in the genome.
Table 1. List of SSR markers used in the genetic stability analysis of the TIB-derived sugarcane seedlings
Marker
|
Forward and reverse primer(s)
|
Ta (C)
|
PIC
|
Reference
|
MCSA068G08
|
CTAATGCCATGCCCCAGAG
GCTGGTGATGTCGCCCATC
|
61
|
0.43
|
Wu et al. 2019
|
SCC06
|
TATTCCACCGGGAACAAGA
GGGATTGTAGCGACGAGTT
|
61
|
0.71
|
Santos et al. 2012
|
SCM15
|
GGAGATGTTTGAGAGGGAA
AGAGTAGCATAAAGGAGG
|
58
|
0.73
|
Singh et al. 2010
|
SCM18
|
CATCAGTATCATTTCATCTT
CAGTCACAGTCGGGTAGA
|
55
|
0.70
|
Singh et al. 2010
|
SCM27
|
TTCTCTGACTTCCAATCCAA
ATCAAGCACGCCCGCCTC
|
55
|
0.48
|
Singh et al. 2010
|
SCM7
|
ACGGTGCTCTTCACTGCT
GGGCATACTTCCTCCTCTAC
|
61
|
0.13
|
Wu et al. 2019
|
SKM09
|
GGTGGCTAACAGACAGGG
TTGCTGCCGAGAGTCATA
|
61
|
0.77
|
Abdullah et al. 2013
|
SKM10
|
GCGCCTATTTAATACCAGA
CTTTCCCTATACCCATGATA
|
52
|
0.88
|
Abdullah et al. 2013
|
SMs12
|
AAATGTCTTCGCACTAACC
AAGGAGATGCTGATGGAGA
|
58
|
0.72
|
Abdullah et al. 2013
|
UGSM351
|
AAGAAGAGCCGTAGAAAC
ATTGAGCGAGGGATGAAC3
|
52
|
0.50
|
Pandey et al. 2012
|
UGSM681
|
ACACATCGCTTTCCCACA
GCATACCTGTCGTCGTCT
|
61
|
0.54
|
Singh et al. 2010
|
SCC05
|
CGGAATCCAATTCGTACGT
CATTGGTTGCACCACAGTTC
|
61
|
0.82
|
Santos et al. 2012
|
SKM01
|
TATGGAGAGAGCAACCTATCA
GACGGAAGATTGGGATTC
|
58
|
0.65
|
Abdullah et al. 2013
|
SKM02
|
GGCCTTCGATTAACCGAT
ACAGGACGCTGCTTCTTG
|
61
|
0.74
|
Abdullah et al. 2013
|
SKM08
|
TTATCCCTTTCGTTCAGTAGAG
ATTTTGCGTAGGGTCTGAG
|
58
|
0.70
|
Abdullah et al. 2013
|
In addition to polymorphism, another factor that was taken into account in the selection of the SSR markers was the position of the marker in the genome. In this study, 9 out of the 15 selected markers were screened from cDNA, unigene, or expressed sequence tags (ESTs) libraries (Singh et al. 2010; Pandey et al. 2012; dos Santos et al. 2012; Wu et al. 2019). The use of SSR markers derived from these libraries is crucial since genetic changes in intragenic regions are more likely to affect plant phenotypes (Kuromori et al. 2006; Vicente-Dólera et al. 2014; Xu et al. 2019). The use of selected SSR markers is expected to cover most of the potential genetic changes in sugarcane that may occur due to micropropagation.
Fig. 3a showed the similarity matrices of SSR pattern from the three sugarcane varieties. The genetic similarity of TIB-derived sugarcane plants relative to the mother plants varied between 88.1 % to 97.6 %. The average genetic similarity of PSKA 942 sugarcane was 96.3 %, while PS 094 was 91.5 %, and PS 091 was 96.1 % (Fig. 3b). The overall genetic similarity of the TIB-derived sugarcane plants was higher than 90 %, which is considerably high. Azizi et al. (2020) reported 94% genetic similarity with the SSR analysis, between sugarcane mother plants and the micropropagated sugarcane which was subcultured up to nine times on the solid regeneration media. Whereas, Hsie et al. (2015) reported no polymorphism of the micropropagated sugarcane with up to fifteen consecutive subcultures in the liquid multiplication media. However, there is still no standard of the maximum level for genetic changes in the sugarcane micropropagation products can be tolerated. Therefore, observations on the phenotypic or morphological changes in the sugarcane micropropagation products should accompany the genetic analysis results.
Overall, SSR analysis is one of the most efficient ways to confirm the uniformity of the micropropagated plants. However, due to its polyploidy, SSR analysis in sugarcane hybrids is not as simple as that in diploid plants. Expensive technique such as capillary electrophoresis with high resolution is required to accurately detect the small genetic changes that are almost impossible to detect with regular gel electrophoresis. To counter said limitation, it is necessary to use as many SSR markers as possible to broaden the genomic coverage and to detect as many changes as possible.
Generally, genetic stability of the micropropagated plants was suggested to be affected by the plant genotype, and the media composition and exposure (Eeuwens et al. 2002; Abass et al. 2017; Garcia et al. 2019). As mentioned, TIB implemented system which limiting plants exposures to the media, thus could prevent SV. Besides, with the high explant multiplication achieved with TIB, the number of subculture cycles could also be reduced. If the traditional solid culture system could achieve maximum of three biomass multiplies per subculture the TIB system could achieve maximum of 30 multiplies (Fig. 1b), so that at least the subculture cycle could be reduced by tenth.
Morphological observations of the TIB-derived sugarcane seedlings
Sugarcane polyploidy limits the understanding between the genetic analysis results and the changes in phenotypes. Therefore, a morphological analysis and comparison are still necessary to accompany the results of SSR-based genetic analysis in sugarcane. Morphological analysis can also be the basis to determine the maximum level of genetic changes detected by SSR markers that can be tolerated or have no effect in plants phenotype.
Morphological observation in the field of the 4 months old sugarcanes showed uniformity or no difference between TIB-derived sugarcanes and the mother plants (Fig. 4). Plant heights of the PSKA 942 ranged from 1.75 – 2.50 m, while PS 094 ranged from 1.80 – 2.30 m, and the PS 091 ranged from 1.45 – 2.20 m. The stalk diameter ranged from 1.80 – 3.00 cm in all varieties. The leaf blade length of the PSKA 942 ranged from 119 – 163 cm, while PS 094 ranged from 131 – 167 cm, and the PS 091 ranged from 117 – 160 cm. The leaf blade width of the PSKA 942 ranged from 4.0 – 6.8 cm, while PS 094 ranged from 2.8 – 5.6 cm, and the PS 091 ranged from 3.6 – 5.4 cm. The overall organ morphological descriptions were also in accordance with the documents of the released varieties (Table 2).
Accordingly, results of the morphological observation supported the SSR genetic analysis, revealing that the TIB-derived sugarcanes were true-to-type. It might also indicate that about 88.1% to 97.6% of genetic similarity based on SSR analysis did not cause phenotypic changes in the TIB-micropropagation products of sugarcanes.
Table 2 Sugarcanes morphological observations based on the varieties description
Organ(s)
|
Descriptions
|
Observations
|
PSKA 942
|
Leaf
|
Auricle
|
Absent; or present, weak, oblique position
|
Absent
|
Leaf colour
|
Dark green
|
In accordance with the variety description
|
Leaf width
|
Medium (4 – 5 cm)
|
In accordance with the variety description
|
Curvature
|
Curved tips
|
In accordance with the variety description
|
Sheath dorsal hair
|
Present, sparse, narrow, oblique, < ¼ blade width
|
In accordance with the variety description
|
Internode
|
Shape
|
Cylindrical
|
In accordance with the variety description
|
Cross-section
|
Circular
|
In accordance with the variety description
|
Zig-zag alignment
|
Absent or very weak
|
Very weak
|
Crack
|
Absent
|
In accordance with the variety description
|
Bud/eye
|
Shape
|
Round
|
In accordance with the variety description
|
Position
|
On the leaf sheath
|
In accordance with the variety description
|
Stem
|
Colour
|
Yellowish-green
|
In accordance with the variety description
|
Waxiness
|
Medium
|
In accordance with the variety description
|
Crack
|
Absent
|
In accordance with the variety description
|
PS 094
|
Leaf
|
Auricle
|
Present, weak, straight position
|
Present, solid, straight position
|
Leaf colour
|
Dark green
|
In accordance with the variety description
|
Leaf width
|
Medium (4 – 5 cm)
|
In accordance with the variety description
|
Curvature
|
Curved tips
|
In accordance with the variety description
|
Sheath dorsal hair
|
Present, dense, narrow, straight, < ¼ blade width
|
In accordance with the variety description
|
Internode
|
Shape
|
Conoidal
|
In accordance with the variety description
|
Cross-section
|
Circular
|
In accordance with the variety description
|
Zig-zag alignment
|
Absent or very weak
|
Absent
|
Crack
|
Present in part of internode
|
In accordance with the variety description
|
Bud/eye
|
Shape
|
Round
|
In accordance with the variety description
|
Position
|
On the leaf sheath
|
In accordance with the variety description
|
Stem
|
Colour
|
Yellowish-green
|
In accordance with the variety description
|
Waxiness
|
Strong
|
In accordance with the variety description
|
Crack
|
Absent
|
In accordance with the variety description
|
PS 091
|
Leaf
|
Auricle
|
Present, solid, straight position
|
In accordance with the variety description
|
Leaf colour
|
Dark green
|
In accordance with the variety description
|
Leaf width
|
Medium (4 – 5 cm)
|
In accordance with the variety description
|
Curvature
|
Absent
|
In accordance with the variety description
|
Sheath dorsal hair
|
Absent
|
In accordance with the variety description
|
Internode
|
Shape
|
Cylindrical
|
In accordance with the variety description
|
Cross-section
|
Circular
|
In accordance with the variety description
|
Zig-zag alignment
|
Absent
|
In accordance with the variety description
|
Crack
|
Present in part of internode
|
In accordance with the variety description
|
Bud/eye
|
Shape
|
Ovate
|
In accordance with the variety description
|
Position
|
On the leaf sheath
|
In accordance with the variety description
|
Stem
|
Colour
|
Yellowish-green
|
In accordance with the variety description
|
Waxiness
|
Medium
|
In accordance with the variety description
|
Crack
|
Absent
|
In accordance with the variety description
|