Direct regeneration and efficient in vitro root development studies in lentil (Lens culinaris Medik)

Lentil is a self-pollinating annual crop with increasing demand all over the world due to its high protein content and easy digestibility. However, like many other crops lentil too needs improvement for which conventional as well as biotechnological tools are to be employed. This study was aimed at development of tissue culture protocol especially targeting improved root development to ensure their establishment in soil in order to use their potential towards genetic manipulation. Two Pakistani lentil cultivars, Masoor-2002 and Manshera-89 were used to obtain cotyledonary nodes, epicotyl and hypocotyl explants. The explants were cultured on shoot regeneration medium containing different concentration of kinetin, BAP and tyrosine with the addition of GA3, with or without charcoal for shoot development. Masoor-2002, showed the highest frequency of shoot development on MS medium containing 5.5 mg/L tyrosine, 0.25 mg/L kinetin, 1.0 mg/L BAP, 0.1 mg/L GA3, using cotyledonary node as explant. The addition of 2 g/L of charcoal in shoot medium resulted in healthier plants, but the number of shoots were reduced. Regarding the effect of age of explants on regeneration frequency, cotyledonary nodes of age 4-6 days had higher regeneration potential. Well-developed shoots were shifted to rooting medium containing different concentration of auxin with or without charcoal. Healthier and more roots were observed on medium containing 4 mg/L IAA with addition of 2 g/L charcoal. Plants were better established (70% survival) in a soil mix containing perlite, vermiculite and peat moss in 1:1:1 ratio.