Embryo rescue from interspecific crosses in apple rootstocks

The objetive of this work was to rescue immature embryos of apple rootstocks Malus prunifolia (Marubakaido) and Malus pumila (M9) after 40–60 days of pollination and to put them into MS culture media supplemented with agar (6 g L-1) and casein hydrolysate (500 mg L-1). Embryos originated from interspecific crosses and open pollination showed differences in the in vitro responses, depending on the female parent, the developmental stage of the embryo, and the culture medium composition. Embryos of the M. pumila rootstock, rescued within 40 days after pollination and put in culture medium supplemented with indolacetic acid (IAA), gibberellic acid (GA3), kinetin and maltose, resulted in a normal development of plantlets. However, embryos originating from hand-pollination, cultivated in medium supplemented with 14 μM IAA, 5 μM kinetin and 1.5 μM GA3 (MS1), mainly those of M. prunifolia x M. pumila, showed a high percentage of rusted embryos (96.2%). Embryos from open pollination of M. prunifolia and M. pumila formed calluses. It was possible to identify the influence of the female parent by the enhanced development of M. pumila shoots derived from open or hand-pollination. The crossing of responsive species and the use of the technique of embryo culture provided a rapid and uniform germination and, consequently, the development of fully normal seedlings.


Introduction
The juvenile period in apples is significantly reduced by grafting on dwarfing rootstocks (Visser, 1970).When the juvenile period is shortened, the time from crossing to seed germination constitutes a relatively larger part of the breeding cycle.It may, therefore, be of interest to investigate whether this period can also be shortened (Hansche, 1983).
The continuous demand for new plant materials requires the upgrading of traditional breeding methods to accelerate the production of new and improved genotypes.Biotechnological tools and traditional breeding techniques may successfully be coupled.These new strategies allow the early selection of useful traits in apple trees, while avoiding the problems of the long juvenile period and the long generation times (Roen, 1994).
The rescue of hybrid embryos resulted from intra and interspecific crosses is commonly applied in apple breeding programs aimed at increasing the efficiency of the seed germination and the number of individuals obtained through sexual hybridization (Rubio Cabetas et al., 1997).
To provide an efficient alternative for dormancy suppression, when isolated embryos can be rescued at different developmental stages (Ramming, 1990;Burgos Ortiz, 1997), the low-temperature treatment can be partly replaced by the use of gibberellin (GA 3 ), alone or in combination with cytokinin or auxin, added to the culture medium as a supplement (Zhang & Lespinasse, 1991;Dantas et al., 2002).
Interspecific hybrids are useful for the transfer of desirable genes from wild to cultivated species.However, in many cases, successful wide crosses between species are difficult to accomplish.Post-zygotic barriers, such as endosperm abortion, are commonly observed, but this has been overcome through the use of embryo rescue (Lu & Bridgen, 1996;Palmer et al., 2002).
The objective of this work was to evaluate the application of embryo rescue technique and the effect of different growth regulators and carbon sources on the origin of embryo and time of rescue of M9 apple rootstocks (Malus pumila Mill.) and Marubakaido (Malus prunifolia Borkh.).

Material and Methods
Forty and sixty days after the open and hand pollinations of M. pumila (M9) and M. prunifolia (Marubakaido), the young fruits were collected at the Experimental Station of Empresa de Pesquisa Agropecuária e Extensão Rural de Santa Catarina (Epagri), in São Joaquim, Santa Catarina State, Southern Brazil.For wide crossing, the flowers were emasculated and immediately hand-pollinated.Flowers were then closed with a paper bag.Flowers that were not handpollinated were subjected to open pollination by bees.
Seeds were removed from the fruits and immersed in ethanol 70% for 30 seconds, followed by treatment in sodium hypochloride 1.25%, for 15 minutes, after which they were washed with sterile distilled water three times and cut open aseptically.Embryos were then dissected out with the use of forceps.Intact embryos were sterilized by the same procedures and were put in test tubes (150x25 mm) containing 15 mL of MS (Murashige & Skoog, 1962) basal medium supplemented with the compounds described below.The cultures were incubated in the darkness at 25±2 o C, for the first ten days, and then transferred to a culture room under a 16-hours light period using a cool-white fluorescent lamp at 40 µmol m -2 s -1 light intensity.
All experiments were arranged in a completely randomized design, with five embryos per experimental unit and five repetitions per treatment.Following traits were evaluated after 60 days in culture: length of shoots and radicle, number of buds, calluses induction, oxidation rates and percentage of embryos germinated.The data were submitted to analysis of variance and SNK multiple range test at 5% of probability.No statistical analysis was carried out for data of the number and percentage of plantlets formed.

Results and Discussion
The rescue of M. pumila embryos after 40 and 60 days of open pollination resulted in values of 48 and 80% of seedling recovering, respectively.The greatest length of shoots (3.7 cm) was observed in response to the culture medium MS 1 (kinetin, GA 3 and IAA), for embryos rescued 40 days after the pollination (Table 1).The culture media MS 1 and MS 3 (kinetin, GA 3 and IAA) promoted an enhanced growth of the shoots, when compared to the results obtained in response to the MS 2 and MS 4 (GA 3 ) media.Maltose was more efficient than sucrose as carbon source (Table 1).
Embryos rescued after 40 days and put in culture medium supplemented with maltose (MS 3 and MS 4 ) resulted in the highest number of buds, differing statistically from the results observed in the culture medium supplemented with sucrose (MS 1 and MS 2 ) (Table 1).MS 1 culture medium produced the longest roots, however, it provoked the highest rate of callus induction in the root (Table 2).
Successful production of seedlings through embryo culture largely depends on both the developmental stage of the embryo and the composition of the culture medium (Sharma et al., 1996).In the present work, immature embryos rescued after 40 days produced plantlets within four weeks of culture.
The best results for radicle emergence and seedling development resulted from the MS culture medium supplemented with Kin, GA 3 and IAA (MS 3 ).For M. pumila, the addition of either cytokinins and auxins or gibberellins enhanced the in vitro development of rescued embryos (Dantas et al., 2002).The promotion of precocious embryo germination promoted by growth regulators has also been reported in other species (Anderson et al., 1990;Bajaj, 1990;Das et al., 1999).
Maltose has proved to be superior to other carbon sources in the culture of isolated microspores (Scott & Lyne, 1995) and for the induction of somatic embryos (Ladyman & Girard, 1992).It is postulated that maltose maintains the osmolarity in the culture medium over the whole culture period due to its slow hydrolysis to glucose, while sucrose is rapidly hydrolyzed (Indrianto et al., 1999).
The rate of the germinated interspecific hybrids was higher in open pollinated M. pumila (60.4%) than in open pollinated M. prunifolia (47.9%).The crossing between M. pumila and M. prunifolia resulted in 26.7% of germinated embryos.For the reciprocal cross, the rate of germination was 12.8%.From open pollination condition, M. pumila resulted in 52% of plantlets (Table 3).
Rescued embryos from the open pollination of M. prunifolia and M. pumila put in MS 1 culture medium showed rates of callus induction of 23.8 and 12.5%, respectively (Table 4).In general, embryos originating from open pollination were more efficient in the calluses induction than those resulting from hand-pollination.
The different in vitro responses of genotypes may be related to the nature of the parental species and the degree of compatibility between parents.Genotypic differences in the capacity of cultured immature embryos were found in wheat (Triticum aestivum) (Maddock  (1) .
(1) Averages followed by the same lower case letter, comparing the medium, and capital letters, comparing 40 and 60 days, do not distinguish each treatment from the test SNK at 5% of probability.

Table 2. Length of roots in embryos of the rootstock
Malus pumila from open pollinated plants and cultivated in vitro (1) .
(1) Averages followed by the same letter are not different by SNK test at 5% of probability.
Table 3. Relative efficiency of embryo cultures from Malus isolated 60 days after pollination.
(1) Averages followed by the same letter are not different by SNK test at 5% of probability.
Table 4. Percentage of callus formed and oxidation in embryos rescued from apple rootstocks in open and hand pollinations (1) .
(1) Averages followed by the same small letter, comparing the pollinations among culture media, and capital letters, comparing the medium inside each pollination type, are not different by SNK test at 5% of probability.et al., 1983).Kapila & Sethi (1993) reported that in vitro responses such as the percentage of embryo germination, callus development and plantlet regeneration were affected by parental wheat genotypes in the embryo rescue of bread wheat x hexaploid triticale hybrid.
In Alstroemeria the percentages of embryo germination, and callus and shoot developments were significantly affected by the parental genotype in the interspecific crosses (Lu & Bridgen, 1996).In the present study, genotypic differences in the in vitro response of immature hybrid embryo were observed.
Embryos rescued from the M. prunifolia and M. pumila cross resulted in a 96.2% oxidation rate in culture medium MS 2 , differing statistically from results observed in the culture medium MS 1 for the same cross and also from open pollinated M. prunifolia (Table 4).
The stem axis length of plantlets obtained from M. pumila x M. prunifolia embryos cultivated in MS 1 , or originating from open pollinated M. pumila cultured in MS 2 , showed the values 2.5 cm and 3.0 cm, respectively (Table 5).For both rootstocks originated from open pollination, the observed values were higher in plantlets cultured in MS 2 compared to the values observed in MS 1 culture medium.
Embryos resulted from M. pumila x M. prunifolia presented the greatest number of buds (11.5) when cultured in MS 1 .Rescued embryos resulting from open pollination demonstrated that they were able to differentiate according to the medium used, since those from M. pumila in MS 2 showed the highest number of buds (4.4) (Table 5).
The results obtained in the present work showed the female progenitor and the genotype influences on the morphogenetic responses.For example, in M. pumila, the presence of normal seeds was observed, while for M. prunifolia a number of aborted embryos were verified.The supplementation of the culture medium with a source of auxin (IAA), cytokinin (kinetin) and gibberellin (GA 3 ) was effective in increasing the germination rates of rescued embryos.However, in some cases, the presence of enlarged hypocotyls, epicotyls and calluses was observed.
The little production of plantlets resulting from the rescue of immature hybrid embryos is to be expected, and a number of previous studies have reported this occurrence in apples (Dantas et al., 2002) as well as in other species (Anderson et al., 1990;Palmer et al., 2002).The failure of many of the immature hybrid Malus rescued embryos to germinate is common in noncompatible interspecific crosses, possibly due to the often reduced viability to rescue embryos, which happens because of the activation of post-zygotic abortion mechanisms (Bajaj, 1990).
Apple is self-incompatible and need allelic polymorphism for the fertilization and subsequent embryo and fruit developments.Several hypotheses have been proposed to explain the failure of the normal embryo development in interspecific crosses, such as endosperm breakdown or collapse (Camadro & Masuelli, 1995).
The results reported here are valuable for breeders, since crossing of responsive species and using of the technique of embryo rescue culture can transfer desirable genes to different species.Further extension of the cross range within the Malus genus can be successfully achieved by embryo rescue techniques, but this rests mainly on the development of techniques to overcome pre-and post-fertilization barriers involved in the interspecific hybridization process (Palmer et al., 2002).

Conclusions
1. Embryos rescued 40 days after the pollination, in culture medium MS 3 perform better development in the number of buds and root lengths.
2. Embryos originated from open pollination are more potent in the induction of calluses than those resulted from hand-pollination.
3. The apple rootstock Malus pumila shows a better number of normal seeds, while M. prunifolia presents a number of aborted embryos.
4. Embryos resulted from interspecific crosses M. pumila x M. prunifolia present the higher number of buds.  (1. (1) Averages followed by the same small letter, comparing the pollinations among each medium, and capital letters, comparing the medium inside each pollination system, are not different by SNK test at 5% of probability.

Table 1 .
Length of shoots and number of buds from embryos of apple rootstock Malus pumila rescued 40 and 60 days after open pollination and cultivated in vitro

Table 5 .
Length of shoots and number of buds derived from embryos rescued from rootstocks in open and hand pollinations