Production of a triple mutant,chlorophyll-deficient,streptomycin-, and kanamycin-resistant Nicotiana tabacum,and its use in intergeneric somatic hybrid formation with Solanum melongena

Summary In order to produce a triple mutant, sexual crosses between a chlorophyll-deficient, streptomycin-resistant mutant of Nicotiana tabacum (SA) and a kanamycin-resistant transformant of N. tabacum (KR.) were carried out. From the offspring of this cross, a triple mutant (KR-SA) was selected. In N. tabacum KR-SA, chlorophyll deficiency is due to recessive mutation in the nuclear genome, streptomycin resistance is due to a dominant mutation in the chloroplast genome, and kanamycin resistance is shown to be a dominant nuclear marker. Cell suspension protoplasts of N. tabacum KRSA were fused with callus protoplasts of Solanum melongena by dextran treatment. Somatic hybrid plants were selected for streptomycin resistance and the ability to produce clorophyll in regenerated plants. By using this selection system, green plants were recovered from two colonies. When these green plants were then tested for kanamycin resistance, all analyzed plants carried this trait. In addition, the hybrid nature of these plants was confirmed by investigation of the peroxidase isozyme. The present results show that the use of N. tabacum KR-SA in studies of somatic hybridization makes it possible to select somatic hybrid plants easily and provides information of the N. tabacum genome.Chemical Regulation of Biomechanism, The Institute of Physical and Chemical Research, Wako 351-01, Japan     

Transmission of paternal chloroplasts in tobacco (Nicotiana tabacum)

Medgyesy et al. (1986, Mol. Gen. Genet. 204, 195–198) have described in Nicotiana plumbaginifolia and in an interspecific cross involving N. plumbaginifolia and N. tabacum a procedure for selecting cell lines derived from seedlings carrying paternal chloroplasts by taking advantage of a plastid-encoded mutation which confers resistance to streptomycin. We have extended their demonstration of occasional transmission of chloroplasts through pollen to the case of an intraspecific cross in N. tabacum. The line used as maternal parent, ITB19(sua), displayed a cytoplasmic male sterility due to the presence of a cytoplasm originating from N. suaveolens. The line used as paternal parent, SR1, was fertile and possessed mutant chloroplasts conferring resistance to streptomycin. From cell lines derived from 204 seedlings, three were regenerated into streptomycin-resistant buds. The plants derived from these three clones were male-sterile. Their progeny, after crossing with a wild type tobacco line, XHFD8, was resistant to streptomycin. Tests of resistance of the seedlings to tentoxin and restriction analyses of the chloroplast DNA indicated that two clones still had the maternal chloroplasts and were thus probably new streptomycin-resistant mutants, whereas the third one had acquired the chloroplasts of the paternal parent, but had retained the mitochondria of the maternal parent.Abbreviations cp-DNA chloroplast DNA - mt-DNA mitochondrial DNA - Np Nicotiana plumbaginifolia - Nt Nicotiana tabacum     

Cybridization in Nicotiana tabacum L. using double inactivation of parental protoplasts and post-fusion selection based on nuclear-encoded and chloroplast-encoded marker genes

An effective selection system preceded by double inactivation of parental protoplasts was used to transfer Nicotiana suaveolens Leh. cytoplasmic male sterility into a commercial tobacco (N. tabacum L.) breeding line. Mesophyll protoplasts from transformed plants of N. tabacum cultivar WZ2-3-1-1 possessing a neomycin phosphotransferase II gene were used as the nuclear donors, while those isolated from N. suaveolens plants carrying a chloroplast mutation for resistance to spectinomycin, induced using nitrosomethyl urea, were the cytoplasm donors in somatic cybridizations. Prior to fusion, nuclear donor protoplasts were inactivated with iodoacetamide or rhodamine 6G, while those of the cytoplasm donor were inactivated by X-irradiation. The resultant microcalli were cultured on a shoot regeneration medium containing both kanamycin and spectinomycin to select cybrids. Only regenerants that had typical characteristics of the N. tabacum cultivar were selected for transfer to the glasshouse. Four putative cytoplasmic male-sterile (CMS) plants, out of a total of 44 regenerated plants transferred to the glasshouse, were obtained. Intraspecific somatic transfers of the CMS trait between N. tabacum cultivars with distinctlydifferent morphologies using single inactivation and nonselective shoot regeneration medium were demonstrated. The implications of the results for practical tobacco breeding as a means of circumventing lengthy backcrossing procedures are discussed.     

A light sensitive recipient for the effective transfer of chloroplast and mitochondrial traits by protoplast fusion in Nicotiana

Summary A light sensitive mutant was used as a recipient in the transfer of chloroplasts from a wildtype donor. Gamma irradiated (lethal dose) mesophyll protoplasts of Nicotiana gossei were fused with mesophyll protoplasts of a N. plumbaginifolia line carrying light sensitive plastids from a N. tabacum mutant. After fusion, colonies containing wild-type plastids from the cytoplasm donor were selected by their green colour. Most of the regenerated plants had N. plumbaginifolia morphology, but were a normal green in colour. The presence of donor-type plastids was confirmed by the restriction pattern of chloroplast DNA in each plant analysed. These cybrids were fully male sterile with an altered flower morphology typical of certain types of alloplasmic male sterility in Nicotiana. The use of the cytoplasmic light sensitive recipient proved to be suitable for effective interspecific transfer of wild-type chloroplasts. The recombinant-type mitochondrial DNA restriction patterns and the male sterility of the cybrids indicated the co-transfer of chloroplast and mitochondrial traits. On leave from: Department of Genetics, Section of Biosciences, Martin Luther University, Domplatz 1, DDR-4020 Halle/ S., German Democratic Republic     

Anther cultures of Nicotiana tabacum L. mutants

Summary The theoretically expected and experimentally observed phenotypic ratios have been compared in populations of haploids derived from chlorophyll mutants of Nicotiana tabacum L. with a known genotypic constitution. The frequencies of mutant genotypes were significantly lower than the expected values, proving the existence of selection in a system of haploid embryoids developing in the anther.The anthers from M₁ plants of a diploidized Nicotiana tabacum haploid cv. Samsun, treated with various concentrations of N-nitroso-N-methylurea and n-butylmethane sulphonate, were cultivated in vitro. The number of anthers which gave rise to haploids (embryogenic anthers) was stimulated by lower concentrations of both the mutagens. The stimulation at the level of M₁ sporophyte is explained by internal genetic heterogeneity induced by adequate mutagen concentration. The average number of haploids per embryogenic anther decreased in all the treatments. The frequency of haploid plants of the mutant phenotypes increased with increasing mutagen concentration.     

Nucleus substitution between Petunia species using gamma ray-induced androgenesis

Summary The ovaries of two different Petunia species: Petunia hybrida (hort) and Petunia parodii (Steere) were irradiated with -ray doses ranging from 50 to 1,000 Gy before pollination. Seed setting occurred after 4 days preculture on a non-sterile medium. Ovaries transformed into fruits were then cultivated aseptically with the following results: (1) -ray doses ranging from 200 to 1,000 Gy led to the development of two types of plants: haploids 2n=x=7 and overdiploids 2n>2x=14. (2) The androgenetic origin of haploids was ascertained by using genetic markers. The origin of overdiploids is discussed. (3) Androgenetic haploids contained the chloroplasts of the irradiated female parent. No visible change of cp DNA patterns was observed after irradiation. (4) The four possible androgenetic events were successfully obtained between the two Petunia species: hybrida haploids with hybrida or parodii cytoplasm, and parodii haploids with parodii or hybrida cytoplasm.     

Development of four tobacco cytoplasmic male sterile sources using in vitro techniques

In the present paper are summarized our results on obtaining tobacco male sterile forms through interspecific hybridization. The wild species Nicotiana velutina, N. benthamiana, N. maritima, N. paniculata were used as cytoplasm donors and N. tabacum as the donor of the nucleus. Completely sterile hybrids from these combinations were obtained whose sterility was overcome through the use of tissue culture. Stem parenchyma grown in vitro on MS medium was used for inducing callus, and for organogenesis and rooting. The regenerants obtained were mixoploid. Male sterile plants were obtained in BC₁P₂ progenies from the combinations between N. velutina × N. tabacum, N. benthamiana × N. tabacum and in R₂ progenies from N. maritima × N. tabacum and N. paniculata × N. tabacum. The observed male sterility was preserved in BC₁P₂-BC₇P₂ progenies and was identified as cytoplasmic male sterility (CMS) because it was inherited only throuth the female parent.     

Evidence for spontaneous diploid androgenesis in Brassica napus L.

Summary Seeds of androgenetic origin were obtained among the F₁ progenies of two crosses between resynthesized and cultivated forms of Brassica napus. The high-erucic, white-flowered, resynthesized line No7076 acted as the female, and the zero-erucic, yellow-flowered, cultivars Topas and Puma, as males. No androgenetic seeds were obtained in the reciprocal crosses. Resynthesized rape could thus be of potential use for the production of androgenetic plants. Of special interest is the high frequency (21%) of spontaneous androgenesis observed in one of the two crosses. One plant, determined from erucic acid content and flower colour analysis as androgenetic, had a diploid chromosome number. Further knowledge about the genetic control of spontaneous androgenesis in the present material and the origin of the cytoplasm in androgenetic plants are required in order to exploit this phenomenon in practical plant breeding.     

Maternal haploids of Petunia axillaris (Lam.) B.S.P. via culture of placenta attached ovules

Summary Hybridization of Petunia axillaris and P. parodii with Nicotiana tabacum was attempted using the method of in vitro pollination and fertilization. Seedlings were produced when the Petunia species and N. tabacum were used as the maternal parents; however, most of these had the identical somatic chromosome complement of the maternal parent. With crosses involving P. axillaris as the maternal parent, a low frequency of haploids was also produced. Due to the potential of haploids in basic and applied genetic research, additional experiments were carried out to determine whether in vitro pollination was necessary to stimulate haploid production and to more closely define the optimal time for ovule excision and culture. Four treatments were applied to accomplish these objectives. They were: placentas cultured prior to the time of anthesis, with and without pollination, and placentas cultured after the time of anthesis, with and without pollination. In vitro pollination had no effect on the frequency of haploids produced. Placenta attached ovules cultured prior to the time of anthesis produced significantly more haploids than those cultured after anthesis. The preanthesis treatment produced a frequency of 6.5 haploids per 100 ovaries cultured. The culture of placenta attached ovules provides an alternative to anther culture as a means for haploid production.The investigations reported herein were supported by USDA/SEA/CRGO Project 59-2213-1-1-613-0 and the paper (No. 84-3-36) is published with the approval of the Director of the Kentucky Agricultural Experiment StationThe authors are Graduate Research Assistant and Professor, respectively, Department of Agronomy, University of Kentucky, Lexington 40546-0091. The research reported in this paper is in partial fulfillment of the PhD requirements for the senior author     

Restoration of fertility in cytoplasmic male sterile (CMS) Nicotiana Sylvestris by fusion with X-irradiated N. tabacum protoplasts

Summary Restoration of male fertility was achieved by fusing protoplasts from male sterile (CMS) Nicotiana sylvestris plants with X-irradiated protoplasts derived from fertile N. tabacum plants. The CMS N. sylvestris plants were derived from a previous somatic hybridization experiment and contained alien (Line 92) cytoplasm. About one quarter of the regenerated plants were found to be cybrids. i.e. they consisted of N. sylvestris nuclei combined with all or some components of N. tabacum cytoplasm. In one half of these cybrids male fertility was restored to different levels. The chloroplasts of the two parental donors differ in respect to tentoxin sensitivity: chloroplasts of CMS N. sylvestris are sensitive while those of N. tabacum are insensitive. It could therefore be demonstrated that there was an independent segregation of chloroplast type and male fertility/sterility: several somatic cybrids were male fertile but tentoxin sensitive and others were tentoxin insensitive yet they were male sterile. Only in about one half of the somatic cybrids was male fertility restored together with restoration to tentoxin insensitivity.     

Several nuclear genes control both male sterility and mitochondrial protein synthesis in Nicotiana sylvestris protoclones

Summary Male sterile plants appeared in the progeny of three fertile plants obtained after one cycle of protoplast culture from a fertile botanical line and two androgenetic lines ofNicotiana sylvestris. These plants showed the same foliar and floral abnormalities as the cytoplasmic male sterile (cms) mitochondrial variants obtained after two cycles of culture. We show that male sterility in these plants is controlled by three independent nuclear genes,ms1, ms2 andms3, while no changes can be seen in the mitochondrial genome. However, differences were found between thein organello mitochondrial protein synthesis patterns of male sterile and parent plants. Two reproducible changes were observed: the presence of a new 20 kDa polypeptide and the absence of a 40 kDa one. Such variations were described previously in mitochondrial protein synthesis patterns of the cms lines. Fertile hybrids of male sterile plants showed normal synthesis patterns. The male sterile plants are thus mutated in nuclear genes involved in changes observed in mitochondrial protein synthesis patterns.     

Subcellular location of lincomycin resistance in Nicotiana mutants

Summary Lincomycin-resistant Nicotiana plumbaginifolia plastid mutants were considered also to carry mitochondrial mutations on the basis of their ability to grow in the dark under selective conditions. To clarify the role of mitochondria, individual protoplasts of the green, lincomycin-resistant N. plumbaginifolia mutant LR400 were microfused with protoplasts of the N. tabacum plastid albino line 92V37, which possesses N. undulata cytoplasm. The production of lincomycin-resistant albino cybrid lines, with N. undulata plastids and recombinant mitochondria, strongly indicated a determining role for mitochondria in the lincomycin resistance. Sequence analysis of the region encompassing putative mutation sites in the 26S rRNA genes from the LR400 and several other lincomycin-resistant N. plumbaginifolia mutants revelaed, however, no differences from the wild-type sequence. As an alternative source of the resistance of the fusion products, the N. tabacum fusion partner was also taken into account. Surprisingly, a natural lincomycin resistance of tobacco was detected, which was inherited as a dominant nuclear trait. This result compromises the interpretation of the fusion data suggested above. Thus, to answer the original question definitively, the mutant LR400 was crossed as a female parent with a N. plumbaginifolia line carrying streptomycin-resistant N. tabacum plastids. Calli were then induced from the seedlings. Occasional paternal plastid transmissions were selected as streptomycin-resistant calli on selective medium. These cell lines were shown by restriction enzyme analysis to contain paternal plastids and maternal mitochondria. They were tested for greening and growing ability in the presence of lincomycin. These resistance traits proved to be genetically linked and exclusively located in the plastids.EMBL accession number X68710     

Effect of ploidy and homozygosity on transgene expression in primary tobacco transformants and their androgenetic progenies

Expression of a transgene is rarely analysed in the androgenetic progenies of the transgenic plants. Here, we report differential transgene expression in androgenetic haploid and doubled haploid (DH) tobacco plants as compared to the diploid parental lines, thus demonstrating a gene dosage effect. Using Agrobacterium-mediated transformation, and bacterial reporter genes encoding neomycin phosphotransferase (nptII) and β-glucuronidase (uidA/ GUS), driven respectively by the mas 1′ and mas 2′ promoters, we have generated more than 150 independent transgenic (R₀) Nicotiana tabacum plants containing one or more T-DNA copies. Transgene analyses of these R₀, their selfed R₁ lines and their corresponding haploid progenies showed an obvious position effect (site of T-DNA insertion on chromosome) on uidA expression. However, transgene (GUS) expression levels were not proportional to transgene copy number. More than 150 haploids and doubled haploids, induced by treatment with colchicine, were produced from 20 independent transgenic R₀ plants containing single and multiple copies of the uidA gene. We observed that homozygous DH plants expressed GUS at approximately 2.9-fold the level of the corresponding parental haploid plants. This increase in transgene expression may be attributed mainly to the increase (2-fold) in chromosome number. Based on this observation, we suggest a strong link between chromosome number (ploidy dosage effect) and transgene expression. In particular, we demonstrate the effect on its expression level of converting the transgene from the heterozygous (in R₀ plants) to the homozygous (DH) state: e.g. an increase of 50% was observed in the homozygous DH as compared to the original heterozygous diploid plants. We propose that ploidy coupled with homozygosity can result in a new type of gene activation, creating differences in gene expression patterns. Received: 27 April 1998 / Accepted: 12 August 1998     

Somatic hybridization in Nicotiana: Segregation of organellar traits among hybrid and cybrid plants

Protoplasts from a nitrate reductase-deficient mutant of Nicotiana tabacum L. were fused with protoplasts from a stamen-less, cytoplasmically malesterile cultivar of tobacco containing the cytoplasm from N. suaveolens Lehm. Plants were regenerated from the fused protoplasts and characterized with respect to stamen development, chromosome number, and chloroplast composition. Of 29 regenerated plants, stamen production was restored in 26 plants and pollen production in 22. One plant was male sterile and two plants have never flowered. Analysis of the electrophoretic mobility of ribulose-1,5-bisphosphate carboxylase (RuBPcase) showed that 19 of the plants contained RuBPcase of the N. suaveolens type, six plants contained enzyme of the N. tabacum type, and four plants contained both types. Analysis of resistance to tentoxin in seedlings from 20 of the plants demonstrated that 14 had N. suaveolens-type chloroplasts, three had N. tabacum type, and three contained both types. Many of the plants which produced stamens and pollen still contained chloroplasts of the N. suaveolens type. Thus, the trait of cytoplasmic male sterility in tobacco is not an expression of the type of chloroplast genetic material.     

Expression of nuclear and chloroplastic genes coding for fraction-1 protein in somatic hybrids of Nicotiana tabacum + rustica

In the sexual interspecific cross, Nicotiana rustica L.xN. tabacum L., N. rustica can serve as the female but not as the male parent. By fusion of protoplasts, the barrier to fertilization was overcome and somatic hybrids containing N. tabacum cytoplasm were produced as shown by isoelectric focusing of the Fraction-1 protein (F-1-protein). All somatic hybrids displayed polypeptides of the large subunit of F-1 protein (which is coded by the chloroplast genome) characteristic of only one or the other parental species. Two hybrids had large subunits of the N. tabacum type and two hybrids had those of the N. rustica type. Three hybrids contained three smallsubunit polypeptides (coded by the nuclear genome), one being characteristic of N. rustica, one characteristic of N. tabacum, and one with an isoelectric point common to both species. A fourth hybrid contained only two small-subunit polypeptides of the N. tabacum type but in a F-1 protein macromolecule whose large subunits were of the N. rustica type. One somatic hybrid was self-fertile and its F₂ progeny contained large- and small-subunit polypeptides indistinguishable in their isoelectric points from those in the parent F₁ hybrid. All somatic hybrids showed an aneuploid chromosome number and morphological characteristics intermediate between those of N. rustica and N. tabacum.     

Transmission of paternal chloroplasts in Nicotiana

Summary Transmission of paternal chloroplasts was observed in Nicotiana, considered to inherit organelles in a strictly maternal way. Plants carrying streptomycin resistant plastids were used as pollen donors. Cell lines with paternal plastids in the offspring were selected as green (resistant) sectors on calli induced from the seedlings on streptomycin-containing media. The presence of paternal plastids in the regenerated plants was confirmed by restriction analysis. In the Nicotiana plumbaginifolia xN. plumbaginifolia Np(SR1)3 and the N. plumbaginifolia Np(gos)29 xN. tabacum SR1 crosses 2.5% and 0.07% of the offspring were found to contain paternal (tabacum) plastids, respectively. These plants, however, carried maternal mitochondria exclusively. This sexual cybridization method offers a simple way to transfer chloroplasts solely, a goal not accessible by protoplast fusion.     

Fertile somatic hybrids between transgenicNicotiana tabacum and transgenicN. debneyi selected by dual-antibiotic resistance

Summary A simple, yet effective selection system was used to produce fertile somatic hybrids betweenNicotiana tabacum andN. debneyi. This approach utilized transgenic antibiotic-resistantN. tabacum andN. Debneyi as donor plants for mesophyll protoplast fusions. Thirteen somatic hybrid plants were regenerated from calli capable of growth on medium containing both antibiotics. The majority of the hybrids displayed a range of leaf and floral morphologies and growth habits that were intermediate to those of the parental species, and had chromosome numbers varying from amphidiploid (2n = 96) to hypoaneuploid (2n = 60). Isoenzyme and RFLP analysis demonstrated the presence and expression of nuclear genes from both parents in all of the hybrids. Most plants are fully fertile. Thus, these plants differ from the malesterile tobacco cybrids and alloplasmic lines produced by transferring theN. debneyi cytoplasm to tobacco. A nonrandom pattern of cytoplasmic segregation in the fusion products occurred with a bias towards the presence ofN. debneyi cp and mtDNA. Evidence for the presence of rearranged or recombinant cp and mtDNA in some of the hybrids was obtained. The somatic hybrids were successfully backcrossed to theN. tabacum parent and are now being tested for immunity to black root rot, a trait specific toN. debneyi, but not existent in theN. tabacum parental line.     

Restoration of Fertility in Cytoplasmic Male-Sterile Nicotiana tabacum (Cytoplasm N. bigelovii) by Protoplast Fusion with X-Irradiated Protoplasts of N. tabacum, SR-1

The ‘donor–recipient’ fusion method was usedto investigate the intraspecific transfer of organelles andorganelle-encoded traits from donor to recipient Nicotiana speciesunder conditions which were selective for chloroplast transfer.An alloplasmic male sterile line of N. tabacum carrying thecytoplasm of N. bigelovii as the recipient and N. tabacum SR-1,a mutant with maternallyinherited streptomycin resistance, asthe cytoplasm donor were used. Organelle composition of 13 cybridplants was investigated by analysis of tentoxin and streptomycinsensitivities, chloroplast and mitochondrial DNA restrictionpatterns, and alloplasmic male sterility Chloroplast DNA analysisand the tentoxin test both showed that all 13 cybrid plantspossessed chloroplasts from the N. tabacum, SR-1 parent only.Analysis of mitochondrial DNA from second generation plantsderived from 11 of the original cybrid plants indicated verylittle heterogeneity with nine of the plants expressing pureparental-type mtDNAs. Although conditions were selective fordonor-type chloroplast transfer, the results indicate the generationof novel cytoplasmic combinations; pure donor chloroplasts incombination with either pure or recombinant-type recipient mitochondria.Our results support previous findings that the ‘donor-recipient’fusion method can be used to restore fertility of CMS linesof Nicotiana Key words: Nicotiana, cytoplasmic male sterility, chloroplast DNA, mitochondrial DNA, somatic hybridization     

Morphological characteristics and chloroplast DNA distribution in different cytoplasmic parasexual hybrids of Nicotiana tabacum

Summary Protoplast fusion makes possible the fusion of two different cytoplasms, allowing genetical analysis of cytoplasmic factors. Two varieties of Nicotiana tabacum differing by their cytoplasms have been used. Techne, the first variety, obtained by an interspecific cross between N. debneyi (female) and N. tabacum (male) is characterized by the nuclear tabacum genome inside the debneyi cytoplasm. Techne plants present abnormal flowers with cytoplasmic male sterility (cytoplasmic marker) and sessile leave (nuclear marker). Techne leaf protoplasts were fused with leaf protoplasts of N. tabacum var. Samsun (or Xanthi). The last variety is characterized by petiolated leaves and normal flowers, because it possesses the nuclear tabacum genome associated with the tabacum cytoplasm. The nuclear marker (leaf shape) and the cytoplasmic one (flower shape inducing male sterility or fertility) have been used to distinguish among the whole regenerated plants the somatic nuclear hybrids and the cytoplasmic hybrids (cybrids) displaying the nuclear phenotype of one of the two parents associated with a modified flower type, intermediate between the parental ones.The chloroplastic (cp) DNA contained in each parent has been specifically identified by using EcoRI restriction nuclease and gel electrophoresis. EcoRI fragment patterns of cp DNA isolated from the first progeny of the regenerated cytoplasmic hybrids revealed that only one of the two parental cp DNAs is present in all cases; neither mixture of both parental cp DNAs nor recombinant cp DNA molecules were observed. This indicates that a specific elimination of one or the other parental cp DNAs occurs after the initial mixing of the cytoplasms. The study of the association of the modified flower type with the cp DNA isolated from the corresponding plant showed that cp DNA seems independent from the mechanism of cytoplasmic male sterility in tobacco.     

Nicotiana tabacum mutants with chloroplast encoded streptomycin resistance and pigment deficiency

Summary Callus ofNicotiana tabacum SRI, a mutant with maternally inherited streptomycin resistance, was induced from leaf sections. Callus pieces were mutagenised with N-ethyl-N-nitrosourea and inoculated onto a shoot-induction medium on which calli are normally green. White callus sectors were observed in the mutagenised cultures, and white and variegated shoots were regenerated from these sectored calli. The SR1-A10 line regenerated a chimeric shoot with white leaf margins. The chimeric shoot was grafted onto a normal green rootstock, grown into a flowering plant in the greenhouse, and crosses were made. The SRI-A15 line was crossed using flowers formed on albino plants grown in sterile culture. Pigment deficiency was maternally inherited in both lines. Physical mapping of the chloroplast genome of the SR1-A15 mutant by SalI, PstI and BamHI restriction endonucleases did not reveal any difference between the SR1-A15 and the parental SRI chloroplast genomes.