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Published in Issue No. 122, page 13 to 15 - (10703) characters

Production of Populus euphratica Oliv. x P. alba L. hybrid poplars through ovary and ovule cultures

Ali Jafari Mofidabadi  Ali Reza Modir-Rahmati  Introduction


Hybridization is currently used to combine desirable traits and to achieve hybrid vigour in many crops and trees. There has been a long and sustained interest in the hybridization of poplars largely because of the benefits derived from capturing heterosis and combining desirable traits to improve the quality and amount of wood production (Stettler 1980). In view of its resistance to drought and salinity, Populus euphratica Oliv. has been chosen as a parental species in Populus breeding before, however, incompatibility has been observed between this species and some other poplar species (Willing and Pryor 1976). During the last decade, a number of observations have been made on pollen-stigma interactions in interspecific crosses of Populus, with emphasis on the sections Aigeiros, Leuce and Tacamahaca (Knox et al. 1972; Heslop-Harrison 1975; Guries and Stettler 1979; Gaget et al. 1984).


Usually incompatibility in tree species is due to premature abscission of flowers, early dehiscence of the capsules, pollen mortality, grafting failure and to unknown physiological disorders occurring within the flowering branches (reviewed in Ramming 1990 for fruit trees). In addition, with interspecific intergeneric crosses, and crosses between diploids and tetraploids, the endosperm often develops poorly or not at all. This problem can be overcome by aseptically culturing the embryo in a nutrient medium, a technique used in many different crosses and which seems to be amenable to large-scale application in poplar hybridization (Li et al. 1983; Kouider et al. 1984; Li and Li 1985; Noh et al. 1986, and Savka et al. 1987). The in vitro method developed for P. alba x P. euphratica hybrid plant induction (Jafari Mofidabadi et al. 1998) was used to produce P. euphratica x P. alba hybrid poplar (reciprocal crosses).



Materials and methods


Artificial pollination was carried out of P. euphratica Oliv. and P. alba L. (in one direction). Pollen grains of P. alba were collected in March from the Research Centre of Alborz - Karaj, Iran. The female inflorescent buds of a P. euphratica tree were covered with transparent paper before anthesis to avoid contamination. Pollination was then carried out on covered female inflorescence by intensively dusting P. euphratica stigma with collected P. alba pollen grains. Artificial pollination was carried out using the bottle grafting, and twig and pot techniques (described by Kouider et al. 1984; see also Rajora and Zsuffa 1984) on a mature tree. A control pollination was made on the same tree in different branches. Catkins were collected from branches 10, 30 and 45 days after pollination. Closed capsules, still attached to the axis of the catkin, were then surface disinfected for 12 min in a 2.6% solution of calcium hypochlorite followed by three 5-min rinses in sterile deionized water. To rescue fertilized embryos, isolated ovaries and ovules were transferred to a 50% MS agar medium in 100 x 15 mm petri dishes. Cultures were incubated at 24°C under a 16 h photoperiod with light provided by cool white 40-watt fluorescent lamps (4000-5000 lux). Plantlets 1-2 cm in height were transferred to jars containing the same medium and kept for two months under the same conditions before being acclimatized.


The tested medium for the embryo germination and plantlet development contained a 50% concentration of MS inorganic salts (Murashige and Skoog 1962) with FeEDTA 10-4M and pH adjusted to 5.8; two concentrations of sucrose (0.06M and 0.17M) were used. No growth regulators were added to the medium, which was autoclaved for 20 min at 120°C and then dispensed in sterile petri dishes and jars with 10 and 20 ml respectively.



Results and discussion


Germination of embryos was observed when half-capsules of ovaries and isolated ovules were placed on the surface of an agar medium. The method used in our experiment differed from the culture techniques described by Li et al. (1983), Kouider et al. (1984), Li and Li (1985) and Savka et al. (1987), as these required the excision of individual embryos. This step is time consuming, requires technical ability and can also damage the integument of the embryos. We simply cultured the intact ovary and ovule and obtained a high number of plantlets. The highest embryo germination rate (more then 90%) was observed on the 50% MS medium containing 0.17M sucrose in both ovule and ovary culture. The same result was reported in the embryo germination of P. alba x P. euphratica hybridization product (Jafari et al. 1998).


Kouider et al. (1984) and Li and Li (1985) used a culture media which although based on MS components was supplemented with growth hormones, namely 3-indole-acetic acid (IAA) and 6-benzylaminopurine (BAP), as well as vitamins. This resulted in the production of calluses and multiple shoots, and made it necessary to transfer embryos to a rooting medium in order to obtain plantlets. To simplify the composition of the media, Raquin and Troussard (1993) only used media that contained basic components, i.e., mineral salts, water and sucrose water, and no growth regulators. In order to simplify and avoid the variation caused by callusing, we used 50% concentration MS agar medium without plant growth regulators.


Embryo age (number of days post-pollination) affected the ability of the embryos to respond to culture media. Due to the long period of P. euphratica embryo development, embryos younger than 45 days (10 and 30 days old) did not respond to the culture media (Table 1). The mean effect of explants (ovary and ovule) and sucrose concentration on embryo germination and plantlet production was compared (Table 2) and significant differences between explants and sucrose concentration (P<0.01) were found. Pollination of P. euphratica x P. alba was successful only on a mature tree in contrast to the P. alba x P. euphratica cross where pollination was successfully carried out by using the twig and pot system (Jafari et al. 1998). The length of time required for embryo development when P. euphratica is used as the maternal plant is the reason why artificial pollination using bottle grafting and twig and pot did not produce plantlets.


In ovary culture, an average of 67% of cultured capsules produced plantlets, while this value was 90% for ovule culture. Compared to ovary culture, ovules produced only one tiny shoot each (Table 1), while due to the induction of two to three plantlets in each cultured ovary, higher numbers and yields of induced plantlets were observed in ovary culture (Table 1). Germination of ovule embryos started three to four days after culture initiation, i.e. transfer to the new media. The highest frequency of embryo germination (90%) was obtained in ovule culture harvested 45 days after fertilization.


Of the cultured ovaries, 32.5% changed to white-yellow within 2 weeks and did not produce plantlets that turned necrotic. Abnormal structures were observed in ovary culture while there were no malformed ovules on ovule culture. Malformed ovules have been described for other tree species such as sour cherry (Furokawa and Bukovac 1989) and fertilized and unfertilized ovules of apricot (Eaton and Jamont 1992). The fastest germination, 10 days in the case of 45-day-old ovaries (45 days after pollination), was observed with the combination of P. euphratica x P. euphratica (control pollination). Thirteen P. euphratica Oliv. x P. alba L. hybrid plants were successfully acclimatized in a greenhouse and transferred to the field (Fig. 1).



References

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Furokawa, Y. and M.J. Bukovac. 1989. Embryo sac development in sour cherry during the pollination period as related to fruit set. Hort. Sci. 24:1005-1013.

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Guries, R.P. and R.F. Stettler. 1979. Pre-fertilization barriers to hybridization in the poplars. Silvae Genet. 25:37-44.

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Raquin, C. and L. Troussard. 1993. Ovary embryo culture as a tool for poplar hybridization. Can. J. Bot. 71:1271-1275.

Savka, M.A., J.O. Dawson, J.J. Jokela and R.M. Skirvin. 1987. A liquid culture method for rescuing immature embryos of eastern cottonwood. Plant Cell, Tissue Organ Cult. 10:221-226.

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