Desiccation tolerance in embryogenic cultures
--F. Locatelli and E. Lupotto
During studies on alternative methods for transformation (MNL 64:22-23, 1990), the capacity of embryogenic cultures to sustain rapid and drastic desiccation has been ascertained. This characteristic, peculiar to the zygotic embryo during maturation in planta, has been already detected in other plant species, where dried somatic embryos were proposed as synthetic seeds (Senaratna et al., In Vitro Cell Dev. Biol. 26:85-90, 1990).
Therefore, the system in maize has been more deeply investigated, in order to characterize the conditions needed for an efficient recovery of the tissues and consequent plant regeneration. Because of the strict homology existing among the Gramineae, it is presumable that results may be extrapolated for other monocot cultures, namely cereals. We already have additional evidence that this is true for sorghum and durum wheat cultures.
Embryogenic tissue cultures were derived from genotypes establishing type 2 highly embryogenic, friable calli: B79, LH126 x B79, Lo907 x B79, and Lo976 x B79 F2 selections. Calli were induced from immature embryos in the presence of 2 mg/l 2,4-D and propagated in the dark on 1 mg/l 2,4-D (ref. Locatelli et al., MNL, this issue). For drying treatments, homogeneous embryogenic calli, approximately of equal size, were placed on a layer of filter paper in petri dish lids in constant air stream (0.45 m/sec) in a sterile flow hood. Treatments were from 1 to 6 hours desiccation. Desiccated calli were then tested for growth and development on N6P and MShf, respectively. Performance of treated tissues was compared to that of control calli non-desiccated and grown in the same conditions. Parameters used for evaluation were: callus growth, somatic embryo germination, and plantlet development. When the effect of storage was tested, dried tissues were stored in the dark in 10ml screw cap plastic tubes at room temperature (22▒2 C) or at 4 C. Alternatively, storage was also tested at -20 C. Desiccation was tested on 50-70mg FWT calli during 6 hours, allowing us to draw the desiccation curve of these tissues. Two hours treatment reduced the fresh weight of the tissue to 35-40%; at 4 hours desiccation, weight was reduced to 15-20% of the initial fresh weight. Further desiccation did not reduce consistently the callus weight. Calli were induced to recover on N6P medium either directly, or with a previous rehydration in a 100Ál drop of liquid medium. Slow rehydration (on agar medium) was more effective than rapid rehydration (on liquid medium). Furthermore, desiccated calli (2 hours treatment) resulted in definitively better growing than control cultures, the highest difference being registered at day 21 of subculture. Fast rehydrated calli partly degenerated, and their growth was far less than control calli. Re-established cultures were consistently similar to the original ones and could be propagated in the same way, thus confirming that desiccation did not interfere either with callus performance or with callus phenotype.
When desiccated calli were placed directly onto MShf medium they proliferated into germinating somatic embryos which subsequently developed into complete plantlets. The whole procedure of regeneration was the three step procedure described in Locatelli et al., MNL, this issue. The number of regenerates was similar or even higher than the total yield of non-desiccated calli. The efficiency of plant regeneration in B79 cultures was 16.21 plants per g FWT in control calli, against 18.47 plants per g FWT in desiccated tissues. Again, the best performing calli were desiccated two hours.
The effect of storage on desiccated calli was investigated; storage up to one year is currently being considered. Results obtained to date indicate a relative resistance of non-treated calli up to 2 weeks either at room temperature or at 4 C in the dark. Growth index was comparable to that of non-stored calli (about 8-9 GI in a 21 day subculture). Conversely, 2 hours-desiccated calli were more efficiently stored at 4 C in the dark. Their GI was about 6-7, compared to the same tissues at room temperature, with GI=3-4.
Furthermore, storage of 2 hours-desiccated tissues stored at 4 C did not affect the recovered callus phenotype, whereas in non-desiccated calli and in 2 hours-desiccated calli stored at room temperature, a change of callus performance, toward a non-embryogenic, rooting type, was registered. Four hours desiccation and storage at 20 C were lethal in all cases. The most interesting observations on the desiccation system were made when desiccated calli (2 and 4 hours) were stored (room temperature, 4 C) and subsequently plated on MShf for direct regeneration. In this case, 2 hours desiccation and storage at 4 C resulted in the best plant regeneration. One hundred percent of the desiccated and stored calli germinated somatic embryos. Among these, a high percentage regenerated into complete plantlets (70-80%). Regenerated plantlets were also more vigorous than control regenerates obtained from non-desiccated calli.
The whole system presents peculiar characteristics which may be particularly interesting as an applicative tool for storage and diffusion of elite maize (or cereal) lines in vitro, and as a model system for studying in vitro response to desiccation.
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