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protoplasts isolated from mesophyll tissue of sweet pea Lofi~yrus odorolus (Razdan et al.,
1980). Protoplasts were isolated from leaf tissue of Lens culinaris by using cellulase,
macerozyme dissolved in 0.5 M mannitol witit pental salts. However, the callus cultures
could not be regenerated into plantlets (Stiff et al., 1991). Genetically variable plants were
obtained from anther derived callus cultures obtained from microspore cultures of Cuja~ius
cajun (Kaur and Bhalla, 1998). Cryopreservation is an excellent method for germplasrn
conservation provided an efficient method is abailable for regeneration. Methods for pollen
embryo cryopreservation and conservation of germplasm of Arachis, Rrossica and
Triticunl sp. were explicitly reviewed (Bajaj, 1983).
2.2.1A Genetic transforniatio~i
h/
Aerobacierii~m-~nediated frat~sJ?~r~t~urio~i: Direct crown galls were induced by
infecting stem explants of Lentil (Le11s a~lbiuris) with four strains of Agrobacieriuni
lurne/iucie~is. Opines were detected in the crown gall and Southern analysis showed that T-
DNA was transfersed (Warkentin and MctIughen, 1991), inclusion of potato suspension
culture in the culturc niediuni cnilanced the trailsformation frequency of the callus obtained
from In vitro grown seedlings ofthe Glyci~~e 1lia.r (Chang and Chan, 1991). An efficient
protocol for Agrobacreriuiii-n~ed~ated transfornlatio~l of cotyledon explants from in vitro
grown seedlings of Amchis 1typogoc.u that resulled in a very h~gh frequency of
transforniation (55%) was reported. The expla~lt, were transformed with binary vectors
pBI12I and pROK1I:IPCVcp that consisted oC1ipt11 as seleclable marker gene and lndlan
Peanur Clump Vlrus coat protein gene as the agrono~llically important gene (Sharma and
Anjiah, 2000). Co-cultivation of cotyledonary node explants of Ari~cir~s Iiypuguea was
done with A. IUIII~~UC~L'IIS strain Ilarbor~ng binary vector containing uidA gene as reporter