Anna Pindel

Improvement in Lupinus luteus (Fabaceae) protoplast culture – stimulatory effect of agarose embedding and chemical nursing on protoplast divisions

Comprehensive studies were undertaken to determine optimal conditions for yellow-lupin (Lupinus luteus L.) protoplast culture. The adopted isolation procedure resulted in a high yield of protoplasts obtained from hypocotyls, cotyledons and young leaves. The usefulness of liquid and agarose-solidified media was evaluated in relation to promoting survivability and morphogenetic responses of protoplasts. The influence of different plant-growth regulators and anti-oxidative agents was also examined. Cultivar ‘Parys’ was the most promising material for manipulations. The solidification of medium enhanced the development of cultures initiated from hypocotyls and cotyledons, significantly increasing the division rate. In both liquid and solid media, mitoses were arrested after the initial division and daughter cells failed to divide. An important breakthrough in the development of cultures appeared in media supplemented with 0.1% activated charcoal. Bypassing the mitosis suppression led to the formation of small aggregates from hypocotyl protoplasts. This is the first report on protocolony formation in protoplast cultures of L. luteus. Notable progress achieved here indicates that, although this species is considered as recalcitrant to in vitro manipulations, it is possible to obtain responsive cultures with higher morphogenetic potential and thus provide new material for breeding programs of lupins.

Early stages of somatic embryogenesis in root callus of grasspea (Lathyrus sativus L.)

Callus cultures from root explants of Lathyrus sativus L. ‘Derek’ were tested for their morphogenic capacity. Primary explants (fragments of roots) were cultivated on three induction media. We obtained three lines of callus tissue among which we identified two non-embryogenic lines and one embryogenic line. Callus originally cultivated on modified MS medium supplemented with 0.05 mg*L-1 picloram, formed embryo-like structures upon transfer to media containing 0.1 mg*L-1 picloram or 0.9 mg*L-12,4-D. Histological examinations confirmed embryogenity of obtained structures. Previous studies had revealed that, notwithstanding efficient callus induction and proliferation, its capacity to differentiate shoots or somatic embryos is limited. Consequently, rhizogenesis was only form of complete organogenesis obtained in our experiments. However attempts to develop the methods for indirect plant regeneration in L. sativus would allow creation of new genetic variations required to improvement of this species.

Protoplast culture utilization in studies on legume crops.

Abstract Plant protoplasts are single cells that have had their cell walls removed by enzymatic digestion. Under appropriate in vitro culture conditions protoplasts rebuilt a new wall and then divide to form cell colonies, from which plantlets can develop. Legume species (Fabaceae) have significantly reduced genetic variability, probably due to their long history of inbreeding, and exhibit relatively strong recalcitrance to in vitro manipulations. Unique properties of protoplasts enable their exploitation in genetic enhancement of crops, involving creation of novel germplasm through direct transformation or somatic hybridization techniques. For these reasons, utilization of protoplast technology for legume improvement focuses on elaboration of efficient plant regeneration protocols, which lay the foundations for more complex research. Progress in the introduction of such technology into legume breeding is still somewhat limited, but recent achievements are strongly encouraging. Successful applications of somatic hybridization and transformation via direct DNA uptake have been already described for several genera, e.g. Medicago or Phaseolus. Isolated legume protoplasts also provide a valuable model in a range of fundamental and experimental studies, involving cell structure and behavior. In this article particularly interesting reports relevant to trends in applications of legume protoplasts are reviewed.

Explant-dependent receptivity to isolation and a cell-wall resynthesis in protoplast culture of recalcitrant yellow lupin.

Cell-wall resynthesis was studied in protoplast culture of yellow lupin (Lupinus luteus L.). We optimized protoplast isolation and found that explants excised from young seedling were more suitable sources of protoplasts, in contrast to callus tissue. Incubation in 2% cellulase R-10, 1% pectinase and 0.5% macerozyme solution for 3h effectively released protoplasts from majority of tested explants. Furthermore, we determined the optimal developmental age of explants which was 4, 21, 25 and 35 days for hypocotyls, cotyledons, in-vitro leaf mesophyll and ex-vitro leaf mesophyll, respectively. Explant type, culture medium and genotype influenced both a rate and a pattern of the cell wall regeneration. After 10 days of culture, the number of regenerated cells reached 44%-59% in hypocotyl, 84%-91% in cotyledonary, and 31%-42% in mesophyll protoplasts. Our results show that the earlier wall regeneration begins, the wall surface will be more incomplete. We suggest that unbalanced and inefficient cell-wall resynthesis likely contributes to recalcitrance of yellow lupin to manipulations in protoplast technology.