Somatic hybridization in mint: identification and characterization of Mentha piperita (+) M. spicata hybrid plants

 Twenty eight somatic hybrid plants were identified following protoplast fusions between peppermint (Mentha piperita L. cv Black Mitcham), producing high-quality oil, and spearmint (Mentha spicata L. cv Native Spearmint), likewise producing high-quality oil and also possessing resistance to verticillium wilt. Prior to fusion, peppermint protoplasts were subjected to iodoacetic acid to inhibit cell division. Protoplasts of peppermint and spearmint were fused using polyethylene glycol plus DMSO. Fusion products were cultured according to an efficient protoplast-to-plant-cycle protocol developed for peppermint. Using this protocol, iodoacetic acid-treated peppermint protoplasts were not able to divide, whereas untreated spearmint protoplasts had the ability to produce callus but not shoots. Therefore, selection of somatic hybrid calli was based on the presumed capability of hybrid cells to form calli and shoots. Shoots in vitro were initially identified as hybrids using RAPD profiles. Subsequently, observations on morphology, chromosome counts, and Southern-hybridization patterns confirmed their hybrid status. The results of verticillium tests revealed that 18 somatic hybrids were more susceptible than Native Spearmint, while hybrid II-14 had a level of susceptibility intermediate between that of the fusion parents. Oil-analysis of hybrid plants indicated that they all have a GC-profile typical of spearmint oil. Received: 8 February 1997 / Accepted: 9 April 1997     

Somatic hybrids of Datura innoxia Mill.+Datura discolor Bernh. and of Datura innoxia Mill.+Datura stramonium L. var tatula L.

Summary Following fusion of protoplasts from a chlorophyll-deficient diploid mutant of Datura innoxia Mill. which can be regenerated to shoots, with green wild-type protoplasts of Datura stramonium L. var. tatula L. which can not, it was possible to isolate 49 green hybrid calli on agar medium. Most of these somatic hybrid calli gave rise to leaves and shoots. The chromosome numbers of the somatic hybrids were determined: 15 were tetraploid (amphidiploid), 24 hexaploid, and the other showed an aneuploid chromosome number.In a similar experiment protoplasts of the Datura innoxia mutant were fused with green wild-type protoplasts of Datura discolor Bernh. which are also not able to be regenerated, four green calli were obtained from which leaves and shoots developed after some transfers on agar medium. Three of them showed the amphidiploid (48) chromosome number, whereas one possessed an aneuploid number of 46 chromosomes.After transfer of rooted shoots to soil flowering plants could be obtained in both combinations. The habits of the somatic hybrids in both combinations were intermediate between the habits of the respective parental plants.Dedicated to my father, Prof. Dr. Theodor Schieder, on the occasion of his 70th birthday.     

Hybridisation experiments with protoplasts from chlorophyll-deficient mutants of some Solanaceous species

Following fusion between protoplasts from two different chlorophyll-deficient diploid mutants of Datura innoxia Mill. it was possible to select 33 green hybrid calli on agar culture medium. Half of the somatic hybrids gave rise to leaves and some to shoots. The chromosome number of 20 somatic hybrids was determined: five were tetraploid, eight hexaploid, three octoploid, and four showed an aneuploid chromosome number. After transfer of the shoots of the five tetraploid hybrids to soil they developed roots. In control experiments in which protoplasts of the two mutants were cultured either as a mixture without being treated with the fusion agent, or cultured separately, no green callus could be obtained. Similar experiments involving protoplasts from one chlorophyll-deficient mutant of Datura innoxia, on the one hand, and those from similar mutants of Nicotiana sylvestris Spegazz. et Comes and Petunia hybrida, on the other, yielded no green somatic hybrid although hybrid protoplasts could be detected.     

Allotriploid somatic hybrids of diploid tomato (Lycopersicon esculentum Mill.) and monoploid potato (Solanum tuberosum L.)

Allotriploid somatic hybrids were obtained from fusions between protoplasts of diploid tomato and monohaploid potato. The selection of fusion products was carried out in two different ways: (1) The fusion of nitrate reductase-deficient tomato with potato gave rise only to hybrid calli if selection was performed on media lacking ammonium. Parental microcalli were rarely obtained and did not regenerate. (2) The fusion of cytoplasmic albino tomato with potato gave rise to albino and green hybrid calli and plants. Allotriploids were identified from the two somatic hybrid populations by counting chloroplast numbers in leaf guard cells and by flow cytometry of leaf tissue. Although some pollen fertility of allotriploids and pollen-tube growth of tomato, potato andLycopersicon pennellii into the allotriploid style were observed, no progeny could be obtained. The relevance of allotriploid somatic hybrids in facilitating limited gene transfer from potato to tomato is discussed.     

Development and evaluation of a storage root-bearing sweetpotato somatic hybrid between <Emphasis Type="Italic">Ipomoea batatas</Emphasis> (L.) Lam. and <Emphasis Type="Italic">I. triloba</Emphasis> L.

A storage root-bearing somatic hybrid was produced for the first time by protoplast fusion between sweetpotato (Ipomoea batatas (L.) Lam.) cv. Kokei No. 14 and its wild relative I. triloba L. Protoplasts isolated from embryogenic suspension cultures of Kokei No. 14 were fused with petiole protoplasts of I. triloba L. using polyethylene glycol-mediated protocol. Fusion products were cultured in a modified Murashige and Skoog medium containing 0.05 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg l⁻¹ kinetin. A total of 176 plants were obtained from 42 out of 134 calluses derived from fused protoplasts, and 91 of these plants were confirmed to be somatic hybrids through peroxidase isozyme, random amplified polymorphic DNA, amplified fragment length polymorphism, and cytological analyses. Upon transfer into soil and grown in the greenhouse and then to the field, 100% survival was observed. A single plant, designated KT1, was found to produce storage roots. Genomic in situ hybridization analysis confirmed presence of chromosomes from both parents and recombinant chromosomes in KT1. Drought tolerance, dry matter content, soluble sugar content, and fertility of this somatic hybrid were evaluated for potential use in sweetpotato breeding.     

Selection of somatic hybrids after fusion of protoplasts from Datura innoxia Mill. and Atropa belladonna L.

After fusion of protoplasts from a diploid (2n=24) and a tetraploid (4n=48) chlorophyll-deficient mutant of Datura innoxia Mill. with diploid (2n=72) green wild-type protoplasts of Atropa belladonna L. thirteen somatic hybrids could be selected, most of which had already started to produce leaves and shoots. Hybrid calli were recognizable by the production of hairs, typical for Datura innoxia, and the green colour, derived from Atropa belladonna. Further proof for the hybrid nature was furnished by cytological investigations. The metaphase chromosomes of both species are easily distinguishable in their size: chromosomes of Datura innoxia are about twice as large as those of Atropa belladonna. The chromosome numbers of the hybrids varied from ca. 84 to ca. 175.Abbreviations BAP 6-benzylaminopurine - NAA -naphthalene acetic acid     

A Lactuca universal hybridizer, and its use in creation of fertile interspecific somatic hybrids

A Lactuca sativa cv. Ardente line heterozygous for a gene encoding resistance to kanamycin, a positive and dominant trait, was crossed with cv. Girelle, which is heterozygous for a recessive albinism marker. The resulting seeds yielded 25% albino seedlings, of which 50% were also resistant to kanamycin. Such plantlets (KR, a) grown in vitro were used for preparation of universal hybridizer protoplasts, since green buds that can develop on kanamycin containing-medium should result from fusion with any wild-type protoplast. To test the practicability of this selection scheme, we fused L. sativa KR, a protoplasts with protoplasts derived from various wild Lactuca as well as various other related species. Protoplast-derived cell colonies were selected for resistance to kanamycin at the regeneration stage. Green buds were regenerated after fusion with protoplasts of L. tatarica and of L. perennis. So far, 9 interspecific hybrid plants have been characterized morphologically. In addition, random amplified polymorphic DNA (RAPD) analysis with selected primers confirmed that these plants are indeed interspecific hybrids. Some plants are female-fertile and production of backcross progenies with L. sativa is in progress. Since many desirable traits such as resistances to viruses, bacteria and fungi (Bremia lactucae) have been characterized in wild Lactuca species, the use of somatic hybridization in breeding programmes now appears a practical possibility.     

Somatic hybridization between birdsfoot trefoil (Lotus corniculatus L.) and L. conimbricensis Willd

Summary Somatic hybrid plants were produced by fusion of birdsfoot trefoil (Lotus corniculatus) cv Leo and L. conimbricensis Willd. protoplasts. Birdsfoot trefoil etiolated hypocotyl protoplasts were inactivated with iodoacetate to inhibit cell division prior to fusion with L. conimbricensis suspension culture protoplasts. L. conimbricensis protoplasts divided to form callus which did not regenerate plants. Thus, plant regeneration from protoplast-derived callus was used to tentatively identify somatic hybrid cell lines. Plants regenerated from three cell lines exhibited additive combinations of parental isozymes of phosphoglucomutase, and L. conimbricensis-specific esterases indicating that they were somatic hybrids. The somatic chromosome number of one somatic hybrid was 36. The other somatic hybrid exhibited variable chromosome numbers ranging from 33 to 40. These observations approximate the expected combination of the birdsfoot trefoil (2n=4x=24) and L. conimbricensis (2n=2x=12) genomes. Somatic hybrid flowers were less yellow than birdsfoot trefoil flowers and had purple keel tips, a trait inherited from the white flowered L. conimbricensis. Somatic hybrids also had inflorescence structure that was intermediate to the parents. Fifteen somatic hybrid plants regenerated from the three callus lines were male sterile. Successul fertilization in backcrosses with birdsfoot trefoil pollen has not yet been obtained suggesting that the hybrids are also female sterile. This is the first example of somatic hybridization between these two sexually incompatible Lotus species.Formerly USDA-ARS, St. Paul, Minn, USA     

Mixing of maize and wheat genomic DNA by somatic hybridization in regenerated sterile maize plants

Intergeneric somatic hybridization was performed between albino maize (Zea mays L.) protoplasts and mesophyll protoplasts of wheat (Triticum aestivum L.) by polyethylene glycol (PEG) treatments. None of the parental protoplasts were able to produce green plants without fusion. The maize cells regenerated only rudimentary albino plantlets of limited viability, and the wheat mesophyll protoplasts were unable to divide. PEG-mediated fusion treatments resulted in hybrid cells with mixed cytoplasm. Six months after fusion green embryogenic calli were selected as putative hybrids. The first-regenerates were discovered as aborted embryos. Regeneration of intact, green, maize-like plants needed 6 months of further subcultures on hormone-free medium. These plants were sterile, although had both male and female flowers. The cytological analysis of cells from callus tissues and root tips revealed 56 chromosomes, but intact wheat chromosomes were not observed. Using total DNA from hybrid plants, three RAPD primer combinations produced bands resembling the wheat profile. Genomic in situ hybridization (GISH) using total wheat DNA as a probe revealed the presence of wheat DNA islands in the maize chromosomal background. The increased viability and the restored green color were the most-significant new traits as compared to the original maize parent. Other intermediate morphological traits of plants with hybrid origin were not found.     

Chromosomes are eliminated in the symmetric fusion between <Emphasis Type="Italic">Arabidopsis </Emphasis><Emphasis Type="Italic">thaliana</Emphasis> L. and <Emphasis Type="Italic">Bupleurum scorzonerifolium</Emphasis> Willd.

In order to investigate chromosome elimination in symmetric somatic hybridization between Bupleurum scorzonerifolium and Arabidopsis thaliana, protoplasts were isolated from suspension cultures of both A. thaliana and B. scorzonerifolium parents. Biparental protoplasts were mixed at a rate of 1.5:1 and fused with PEG-method. After protoplast fusion, the products were cultured in the P5 liquid medium for microcallus formation. Single cell lines formed from microcalli after subculturing on the MB1 (Xia and Chen, Plant Sci 120:197–203, 1996) solid medium. The putative somatic hybrid cell lines were identified by cytological and molecular analysis. Of the 132 somatic cell lines generated, 16 were identified as somatic hybrids, with the phenotypes resembled B. scorzonerifolium parent. These hybrids showed a complete set of B. scorzonerifolium chromosome and 0–2 small chromosome(s) of A. thaliana. A few of them showed nuclear and cytoplasmic SSR fragments of A. thaliana. These hybrid cell lines could differentiate to green spots, buds/leaves through complementation of regeneration ability. The chromosomes elimination of A. thaliana was discussed. Wang Minqin and Zhao Junsheng contributed equally to the work.     

Somatic hybridization between Nicotiana rustica and N. sylvestris

Protoplasts isolated from cell cultures of chlorophyll-deficient Nicotiana rustica cv. chlorotica and wild-type N. sylvestris were fused. The scheme for selection of somatic hybrids was based on the inability of the protoplast-derived colonies of the parental species to turn green; N. sylvestris protoplasts also had a very low plating efficiency in the medium employed. A total of 777 green colonies which were presumptive hybrids were isolated within four weeks of the fusion experiments. One hundred and eight green colonies formed shoots in vitro and 16 lines were rooted and grown in the greenhouse. Each of these hybrid plants displayed vegetative and floral traits intermediate to those of the parental species, except for plant height which in almost all cases was greater in the hybrids. Isozyme analyses by gel electrophoresis and isoelectric focussing of the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (RUBPCase) demonstrated that the nuclear genomes of both parents were expressed by the hybrids. Each of the eight somatic hybrid plants analyzed expressed only the N. rustica chloroplast genome as shown by isoelectric focussing of the large subunit of RUBPCase. This study demonstrated the value of N. rustica cv. chlorotica as a parental line in somatic hybridization with N. sylvestris and it might have widespread use with wild-type lines of other species.     

Regeneration of somatic hybrid plants formed between Lycopersicon esculentum and L. pennellii

Summary Somatic hybrid plants have been regenerated following polyethylene glycol mediated fusion of leaf mesophyll protoplasts from tomato and protoplasts from Lycopersicon pennellii callus. Three different cultivars of tomato were used as sources of protoplasts: Early Girl, Manapal, and UC82B. Fusions were performed between protoplasts of these tomato cultivars and protoplasts of L. pennellii, and between protoplasts of the cultivars and protoplasts of L. pennellii that had been exposed to 3 or 6 krads of gamma radiation. Somatic hybrid plants were identified on the basis of heterozygous isozyme banding patterns, and leaf and flower morphology. Somatic hybrid plants were regenerated following fusion of tomato protoplasts with either untreated or irradiated L. pennellii protoplasts. All were heterozygous for isozyme loci on five different chromosomes. Regenerated somatic hybrids showed inheritance of either or both parental chloroplast genomes, but predominantly the L. pennellii mitochondrial genome. The regenerated somatic hybrid plants exhibited reduced fertility, less than 20% viable pollen. A total of 34 somatic hybrid calli were identified. Of these, 21 regenerated shoots, and 7 produced seed following manual pollinations.     

Regeneration of plants from Ipomoea cairica L. protoplasts and production of somatic hybrids between I. cairica L. and sweetpotato, I. batatas (L.) Lam.

Plants were regenerated from mesophyll protoplasts of Ipomoea cairica L., a wild relative of sweetpotato (Ipomoea batatas (L.) Lam.), and somatic hybrids between I. cairica L. and sweetpotato cv. Xushu 18 were obtained by PEG-mediated method. I. cairica L. protoplasts were isolated from the leaves of in vitro grown plants and cultured in a modified MS medium containing 0.05 mg l⁻¹ 2,4-D and 0.5 mg l⁻¹ kinetin. Nine weeks after plating, the obtained small calluses up to about 2 mm in diameter were transferred to solid MS medium supplemented with 0.05 mg l⁻¹ 2,4-D and 0.5 mg l⁻¹ kinetin for callus proliferation. Three weeks after transfer, the calluses were transferred to MS medium supplemented with 0–1.0 mg l⁻¹ IAA and 1.0–3.0 mg l⁻¹ BAP and further to hormone-free MS medium for plant regeneration. The frequencies of calluses forming plants ranged from 6.0% to 41.3% based on the different concentrations of IAA and BAP, and 2.0 mg l⁻¹ BAP gave the highest regeneration frequency of protoplast-derived calluses in I. cairica L.. The regenerated plants, when transferred to soil, showed 100% survival. No morphological variations were observed. Mesophyll protoplasts of I. cairica L. were fused with protoplasts isolated from embryogenic suspension cultures of Xushu 18 by PEG-mediated method. The fused products were cultured with the best protoplast culture system of I. cairica L.. Finally, 114 plants were produced from 63 of the 182 calluses derived from the fused protoplasts, and 46 plants of them were confirmed to be somatic hybrids through peroxidase isozyme, RAPD, morphological and cytological analyses.     

Intergeneric Somatic Hybrid Plants of Nicotiana tabacum L. and Lycium barbarum L. by Protoplast Electrofusion

Mesophyll protoplasts of Nicotiana tabacum L. and protoplasts from cell suspension of Lycium barbarum L. were heterofused by electrofusion with a frequency of ca. 4%--5%. One hundred cell lines were selected at random identified by isozyme analysis with peroxidase and superoxide dismatase, and the differences from their parent were found. Results indicated that 9 cell lines expressed enzymatic bands characteristic of both parents. Five of the 9 cell lines were highly morphogenic and regenerated numerous young shoots that manifested morphological traits specific to both parents. However, these shoots never grew up or regenerate roots. Esterase analysis of leaf material from the regenerants of 5 morphogenic hybrid cell lines demonstrated that two of them (NL4 and NL8) expressed an unique hybrid band which were not shown in either parents. Cytological observation on parental and NL4 hybrid cell lines revealed that the somatic chromosome number of NL4 varied from 58 to 80, significantly higher than that of either parents. Ribosomal DNA analysis of NL4 and NL8 showed that NL8 covered all fragments of both parents: NL4 did not have the fragments characteristic of Lyciurn barbarurn L. Both hybrids had new fragments, suggestive of intermolecular recombination of rDNAs of the parents. Four normal plants morphologically similar to tabacco parent were obtained from hybrid cell hne NL4, which survived after being transferred to soil. Cytological analysis of root-tips from one of the plants indicated that it has ca. 58 chromosomes. This paper also discussed the problems on the production frequency and incompatibility of somatic cell hybrid.     

Regeneration of protoplasts of dihaploid potato plants bleached by a herbicide (SAN 6706)

Summary Bleaching of dihaploid potato shoots by application of the herbicide SAN 6706 did not affect the ability for cell division of subsequently isolated mesophyll protoplasts. The development of green calluses from bleached protoplasts shows that bleaching is reversable. The positive effects of SAN 6706 on cell division activity in protoplasts are discussed and the application of the bleaching method in experiments to select somatic hybrids after protoplast fusion.     

Molecular and morphological characterization of somatic hybrids between <Emphasis Type="Italic">Solanum tuberosum</Emphasis> L. and <Emphasis Type="Italic">S. etuberosum</Emphasis> Lindl.

Interspecific potato somatic hybrids between Solanum tuberosum L. (di)haploid C-13 and 1 endosperm balance number non-tuberous wild species S. etuberosum Lindl. were produced by protoplasts electrofusion. The objective was to transfer virus resistance from this wild species into the cultivated potatoes. Post-fusion products were cultured in VKM medium followed by regeneration of calli in MS₁₃ K medium at 20°C under a 16-h photoperiod, and regenerants were multiplied on MS medium. Twenty-one somatic hybrids were confirmed by RAPD, SSR and cytoplasm (chloroplast/mitochondria) type analysis possessing species-specific diagnostic bands of corresponding parents. Tetraploid nature of these somatic hybrids was determined through flow cytometry analysis. Somatic hybrids showed intermediate phenotypes (plant, leaves and floral morphology) to their parents in glass-house grown plants. All the somatic hybrids were male-fertile. ELISA assay of somatic hybrids after artificial inoculation of Potato virus Y (PVY) infection reveals high PVY resistance.     

Improved isolation of dihaploidsolanum tubersoum protoplasts and the production of somatic hybrids between dihaploidS. tuberosum andS. brevidens

Summary A modified protoplast isolation technique, applicable to a range of dihaploidSolanum tuberosum genotypes, has been developed. A combination of high calcium and high pH was used to fuse mesophyl protoplasts of dihaploidS. tuberosum (PDH40) and the diploid wild speciesS. brevidens. Large numbers of colonies were obtained after fusion and putative hybrids selected on the basis of phenotype from regenerated shoots. From these, 11 somatic hybrid plants have been identified by their isoenzyme patterns and morphologic characteristics. Four of these hybrids had the expected chromosome number of 48. The approach of mass culture after fusion followed by selection of hybrids from regenerated shoots and the application of somatic hybridization to potato breeding are discussed.     

Interspecific somatic hybrids between wild potato Solanum acaule Bitt. and anther-derived dihaploid potato (Solanum tuberosum L.)

Solanum acaule Bitt. is a disomic tetraploid (4x) wild potato species which is resistant to several potato diseases. Introgression of disease resistance and abiotic stress tolerance to the tetrasomic tetraploid (4x) cultivated potato (S. tuberosum L.) gene pool via crossing has been limited due to the difference in the endosperm balance number. In the present study, protoplast fusion was applied to produce hexaploid (6x) somatic hybrids between the parental lines, tetraploid (4x) S. acaule and two anther-derived dihaploid (2x) lines of S. tuberosum cv. White Lady. One callus (0.4%) of a total of 229 calli obtained regenerated into shoots in the fusion combination S. acaule (+) White Lady 15.dh.8.2.2. All the regenerated shoots were confirmed to be interspecific somatic hybrids using species-specific RAPD markers. In another fusion combination, S. acaule (+) White Lady 7.dh.23.1.1, fifteen calli (5%) regenerated into a total of sixteen shoots from 289 calli. All the analysed somatic hybrids between S. acaule and S. tuberosum were hexaploid. The mean DNA content (2C value) of the combination S. acaule (+) White Lady 15.dh.8.2.2 somatic hybrids (4.55 pg), was approximately the sum (4.69 pg) of the DNA contents of the parental lines, S. acaule (2.95 pg) and S. tuberosum (1.74 pg). In the greenhouse, the two somatic hybrids analysed were normal in their morphological characteristics and more vigorous than their parental lines. Most of the morphological characteristics were closer to the tetraploid S. acaule than to the dihaploid S. tuberosum. The interspecific somatic hybrids are currently being tested for frost tolerance and glycoalkaloid composition. Received: 19 January 1998 / Revision received: 27 March 1998 / Accepted: 20 April 1998     

Genome composition of asymmetric hybrids in relation to the phylogenetic distance between the parents. Nucleus-chloroplast interaction

Summary A series of fusion experiments were performed between protoplasts of a cytoplasmic albino mutant of tomato, Lycopersicon esculentum (ALRC), and gamma-irradiated protoplasts of L. hirsutum and the Solanum species S. commersonii, S. etuberosum and S. nigrum. These species were chosen for their different phylogenetic relationships to tomato. In all fusion combinations except from those between ALRC and S. nigrum, green calli were selected as putative fusion products and shoots regenerated from them. They were subsequently analyzed for their morphology, nuclear DNA composition and chloroplast DNA origin. The hybrids obtained between ALRC and L. hirsutum contained the chloroplasts of L. hirsutum and had the flower and leaf morphology of L. esculentum. After Southern blot analysis, using 13 restriction fragment length polymorphisms (RFLPs) randomly distributed over all chromosomes, all hybrids showed L. esculentum hybridization patterns. No chromosomes of L. hirsutum were found. These results indicate that these hybrids were true cybrids.The putative asymmetric hybrids, obtained with S. commersonii and S. etuberosum, showed phenotypic traits of both parents. After hybridization with species-specific repetitive nuclear DNA probes it was found that nuclear material of both parents was present in all plants. In the case of S. nigrum, which combination has the greatest phylogenetic distance between the fusion parents, no hybrid plants could be obtained. The chloroplast DNA of all hybrid plants was of the donor type suggesting that chloroplast transfer by asymmetric protoplast fusion can overcome problems associated with large phylogenetic distances between parental plants.     

Interspecific somatic hybridization between lettuce (Lactuca sativa) and wild species L. virosa

Somatic hybrids between cultivated lettuce (Lactuca sativa) and a wild species L. virosa were produced by protoplast electrofusion. Hybrid selection was based on inactivation of L. sativa with 20mM iodoacetamide for 15 min, and the inability of L. virosa protoplasts to divide in the culture conditions used. Protoplasts were cultured in agarose beads in a revised MS media. In all 71 calli were formed and 21 of them differentiated shoots on LS medium containing 0.1mg/l NAA and 0.2mg/l BA. Most regenerated plants exhibited intermediate morphology. These plants were confirmed as hybrids by isoenzyme analysis. The majority of somatic hybrids had 2n=4x=36 chromosomes, and had more vigorous growth than either parent. Hybrids had normal flower morphology, but all were sterile.