Improved in vitro embryo development of stenospermic grape by putrescine

The goal of this study was to determine the effect of putrescine, added to the culture medium, on the in vitro development of stenospermic grape (Vitis vinifera L) embryos. The cross breedings of Perlón x G.C88552 and Perlón x Argentina were used. 0 (control), 2 and 4 mM of putrescine were added to the immature seed’s culture medium. In Perlón x Argentina, 2mM of putrescine statistically increased the percentage of total embryos, direct germination, polyembryos and normal plants. In Perlón x G.C88552, only 2 mM of putrescine increased all the variables considered, eventually tripling the percentage of normal plants obtained. The results suggest that the endogenous concentration of putrescine may be a growth limiting factor. Adding putrescine to the culture medium of immature grape seeds is a legitimate resource to significantly increase the results of this technique. Abbreviations: put-putrescine, G.C.-Gargiulo’s Crossing BIOCELL 2002, 26(2): 263-266 ISSN 0327 9545 PRINTED IN ARGENTINA


Introduction
One of the main objectives of table grape breeding is to develop new seedless cultivars.In the so-called stenospermic grapes, fertilization takes place, but the embryo aborts (Stout, 1936).The causes for this abortion are not entirely known.Cain et al. (1983) hypothesized a hormonal imbalance in the maternal tissues during the first stages of the seeds ontogeny.
The embryo rescue technique in grape allows us to obtain plants from direct crossing between seedless cultivars (Cain et al., 1983;Spiegel-Roy et al., 1985;Bou-quet and Davis, 1989;among others).The yield of this technique is conditioned by maternal genotype, harvest date, pollinator and culture medium, (Bouquet and Davis, 1989;Gribaudo et al., 1993 andPonce et al., 2000).The proportion of normal plants obtained is relatively low: from 7 to 10% (Barlass et al., 1988).
Efforts to increase the efficiency of the technique have had only relative success.Different combinations and concentrations of hormones (Ponce et al., 1995), the effects of cold, gibberellic acid and manipulation of the chalazal (Agüero et al., 1996), the use of light enriched in the long red wave lengths, and frequent changes to fresh medium, (Ponce et al., 1997) have been studied.
On the basis of the results of some of the studies previously mentioned, it has been concluded that adding different auxins (indol acetic acid, indol butyric acid, naphtalen acetic acid) and gibberellic acid to the culture medium does not improve the proportion of embryos obtained.
Polyamines (putrescine, spermidine, spermine, among others) are growth regulators present in all living organisms, essential for growth and development of tissues due to their role in cell reproduction, transduction of signals, and protein synthesis (Tiburcio et al., 1993).
Putrescine added to the culture medium increased hairy root growth 2.3 times in Chicorium intybus (Bais et al., 1999), remarkably increased the embryogenic capacity of leaf discs of Solanum melongena L. (Yadav and Rajam, 1998), stimulated the growth and development of microcuttings of Fragaria x ananassa (Tarenghi et al., 1995), increased the growth of stems obtained from cotyledons of Brassica campestris (Chi et al., 1994), and in Vitis vinifera increased micropropagation rate, number of roots, number of nodes, length of root and stem, and fresh and dry weight (Martin-Tanguy and Carre, 1993).
One proof of polyamines intervention in plant development is that when putrescine synthesis inhibitors are used on rice plantlets, their development is detained, whereas it will be reestablished when added exogenously (Bonneau et al., 1994).
The goal of this study was to determine the effects of putrescine, added to the culture medium, on the in vitro development of stenospermic grape embryos.

Materials and Methods
The crossings of stenospermic cultivars Perlón x G.C. 88552 and Perlón x Argentina were used.The treatments consisted of adding 0 (control), 2 and 4 mM of putrescine to the culture medium, by sterile filtration (0.2 µ cellulosa acetate filter).The clusters were harvested the eighth week after set.The berries were sterilized with 20% sodium hypochlorite (60g.L -1 active chlorine) for 20 minutes.The immature seeds were aseptically extracted from the berries and put in 9 cm diameter Petri dishes (30 seeds per dish), with Nistch and Nistch (1969) medium, suplemented with 20 g.L -1 sucrose, 2.8 g.L -1 Phytagel (Sigma, St. Luis, USA) and different concentrations of putrescine.These dishes were taken to growth chambers at 25 ± 2ºC with 16 hours of fluorescent light (50 µmol.m -2 .s - ).
After three months of growth under these conditions, the embryos that had germinated were extracted, considering germinated only those in which the radicule had emerged from the inside of the seed, and they were called direct germinations.The embryos that had not germinated were dissected under magnifying glass, and called rescued embryos.The sum of direct germinations and rescued embryos constituted the total embryos.The plants that originated from direct germinations and rescued embryos were put in test tubes with 15 ml of Murashige Skoog (1962) medium, with halfstrength macronutrients and without hormones, complemented with 20 g.L -1 sucrose and solidified with 2.8 g.L -1 Phytagel.Afterwards, they were put in growth chambers at 25 ± 2ºC and 50 µmoles.cm - .s - , with a 16 hour photoperiod.After two months, the number of plants obtained with each treatment was counted.
In the crossing of Perlón x G.C. 88552, 456, 508 and 463 immature seeds were planted in vitro for the control group, the 2 mM and 4mM putrescine respectively, and 1649, 2023 and 1269 in the crossing of Perlón x Argentina.
In the statistical analysis percentages were analysed by Chi squared test.When differences at the 5% level of significance were found they were compared with the Arcsine test, according to Capelletti (1982).

Results
The response to the inclusion of putrescine differed for each of the crossings studied.If we compare the control groups, in the crossing of Perlón x G.C. 88552, 26.8% total embryos were obtained, and in the crossing of Perlón x Argentina this quantity almost doubled to 48.8% (Table 1).
In the crossing of Perlón x G.C. 88552, adding 2mM of putrescine to the culture medium statistically increased the percentage of total embryos and the percentage of direct germinations, 49.7% and 28.4% respectively in relation to the number of seeds planted, whereas when the dose of putrescine was of 4 mM, there was no difference with the control group.This same relationship was observed in the percentage plants, when 2mM of putrescine were added to the culture medium 35.2% of the seeds originated normal plants, percentage that was markedly lower (13.1%) in the control group and the higher putrescine dose (4mM).(Table 1) In the crossing of Perlón x Argentina, the 2mM treatment of putrescine statistically increased the percentage of total embryos (56.4%), the direct germinations, the frequency of polyembryos, and the percentage of plants.In this crossing, the higher dose of putrescine was detrimental to embryo development, producing lower values than the control group for all the measured variables.When 2mM of putrescine were added to the culture medium, 43% germinated directly and 18.3% produced normal plants, whereas in the control group 12.6% of the seeds originated plants and the higher dose of putrescine decreased this value to 6.7%.(Table 1)

Discussion
First, the effect of the pollinator is obvious, since comparing the response of the control groups of both crossings, the crossing of Perlón x Argentina had 100 to 200% more total embryos, direct germinations and polyembryos than the crossing of Perlón x G.C. 88552.This effect of the pollinator has already been described by Ponce et al (2000).
Polyamines have been shown to be stimulators of growth in several species (Tarenghi et al., 1995).Our results suggest that the endogenous concentration of these substances may limit growth, and would explain the fact that in both crossings putrescine favored the development of a greater number of embryos and their subsequent growth, thus allowing a greater number of direct germinations and the development of a larger number of plants.
Both crossings had a spontaneous polyembryo frequency, which could be observed in the control group.This phenomena has already been reported by Valdez and Ulanovsky (1997) in the in vitro growth of immature seeds of the crossing of Superior x Ruby Seedless and its reciprocal.The presence of putrescine (2 mM) in the culture medium remarkably increased (200 to 300%) the frequency of polyembryos in both crossings.Yadav and Rajam (1998) have demonstrated that polyamines are involved in cell differentiation that occurs in somatic embryogenesis, and they propose the use of putrescine to improve this process in recalcitrant species.
On the basis of the results obtained, we conclude that including putrescine in the culture medium of immature grape seeds is a valid resource to considerably increase the yield of this technique.

TABLE 1 .
Effect of putrescine in the culture medium on the in vitro development of embryos of the crossings of Perlón x G.C. 88552 and Perlón x Argentina.
Note: different letters in a column indicate significant differences between treatments for each crossing (p ≤ 0,05)