High throughput in vitro micropropagation of sugarcane (Saccharum officinarum L.) from spindle leaf roll segments: Cost analysis for agri-business industry

Abstract

High throughput micropropagation (HTM) protocol in sugarcane through direct shoot regeneration comprising five stages, was developed for agri-business industry. The distinction of the protocol lies in direct adventitious shoot regeneration without an intervening callus phase and liberty over the number of subculture passages leading to high rates of synchronous plant production. Stage 0 dealt with selection and maintenance of field-grown stock plants; Stage I marked the initiation of in vitro propagation on culture initiation medium using spindle leaf roll segments from apical tops; Stage II involved five passages (of 14 days each) on shoot multiplication medium followed by one additional passage (of 10 days) on shoot multiplication-cum-elongation medium. The shoot multiplication rates ranged from 4 to 25-fold in CoPb 91 and CoJ 83 cultivars, respectively; Stage III involved in vitro rooting on root induction medium and hardening for 14 days at each step; Stage IV comprised separation of clumps into single plantlets and transfer to soil. The complete plants were produced in 157 days with 97 percent survival rate. The fidelity of the protocol for agri-business industry was tested by producing approximately 0.1 million saleable HTM sugarcane plants in a small scale (150 m 2 ) tissue culture unit. The recurring (energy, labour, media, culture container, transplanting material) and non-recurring (laboratory infrastructure, glasshouse, equipment) parameters accounted for 75.56 and 24.44 percent production costs, respectively. The lowcost options for media, culture containers and transplanting material were incorporated and cost per plant was calculated to be US$ 0.13. The HTM protocol was adopted by two sugar mills, and the HTM plants produced in our tissue culture unit were sold for seed multiplication to eight sugar mills and 430 farmers throughout North India.

Plant regeneration from direct and indirect organogenesis and assessment of genetic fidelity in Saccharum officinarum using DNA-based markers

“…The present study was conducted to screen invitro plant regenerationin sugarcane from direct (axillary buds, apical meristem, leaf whorl disk) and indirect (callus) organogenesis and to confi rm genetic stability, in-vitro regenerated plants were evaluated through RAPD and ISSR markers. Micropropagation protocol for sugarcane was standardized in an earlier report through apical meristem (Sawant and Tawar, 2011), axillary buds (Thorat et al 2016), direct shoot regeneration from leaf whorl disk (Kaur and Sandhu, 2015) and indirect organogenesis from embryogenic callus (Gill et al 2004). Shoot formation was observed from all types of explants in sugarcane var.…”

“…After surface sterilization treatment, leaf whorls were washed thoroughly in sterilized distilled water for 3-4 times and external layers of leaf sheath were pulled out one by one until the cylinder reached approximately 0.5-1 cm in diameter. The 50 cylinders were cut transversely into thin slices (1-1.5 mm thick) and inoculated on MS medium supplemented with 5.0 mg/l naphthaleneacetic acid (NAA) with 0.5 mg/l kinetin (Kaur and Sandhu, 2015). About 30 explants were taken per treatment and number of replications per treatment was three.…”

“…Generally, plant tissue culture technique is used for providing the highest number of healthy plantlets within the short duration, which is useful for rapid spreading of newly, released sugarcane varieties in short duration (Fildmann et al 1994). In sugarcane, various explants like apical meristem (Devarumath et al, 2007), axillary buds (Thorat et al 2016), leaf whorl disk (Gill et al 2006;Kaur et al 2015), callus (Behara and Sahoo, 2009), and cell suspension culture (Thorat et al 2017) were used for rapid multiplication. During the process, to maintain genetic fi delity of the micro-propagated plantlets with respect to the mother plants are major concerns.…”

In Vitro Propagation of Sugarcane for Certified Seed Production

“…Conventional micropropagation of sugarcane in semisolid media has been reported [10,11]. However, to reduce the labor required and increase efficiency, temporary immersion systems (TISs) have been successfully used to improve in vitro sugarcane multiplication [12][13][14][15][16].…”

In vitro multiplication of sugarcane with different nitrogen and sucrose concentrations

“…Due to its economic importance in Brazil and its worldwide prominence, many studies are directed at the development of sugarcane genetic breeding programs (Oliveira et al, 2016). Within this scenario, tissue culture techniques have been applied in plant propagation, allowing the production of good quality, homogeneous, and disease-and pest-free seedlings (Kaur & Sandhu, 2015). Other advantages are the large-scale production of seedlings in a short period of time, using a smaller area compared to conventional cultivation, and the possible increase in yield and longevity of sugarcane plantations.…”