Ajinder Kaur

Enhanced in vitro shoot multiplication and elongation in sugarcane using cefotaxime

Antibiotic cefotaxime has been found to be a growth promoting substance in sugarcane tissue culture. Young leaf segments (1–2 cm long) of three commercially important Indian sugarcane varieties viz., CoJ 83, CoJ 85 and Co 89003, regenerated into shoots and multiplied on semi-solid MS medium supplemented with 6-benzylaminopurine (BA, 0.2 mg/l) and kinetin (0.2 mg/l) for 2 cycles of 2 weeks each. Shoots (2 cm long) were separated and cultured on semi-solid half-strength MS medium supplemented with NAA (2 mg/l) and cefotaxime at various concentrations viz., 0, 250, 500 and 750 mg/l. Maximum shoot multiplication and elongation with respect to number of microtillers, shoot length and plantlet fresh weight in all the genotypes was obtained with cefotaxime used at the rate of 250 and 500 mg/l in the medium. Among the different varieties, on the basis of mean of 50 shoot cultures, number of microtillers per culture was highest in Co 89003 (7.50, 145.90% increase over control), whereas, shoot length and plantlet fresh weight were highest in CoJ 83, i.e. 10.30 cm (60.94% increase over control) and 360.45 mg (50.75% increase over control), respectively after 2 weeks of culturing with cephotaxime used at the rate of 500 mg/l in the medium. Statistical analysis of data revealed significant differences among varieties and media for different shoot multiplication and elongation parameters. Therefore, use of cefotaxime during tissue culture and genetic transformation of sugarcane can improve frequency of shoot multiplication and transformation, respectively.

Red rot resistant transgenic sugarcane developed through expression of β-1,3-glucanase gene

Sugarcane (Saccharum spp.) is a commercially important crop, vulnerable to fungal disease red rot caused by Colletotrichum falcatum Went. The pathogen attacks sucrose accumulating parenchyma cells of cane stalk leading to severe losses in cane yield and sugar recovery. We report development of red rot resistant transgenic sugarcane through expression of β-1,3-glucanase gene from Trichoderma spp. The transgene integration and its expression were confirmed by quantitative reverse transcription-PCR in first clonal generation raised from T0 plants revealing up to 4.4-fold higher expression, in comparison to non-transgenic sugarcane. Bioassay of transgenic plants with two virulent C. falcatum pathotypes, Cf 08 and Cf 09 causing red rot disease demonstrated that some plants were resistant to Cf 08 and moderately resistant to Cf 09. The electron micrographs of sucrose storing stalk parenchyma cells from these plants displayed characteristic sucrose-filled cells inhibiting Cf 08 hyphae and lysis of Cf 09 hyphae; in contrast, the cells of susceptible plants were sucrose depleted and prone to both the pathotypes. The transgene expression was up-regulated (up to 2.0-fold in leaves and 5.0-fold in roots) after infection, as compared to before infection in resistant plants. The transgene was successfully transmitted to second clonal generation raised from resistant transgenic plants. β-1,3-glucanase protein structural model revealed that active sites Glutamate 628 and Aspartate 569 of the catalytic domain acted as proton donor and nucleophile having role in cleaving β-1,3-glycosidic bonds and pathogen hyphal lysis.

Optimization of gamma radiation dose for induction of genetic variation in sugarcane (Saccharum spp.) callus and regenerated shoot cultures.

Callus cultures were established in three commercial sugarcane varieties viz., CoJ 64, CoJ 83 and CoJ 86 from spindle explants on MS + 2,4-D (4 mg l−1) + BAP (0.5 mg l−1) medium. Shoots were regenerated from two-month-old calli on MS + BAP (0.5 mg l−1) medium. Callus and callus derived shoots were treated with gamma (γ) radiation at 20, 40, 60 and 80 Gray (Gy). Per cent shoot regeneration from y-irradiated calli in the three varieties ranged from 90 to 93.8 at 20 Gy, 83.3 to 87.5 at 40 Gy, 30 to 36.4 at 60 Gy and 0 at 80 Gy. Upon irradiating shoots, subsequent shoot proliferation in the three varieties ranged from 90.9 to 93.1% at 20 Gy, 82.6 to 84.0% at 40 Gy and 27 to 32.3% at 60 Gy, whereas 80 Gy dose was 100% lethal. Thus, 60 Gy dose of y-radiation was found to be optimum for carrying out mutagenesis of both callus and callus derived shoots. In the field, different irradiated clones of the same variety exhibited huge variability with respect to number of canes, cane girth, cane height and sucrose content.

Micropropagation and Somatic Embryogenesis in Sugarcane

Sugarcane propagation through conventional means does not provide sufficient planting material of a variety, particularly desirable in case of newly released varieties to achieve large-scale dissemination; this is attributed to slow rate of seed multiplication by conventional sett planting. On the other hand, micropropagation technique of tissue culture ensures production of disease-free and true-to-type planting material of popular (new as well as old) varieties in an abundant quantity in a short period of time. The cultures of meristematic buds or spindle leaves, collected from healthy plants, are established aseptically under controlled nutritional and environmental conditions in vitro, followed by multiplication of shoots and induction of roots; the plantlets are hardened and supplied to growers. Somatic embryogenesis is the process of embryo formation and development from somatic cells of an explant under in vitro conditions. The somatic cells in culture can follow two pathways for somatic embryogenesis, either direct or indirect. The plants regenerated through direct somatic embryogenesis are often uniform; thus, the pathway finds use in clonal propagation and genetic transformation of sugarcane genotypes. In indirect somatic embryogenesis pathway, first callus is induced from cultured explants under the influence of an auxin (mostly 2, 4-D) which is then regenerated into plants; such plants may exhibit somaclonal variation.

Somaclonal Variation for Sugarcane Improvement

Cell and tissue culture approaches serve as an important and an easily accessible source for the creation and utilization of variability in sugarcane improvement programmes. This variation arising from cell and tissue cultures is called somaclonal variation that can be genetic/epigenetic in nature. Somaclonal variation is considered as a complex phenomenon resulting from various genetic and cellular mechanisms under in vitro conditions. The induction of variation under in vitro conditions either through shock treatment or in stepwise manner generates useful variability without sexual recombination. Due to limited genetic system and/or narrow genetic base, somaclonal variation is more rewarding in sugarcane. During plant propagation process under laboratory conditions, the genomic shock is induced which can result in the activation of various transposons, retrotransposons, chromosomal changes, methylation and demethylation of DNA leading to somaclonal variation. Desirable variants (disease resistant, herbicide resistant, drought tolerant, salt tolerant, antibiotic resistant, etc.) have been isolated in sugarcane through in vitro selection. The frequency of variation can be further enhanced using physical and chemical mutagens. ‘Ono’, ‘Phule Savitri’ and ‘VSI 434’ are the sugarcane varieties released through the process of somaclonal variation.