Ex situ plant germplasm conservation revised at the light of mechanisms and methods of genetics / Conservación de germplasma Ex situ revisada a la luz de mecanismos y métodos de genética

ABSTRACT Plant genetic resources for food and agriculture are ex situ conserved in germplasm banks as samples (accessions) of natural or naturalized populations, either as the originally sampled propagules (mainly seeds) or their multiplications. The premises underlying ex situ conservation are that (a) it is the safest and cheapest alternative for germplasm preservation for future generations and (b) accessions are representative of the genetic diversity encountered in nature. In the past decades, ideas, alternatives and considerations have been put forward on the topic, and protocols have been devised for plant germplasm sampling, conservation and multiplication. However, limitations in the management efficiency of germplasm banks have been pointed out by international organizations. In our opinion, germplasm banks in general need to revise their functioning and management at the light of principles and methods of Genetics. To that end, it is necessary to consider the reproductive biology of higher plants -whose genetic consequences at both the individual plant and the population levels are not always either fully understood or taken into account in devising the protocols-, the genetic structures of wild and cultivated populations, and the course of the genetic material in the populations. In this paper, we discuss the three topics and provide an example of a national forage breeding program, from germplasm bank accessions as the germplasm of origin to the obtainment of commercial cultivars. Finally, we present a proposal as a base for discussion among curators, researchers and breeders.

RESUMEN Los recursos genéticos vegetales para la alimentación y la agricultura se conservan ex situ en bancos de germoplasma como muestras (introducciones) de poblaciones naturales o naturalizadas ya sea como propágulos originales (mayoritariamente semillas) o sus multiplicaciones. Las premisas subyacentes son que (a) es la alternativa más segura y barata de preservación de germoplasma para futuras generaciones y (b) las introducciones son representativas de la diversidad genética que se encuentra en la naturaleza En las últimas décadas, se han presentado ideas, alternativas y consideraciones sobre el tema y se han elaborado protocolos para el muestreo, conservación y multiplicación de germoplasma. Sin embargo, organizaciones internacionales han señalado limitaciones en la eficiencia del manejo de los bancos de germoplasma. En nuestra opinión, se necesita revisar el funcionamiento y manejo de dichos bancos en general a la luz de los principios y métodos de Genética. Para tal fin, es necesario considerar la biología reproductiva de las plantas superiores -cuyas consecuencias genéticas a nivel de planta individual y de población no se comprenden en su totalidad o no se consideran al idear los protocolos -, las estructuras genéticas de poblaciones naturales y cultivadas, y el curso del material genético en las poblaciones. En este trabajo discutimos los tres temas y proveemos un ejemplo de un programa nacional de mejoramiento de forrajeras, desde las introducciones como germoplasma de origen hasta la obtención de cultivares comerciales. Finalmente, presentamos una propuesta como base de discusión entre curadores, investigadores y mejoradores.

Pollen viability and meiotic irregularities in a collection of Stevia rebaudiana (Bertoni) Bertoni cultivated in Tucumán, Argentina / Viabilidad de polen e irregularidades meióticas en una colección de Stevia rebaudiana (Bertoni) Bertoni cultivada en Tucumán, Argentina

Stevia rebaudiana (Bertoni) Bertoni (2n=2x=22) (Asteraceae family) is a species of economic value due to the presence of steviol glycosides in leaves -mainly stevioside and rebaudioside A- which are non-caloric sugars. In 2013, a collection of plants (genotypes) from four contrasting environments was established in Tucuman, Argentina, for evaluation under local conditions and, eventually, breeding purposes. As a first step, pollen viability and meiosis was studied in a sample of 56 plants. The percentage of pollen viability varied from medium (69.4%) to high (99.6%) in 52 of them, but from low (36.5%) to relatively low (51.5%) in the remaining four. The latter four plants also presented pollen grains of heterogeneous size, which were classified as n (normal, the most frequent size in the sample),

Stevia rebaudiana (Bertoni) Bertoni (2n=2x=22) (familia Asteraceae) es una especie de valor economico debido a la presencia de glucosidos de esteviol en sus hojas, principalmente esteviosido y rebaudiosido A, que son azucares no caloricos. En 2013, se establecio una coleccion de plantas (genotipos) provenientes de cuatro ambientes contrastantes en Tucuman, Argentina, para su evaluacion en condiciones locales y, eventualmente, con fines de mejoramiento genetico. Como primer paso, se estudio la viabilidad del polen y la meiosis en una muestra de 56 plantas. El porcentaje de viabilidad del polen fue de medio (69,4%) a alto (99,6%) en 52 de ellas, pero de bajo (36,5%) a relativamente bajo (51,2%) en las cuatro restantes. Las ultimas cuatro plantas tambien presentaron granos de polen de tamano heterogeneo, que se clasificaron como n (normal, el tamano mas frecuente en la muestra),

Molecular bases of the postzygotic barriers in interspecific crosses between the wild potato species Solanum acaule and Solanum commersonii.

To investigate the molecular bases of postzygotic hybridization barriers in tuber-bearing Solanums, the wild species Solanum commersonii Dunal ex Poir. (cmm, 2n = 2x = 24, 1EBN) and Solanum acaule Bitter (acl, 2n = 4x = 48, 2EBN) were crossed in intra- and interspecific genotypic combinations, and the transcriptome of immature seeds was analyzed by using the cDNA-AFLP technique. From a total of 423 analyzed cDNA fragments, 107 (25.3%) were differentially regulated in the compatible (acl × acl and cmm × cmm) versus incompatible (acl × cmm) crosses. DNA sequence data were obtained from 21 fragments and RT-PCR analyses were carried out with five fragments to validate the cDNA-AFLP differential pattern. Sequence analysis suggested a possible role for the differentially expressed sequences in cytokinesis, cell cycle, secondary and hormonal metabolism, biodegradation, and transport. In situ hybridization experiments with fragments encoding an ubiquitin-fold modifier 1 precursor and a possible vesicle transport protein revealed expression of these genes in the embryo and endosperm. The results suggest that the collapse of the embryo and endosperm in incompatible crosses may be related to alterations in cell cycle and cytokinesis.

A genetic approach to the species problem in wild potato.

Wild potatoes are native to the Americas, where they present very wide geographical and ecological distribution. Most are diploid, obligate out-crossers due to a multiallelic gametophytic self-incompatibility (S) locus that prevents self-fertilisation and crossing between individuals carrying identical S-alleles. They have two alternative modes of reproduction: sexual (by seeds) and asexual (by stolons and tubers), which provide, respectively, for genetic flexibility in changing environments and high fitness of adapted genotypes under stable conditions. Since the early twentieth century, their taxonomic classification has been mostly based on morphological phenotypes (Taxonomic Species Concept). More recently, attempts have been made to establish phylogenetic relationships, applying molecular tools in samples of populations (accessions) with a previously assigned specific category. However, neither the reproductive biology and breeding relations among spontaneous populations nor the morphological and genetic variability expected in obligate allogamous populations are considered when the taxonomic species concept is applied. In nature, wild potato populations are isolated through external and internal hybridisation barriers; the latter, which are genetically determined, can be either pre-zygotic (pollen-pistil incompatibility) or post-zygotic (abortion of embryo, endosperm or both tissues, sterility, and hybrid weakness and breakdown in segregating generations). The internal barriers, however, can be incomplete, providing opportunities for hybridisation and introgression within and between populations and ploidy levels in areas of overlap. The widespread occurrence of spontaneous hybrids in nature was recognised in the mid-twentieth century. Using genetic approaches, results have been obtained that provide strong support to the assertion that populations are at different stages of genetic divergence and are not at the end of the evolutionary process, as presupposed by the Taxonomic Species Concept. Furthermore, since wild potatoes have uniparental and biparental overlapping generations, the Biological Species Concept - developed for sexually reproducing biparental organisms - cannot be applied to them. In this paper, morphological, genetic, molecular and taxonomic studies in wild potato are reviewed, considering the genetic consequences of their reproductive biology, in an attempt to shed light on the species problem, because of its relevance in germplasm conservation and breeding.

Meiotic abnormalities underlying pollen sterility in wild potato hybrids and spontaneous populations.

Wild potato species are widely distributed in the Americas, where they spontaneously grow in very diverse habitats. These species - with low chromosome differentiation - form polyploid series with 2n = 2x, 3x, 4x and 6x (x =12). They are isolated in nature by external and internal hybridisation barriers that can be incomplete, allowing hybridisation in areas of sympatry. Nevertheless, most accessions in germplasm banks, regardless of genetic background of the sampled spontaneous populations, have been assigned specific categories based on morphological characters. To further investigate the extent of hybridisation in the group and for comparative purposes, pollen viability was estimated in (i) artificial hybrids between a commercial cultivar (Calén INTA) of the common potato (tetraploid Solanum tuberosum ssp. tuberosum) and the tetraploid cytotype of the related wild species S. gourlayi, and (ii) samples of plants (accessions) and inflorescences of natural populations from Argentina, tentatively classified as 'presumed hybrids' (S. infundibuliforme-S. gourlayi) and 'species' (S. infundibuliforme, S. gourlayi and S. chacoense). Regardless of origin, 98 out of 103 plants analysed had zero to 70% pollen viability (zero to 40% in eight of them). Pollen grains were of variable size and morphology and, in mostly male sterile plants, the only viable pollen grains were 2n and/or 4n. Furthermore, male sterile plants shared various abnormalities in meiosis I and II (unpaired chromosomes, unequal chromosome distribution, precocious/lagging chromosomes, parallel, tripolar, fused and multiple spindles, unequal size nuclei, dyads, triads and pentads in addition to normal tetrads, among others). These results provide novel evidence to support field observations of early potato botanists on the extent of spontaneous hybridisation in wild Argentinian potato populations, which is not reflected in the current taxonomy and has significant consequences for germplasm conservation and breeding.

Sensitivity of Bidens laevis L. to mutagenic compounds. Use of chromosomal aberrations as biomarkers of genotoxicity.

The wetland macrophyte Bidens laevis possesses suitable cytological characteristics for genotoxicity testing. To test its sensitivity as compared to terrestrial plants species currently in use in standardized assays, Methyl Methanesulfonate (MMS), N-ethyl-N-nitrosourea (ENU) and Maleic Hydrazide (HM) were used. On the other hand, the insecticide Endosulfan (ES)--an environmentally relevant contaminant--was assayed in seeds and two-month old plants. Mitotic Index (MI), frequency of Chromosome Aberrations in Anaphase-Telophase (CAAT) and frequency of Abnormal Metaphases (AM) were analyzed. MH, MMS and ENU caused a significant decrease of the MI. MMS was aneugenic whereas MH and ENU were both aneugenic and clastogenic. ES caused a significant concentration-dependent increase of total- and aneugenic-CAAT in roots and a significant high frequency of AM at high concentrations. Because of its sensitivity to mutagenic substances, B. laevis can be regarded as a reliable and convenient species for genotoxicity assays especially if aquatic contaminants are evaluated.

The importance of Arabidopsis seed mutants in the elucidation of the molecular basis of Endosperm Balance Number in tuber-bearing Solanum species

The Endosperm Balance Number (EBN) is an important concept for potato breeding and has evolutionary importance in tuber-bearing Solanum species. The EBN is part of the post-zygotic hybridization barriers in the group and represents a reproductive isolating mechanism. Few genes have been proposed to be involved in its genetic control; until now, however, neither specific genes nor its molecular basis have been well established. Histological observations of embryo and endosperm development in inter-EBN crosses in tuber-bearing Solanum revealed phenotypes similar to those recently described in Arabidopsis seed mutants. The common feature between them is that the endosperm nuclei become greatly enlarged and that embryos are arrested at the globular stage. The proteins encoded by the Arabidopsis TITAN genes are related to chromosome dynamics and cell division. Based on the sequence of titan mutants, genes in potato species related to cell cycle and microtubule assembly were isolated. In this article a perspective model is proposed to explore the utility of Arabidopsis mutants associated with cell cycle control as a tool to elucidate the molecular basis of EBN in potato. Further research focused on the expression pattern of these genes in intra- and inter-EBN crosses in potato species will be performed.

The importance of Arabidopsis seed mutants in the elucidation of the molecular basis of Endosperm Balance Number in tuber-bearing Solanum species

The Endosperm Balance Number (EBN) is an important concept for potato breeding and has evolutionary importance in tuber-bearing Solanum species. The EBN is part of the post-zygotic hybridization barriers in the group and represents a reproductive isolating mechanism. Few genes have been proposed to be involved in its genetic control; until now, however, neither specific genes nor its molecular basis have been well established. Histological observations of embryo and endosperm development in inter-EBN crosses in tuber-bearing Solanum revealed phenotypes similar to those recently described in Arabidopsis seed mutants. The common feature between them is that the endosperm nuclei become greatly enlarged and that embryos are arrested at the globular stage. The proteins encoded by the Arabidopsis TITAN genes are related to chromosome dynamics and cell division. Based on the sequence of titan mutants, genes in potato species related to cell cycle and microtubule assembly were isolated. In this article a perspective model is proposed to explore the utility of Arabidopsis mutants associated with cell cycle control as a tool to elucidate the molecular basis of EBN in potato. Further research focused on the expression pattern of these genes in intra- and inter-EBN crosses in potato species will be performed.(AU)

The importance of Arabidopsis seed mutants in the elucidation of the molecular basis of Endosperm Balance Number in tuber-bearing Solanum species.

The Endosperm Balance Number (EBN) is an important concept for potato breeding and has evolutionary importance in tuber-bearing Solanum species. The EBN is part of the post-zygotic hybridization barriers in the group and represents a reproductive isolating mechanism. Few genes have been proposed to be involved in its genetic control; until now, however, neither specific genes nor its molecular basis have been well established. Histological observations of embryo and endosperm development in inter-EBN crosses in tuber-bearing Solanum revealed phenotypes similar to those recently described in Arabidopsis seed mutants. The common feature between them is that the endosperm nuclei become greatly enlarged and that embryos are arrested at the globular stage. The proteins encoded by the Arabidopsis TITAN genes are related to chromosome dynamics and cell division. Based on the sequence of titan mutants, genes in potato species related to cell cycle and microtubule assembly were isolated. In this article a perspective model is proposed to explore the utility of Arabidopsis mutants associated with cell cycle control as a tool to elucidate the molecular basis of EBN in potato. Further research focused on the expression pattern of these genes in intra- and inter-EBN crosses in potato species will be performed.

Plant regeneration after long-term callus culture in clones of Asparagus officinalis L

Callus growth and plant regeneration from long-term callus cultures were studied in two elite clones of Asparagus officinalis cv. Argenteuil, to establish a suitable protocol for a prospective in vitro selection program. Callus initiation and growth was evaluated on MS medium with 3

agar, 1 mg x l(-1) kinetin, and three levels of 2,4-D. The highest callus relative growth was obtained on medium with 1.5 mg x l(-1) 2,4-D and 1 mg x l(-1) kinetin. Shoot primordia (SP) induction from > 18-months-old calluses was evaluated on several media; the highest percentage of SP induction (89

) and average number of SP per callus (8.6) were obtained with clone [quot ]265[quot ] on MS medium with 5 mg x l(-1) 2iP, 1 mg x l(-1) IAA, 3

sucrose and 0.9

agar. The highest percentage of root induction (100

) was achieved with clone ’265’ on MS medium with 0.1 mg x l(-1) kinetin, 0.1 mg x l(-1) NAA, 1.32 mg x l(-1) ancymidol, 7

glucose and 0.8

agar. Important medium x genotype interactions were detected, pointing to the need of adjusting this and other in vitro protocols for specific asparagus genotypes.

Plant regeneration after long-term callus culture in clones of Asparagus officinalis L.

Callus growth and plant regeneration from long-term callus cultures were studied in two elite clones of Asparagus officinalis cv. Argenteuil, to establish a suitable protocol for a prospective in vitro selection program. Callus initiation and growth was evaluated on MS medium with 3% sucrose, 0.9% agar, 1 mg x l(-1) kinetin, and three levels of 2,4-D. The highest callus relative growth was obtained on medium with 1.5 mg x l(-1) 2,4-D and 1 mg x l(-1) kinetin. Shoot primordia (SP) induction from > 18-months-old calluses was evaluated on several media; the highest percentage of SP induction (89%) and average number of SP per callus (8.6) were obtained with clone "265" on MS medium with 5 mg x l(-1) 2iP, 1 mg x l(-1) IAA, 3% sucrose and 0.9% agar. The highest percentage of root induction (100%) was achieved with clone '265' on MS medium with 0.1 mg x l(-1) kinetin, 0.1 mg x l(-1) NAA, 1.32 mg x l(-1) ancymidol, 7% glucose and 0.8% agar. Important medium x genotype interactions were detected, pointing to the need of adjusting this and other in vitro protocols for specific asparagus genotypes.

Plant regeneration after long-term callus culture in clones of Asparagus officinalis L.

Callus growth and plant regeneration from long-term callus cultures were studied in two elite clones of Asparagus officinalis cv. Argenteuil, to establish a suitable protocol for a prospective in vitro selection program. Callus initiation and growth was evaluated on MS medium with 3

sucrose, 0.9

agar, 1 mg x l(-1) kinetin, and three levels of 2,4-D. The highest callus relative growth was obtained on medium with 1.5 mg x l(-1) 2,4-D and 1 mg x l(-1) kinetin. Shoot primordia (SP) induction from > 18-months-old calluses was evaluated on several media; the highest percentage of SP induction (89

) and average number of SP per callus (8.6) were obtained with clone [quot ]265[quot ] on MS medium with 5 mg x l(-1) 2iP, 1 mg x l(-1) IAA, 3

sucrose and 0.9

agar. The highest percentage of root induction (100

) was achieved with clone 265 on MS medium with 0.1 mg x l(-1) kinetin, 0.1 mg x l(-1) NAA, 1.32 mg x l(-1) ancymidol, 7

glucose and 0.8

agar. Important medium x genotype interactions were detected, pointing to the need of adjusting this and other in vitro protocols for specific asparagus genotypes.

Substitutes for genome differentiation in tuber-bearing Solanum: interspecific pollen-pistil incompatibility, nuclear-cytoplasmic male sterility, and endosperm.

The cultivated potato, Solanum tuberosum L. (2n=4x=48), has a very large number of related wild and cultivated tuber-bearing species widely distributed in the Americas. These species, grouped in 16 taxonomic series, range from the diploid to the hexaploid level. Polyploid species are either disomic or polysomic, and sexual polyploidization via genetically controlled 2 n gametes has played a major role in their evolution. Species are separated in nature by geographical and ecological barriers. However, there are several examples of sympatric species that share the same niches but do not readily cross (i.e., the diploids S. commersonii and S. chacoense in certain areas of Argentina). External barriers alone are, therefore, not sufficient to explain species integrity. In addition, there is no strong evidence indicating that genome differentiation is important in the group. In this review we present evidence supporting the assertion that interspecific pollen-pistil incompatibility, nuclear-cytoplasmic male sterility, and the endosperm are major forces that strengthen the external hybridization barriers allowing, at the same time and under specific circumstances, a certain amount of gene exchange without jeopardizing the integrity of the species.

Somatic embryogenesis in Asparagus officinalis L. cv. Argenteuil: interactions between genotype, explant type and growth regulators on callus induction, growth and embryogenic differentiation.

Callus induction, growth and embryogenic differentiation were studied in two selected genotypes of Asparagus officinalis L. cv. Argenteuil, to develop a protocol for somatic embryogenesis. Two experiments were carried out. In the first, four explant types (spear sections and lateral buds, both of in vivo and in vitro origin), and three levels of 2,4-D (1.5, 5 and 10 mg.l-1) and two of KIN (0 and 1 mg.l-1) added to MS basal medium, were tested during the three initial culture stages (90 d period). All factors were included in one factorial design to statistically analyze interaction effects. Interactions between genotype, explant type and time of culture initiation were significant for callus induction and growth during the two initial culture stages. The addition of kinetin enhanced callus induction and growth, and high levels of 2,4-D depressed callus growth. The average embryogenic differentiation was < 2% overall (11% with the best factor combination). In the second experiment, another explant type (bud clusters) was tested; the average embryogenic differentiation was 1.9% (best combination: 5.6%). For the two genotypes studied, the best factor combinations were the culture of in vivo lateral buds or bud clusters on MS basal medium with 1.5 mg.l-1 2,4-D and 1 mg.l-1 KIN.

Somatic embryogenesis in Asparagus officinalis L. cv. Argenteuil: interactions between genotype, explant type and growth regulators on callus induction, growth and embryogenic differentiation.

Callus induction, growth and embryogenic differentiation were studied in two selected genotypes of Asparagus officinalis L. cv. Argenteuil, to develop a protocol for somatic embryogenesis. Two experiments were carried out. In the first, four explant types (spear sections and lateral buds, both of in vivo and in vitro origin), and three levels of 2,4-D (1.5, 5 and 10 mg.l-1) and two of KIN (0 and 1 mg.l-1) added to MS basal medium, were tested during the three initial culture stages (90 d period). All factors were included in one factorial design to statistically analyze interaction effects. Interactions between genotype, explant type and time of culture initiation were significant for callus induction and growth during the two initial culture stages. The addition of kinetin enhanced callus induction and growth, and high levels of 2,4-D depressed callus growth. The average embryogenic differentiation was < 2

overall (11

with the best factor combination). In the second experiment, another explant type (bud clusters) was tested; the average embryogenic differentiation was 1.9

(best combination: 5.6

). For the two genotypes studied, the best factor combinations were the culture of in vivo lateral buds or bud clusters on MS basal medium with 1.5 mg.l-1 2,4-D and 1 mg.l-1 KIN.

Preliminary results on embryo rescue for circumventing hybridization barriers in Asparagus

Garden asparagus, Asparagus officinalis, is reproductively isolated from a related ornamental species with potential breeding value, Asparagus densiflorus cv. Sprengeri, by pre- and post-zygotic barriers. The latter barrier operates at the endosperm level five days after pollination in A. officinalis x A. densiflorus crosses. To try to circumvent this barrier, in vitro embryo rescue using ovule and ovary cultures was tested. Controlled interspecific crosses were made and 2,032 ovules and 826 ovaries were cultured three days after pollination under various culture media and incubation conditions. Ovaries cultured for 60 days became red (similar to mature fruits), but seed formation was incomplete. Transfer of ovules to other media was necessary to promote embryo development. The interspecific embryos increased their length from 35 microns at the initiation of culture to 1,900 microns after 120 days of culture, but seedlings were not obtained. Histological studies revealed differentiation of protoderm only. The possible causes of the failure of the embryos to complete differentiation and morphogenesis are discussed.

Preliminary results on embryo rescue for circumventing hybridization barriers in Asparagus

Garden asparagus, Asparagus officinalis, is reproductively isolated from a related ornamental species with potential breeding value, Asparagus densiflorus cv. Sprengeri, by pre- and post-zygotic barriers. The latter barrier operates at the endosperm level five days after pollination in A. officinalis x A. densiflorus crosses. To try to circumvent this barrier, in vitro embryo rescue using ovule and ovary cultures was tested. Controlled interspecific crosses were made and 2,032 ovules and 826 ovaries were cultured three days after pollination under various culture media and incubation conditions. Ovaries cultured for 60 days became red (similar to mature fruits), but seed formation was incomplete. Transfer of ovules to other media was necessary to promote embryo development. The interspecific embryos increased their length from 35 microns at the initiation of culture to 1,900 microns after 120 days of culture, but seedlings were not obtained. Histological studies revealed differentiation of protoderm only. The possible causes of the failure of the embryos to complete differentiation and morphogenesis are discussed.(AU)

Preliminary results on embryo rescue for circumventing hybridization barriers in Asparagus.

Garden asparagus, Asparagus officinalis, is reproductively isolated from a related ornamental species with potential breeding value, Asparagus densiflorus cv. Sprengeri, by pre- and post-zygotic barriers. The latter barrier operates at the endosperm level five days after pollination in A. officinalis x A. densiflorus crosses. To try to circumvent this barrier, in vitro embryo rescue using ovule and ovary cultures was tested. Controlled interspecific crosses were made and 2,032 ovules and 826 ovaries were cultured three days after pollination under various culture media and incubation conditions. Ovaries cultured for 60 days became red (similar to mature fruits), but seed formation was incomplete. Transfer of ovules to other media was necessary to promote embryo development. The interspecific embryos increased their length from 35 microns at the initiation of culture to 1,900 microns after 120 days of culture, but seedlings were not obtained. Histological studies revealed differentiation of protoderm only. The possible causes of the failure of the embryos to complete differentiation and morphogenesis are discussed.

Preliminary results on embryo rescue for circumventing hybridization barriers in Asparagus.

Garden asparagus, Asparagus officinalis, is reproductively isolated from a related ornamental species with potential breeding value, Asparagus densiflorus cv. Sprengeri, by pre- and post-zygotic barriers. The latter barrier operates at the endosperm level five days after pollination in A. officinalis x A. densiflorus crosses. To try to circumvent this barrier, in vitro embryo rescue using ovule and ovary cultures was tested. Controlled interspecific crosses were made and 2,032 ovules and 826 ovaries were cultured three days after pollination under various culture media and incubation conditions. Ovaries cultured for 60 days became red (similar to mature fruits), but seed formation was incomplete. Transfer of ovules to other media was necessary to promote embryo development. The interspecific embryos increased their length from 35 microns at the initiation of culture to 1,900 microns after 120 days of culture, but seedlings were not obtained. Histological studies revealed differentiation of protoderm only. The possible causes of the failure of the embryos to complete differentiation and morphogenesis are discussed.

Cross-incompatibility between two sympatric polyploid Solanum species.

Preliminary results from a large number of reciprocal crosses between the closely related sympatric species S. gourlayi Hawkes (2n=4x=48) and S. oplocense Hawkes (2n=6x=72) indicated that they are difficult to hybridize. Pollen-pistil incompatibility barriers were detected via fluorescent microscopy. The cross incompatibility reaction occurred at three sites in 6x×4x crosses; on the stigma, in the first one-third of the style, and in the first two-thirds of the style. In the reciprocal 4x×6x crosses the incompatibility reaction invariably occurred in the ovary. Backcrosses of interspecific pentaploid hybrids (that were occasionally formed) to both parental populations were fully compatible, partially compatible, and fully incompatible with three sites of cross-incompatibility reaction similar to those observed in 6x×4x crosses, respectively. Both polyploid species were found to be selfcompatible, whereas their F1 hybrids were found to be self-incompatible. An hypothesis based on interactions of dominant cross-incompatibility (CI) genes in pistils and dominant specific complementary genes in pollen grains is postulated to explain these observations. The cross-incompatibility system that appears to be operating in nature between 4x S. gourlayi and 6x S. oplocense provides a way for gene exchange between sympatric populations without threatening the identity of either species.