Note on the presence of the sym-1 gene in Vicia faba hampering its symbiosis with Rhizobium leguminosarum

Summary A symbiotically defective mutant in Vicia faba is described. This character is controlled by a recessive gene named sym-1. Reciprocal graft experiments indicate that only the root system controls its expression. Depending on the strain of Rhizobium, the root phenotype can be either Fix⁺ Nod^- or Nod^-.     

The effect of exogenous DNA on the nodulation of a non-nodulating line of Pisum sativum L.

Summary The nodulation response of a non-nodulating field pea line (Pisum sativum L., PRL-H722) following treatment with DNA from an effectively nodulating cultivar (Trapper) has been investigated. DNA was introduced to the non-nodulating cultivar via imbibition and germination of seeds in DNA solutions under aseptic conditions. The DNA preparations used were >5×10⁶ Daltons, had a smooth melting profile showing 40% hyperchromicity with a Tm=70–72°C in 0.1×SSC⁰ buffer, and 260/280 nm ratio of >1.8. A significant correction (²(I d.f.) = 2032^**) of the mutant phenotype was observed in seeds treated with wild-type DNA. The nodulation response in treated plants was not observed in subsequent generations suggesting that the change observed did not involve integration of genetic information into the genome of germ line cells.NRCC no. 20986     

Cytological and microsatellite mapping of the genes determining liguleless phenotype in durum wheat

Liguleless phenotypes of wheat lack ligule and auricle structures on all leaves of the plant. Two recessive genes principally control the liguleless character in tetraploid wheat. The F₂ progenies of k17769 (liguleless mutant)/Triticum dicoccoides and k17769/T. dicoccum segregated in a 15:1 ratio, whereas the F₂ progenies of k17769/T. durum and k17769/T. turgidum segregated in a 3:1 ratio. A new gene, lg₃, was found on chromosome 2A. Segregation of F₂ progenies between k17769 and chromosome substitution lines for homoeologous group 2 chromosomes suggested that the liguleless genotype had occurred by mutation at the lg₃ locus on chromosome 2A, and then by mutation at the lg₁ locus on chromosome 2B, in the process of domestication of tetraploid wheat. The gene (lg₁) was linked to Tc₂ (11.9 cM), which determines phenol colour reaction of kernels, on the long arm of chromosome 2B. The distance of lg₁ to the centromere was found to be 60.4 cM, and microsatellite mapping established the gene order, centromere – Xgwm382 – Xgwm619 – Tc₂ – lg₁ on the long arm of chromosome 2B.     

Two host genes in Vicia faba for nodulation deficiency with strain specificity for Rhizobium leguminosarum

In greenhouse tests, two inbred lines of faba bean, I 40 and I 25, proved to be nodulation resistant and F₂ progenies were used to determine the genetic control of this nodulation deficiency. The defect is strain-specific for the Rhizobium strains St 48 and St 53. In inbred line I 40, the deficient nodulation character is controlled by a dominant gene which is called Sym-2, while another recessive gene sym-3 is assumed for line I 25; Sym-2 is epistatic over sym-3.     

Pattern analysis and genotypic x environment interactions in faba bean (Vicia faba L.) populations

Summary Twenty-nine populations of faba bean (Vicia faba L. subsp. major, equina and minor) of mediterranean origin were evaluated for the genetic variability of 12 biomorphological characters at four locations in Apulia and Basîli cata regions (Italy) in 1983–84.A pattern analysis appoach, based on classification and ordination procedures is presented for to characterize differences among population in mean performance and response across environments. Informations about G × E interaction were obtained.Research work supported by CNR, Italy. Special grant I.P.R.A. Sub-project 1. Paper N. 1099.     

Investigation of the fenugreek (Trigonella foenum-graecum L.) genotypes for fresh weight, seed productivity, symbiotic activity, callus formation and aceumulation of steroids

Summary As a result of the analysis of 31 fenugreek (Trigonella foenum-graecum L.) genotypes we demonstrated that subsp. indica surpass significantly subsp. foenum-graecum in fresh weight, seed yield, nodulation ability, resistance to coot rot and the duration of the interstage and growing periods. There was no difference between the subspecies for leafiness and biochemical composition of seeds (concentrations of diosgenin and steroid glucosides, nitrogen, fat, inhibitors of tripsin and hemotripsin). The most promising genotypes (as compared with the standard cv. Nakhichevanskaya Shambala) yield 1650–1938 g/m² of fresh weight, 98.2–116.1 g/m² of seeds and accumulate 1.45–1.64% of diosgenin in seeds. High positive correlations (r=+0.53 ... +0.84; P_o<0.01) were found between fresh weight, seed yield, plant leafiness and height. Inoculation of the cv. Nakhichevanskaya Shambala with Rhizobium meliloti strains 851 and 852 increased its fresh weight by 94.5% and 93.6% and seed yield by 83.1% and 60.5%, respectively. Significant correlations of the nodule number with the fresh weight and seed yield (r=+0.70 and +0.77; P_o<0.01) allow us to assume that selection for increased symbiotic activity may be useful for improving the fenugreek productivity. The intersubspecies hybrids (four genotypes) surpass the subsp. indica (six genotypes) for the sizes of calli formed in presence of 2.4-D and kinetin. The sizes of calli formed by ten analysed genotypes in presence of BAP and kinetin (or 2.4-D) were negatively correlated with the nodule number (or fresh weight).     

Oil content, tocopherol composition and fatty acid patterns of the seeds of 51 Cannabis sativa L. genotypes

The oil content, the tocopherol composition, the plastochromanol-8 (P-8) content and the fatty acid composition (19 fatty acids) of the seed of 51 hemp (Cannabis sativa L.) genotypes were studied in the 2000 and 2001 seasons. The oil content of the hemp seed ranged from 26.25% (w/w) to 37.50%. Analysis of variance revealed significant effects of genotype, year and of the interaction (genotype × year) on the oil content. The oil contents of the 51 genotypes in 2000 and 2001 were correlated (r = 0.37**) and averaged 33.19 ± 1.45% in 2000 and 31.21 ± 0.96% in 2001. The -tocopherol, -tocopherol, -tocopherol, P-8- and -tocopherol contents of the 51 genotypes averaged 21.68 ± 3.19, 1.82 ± 0.49, 1.20 ± 0.40, 0.18 ± 0.07 and 0.16 ± 0.04 mg 100g^–1 of seeds, respectively (2000 and 2001 data pooled). Hierarchical clustering of the fatty acid data did not group the hemp genotypes according to their geographic origin. The -linolenic acid yield of hemp (3–30 kg ha^–1) was similar to the -linolenic acid yield of plant species that are currently used as sources of -linolenic acid (borage (19–30 kg ha^–1), evening primrose (7–30 kg ha^–1)). The linoleic acid yield of hemp (129–326 kg ha^–1) was similar to flax (102–250 kg ha^–1), but less than in sunflower (868–1320 kg ha^–1). Significant positive correlations were detected between some fatty acids and some tocopherols. Even though the average content of P-8 in hemp seeds was only 1/120^th of the average -tocopherol content, P-8 content was more closely correlated with the unsaturated fatty acid content than -tocopherol or any other tocopherol fraction. The average broad-sense heritabilities of the oil content, the antioxidants (tocopherols and P-8) and the fatty acids were 0.53, 0.14 and 0.23, respectively. The genotypes Fibrimon 56, P57, Juso 31, GB29, Beniko, P60, FxT, Félina 34, Ramo and GB18 were capable of producing the largest amounts of high quality hemp oil.     

Effect of Gluconacetobacter diazotrophicus and Trichoderma viride on soil health, yield and N-economy of sugarcane cultivation under subtropical climatic conditions of India

Intensive cropping and exhaustive nature of sugarcane–wheat–rice cropping system in the Indo-Gangetic Plains of South Asia have led to the depletion of soil organic carbon content and inherent soil fertility resulting in a serious threat to the sustainability of these production systems. Bioagents like Gluconacetobacter diazotrophicus and Trichoderma viride have great potential to restore soil fertility and promote sugarcane growth. Field experiments, therefore, have been conducted to study the integrated effect of bioagents (G. diazotrophicus and T. viride), Farm Yard Manure (FYM) and fertilizer N on sugarcane rhizosphere, crop yield and N economy for two crop cycles during 2004–2006 and 2005–2007 crop seasons at Lucknow, in the middle Indo-Gangetic plain region. Both bioagents could survive and colonize sugarcane rhizosphere and FYM improved their colonization. Enhanced soil microbial population and microbial carbon (SMC) and nitrogen (SMN) with increasing N level were probably due to more available N in the soil. FYM/bioagents amendment further enhanced the microbial carbon. The uniform increase in the fraction of SMC and SMN of total organic carbon indicated that immobilization/mineralization was being maintained in the soil where enhanced microbial biomass might act later as a source of nutrients.Bioagents ammended FYM enhanced the uptake of N, P and K in sugarcane at all the levels of fertilizer N. It was mainly due to the enhanced nutrient availability in the rhizospheric soil as the soil organic C and available N, P and K content increased with the application of bioagents/FYM. A saving of 76.3 kg N ha⁻¹ was envisaged by the use of G. diazotrophicus inoculated FYM with marginal (2.4 t ha⁻¹) decline in the cane yield. Application of T. viride enriched FYM, however, brought economy in the use of fertilizer N by 45.2 kg ha⁻¹ and also increased the yield by 6.1 t ha⁻¹compared to the control treatment. Overall, strategic planning in terms of an integrated application of these bioagents/manures with fertilizer N will not only sustain soil fertility but will also benefit farmers in terms of reducing their dependence and expenditure on chemical fertilizers.     

Genetic specificity of nodulation

Summary A genetic system is proposed for establishing desirable strains of Rhizobium japonicum in soybean fields against the competition of aggressive but less productive indigenous strains. This system requires soybean cultivars which substantially exclude the indigenous strains while selectively admitting specific introduced strains. Rhizobium strains were identified which were infective, both in sand and in soil culture, with a host genotype (rj₁rj₁) which excludes most indigenous strains. Rhizobium infectivity was associated, for the first time, with the propensity to induce symptoms caused by rhizobitoxine production by the bacteria. The basis of this association is unknown. A possible enabling role for rhizobitoxine, or a precursor or derivative of rhizobitoxine, in the infection process is discussed.     

A single dominant gene in McCall soybean prevents effective nodulation with Rhizobium fredii USDA257

Summary Rhizobium fredii USDA257 will effectively nodulate Asiatic and unimproved soybean cultivars, such as Peking, but most of the highly selected North American cultivars, such as McCall, produce at most rudimentary, ineffective nodules. In R. fredii USDA 257, a locus containing 6 open reading frames is responsible for this cultivar specific incompatibility. To examine the genetic control of incompatability on the part of the host, the soybean cultivars Peking and McCall were crossed to produce five F₁ progeny. These plants and their selfed progeny were tested for nodulation with USDA257. Resistance to nodulation was found to be conditioned by a single dominant gene. These results indicate that, in soybean, strain specific resistance to nodulation can result from gene(s)-for-gene interactions.     

Nano-silver pulse treatments inhibit stem-end bacteria on cut gerbera cv. Ruikou flowers

Nanometer-sized silver (Ag⁺) particles (NS) are used in various applications as an anti-microbial. Effects of NS (2–5 nm diam.) pulse solution treatments on vase life of cut gerbera (Gerbera jamesonii) cv. Ruikou flowers were investigated. Compared with the control [pulsed with deionised water (DI) and subsequently held in DI] pulsing for 24 h with 5 mg/L NS solution followed by holding in DI maintained water uptake and extended vase life. From in vitro and microscopy assessments, NS pulse treatment inhibited bacteria growth in the vase solution and at cut stem ends during the first 2 d of the vase period.     

Changes in external and internal color during postharvest ripening of ‘Manila’ and ‘Ataulfo’ mango fruit and relationship with carotenoid content determined by liquid chromatography–APcI-time-of-flight mass spectrometry

High-performance liquid chromatography–atmospheric pressure chemical ionization (APcI⁺)-time-of-flight mass spectrometry studies revealed that all-trans-β-carotene and the dibutyrates of all-trans-violaxanthin and 9-cis-violaxanthin were the main carotenoids in ‘Ataulfo’ and ‘Manila’ mango fruit mesocarp. The concentration of these carotenoids in the mesocarp was measured during fruit ripening and correlated with colorimetric changes of mesocarp and epidermis. The lowest and highest concentrations of all-trans-β-carotene, all-trans-violaxanthin and 9-cis-violaxanthin (as dibutyrates) during the ripening of ‘Manila’ mango were 0.25 × 10⁻³ to 35.57 × 10⁻³, 0.40 × 10⁻⁵ to 31.97 × 10⁻³ and 0 to 16.81 × 10⁻³ g kg⁻¹ of fresh mesocarp, respectively. For ‘Ataulfo’ they were 2.55 × 10⁻³ to 39.72 × 10⁻³, 0.16 × 10⁻³ to 15.00 × 10⁻³ and 0.21 × 10⁻³ to 7.48 × 10⁻³ g kg⁻¹ of fresh mesocarp, respectively. The concentration of these carotenoids increased in an exponential manner during fruit ripening in ‘Ataulfo’ and in an exponential or second-order polynomial manner in ‘Manila’. The highest correlation coefficients were obtained for the relationships between the mesocarp and epidermis a* and h° color values and the concentration of the evaluated carotenoids in both mango cultivars (R = 0.81–0.94). Equations to predict the concentration of the most important carotenoids in ‘Manila’ and ‘Ataulfo’ mango fruit on the basis of their mesocarp and epidermis color values were obtained.     

Influence of genotype and culture conditions on the production of embryos from anthers of tetraploid wheat (Triticum turgidum)

Summary Anther culture of 10 tetraploid wheat (Triticum turgidum) genotypes and two backcross lines representing a wide range of genetic variation was studied in a randomized block design with three replications. Each replication consisted of 2 pots with 3 plants. The day length was 16h and temperature 25° C/15°C for day/night in a controlled greenhouse where the anther donor plants were grown. Two different treatments were used for anther culture. The first one was potato 2 medium (Chuang et al., 1978) modified by adding 0.5 mg/l glutamine and solidified by gelrite (4g/l) (Henry & De Buyser, 1981). Cultures were incubated in light (15 E m^–2 S^–1) at 26°C at 16h day length. The second medium was described by Fadel & Wenzel (1990), differing from the first by the nature of the sugar (maltose) and consistency of the medium (semiliquid by ficoll). Anther cultures were incubated in the dark at 28°C. The study of about 1300 anthers per genotype and treatment showed that both genotype and treatment affected embryo formation of tetraploid wheat. The backcross lines exhibited significant differences for androgenic abilities when compared to their common parent. Most of the genotypes were medium dependent for androgenesis and revealed significant interactions with the two treatments. Five green plantlets were regenerated and fertile doubled haploid plants were obtained from three out of the 12 studied genotypes.     

Resynthesizing <Emphasis Type="Italic">Brassica napus</Emphasis> from interspecific hybridization between <Emphasis Type="Italic">Brassica rapa</Emphasis> and <Emphasis Type="Italic">B. oleracea</Emphasis> through ovary culture

Using three varieties of Brassica rapa, cv. Hauarad (accession 708), cv. Maoshan-3 (714) and cv. Youbai (715), as the maternal plants and one variety of B. oleracea cv. Jingfeng-1 (6012) as the paternal plant, crosses were made to produce interspecific hybrids through ovary culture techniques. A better response of seed formation was observed when ovaries were cultured in vitro at 9–12 days after pollination on the basal MS and B5 media supplemented with 6-benzylaminopurine (BA) and naphthylacetic acid (NAA). The best response was observed for cross 714×6012 with the rate of seeds per ovary reaching 43.0%. Seeds for cross 715×6012 showed the best germination response (66.7%) on the regeneration medium (MS+1.0 mg l^–1 BA+0.05 mg l^–1 NAA). In all three cross combinations, good response in terms of root number and length of plants was observed on the root induction medium (MS+1.0 mg l^–1 BA+0.1 mg l^–1 NAA). A better response was observed for the regenerated plants cultured for 14 days than for 7 days. The ovary-derived plants with well-developed root system were hardened for 8 days and their survival rate reached over 80%. Cytological studies showed that the chromosome number of all plants tested was 19 (the sum of both parents), indicating that these regenerated plants were all true hybrids of B. rapa (n = 10) × B. oleracea (n = 9). The regenerated plants were doubled with colchicine treatment, and the best response in the crosses 708×6012, 714×6012 and 715×6012 was observed when treated with 170 mg l^–1 colchicine for up to 30 h and their doubling frequency reached 52, 56 and 62%, respectively.     

Root peroxidases and Rhizobium, Bradyrhizobium nodulation affinities of Phaseolus vulgaris L. and P. acutifolius A. Gray congruity backcross populations

Four parents [P. acutifolius var.acutifolius (A₁₉), P. a.var. latifolius `Serowi' (A<sub>9</sub>),P. a. var. latifolius(A<sub>10</sub>), and P. vulgaris `Red Cloud' kidney (V₁)] were used to create six interspecific recurrent and congruity backcross pedigrees (V₁ × A₉, A₉ × V₁, V₁ × A₁₀, A₁₀ × V₁, V₁ × A₁₉, A₁₉ × V₁) for evaluation of Rhizobium, Bradyrhizobium nodulation affinities and root peroxidase banding patterns. Most primary hybrids nodulated with all Rhizobium, Bradyrhizobium strains, while recurrent generations nodulated with strains of the recurrent parent, suggesting a number of independent loci and random assortment for strain-specific nodulation. A proximity matrix of nodulation phenotypes produced a cluster with V₁, two recurrent and two congruity backcross hybrids with V₁ as the cytoplasmic parent; a separate cluster with all primary interspecific and balanced, congruity backcross hybrids. A₉ and A₁₀were clustered the farthest away from V₁. Root peroxidase zymograms of primary hybrids were the summation of the parents and controlled by independent loci. Advanced hybrids exhibited new root peroxidases, matching those found for otherPhaseolus spp., including ancestralP. coccineus. A proximity matrix of root peroxidase phenotypes produced a cluster with only A₉ ³V₁F₁, a cluster containing only A₁₉ ³V₁ F₂, and a large cluster with all P. acutifoliusparents and hybrids with P. acutifolius as the cytoplasmic parent in balanced hybrids or the majority parent in unbalanced hybrids. Estimation of genetic distances showed primary, interspecific hybrids to be more closely related with the cytoplasmic parent. Recurrent and congruity backcross generations varied in genetic distances between each respective parent. This is the first report of congruity backcrossing creating hybrids with biochemical traits of nonparental species. This revised version was published online in August 2006 with corrections to the Cover Date.     

A stability analysis of time to flowering as a screen for responsiveness to temperature and photoperiod in cowpea (Vigna unguiculata)

Summary Twenty-one genotypes of cowpea (Vigna unguiculata), comprising landraces and varieties, were grown in 22 photothermal environments in Nigeria and Niger, West Africa, and a stability analysis of days from sowing to flowering (f) was carried out. Cowpeas are rarely insensitive to photoperiod; they are typically quantitative shortday plants wherein f is delayed when photoperiod (P) is longer than the critical photoperiod (P _c ). Therefore, in order to quantify genotypic variation in temperature sensitivity, genotype f was regressed against the mean trial f in circumstances where P c (i.e. approximately 13 hd^-1) and mean temperature (T) was between 19° and 28° C. Correspondingly, in order to assess genotypic variation in photoperiod sensitivity, trials where T was near optimal (25°–28° C) but where P ranged from 10–14.5 hd^-1 were used. These stability analyses detected no significant differences (P>0.05) between genotypes 9n temperature sensitivity but revealed significant differences (P<0.001) in photoperiod sensitivity. Regression coefficients from the stability analysis were strongly correlated (r=0.94, 19df) with a photoperiod sensitivity constant, c, determined from a photothermal flowering model. A stability analysis of f from field trials can therefore identify and quantify genotypic variation in response to temperature and photoperiod in cowpea.Abbreviations f days from sowing to flowering - P mean photoperiod - P _c critical photoperiod - P _ce ceiling photoperiod - T mean temperature - T _b base temperature - T _o optimum temperature - SDP short-day plant     

Influence of rare alleles of β‐carotene hydroxylase and lycopene epsilon cyclase genes on accumulation of provitamin A carotenoids in maize kernels

Vitamin A deficiency is one of the major health problems worldwide. Traditional yellow maize possesses very low provitamin A (proA) concentration in endosperm. The influence of rare alleles of β‐carotene hydroxylase (crtRB1) and lycopene epsilon cyclase (lcyE) genes capable of enhancing proA concentration was studied in four BC₂F₂ populations generated using subtropical inbreds and CIMMYT‐HarvestPlus lines. The occurrence of severe segregation distortion for crtRB1 gene was observed, while lcyE gene was segregated as per Mendelian ratio. Genotype with favourable allele of crtRB1 (CC) had a significant effect on β‐carotene (BC) (7.9‐fold), β‐cryptoxanthin (BCX) (twofold) and proA (5.5‐fold) accumulation, compared to unfavourable genotype (C⁺C⁺). Genotype with favourable allele of lcyE (LL) showed 2.1‐fold, 1.6‐fold and twofold significant enhancement in BC, BCX and proA, respectively, over unfavourable genotype (L⁺L⁺) in pooled analysis. Of the nine genotypes, double homozygote (CC/LL) had the highest mean BC (12.60 μg/g), BCX (4.44 μg/g) and proA (14.82 μg/g), and combined effect was significantly better than individual gene effects or any other combinations. The information generated here would be useful in designing strategy for proA enrichment in subtropical maize.     

Somatic in vitro culture response of Lolium perenne L.: genetic effects and correlations with anther culture

Summary Genotypic effects on callus induction and plant regeneration in callus, suspension and protoplast culture, and their correlations with both phenotypic and GCA-values for anther culture response, were studied using 21 genotypes of perennial ryegrass. Differences between genotypes accounted for approximately 40% of the total variation for callus induction and initial callus growth, and 59 and 83% of the variation in callus culture for regeneration percentage and percentage of green plants. Effects of genotypes were less pronounced in suspension culture, where suspensions from the same genotype often behaved differently. Some suspension cultures retained their capacity for green plant regeneration for almost two years, repeatedly producing 80–100% green regenerants during this period. Genotypes with high regeneration percentage and a large proportion of green plants from callus culture were also superior in suspension culture for both regeneration performance and longevity. Regeneration percentage and percentage of green plants were uncorrelated, and probably under different genetic control. While capacity for green plant formation from the different genotypes showed no correlation between anther culture and somatic in vitro culture, a positive correlation was observed between the regeneration percentages in somatic in vitro culture and anther culture (r=0.44^*–0.85^***), suggesting some common genetic control of the two systems.     

Associations and genetics of three components of slow rusting in leaf rust of wheat

Summary Forty F₆ lines, the two parental lines, and a susceptible check cultivar of wheat (Triticum aestivum L.) were inoculated in the young flag leaf stage with leaf rust (Puccinia recondita f.sp. tritici) and evaluated for latent period, receptivity, and uredinium size in a greenhouse experiment. Genotypic (r_g) and phenotypic (r_p) correlations between latent period and uredinium size were –0.81 and –0.62, respectively. A negative correlation (r_g=–0.50, r_p=–0.41) was found between latent period and receptivity and a positive correlation (r_g=0.28, r_p=0.26) between uredinium size and receptivity was found. Area under the disease progress curve (AUDPC) and final rust severity (FRS) obtained from a subsequent field study with common entries were negatively correlated with latent period and positively correlated with uredinium size. Correlations of receptivity with both AUDPC and FRS were not significant. The distributions of F₆ family mean uredinia size and latent period were continuous between slow rusting and fast rusting parents: however, the distribution for receptivity was discrete. Narrow-sense heritability estimates were 63%, 57%, and 47% for uredinium size, latent period, and receptivity, respectively. Estimates of the minimum number of effective factors were three for latent period and three or four for the uredinium size and receptivity. The components are controlled by closely linked genes or due to pleotropic effects of the same gene.Abbeviations AUDPC - Area under the disease progress curve - FRS - Final rust severity     

Production of intergeneric hybrid plants between <Emphasis Type="Italic">Sandersonia aurantiaca</Emphasis> and <Emphasis Type="Italic">Gloriosa rothschildiana</Emphasis> via ovule culture (Colchicaceae)

Intergeneric hybrid plants between Colchicaceous ornamental plants, Sandersonia aurantiaca and Gloriosa rothschildiana, have successfully been produced via ovule culture. After 5 days of reciprocal cross-pollination, a few pollen tubes were observed in the ovary. Although seeds were obtained in both reciprocal cross-combinations, they did not germinate under ex vitro conditions. Ovules with placental tissues isolated 14 days after cross-pollination of S. aurantiaca × G. rothschildiana were cultured on a medium containing 0.01 mg l^–1 each of -naphthaleneacetic acid (NAA) and 6-benzyladenine (BA), on which 41.5% of ovules swollen and produced callus-like structures within 10 weeks. When such swollen ovules were transferred to a medium containing 0.1 mg l^–1 each of NAA and BA, 7.5% of the initially cultured ovules produced rhizome-like structures within 6 weeks. Among the rhizome-like structures, those derived from two independent ovules (3.7% of the initially cultured ovules) produced multiple shoots following transfer to a medium containing 0.25 mg l^–1 NAA and 2.5 mg l^–1 BA. Multiple shoot-derived plantlets were established on a plant growth regulator-free medium, and they were successfully transplanted to pots. Early verification of their hybridity was accomplished by flow cytometry (FCM) analysis, chromosome observation and rDNA analysis.