Establishment of in vitro shoot multiplication and short-term embryo storage protocols for single genetic modification events in sugarcane.
Sugarcane (Saccharum spp. hybrids) is an important economic crop that accounts for more than 80% of world sugar production. Genetic improvement and selection to produce cultivars with traits of interest is difficult in sugarcane because of its complex polyploid and aneuploid genome. Genetic transformation and in vitro mutagenesis techniques are, therefore, being investigated for such purposes. However, it takes a long period to multiply plants regenerated from such events as: 1) the in vitro plants have to establish well in vitro; 2) then they need to acclimatize in the greenhouse and 3) then multiplied using setts, before they can be tested for traits of interest. Furthermore, once a plant is lost in culture, the whole genetic event is lost, which can occur due to lack of labour resources at the time of subculture. The aim of this study was, therefore, two-fold: 1) to develop a protocol to multiply single shoots regenerated from single transformation or mutation events in vitro; and 2) to establish a strategy for short term storage of somatic embryos developed from such events. Preliminary investigations were undertaken using sugarcane varieties NCo310 and NCo376. Two approaches were employed for shoot multiplication, viz. either multiply the shoots when they were well developed or multiply them immediately after embryo germination. In the former, shoots (2 cm in height), produced via indirect somatic embryogenesis, were cultured on six different media, each containing full strength MS salts and vitamins, 20 g l-1 sucrose and different combinations and concentrations of plant growth regulators (PGRs), viz. M1 (no PGRs), M2 (0.1 mg l-1 BAP and 0.015 mg l-1 kinetin), M3 (6 mg l-1 BAP and 1 mg l-1 kinetin), M4 (0.5 mg l-1 BAP and 0.25 mg l-1 kinetin), M5 (1 mg l-1 BAP, 0.1 mg l-1 kinetin and 1 mg l-1 NAA) and M6 (1 mg l-1 IBA, 1 mg l-1 kinetin and 0.5 mg l-1 GA3). The shoots were subcultured two or three times onto same medium. At 6 w, the total shoot yield (no. shoot/original shoot explant) was significantly highest on medium M5 for both NCo376 (12.7 ± 4.0) and NCo310 (7.2 ± 3.4). Similarly, this medium resulted in the highest total number of shoots per embryo at 6 w (11.0 ± 2.0) when applied to germinating embryos of NCo376. Modifications of this medium were investigated but none was found to be better than M5 (P < 0.05). After multiplication, the shoots were transferred to rooting liquid medium containing ½ strength MS salts and vitamins, 20 g l-1sucrose and 1 mg l-1 IBA. More than 80% of the shoots from each medium rooted within 3 w. The developed protocol was then applied to varieties N41, N50 and transgenic lines of NCo376. The results confirmed that M5 can be used for high yielding shoot multiplication for those varieties producing 6.6 ± 0.9 and 4.3 ± 1.3 shoots per shoot at 6 w for N41 and N50, respectively. Subculturing for a further 2 w increased the shoot yields to 18.6 ± 2.3 for N41 and 8.0 ± 0.3 for N50. Transgenic shoots multiplied using the developed protocol, were used to investigate the stability of the transgene in in vitro culture. This was done by testing for the presence of the gene in those shoots using end point PCR. The results showed the presence of the transgene in all the transgenic shoots indicating the protocol did not have a negative effect on the stability of the transgene. To establish a protocol for slow growth storage of somatic embryos, mature embryos of variety NCo376 were encapsulated in alginate beads and placed on semi-solid medium containing ½ strength MS salts and vitamins, 5 g l-1 sucrose and 9 g l-1 agar. The cultures were kept in the dark at room temperature for a month and at 18 oC for 1, 2 and 3 months. The embryos were assessed for germination capacity at the end of each period by transferring them to medium containing full strength MS salts and vitamins, 20 g l-1 sucrose, 0.5 g l-1 casein hydrolysate and 8 g l-1 agar. Embryos that were stored at 18 oC for 1 month had the highest survival percentage (66 ± 5.7% germination) compared with the other treatments and control (53.3 ± 6.7% germination).