REPRESENTATIVE CDNA LIBRARIES FROM FEW PLANT-CELLS

A reverse transcriptase/polymerase chain reaction (RT/PCR) method for the construction of representative cDNA libraries originating from few isolated cells is described. Poly(A)(+) RNA was extracted from an ave rage of 100 maize cells and reverse transcribed into sscDNA. The sscDN A was dG-tailed at its 3' end and amplified during a two-step PCR reac tion. The generated PCR products were analysed and the majority less t han or equal to 2 kbp were full-size cDNAs. A fraction of the amplifie d cDNA from 128 isolated maize egg cells was cloned into the lambda Un i-ZAP XR vector and a primary library of 6.8 x 10(6) p.f.u. was obtain ed. The average insert size is 860 bp. It was further determined, that 0.31% of the clones hybridized to a cytosolic GAPDH probe. It is thou ght that, with this method, the first cDNA library of egg cells in hig her plants was generated.