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cDNA library preparation from a single wheat kernel

100%

Takacs I. , Koszegi D. , Barnabas B.

nr 1

A simplified cDNA cloning protocol was elaborated, easily scalable to very different sizes of plant tissue. The mRNA fraction was extracted from a single wheat kernel with oligo dT magnetic beads. The solid-phase mRNA was transcribed into single-strand cDNA by reverse transcription. Then DNA tags were incorporated into the ds cDNA fragments prior to PCR amplification. The amplified DNA was ligated to a T overhang cloning vector and some of the resulting clones were sequenced. These sequences were identified by BLASTN search in wheat EST databases.

Induced embryogenesis via egg cell activation in cereals

100%

Barnabas B. , Bakos F. , Feher A. , Jager K.

tom 47

nr 1

Regeneration of haploid plants after distant pollination of wheat via zygote rescue

100%

Bakos F. , Jager K. , Barnabas B.

nr 1

Haploid wheat plants have been produced by a new method of zygote rescue carried out after distant pollination. Wheat stigmas were pollinated with maize pollen and subsequently the activated egg cells from the elongated ovaries were rescued for in vitro plant development in single cell culture. As the control, 2-week-old embryos were also dissected and then cultured. The efficiency of both techniques was comparable. Wheat was also pollinated with rice, and the further development of rescued zygotes into multicellular structures is reported here for the first time. Because the lack of a normal endosperm hampers embryo development even in the early stages, early zygote rescue (two days after distant pollination) may represent a more efficient way of producing double haploid (DH) plants in cultivars that are recalcitrant in androgenic cultures, after further optimization of in vitro culture of isolated single cells.

Application of wheat [Triticum aestivum L.] microspore culture and ovaries to raise wheat zygotes in vitro

100%

Bakos F. , Darko E. , Ponya Z. , Barnabas B.

nr 1

Zygotes fertilized in planta developed into fertile plants in vitro. Microspore cultures and ovaries derived from the same species were tested as nurse cells. With both types of feeder system about 20% of the isolated zygotes were able to regenerate into plants. The morphology, cytological properties and development rhythm of the zygotes resembled those of the in vivo course of zygotic development, except that the first division appeared symmetrical in contrast to the asymmetrical division observable in planta. The results indicate that ovaries may have the same nurse effect as microspores on zygotes cultured in vitro. Using ovaries as a nurse system is much less time-consuming than using isolated microspore cultures.