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Sel’skokhozyaistvennaya Biologiya [Agricultural Biology], 2010, № 5, p. 66-71.
UDK 635.646:631.52:575.222.7:581.481:57.086.83
ISOLATION OF EGGPLANT INTERSPECIFIC HYBRIDS BY THE METHOD OF EMBRYO CULTURE
V.M. Verba, M.I. Mamedov, O.N. Pyshnaya, T.N. Suprunova, N.A. Shmykova
All-Russia Research and Development Institute of Vegetable Crops Selection and Seed Growing,
Moscow province, Odintsovo region, settlement Lesnoy Gorodok - VNIISOK143080, Russia,
e-mail: [email protected]
Received May 12, 2010
S u m m a r y
The authors made interspecific reciprocal crossings between three species of eggplant: Solanum melongena, S. aethiopicum and S. integrifolium. By the using of embryo culture in vitro the authors obtained the interspecific hybrids of following combinations: S. melongena x S. aethiopicum, S. melongena x S. integrifolium, S. aethiopicum x S. melongena, S. aethiopicum x S. integrifoliumn S. integrifolium x S. melongena. The stage of embryonic development, optimal for isolation, was determined, the composition of nutrient media for embryo development and rootage of seedlings was optimized.
Key words: eggplant, interspecific hybridization, embryo culture.
Interspecific hybridization is the technique providing wide range of initial material for creation of new varieties and hybrids; it has great practical importance as the effective way to raise genetic variability of cultivated plants.
The species Solanum aethiopicum L. is a possible source of resistance genes to Fusarium oxysporum (1), F. solani (2), Phy-tophtora parasitica (3); S. aethiopicum sp. Gilo - a source of resistance to the fungus Phomopsis vexans (4). S. integrifolium was used to increase the resistance to stem and flower weevil, as well as to viruses of small-leaved disease and fruit rot (6). It has been reported about possibility of transference of resistance genes to double-spotted spider mites from S. macrocarpon (syn. S. integrifolium) to S. melongena (7). S. aethiopicum and S. macrocarpon were found to be less damaged by bacterial wilt (5).
Complications of interspecific crosses include non-crossing of initial forms, sterility and disturbances of formation processes in interspecific hybrids. Thus, S. aethiopicum and S. integrifolium in crosses with S. melongena showed their incompatibility in postzygotic and postembryonic stages (8).
The purpose of this study was obtaining interspecific hybrids of eggplant using the method of embryo culture.
Technique. The subject of work - eggplant samples of breeding and collection origin from the gene pool provided by the Laboratory for breeding and seed growing of solanaceous crops (the All-Russia Research and Development Institute of Vegetable Crops Selection and Seed Growing -VNIISSOK) - S. melongena (lines L-Almaz and L-Brilliant), S. aethiopicum and S. integrifolium. Hybridization (complete diallele scheme) was performed in a greenhouse on plants grown along a small-volume hydroponic technology.
Pollination with a consequent isolation of pollinated flowers was performed from 1000 to 1200. The fruits of S. aethiopicum and S. integrifolium were collected on the 20th, 25th, 30th, 35th, 40th and 45th days after pollination (depending on combination of crosses and considering biological peculiarities of embryogenesis in parent species), S. melongena - on the 20th, 23th, 27th, 31th, 35th and 40th days. The seeds derived from these fruits were disinfected with 50% commercial preparation "Belizna" (AI - sodium hypochlorite) for 10 minutes with next repeated washing with sterile distilled water.
For embrioculture, the Murashige-Skoog medium (MS) (9) with addition of growth regulators - thidiazuron (TDZ) (0,1 mg/l) and naphthylacetic acid (NAA) (10 mg/l) was used (10, 11). Explants were cultivated in glass vessels volume 100 ml with 20 ml agar-ized medium. Cultivation conditions: illumination - 1,5-2,0 klx, photoperiod - 14 h, temperature - 20-22 °C.
The seedlings of 0,3-1,0 cm length (10-15-days-old) were placed for rooting on agarized MS medium keeping half contents of main components (1/2 MS) without addition of growth regulators. The plants having first pair of true leaves and well-developed roots (days 15-25th) were transplanted from the vessels in peat pots filled with ground and covered with perforated plastic cups.
To determine optimal terms for isolation of hybrid embryos, the authors studied embryo development stages in parental species and in hybrids derived from their crosses. The following stages were detected: globular, heart- and torpedo-shaped embryoid, nearly-formed and a completely formed embryo. In each hybrid combination, 15-20 embryos were measured with an eyepiece micrometer of the stereomicroscope (STEMI-SV8, “OpTON”, Germany).
A morphological description of the obtained interspecific hybrids was performed, as well as verification of their hybrid nature by polymerase chain reaction (PCR) using ISSR primers (Inter Simple Sequence Repeat - polymorphism of DNA fragments flanked by microsatellite sequences) and IRAP primers (Inter Retrotranspozon Amplified Polymorphism - polymorphism of DNA fragments flanked by inverted terminal sites of retrotranspozon).
Total DNA of parental lines and hybrids were isolated from young leaves as described (12), with minor modifications: plant samples were grinded directly in extraction buffer, then the tubes were incubated for 20 min at 65 °C. DNA amplification was performed in reaction mixture of 25 ml volume containing 1 x Taq-buffer (50% glycerol; 20 mM Tris-HCl, pH 8,0; 100 mM KCl; 0,1 mM EDTA; 1mM 1,4-dithio-DL-threitol; 0,5% Tween 20) from the corresponding set of reagents, 1,5 mM MgCl2, 0,2 mM of each dNTP, 20 pmol primer, 0,2 units Taq-polymerase (“Fermentas”, Lithuania) and 10-50 ng DNA. The thermocycler MyCycler (“BioRad”, USA) was operated under the following regime: preliminary denaturation - 4 min at 94 °C, denaturation - 40 min at 94 °C; annealing of primers - 40 s at 50-62 °C (depending on primers); DNA synthesis - 1 min at 72 °C, 30 cycles; final elongation - 10 minutes at 72 °C. A control - PCR mixture without DNA. Amplification products were separated by electrophoresis in 1,5% agarose gel with 0,5 x TBE buffer (0,89 M Tris-HCl, 0,89 M boric acid, 20 mM EDTA, pH 7,5) and stained with ethidium bromide. The photographs of results were made using the system CN-1500 Darkroom (“Vilber Lourmat”, France). Eight ISSR-primers and five IRAP-primers (“Syntol”, Russia) were used for amplification.
The limiting error of sample average was determined by a standard variation-statistical method using Microsoft Excel (13).
Results. The accounted stages of embryo development in all three eggplant species occurred in the period 20th - 45th days from the date of pollination (Fig. 1). The fastest formation of embryo was established in the species S. aethiopicum, slowest - in the species S. integrifolium (Table 1).
1. Stages of embryo development in the species of Solanum L
Species Time after pollination, days Length of embryo, mm Stages of embryo development
S. melongena 20th 0,032±0,001 Globular embryoid
23rd 0,100±0,010 Heart-shaped embryoid
27th 0,600±0,060 Torpedo-shaped embryoid
31st 1,200±0,060 Torpedo-shaped embryoid
35th 3,800±0,090 Nearly formed embryo
40th 4,300±0,090 Completely formed embryo
S. aethipicum 20th 0,080±0,007 Globular embryoid and heart-shaped embryoid
25th 2,200±0,300 Torpedo-shaped embryoid / Nearly formed embryo
30 th 4,100±0,050 Completely formed embryo
S. integrfolium 20 0,070±0,005 Globular embryoid
25 th 0,150±0,030 Heart-shaped embryoid
30 th 1,100±0,100 Torpedo-shaped embryoid
35 th 1,800±0,200 Torpedo-shaped embryoid
40 th 3,900±0,200 Nearly formed embryo
45 th 4,500±0,100 Completely formed embryo
Fig. 1. Stages of embryo development in genera Solanum L.: a — globular embryoid, b — heart-shaped embryoid, c — torpedo-shaped embryoids, d — nearly formed embryo, e — completely formed embryo.
The in vitro embrioculture of hybrids between S. melongena and wild species demonstrated earlier transition to completely formed embryo compared with the maternal component S. melongena. In the hybrid S. melongena x S. aethiopicum, the completely formed embryo of 4,4-4,5 mm length was detected on the 35th day after pollination. In the hybrid S. melongena x S. integrifolium, the completely formed embryo of 4,2-4,3 mm length was observed on the 40th day (similar with S. melongena).
In other hybrid combinations with participation of the wild species’ maternal component, dynamics of embryo development and size of embryos corresponded to that of initial maternal forms.
2. The development of embryo of interspecies hybrids (Solanum melongena — maternal component) in 14-28 days after the start of in vitro cultivation in medium with different growth regulators
Hybrid combination Time after Growth regulator
pollination, days TDZ (0,1 mg/l) NAA (10 mg/l)
S. melongena (L-Brilliant) x 25“ Planting in embryo sacs, no development Planting in embryo sacs, no development
S. integrifolium 30th Abnormal cotyledons, thick main root Weak white callus with green bud in the
without lateral roots center
35th Development of normal shoots, adventive Weak development, growth of white par-
40th buds on the hypocotyl enchymal tissue on the hypocotil
Development of normal shoots, beginning of rooting, adventive buds on the hypocotyl White and small green callus with buds
20th
S. melongena (L-Brilliant) x Planting in embryo sacs, no development Planting in embryo sacs, no development
S. aethiopicum 25th Planting in embryo sacs, no development Planting in embryo sacs, no development
30th Development of normal shoots Development of shoots with roots, large white callus
35th Continuation of Table 2
Development of normal shoots, on some of White callus, abnormally developed
25th them - green callus with buds cotyledons, no shoots and roots
S. melongena (L-Almaz) x Planting in embryo sacs, no development Planting in embryo sacs, no development
S. integrifolium 30th Development of shoots, abnormal cotyle- White callus with a weak green shoot in
35th dons, powerful main root the center
Development of shoots, adventive buds on Development of a weak shoot, growth of
40th the hypocotyl white parenchymal tissue, white callus
Development of normal shoots, beginning of rooting, adventive buds on the hypocotyl White callus with buds
20th
S. melongena (L-Almaz) x Planting in embryo sacs, no development Planting in embryo sacs, no development
S. aethiopicum 25th Planting in embryo sacs, no development Planting in embryo sacs, no development
30th Development of normal shoots White callus, abnormally developed leaf rosettes without the hypocotyl
35th Development of normal shoots White callus, weak abnormally developed leaf rosettes
Note: TDZ - thidiazuron, NAA - naphthylacetic acid.
All three species showed better rates of embryo development in the medium MS + TDZ (0,1 mg/l). 30-40-days-old embryos developed into viable plants with normally shaped cotyledons and the root system. In the medium MS + NAA (10 mg/l), embryos usually formed a loose white callus with abnormally developed cotyledons, without stem and with a powerful root system (or, conversely, without any roots). These embryos didn’t develop into normal plants.
A
B
Fig. 2. The development of embryos (aged 35 days) of the hybrid Solanum melongena (L-Brilliant) X S. aethiopicum in 7-14 days after transference into induction medium: A —
MS medium + thidiazuron (0,1 mg/l), B — MS medium + naphthylacetic acid (10 mg/l).
А
The dynamics of embryo development in all hybrid combinations was similar to that of parental species (Table 2, Fig. 2, A). In the medium with NAA (10 mg/l), 30- and 35-day-old embryos formed a loose white callus with abnormal cotyledons and no roots (Fig. 2, B).
In the combination S. melongena x S. integrifolium, the development into normal plants was observed after 35-days age in the MS medium with TDZ (0,1 mg/l). Accounting of survival rates in this medium of 30-40-days-old embryos (torpedo embryoids - completely formed embryo) averaged to 86,4%, in 20-25-days-old ones (globular embryoid - torpedo embryo) - only 7,9%. The exception - the combination S. melongena (L-Brilliant) x S. aethiopicum (35 days) whose embryos developed strong stems and roots along with white callus.
Average proportion of resulting rooted plants suitable for planting in soil amounted to 69,0% for embryos cultivated in the medium with TDZ (0,1 mg/l) and 9,6% - with NAA (10 mg/l). The frequency of rooting in embryos aged 35 days or more exceeded 80,0% in most of combinations.
The most informative primers were ISSR-primer (CAG)5 and IRAP-primer AACGAGGGGTTCGAGGCC, which provided amplification of the highest number of polymorphic DNA fragments. These primers were used to study the purity of hybrid nature of plants obtained by interspecific crosses. The spectra of amplified ISSR- and IRAP-fragments of individual Fi plants included components of both parents (Fig. 3). The hybrid nature of all individual F1 plants obtained by the method of embryo culture in vitro was confirmed by results of molecular analysis of three hybrid combinations.
B C
Fig. 3. The electrophoregram of products of PCR-amplification using ISSR (A and B) and IRAP (C) primers in three parental forms of eggplant— Solanum integrifolium (1), S. melongena (L-Almaz) (2), S. aethiopicum (3) and their interspecific Fx hybrids obtained by in vitro embryoculture. М — molecular weight marker (GeneRuler, 100 bp DNA Ladder, “Fermentas”, Lithuania).
Thus, it has been found that embryos of Solanum melongena, S. aethiopicum and S. integrifolium pass main stages of development (from globular embryoid to completely developed embryo) during the period 20th - 45th days after pollination of maternal flower. The fastest embryo development among the studied species has been detected in S. aethiopicum, slowest - in S. integrifolium. The most suitable for isolation were the well-developed torpedo-shaped embryos (> 2 mm) in later stages of development (30th - 45th days). In the first phase of embryo formation, the optimal induction medium was MS medium with addition of thidiazuron (0,1 mg/l) providing the period of embryo development not exceeding 7-14 days. The MS medium keeping half content of major components and no growth regulators was found to be suitable for rooting of shoots. Under these conditions, hybrid forms have been obtained in short terms for several combinations of crosses: S. melongena x S. aethiopicum, S. melongena x S. integrifolium, S. aethiopicum x S. melongena, S. aethiopicum x S. integrifolium and S. integrifolium x S. melongena.
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