Explants and Media
In vitro cultures from underground rhizomes are the main methodology employed by most investigators, where contamination has been a common serious problem (Islam et al., 2004). Almost 50% contamination has been reported by Balachandran et al. (1990). To contain contamination, mercuric chloride HgCl2 is used as a disinfectant. Rahman et al. (2004) used a pretreatment with Savlon (a patented disinfectant in the Indian market) at 5% before treating with HgCl2. Sodium hypochlorite and Tween 80 (another patented disinfectant) were also used for treatment of terminal bud explants to reduce contamination (Prathanturarug et al., 2005).
For multiple shoot production, mostly vegetative bud explants (Salvi et al., 2002), sprouting buds from rhizomes (Balachandran et al., 1990), axillary buds from unsprouted rhizomes (Panda et al., 2007), pseudostem (Gayatri et al., 2005), leaf (Salvi et al., 2001), shoot tips (Salvi et al., 2001), immature inflorescence (Salvi et al., 2000), and nodal explants (Roy and Ray Chaudhuri, 2004) were used.
A number of investigators used full-strength Murasighe and Skog (MS) media, while Sit and Tiwari (1996) reported suitability of half-strength MS medium for better micropropagation. Cultures were maintained at 2000–3000 lux with a photoperiod of 16/8 (light/dark) cycle at 22–25°C and 55–60% relative humidity. In most cases, MS medium is used except in investigations by Nadgauda et al. (1978, Smith medium), Mukhri and Yamaguchi (1986, Ringe and Nitsch media), Nasirujjaman et al. (2005, woody plant medium (WPM)), and Gayatri et al. (2005, Linsmaier and Skoog (LS) medium).
Within 5–10 days of inoculation, shoot initiation took place. Phytohormones such as 6-benzylaminopurine (BAP), 1,-naphthaleneacetic acid (NA), and kinetin were the most commonly used by majority of the workers for shoot initiation, multiplication, and development, but in some cases gibberellic acid (GA) was used for initial establishment of cultures (Meenakshi et al., 2001). Some investigations point out use of thidiazuron (TDZ) containing media to improve the multiple shoot-inducing ability (Prathanturarug et al., 2005). Salvi et al. (2000) have reported use of indoleacetic acid (IAA) along with TDZ for direct shoot development from immature inflorescence explants. Praveen (2005) reported direct regeneration from leaf explants on TDZ and BAP media. A perusal of the results of investigations indicates that BAP is the most preferred phytohormone for shoot elongation and multiplication, used singly (Balachandran et al., 1990) irrespective of the kind of basal media employed. Majority of the investigators had employed a combination of BAP and NAA (Nasirujjaman et al., 2005), while some used a combination of kinetin and BAP (Balachandran et al., 1990), while Sunitabala et al. (2001) reported use of kinetin and NAA. Use of adjuvants such as coconut water (Nadgauda et al., 1978) has been found to be more preferable, resulting in better bud elongation and generation of multiple shoots.
Salvi et al. (2002), in an elaborate investigation on the effect of various physical and biochemical factors on in vitro response of turmeric, employed young rhizome buds and shoot tips to evaluate a range of plant growth regulators and found that adenine sulfate is also equally effective for shoot multiplication. The effect of various cytokinins on shoot multiplication was investigated by culturing the young shoot buds from sprouting rhizomes on MS liquid medium supplemented with benzyladenine, benzyladenine riboside, kinetin, kinetin riboside, zeatin, 6-yy-dimethylallylaminopurine, adenine, adenine sulfate, or metatopolin in combination with NAA. Among the different carbon sources examined, fructose, glucose, sugar cubes, maltose, levulose, and market sugar were all found to be equally effective in shoot multiplication. However, xylose, rhamnose, lactose, and soluble starch were found to be inhibitory. Tyagi et al. (2007) reported no adverse effect of commercial or market sugar on regeneration and in vitro conservation. Up to 73% reduction in the cost of making media could be achieved by using cheap alternative sources of carbon and gelling agent.
Most of the reports suggest use of 3% sucrose for multiple shoot induction with some exceptions. Keshavchandran and Khader (1989) and Shetty et al. (1982) report a higher level of 4% sucrose for multiple shoot induction and Shirgurkar et al. (2001) report 2% sucrose for bud elongation. Semisolid (0.8% agar) media is reported to be as appropriate for multiple shoot induction by most of the investigators. However, Salvi et al. (2002) report multiple shoots on low-agar (0.4–0.6%) medium. Prathanturarug et al. (2005) also used 0.55% agar gel for micropropagation from dissected terminal buds. As a cheap alternative to agar, isabgol was identified by many investigators like Praveen (2005) and Anuradha et al. (2008), who reported no adverse effect on plantlet regeneration. A better survival of plantlets from 33% to 44% was observed after 12 months in the presence of isabgol than on agar medium (Anuradha et al., 2008). Some investigators have tried media without agar and observed better results. Shirgurkar et al. (2001) and Salvi et al. (2003) report better induction of multiple shoots on liquid MS and Nadgauda et al. (1978) on liquid Whites media.
Production of an increased number of multiple shoots on liquid MS medium has been reported by Chang and Thong (2004). Ali et al. (2004), Winnaar (1989), and Rahman et al. (2004) corroborate the above findings through their own investigations on C. longa. Prathanturarug et al. (2005) report a preculture on liquid MS with TDZ prior to transfer onto plain MS media to enhance multiple shootlet formation and also the application of a temporary immersion culture system to reduce the cost of plantlet production during mass multiplication. Adelberg and Cousins (2006) report that use of liquid media in large culture vessel with gentle tilting agitation can give bigger plantlets, while small vessels on a shaker gave higher number of plantlets. Increased biomass formation was thus reported in liquid medium as compared to solid medium.
The number of shoots regenerated per explants ranged from 2 to 25 plantlets per explants, when all the different results of the investigation were compared. The multiple shoot regeneration reported by various investigators are 10–12 (Shetty et al., 1982); 18 and 11 in 1 year (Prathanturarug et al., 2005); 25 (Praveen, 2005); and as low as 2 (Keshavchandran and Khader, 1989) and 4 (Tule et al., 2005).
Rooting in turmeric was observed to be either spontaneous or induced as reported by several investigators. Mostly NAA alone up to 1 mg/l is employed (Salvi et al., 2000). A combination of NAA with BAP in the initial culture media (Nasirujjaman et al., 2005) was also observed to induce rooting. Meenakshi et al. (2001) also tried IBA as rooting medium. Rahman et al. (2004) reported that NAA, IBA, IAA in the range 0.1–1 mg/l is effective for root induction. It was observed that IBA was best for rooting. These reports are substantiated by Tule et al. (2005), who suggest that IBA up to 2 mg/l to give maximum number of roots. Tule et al. (2005) report that NAA or 2,4-D can be used along with BAP for induction of rooting in vegetative bud explants derived multiple shoots.
Spontaneous rooting was reported by Prathanturarug et al. (2005) in regenerants derived from bud explants, on MS liquid supplemented with TDZ and also by Gayatri et al. (2006) on LS medium containing 2,4-D, where complete plantlets were regenerated. Nasirujjaman et al. (2005) reported spontaneous rooting in the presence of BAP and NAA. Mukhri and Yamaguchi (1986) reported development of complete plantlets on Ringe and Nitsch medium with BAP. Similarly, Nadgauda et al. (1978) also reported root induction in the presence of BA and CM. Nirmal Babu et al. (1997) and Balachandran et al. (1990) also report simultaneous shooting and rooting in the presence of BA alone.
Field survival in the range of 95–100% can be considered as good survival of hardened plantlets. A sand–soil–farmyard mixture in the ratio of 1:1:1 was found to give 95% survival after hardening, according to most investigators. Up to 95% survival was obtained in sterilized soil (Salvi et al., 2002). Sand–rice shell ash in the ratio of 1:1 was also found to be good for hardening (Prathanturarug et al., 2003). Ali et al. (2004) and Prathanturarug et al. (2005) reported 100% survival on sand:clay:compost mixture in the ratio of 1:1:1. Micropropagated plants showed increase in shoot length, number of tillers, length and number of leaves, number of fingers, and total fresh rhizome compared to conventional propagated ones. Also, the former plants were reported to show no change in morphological characters (Balachandran et al., 1990), while Salvi et al. (2002) reported that among 48 plants, 2 showed variegated leaves on the tillers. Genetic fidelity was confirmed by some investigators who reported no variation in the RAPD profiles (Salvi et al., 2002). Panda et al. (2007) have confirmed genetic fidelity of micropropagated plantlets, both through cytometry and RAPD. Metabolic profiling, using GC–MS and liquid chromatography–electrospray ionization tandem–mass spectrometry (LC–ESI–MS), to analyze differences in curcuminoids and mono- and sesquiterpenoids indicated no significant differences between conventional greenhouse-grown and in vitro propagation-derived plants (Ma and Gang, 2006).
Сначала используемые пароли были довольно короткими, что давало возможность компьютерам АНБ их «угадывать». Если искомый пароль содержал десять знаков, то компьютер программировался так, чтобы перебирать все комбинации от 0000000000 до 9999999999, и рано или поздно находил нужное сочетание цифр.
Этот метод проб и ошибок был известен как применение «грубой силы». На это уходило много времени, но математически гарантировало успех. Когда мир осознал возможности шифровки с помощью «грубой силы», пароли стали все длиннее и длиннее.