1. Hardening protocol of in vitro plantlets of Cassava (Manihot esculenta Crantz.) varieties Sree Vijaya, H-226 and H-165 R. Shiji, James George, S. Sunitha and R. Muthuraj AICRPTC, Central Tuber Crops Research Institute, Sreekariyam, Thiruvananthapuram, Kerala, India Hardening protocol of in vitro plantlets of Cassava (Manihot esculenta Crantz.) varieties Sree Vijaya, H-226 and H-165 R. Shiji, James George, S. Sunitha and R. Muthuraj AICRPTC, Central Tuber Crops Research Institute, Sreekariyam, Thiruvananthapuram, Kerala, India Different stages of hardening of in vitro regenerated cassava plantlet. Fig. 1a. Effect of different potting mixture with Hoagland solution on survival of in vitro plantlets of cassava var. “Sree Vijaya”  Cassava (Manihot esculenta Crantz.) is an important tropical tuber crop, used as a major staple food cultivated in several developing countries, providing the basic diet for around 500 million people.  Cassava is usually propagated vegetatively from stem cuttings and the multiplication rate of planting materials is less with a multiplication ratio of approximately 1:10 per annum. It also means the rate of multiplication of new, improved varieties is slow; for their adoption.  Several protocol have been developed for in vitro establishment and rapid multiplication of cassava, but huge losses occur during transfer from in vitro laboratory to ex vitro field conditions.  The present study attempts to establish a hardening protocol of in vitro plantlets of cassava in three varieties Sree Vijaya, H-226 and H-165.  Cassava (Manihot esculenta Crantz.) is an important tropical tuber crop, used as a major staple food cultivated in several developing countries, providing the basic diet for around 500 million people.  Cassava is usually propagated vegetatively from stem cuttings and the multiplication rate of planting materials is less with a multiplication ratio of approximately 1:10 per annum. It also means the rate of multiplication of new, improved varieties is slow; for their adoption.  Several protocol have been developed for in vitro establishment and rapid multiplication of cassava, but huge losses occur during transfer from in vitro laboratory to ex vitro field conditions.  The present study attempts to establish a hardening protocol of in vitro plantlets of cassava in three varieties Sree Vijaya, H-226 and H-165.  One month old in vitro plantlets of cassava grown from nodal explants using MS media supplemented with 0.1 mg/l NAA or 0.3 mg/l IBA for rooting with shoot regeneration along with a control were used for hardening studies.  To optimize the agar concentration of in vitro growth medium causing least root damage during washing of in vitro plantlets for hardening, the nodal explants were transferred to in vitro growth medium containing different growth regulator supplements (0.1 mg/l NAA or 0.3 mg/l IBA)and agar concentration ranging from 0.6- 0.8%.  In vitro rooted plantlets of about 5-7 cm height were transferred to plastic cups containing different potting mixtures viz., 1.sand+soil (3:1); 2. vermiculite+soil (3:1); 3. sand+vermiculite+soil (1:1:1) and 30 ml of Hoagland or phosphorous rich fertilizer (e.g., 10-52-10) solution prepared in 0.1% bavistin was poured and covered with transparent polythene cover having perforation at the top.  One corner of plastic covers was slightly opened after one week, fully opened and removed after 2 to 3 weeks and 5 ml of water and Hoagland solution was poured on alternate days.  One month old plantlets were transplanted to grow bags containing potting media (sand+vermiculite+soil; 1:1:1).  After two months of hardening the plantlets were planted in the field maintaining adequate management conditions.  One month old in vitro plantlets of cassava grown from nodal explants using MS media supplemented with 0.1 mg/l NAA or 0.3 mg/l IBA for rooting with shoot regeneration along with a control were used for hardening studies.  To optimize the agar concentration of in vitro growth medium causing least root damage during washing of in vitro plantlets for hardening, the nodal explants were transferred to in vitro growth medium containing different growth regulator supplements (0.1 mg/l NAA or 0.3 mg/l IBA)and agar concentration ranging from 0.6- 0.8%.  In vitro rooted plantlets of about 5-7 cm height were transferred to plastic cups containing different potting mixtures viz., 1.sand+soil (3:1); 2. vermiculite+soil (3:1); 3. sand+vermiculite+soil (1:1:1) and 30 ml of Hoagland or phosphorous rich fertilizer (e.g., 10-52-10) solution prepared in 0.1% bavistin was poured and covered with transparent polythene cover having perforation at the top.  One corner of plastic covers was slightly opened after one week, fully opened and removed after 2 to 3 weeks and 5 ml of water and Hoagland solution was poured on alternate days.  One month old plantlets were transplanted to grow bags containing potting media (sand+vermiculite+soil; 1:1:1).  After two months of hardening the plantlets were planted in the field maintaining adequate management conditions.  Least root damage (3%) was obtained using 0.6% agar in the in vitro growth medium with 0.1 mg/l NAA supplement during washing of in vitro plantlets before hardening (Table 1).  All the varieties grown from in vitro medium supplemented with 0.1 mg/l NAA showed maximum survival during hardening than 0.3 mg/l IBA supplemented growth medium (Fig. 1., Fig. 2. and Fig. 3.).  But cassava var. H-165 showed best result from the potting media sand+vermiculite+soil using both Hoagland and fertilizer solution, which gave 100% success (Fig. 3.).  Cassava varieties Sree Vijaya and H-226 grown in vermiculite+soil using both Hoagland and fertilizer solution showed maximum survival of 87% and 80% respectively (Fig. 1. and Fig. 2.).  The hardened plantlets could be successfully acclimatized in the green house and transplanted to open field conditions.  Least root damage (3%) was obtained using 0.6% agar in the in vitro growth medium with 0.1 mg/l NAA supplement during washing of in vitro plantlets before hardening (Table 1).  All the varieties grown from in vitro medium supplemented with 0.1 mg/l NAA showed maximum survival during hardening than 0.3 mg/l IBA supplemented growth medium (Fig. 1., Fig. 2. and Fig. 3.).  But cassava var. H-165 showed best result from the potting media sand+vermiculite+soil using both Hoagland and fertilizer solution, which gave 100% success (Fig. 3.).  Cassava varieties Sree Vijaya and H-226 grown in vermiculite+soil using both Hoagland and fertilizer solution showed maximum survival of 87% and 80% respectively (Fig. 1. and Fig. 2.).  The hardened plantlets could be successfully acclimatized in the green house and transplanted to open field conditions. Fig. 2b. Effect of different potting mixture with fertilizer solution on survival of in vitro plantlets of cassava var. “Sree Vijaya” Fig. 2a. Effect of different potting mixture with Hoagland solution on survival of in vitro plantlets of cassava var. “H-226” Fig. 2b. Effect of different potting mixture with fertilizer solution on survival of in vitro plantlets of cassava var. “H-226” Fig. 3a. Effect of different potting mixture with Hoagland solution on survival of in vitro plantlets of cassava var. “H-165” Fig. 3b. Effect of different potting mixture with fertilizer solution on survival of in vitro plantlets of cassava var. “H-165” Table 1. Effect of varying concentrations of agar and different growth regulators on root damage during washing before hardening. Growth media of in vitro plantlets Conc. of agar (%)No. of roots No. of in vitro plantlets washed for hardening No. of plantlets discarded due to root damage % of root damage Control (MS) 0.61-330827 0.71-3301550 0.81-3302583 MS + 0.1 mg/l NAA 0.62-43013 0.72-430517 0.82-4301550 MS + 0.3 mg/l IBA 0.63-530620 0.73-5301137 0.83-5302067  Standardized the protocol for hardening of cassava varieties Sree Vijaya, H-226 and H-165.  Among the potting mixtures, H-165 showed the best result in Sand+Vermiculite+Soil mixture, whereas Vermiculite+ Soil mixture was the best for Sree Vijaya and H-226.  Standardized the protocol for hardening of cassava varieties Sree Vijaya, H-226 and H-165.  Among the potting mixtures, H-165 showed the best result in Sand+Vermiculite+Soil mixture, whereas Vermiculite+ Soil mixture was the best for Sree Vijaya and H-226. In vitro plantlets grown in (a) Control (MS), (b) MS+0.1 mg/l NAA and (c) MS+0.3 mg/l IBA. Sree Vijaya H-226 H-165 abc