Dihaploid Line Research Articles

Estimation of the efficiency of developing regenerative winter bread wheat lines in anther culture <i>in vitro</i>

The efficiency of the anther culture methodin vitrolargely depends on the responsiveness of varieties and hybrids to the induction of androgenesis. The purpose of the current study was to estimate the efficiency of haploproduction of regenerated lines (R0) of winter bread wheat developed by anther culturein vitroand grown in a greenhouse. To achieve this goal, there was set a task to conduct a morphometric estimation of the developed heads (R0) to identify their fertility level. The study was carried out in the laboratory of cell breeding of the FSBSI Agricultural Research Center “Donskoy” in 2021–2022. The objects of the study were 7 hybrid combinations of winter wheat, which were responsive to androgenesisin vitroin the culture of isolated anthers. As a result of induced androgenesis on an artificial nutrient medium, there were identified 333 neoplasms, represented by calluses, embryoids and embryogenic complexes. Green regenerated plants were vernalized in test tubes, cloned, diloidized, then planted into soil. There were obtained 13 haploid lines, after diploidization only 34 plants survived. The highest haploproductive capacity was established in the hybrid combination F3‘Rostovchanka 7xBezostaya 100’. In this combination, there were formed 8 green regenerants, 21 plants of which were developed together with clones, 18 of which survived. The cytometric estimation has shown, that 25 dihaploid (2n= 42) and 16 haploid (n= 21) wheat plants have been identified by hybrid combinations, including 13 dihaploids from the combination ‘Rostovchanka 7xBezostaya 100’. As a result of the structural analysis and morphometric estimation of the head there has been found a different fertility degree of androgenic regenerated plants. There have been established prospects of using the dihaploid line ‘79.2.1.2’ in further breeding work based on the hybrid combination F3‘Rostovchanka 7xBezostaya 100’ with the highest values of such yield elements as ‘number of heads’, ‘number and weight of grains per head’.

Green fluorescent protein (GFP) supports the selection based on callus vigorous growth in the somatic hybrids Solanum tuberosum L. + S. chacoense Bitt

Somatic hybridization based on protoplast fusion is a biotechnological tool enabling multiple resistance genes or traits to be transferred from wild relatives into crops. A key step for successful regeneration of hybrids is the selection of the heterokaryons. Selection can be done on specific culture media, by using intrinsic cellular or fluorescent markers, or specific traits of the fusion products, such as vigorous growth. More vigorous growth is one of the most common used criteria, since it does not require the use of mutants and allows faster selection of putative somatic hybrids. However, vigorous growth does not occur in all fusion combinations and often additional approaches have to be used (molecular markers, flow cytometry, chromosome counts, etc.) which are more costly and time consuming. There is, therefore, a need for the development of a faster, simpler, more cost-effective and reliable method for the selection of the heterospecific fusion products. Our study reports the effective use of the reporter gene gfp to confirm the vigorous growth as a reproducible selection for the somatic hybrids between Solanum tuberosum dihaploid line and the wild species S. chacoense. In vitro shoots of transgenic S. chacoense expressing the reporter gene gfp and a wild type dihaploid line of potato were used to isolate the mesophyll protoplasts. Electrofusion of mesophyll protoplasts and standard culture methods were applied. The S. chacoense gfp+ protoplasts were unable to regenerate and when parental protoplast and putative somatic hybrid cultures were monitored, the GFP was expressed and visualized microscopically only in hybrid regenerants showing vigorous growth. The results confirmed that vigorous growth can be used to select the fusion products between these two species. This strategy might be applied to other fusion combinations to improve somatic hybrid selection.

Genetic diversity among Italian melon inodorus (Cucumis melo L.) germplasm revealed by ISSR analysis and agronomic traits

The genetic relationships among 13 melon inodorus populations that were collected in southern Italy were assessed using 100 inter-simple-sequence repeat (ISSR) primers and 15 morphological traits. The dihaploid line Nad-1 and the cultivar Charentais-T, both of which belong to the botanical variety cantalupensis, were used as reference accessions in the molecular analysis. A total of 358 polymorphic bands were obtained from 39 of the 100 ISSR primers used, and 15 phenotypic traits were scored and used for genetic-similarity calculations and cluster analysis. The resulting dendrograms based on the ISSR and phenotypic data allowed almost all of the melon genotypes to be distinguished on the basis of the skin colour of the fruits. Mantel's test revealed a good correlation between the morphological and molecular data in their ability to detect genetic relationships among melon ecotypes (r = 0.50, P = 0.99). The data obtained confirm the effectiveness of this approach, and open new perspectives to reveal possible molecular associations with the phenotypic traits analysed.

The usefulness of the gfp reporter gene for monitoring Agrobacterium-mediated transformation of potato dihaploid and tetraploid genotypes

Potato is one of the main targets for genetic improvement by gene transfer. The aim of the present study was to establish a robust protocol for the genetic transformation of three dihaploid and four economically important cultivars of potato using Agrobacterium tumefaciens carrying the in vivo screenable reporter gene for green fluorescent protein (gfp) and the marker gene for neomycin phosphotransferase (nptII). Stem and leaf explants were used for transformation by Agrobacterium tumefaciens strain LBA4404 carrying the binary vector pHB2892. Kanamycin selection, visual screening of GFP by epifluorescent microscopy, PCR amplification of nptII and gfp genes, as well as RT-PCR and Southern blotting of gfp and Northern blotting of nptII, were used for transgenic plant selection, identification and analysis. Genetic transformation was optimized for the best performing genotypes with a mean number of shoots expressing gfp per explant of 13 and 2 (dihaploid line 178/10 and cv. 'Baltica', respectively). The nptII marker and gfp reporter genes permitted selection and excellent visual screening of transgenic tissues and plants. They also revealed the effects of antibiotic selection on organogenesis and transformation frequency, and the identification of escapes and chimeras in all potato genotypes. Silencing of the gfp transgene that may represent site-specific inactivation during cell differentiation, occurred in some transgenic shoots of tetraploid cultivars and in specific chimeric clones of the dihaploid line 178/10. The regeneration of escapes could be attributed to either the protection of non-transformed cells by neighbouring transgenic cells, or the persistence of Agrobacterium cells in plant tissues after co-cultivation.

Linhagens diaplóides de trigo: desempenho agronômico em dois locais do estado de São Paulo e tolerância à toxicidade de alumínio em laboratório

Foram avaliados 20 genótipos (18 linhagens diaplóides e as cultivares IAC-24 e IAC-370) de trigo em ensaios instalados em condição de sequeiro e solo ácido em Capão Bonito e em condição de irrigação por aspersão e solo corrigido em Tatuí, no período 2001-2003. A produção de grãos, outros componentes da produção, características agronômicas e resistência às doenças foram analisados. Estudou-se também a tolerância à toxicidade de alumínio em soluções nutritivas, em laboratório. A cultivar IAC-24 e as linhagens diaplóides (L3, L4, L5, L8, L14, L15, L16, L17, L18, L19 e L20), em Capão Bonito, destacaram-se quanto à produção de grãos, e em soluções nutritivas, foram as mais tolerantes à toxicidade de alumínio. Todos os genótipos exibiram porte semi-anão. As linhagens diaplóides L15 e L16 foram resistentes resistência ao acamamento; na cultivar IAC-370 observaram-se espigas mais compridas e maior número de grãos por espiga e por espigueta, a L8 o maior número de espiguetas por espiga e na L6 e L7, os grãos mais pesados. Todos os genótipos avaliados foram suscetíveis à mancha-da-folha. As linhagens L4, L14, L15, L16, L17 e L18 oriundas do cruzamento R27/GHL121//KAL/BB/3/BUC/BUL/4/IAC-24 foram resistentes à ferrugem-da-folha. Os genótipos mais produtivos em condição de sequeiro e solo ácido sem aplicação de calcário foram associados a plantas de porte semi-anão mais altas, exibindo tolerância à toxicidade de alumínio. Em solo corrigido e com irrigação por aspersão, os genótipos mais produtivos não estavam associados às plantas altas, com porte semi-anão, nem à tolerância ao alumínio.

Evaluation of Components of Fusarium Head Blight Resistance in Soft Red Winter Wheat Germ Plasm Using a Detached Leaf Assay

A large environmental influence on phenotypic estimates of disease resistance and the complex polygenic nature of Fusarium head blight (FHB) resistance in wheat (Triticum aestivum) are impediments to developing resistant cultivars. The objective of this research was to investigate the utility of a detached leaf assay, inoculated using inoculum from isolates of Microdochium nivale var. majus, to identify components of FHB resistance among 30 entries of U.S. soft red winter wheat in the 2002 Uniform Southern FHB Nursery (USFHBN). Whole plant FHB resistance of the USFHBN entries was evaluated in replicated, mist-irrigated field trials at 10 locations in eight states during the 2001-2002 season. Incubation period (days from inoculation to the first appearance of a dull gray-green water-soaked lesion) was the only detached leaf variable significantly correlated across all FHB resistance parameters accounting for 45% of the variation in FHB incidence, 27% of FHB severity, 30% of Fusarium damaged kernels, and 26% of the variation in grain deoxynivalenol (DON) concentration. The results for incubation period contrasted with previous studies of moderately resistant European cultivars, in that longer incubation period was correlated with greater FHB susceptibility, but agreed with previous findings for the Chinese cultivar Sumai 3 and CIMMYT germ plasm containing diverse sources of FHB resistance. The results support the view that the detached leaf assay method has potential for use to distinguish between specific sources of FHB resistance when combined with data on FHB reaction and pedigree information. For example, entry 28, a di-haploid line from the cross between the moderately resistant U.S. cultivar Roane and the resistant Chinese line W14, exhibited detached leaf parameters that suggested a combination of both sources of FHB resistance. The USFHBN represents the combination of adapted and exotic germ plasm, but four moderately resistant U.S. commercial cultivars (Roane, McCormick, NC-Neuse, and Pat) had long incubation and latent periods and short lesion lengths in the detached leaf assay as observed in moderately FHB resistant European cultivars. The dichotomy in the relationship between incubation period and FHB resistance indicates that this may need to be considered to effectively combine exotic and existing/adapted sources of FHB resistance.

Influence of near null magnetic field on in vitro growth of potato and wildSolanum species

The influence of near null magnetic field on in vitro growth of different cultures of potato and related Solanum species was investigated for various exposure times and dates. Potato (Solanum tuberosum L. cv. Désirée) in vitro cultures of shoot tips or nodal segments were used. Three different exposure periods revealed either stimulation or inhibition of root, stem, or leaf in vitro growth after 14 or 28 days of exposure. In one experiment the significant stimulation of leaf growth was also demonstrated at biochemical level, the quantity of chlorophyll a and b and carotenoids increasing more than two-fold. For the wild species Solanum chacoense, S. microdontum, and S. verrucosum, standardized in vitro cultures of nodal stem segments were used. Root and stem growth was either stimulated or slightly inhibited after 9 days exposure to near null magnetic field. Callus cultures obtained from potato dihaploid line 120/19 were maintained in near null magnetic field in 2 different months. For these experiments as well as for Solanum verrucosum, callus cultures recorded either slight inhibition or no effect on fresh weight. For all experiments significant growth variation was brought about only when geomagnetic activity (AP index) showed variations at the beginning of in vitro growth and when the explant had at least one meristematic tissue. Moreover longer maintenance in near null magnetic field, 28 days as compared to 14 days or the controls, can also make a difference in plant growth in response to geomagnetic field variations when static component was reduced to zero value. These results of in vitro plant growth stimulation by variable component of geomagnetic field also sustain the so-called seasonal "window" effect.

Comportamento de linhagens diaplóides de trigo em dois locais do Estado de São Paulo

Compararam-se 20 genótipos (18 linhagens diaplóides e as cultivares IAC-24 e IAC-289) de trigo em experimentos instalados em condição de sequeiro e solo ácido de Capão Bonito (1997-1999), e em condição de irrigação por aspersão e solo ácido com aplicação de calcário de Tatuí (1997-2000). Analisaram-se produção de grãos, outros componentes da produção, características agronômicas e resistência às doenças. Estudou-se também a tolerância ao alumínio em soluções nutritivas, em laboratório. As linhagens diaplóides 10 e 11 e a cultivar IAC-24, em Capão Bonito, e a cultivar IAC-24 e a linhagem diaplóide 19, em Tatuí, destacaram-se quanto à produção de grãos. A linhagem diaplóide 5 foi resistente ao acamamento, a 20 apresentou porte baixo e ciclo precoce para maturar, e a cultivar IAC-289 exibiu maior número de espiguetas por espiga e grãos mais pesados. Todos os genótipos avaliados mostraram-se suscetíveis à mancha-da-folha. Em relação à ferrugem-da-folha, todas as linhagens foram resistentes, exceto a 20 que foi moderadamente resistente. A cultivar IAC-24 e todas as linhagens diaplóides avaliadas apresentaram-se tolerantes à toxicidade de alumínio. Os genótipos mais produtivos em solo ácido e em cultivo de sequeiro apresentaram-se associados às plantas altas, com espigas compridas, maior número de grãos por espiga e por espigueta e tolerância ao alumínio. Em solo ácido, com aplicação de calcário e condição de irrigação por aspersão, os genótipos mais produtivos estavam associados às plantas altas (porte semi-anão) com os grãos pesados, não se correlacionando com a tolerância ao alumínio.

Production and characterization of “second generation” somatic hybrids derived from protoplast fusion between interspecific somatohaploid and dihaploidSolanum tuberosum L.

Protoplasts were fused to produce somatic hybrids between a triploid (2n=3x=32-34) interspecific somatohaploid betweenSolanum brevidens Phil. andS. tuberosum L., and a dihaploid (2n=2x=24) anther-derived line ofS. tuberosum cv. Van Gogh. A total of 265 plants were regenerated from protoplast fusion derived calli and their hybridity was verified using fusion partner specific RAPD markers. These “second generation” somatic hybrids were aneuploid pentaploids (2n=5x=51-65) with a 2C DNA content ranging from 3.36 to 4.43 pg, which corresponded to the sum of the 2C values of each of the fusion partners (somatohaploid: 2.22 pg; and the dihaploid line of cv. Van Gogh: 1.87 pg). Most of the “second generation” somatic hybrids were vigorous, but variable in morphology. They were extremely resistant to PLRV and they had tolerance to PVY infection derived from the somatohaploid fusion partner. Even though most of the “second generation” hybrids tuberized, the tuber morphology was variable and most were poorly shaped. InErwinia soft rot resistance tests, the tubers showed higher level of resistance than the tetraploidS. tuberosum cultivars, the dihaploidS. tuberosum fusion partners and the hexaploid somatic hybrids betweenS. brevidens andS. tuberosum. The “second generation” somatic hybrids were all male sterile and failed to produce berries or seeds.

Linhagens diaplóides de trigo: produção de grãos, características agronômicas e tolerância à toxicidade de alumínio

Compararam-se 18 linhagens de trigo diaplóides obtidas via cultura de anteras de plantas híbridas, em geração F1, e os cultivares Al Res 102/84 e IAC-24, em quatro ensaios instalados em condições de irrigação por aspersão e de sequeiro. Analisaram-se a produção de grãos, outros componentes da produção, características agronômicas e resistência à ferrugem-da-folha. Estudou-se também a tolerância ao alumínio em soluções nutritivas, em condição de laboratório. A linhagem diaplóide 5, provinda do cruzamento IAS-63/ALDAN "S"//GLEN/3/IAC-24, de porte baixo, mostrou resistência ao acamamento e ao agente causal da ferrugem-da-folha e tolerância à toxicidade de alumínio, destacando-se, ainda, quanto à produção de grãos. A linhagem 6 identificou-se como fonte genética de maior número de grãos por espigueta, porte baixo, resistência ao acamamento e à ferrugem-da-folha, e a linhagem 8 apresentou espigas mais compridas e maior número de espiguetas por espiga. Todos os genótipos avaliados, com exceção do cultivar IAC-287 (controle sensível) exibiram elevada tolerância à toxicidade de alumínio.

Transfer of resistance to potato leafroll virus, potato virus Y and potato virus X from Solarium brevidens to S. tuberosum through symmetric and designed asymmetric somatic hybridisation

SummaryResistance to potato leafroll virus (PLRV), potato virus Y (PVYo) and potato virus X (PVX) was studied in symmetric and asymmetric somatic hybrids produced by electrofusion between Solanum brevidens (2n=2×=24) and dihaploid S. tuberosum (2n=2×=24), and also in regenerants (B‐hybrids) derived through protoplast culture from a single somatic hybrid (chromosome number 48). All of the somatic hybrids between 5. brevidens and the two dihaploid lines of potato cv. Pito were extremely resistant to PLRV and PVYoand moderately resistant to PVX, irrespective of their chromosome number and ploidy level (tetraploid or hexaploid). Most (56%) of the asymmetric hybrids of irradiated S. brevidens and the dihaploid line of potato cv. Pentland Crown (PDH40) had high titres of PVYosimilar to those of PDH40, whereas the rest of the hybrids had PVYotitres less than a tenth of those in PDH40. Three B‐hybrids had a highly reduced chromosome number (27, 30 and 34), but were however as resistant to PLRV, PVYoand PVX as 5. brevidens. Two asymmetric hybrids and one B‐hybrid were extremely resistant to PLRV but susceptible to both PVY and PVX. The results suggested that resistance to PLRV in 5. brevidens is controlled by a gene or genes different from those controlling resistance to PVY and PVX, and the gene(s) for resistance to PVY and PVX are linked in S. brevidens.

Analysis of the mitochondrial DNA of the somatic hybrids of Solanum brevidens and S. tuberosum using non-radioactive digoxigenin-labelled DNA probes

Mitochondrial (mt) DNAs of somatic hybrids obtained by electrical and chemical fusion of mesophyll protoplasts of S. brevidens and a dihaploid line of S. tuberosum PDH 40 were analysed by Southern hybridization using the digoxigenin-labelled mtDNA sequences nad5 or orf25. In the Southern analysis of the hybrid mtDNA probed with nad5, most of the 19 hybrids analyzed had an RFLP pattern similar, but not identical, to one of the parents, S. tuberosum, PDH40. Nineteen percent of the hybrids had most of the S. brevidens fragments. Five of the hybrids had an identical RFLP pattern to either one of the parents while another two hybrids had novel RFLP patterns. Similar results were obtained by Southern analysis with orf25. These results clearly show that mtDNA rearrangements had occurred at a high frequency in the somatic hybrids. There were no differences in the frequencies of rearrangements observed between the hybrids regenerated from chemical and electrical fusions.

Species-specific sequences in the genus Solanum: identification, characterization, and application to study somatic hybrids of S. brevidens and S. tuberosum

To aid in the identification and analysis of somatic hybrids between potato (Solanum tuberosum, dihaploid line PDH 40) and the non tuber-bearing wild species S. brevidens, a series of species-specific repetitive DNA sequences have been isolated. This was accomplished by making libraries of HaeIII-digested total DNA of S. tuberosum and S. brevidens, by cloning fragments into the SmaI site of plasmid pUC18 and transforming them into E. coli (JM83). The S. brevidens library consisted of 1,000 recombinant clones, and that of S. tuberosum, 700. Nitrocellulose filters with recombinant clones were hybridised to nick-translated total DNA of S. brevidens and also S. tuberosum, and, following autoradiography, clones that hybridised strongly to the DNA of only one of the species were chosen. Two highly repeated S. brevidens clones (pSB1, 400 bp and pSB7,210 bp), one highcopy-number s. tuberosum clone (pST10, 200 bp) and one low-copy-number sequence of S. tuberosum (pST3, 1.5 kbp) were selected for further analysis by Southern hybridisation to digested total DNA. Clone pSB7 gave a ladder pattern on hybridisation to EcoR1-digested total DNA of S. brevidens, with signals at multiples of 200 bp DNA. Using these probes it was possible to verify the hybridity of putative hybrids of dihaploid S. tuberosum and S.brevidens, and to confirm by Southern analysis and by slot blots the parental genome dosage of hexaploid hybrids (two s. brevidens: one S. tuberosum, and vice-versa). The S. tuberosum-specific probe, pSTIO, hybridised with DNA of three other tuber-bearing wild species (S. hjertingii, S. capsicibaccatum and S. berthaultii). A squash-blot procedure was developed using the probes that would allow early identification of somatic hybrid callus. There are a number of useful applications of such species-specific probes in the identification and analysis of somatic hybrids.

Field assessment of dihaploid Solatium tuberosum and S. brevidens somatic hybrids

Following both chemical and electrical fusion of protoplasts of a dihaploid line of potato (Solanum tuberosum), (PDH40), with those of the wild species, Solanum brevidens, 11 and 40 somatic hybrid plants, respectively were obtained. Fifteen of these somatic hybrid genotypes and the two parents were studied further in a small field trial to assess field performance and phenotypic variability. In the UK, somatic hybrid plants are classified as genetically engineered organisms, and the UK Advisory Committee on Genetic Manipulation have imposed various restrictions on field experiments. Examination of the somatic hybrids in the field showed extensive phenotypic variability, and no two genotypes were identical. Some of the variation reflected changes in chromosome numbers, but other factors were also involved. Half the somatic hybrid genotypes produced tubers in the field, although the tubers were smaller and differed morphologically from those of PDH40. The results of the study suggest that the extent of somaclonal variation manifested in somatic hybrids is greater than that found in protoplast-derived plants of potato. The implications of this observation and the current regulations concerning field experiments of somatic hybrid plants in the UK are discussed.

Elimination of Solanum phureja nucleolar chromosomes in S. tuberosum + S. phureja somatic hybrids

The karyotype of the dihaploid SVP1 line of S. tuberosum (2n=2x=24) showed two nucleolar chromosomes with differently sized satellites. The diploid SVP5 line (2n=2x=24) and tetraploid regenerants of S. phureja had larger but similar satellites. Somatic hybrids between the diploid lines of these potato species with genome combinations 4 tub + 2 ph (plants 1-3), 2 tub + 4 ph (plants 4-7) and 4 tub + 4 ph (plant 8) had lost 2 phureja nucleolar chromosomes if 4 phureja genomes were present. One phureja nucleolar chromosome of plants 1-3 and both of plants 5 and 7 had rearranged satellites. Elimination of the two nucleolar chromosomes occurred preferentially, was under genetic control, and probably took place during early callus development. NOR activity resulting in rear-rangements between NORs may have caused the elimination.

Polyethylene glycol and electric field treatment of plant protoplasts: characterization of some membrane properties

Petiolar protoplasts of a dihaploid line of winter oilseed rape Brassica napus L. ssp. oleifera were exposed to fusogenic polyethylene glycol (PEG) and electric field treatments. The surface properties and stability of membrane components of the treated protoplasts were investigated by contact angle measurements in aqueous two‐phase systems and differential scanning calorimetry, respectively. The leakage of intracellular components was estimated with respect to amino acids, proteins and DNA. Both fusogenic treatments resulted in the same apparent changes in membrane surface hydrophobicity and the same destabilization of membrane components. However, the PEG‐treated protoplasts were more leaky than both the control and the electric field‐treated protoplasts. The results indicate that the molecular mechanisms of PEG‐ and electrical field‐induced fusion are similar. However, the effects of the latter appear to be less harmful presumably because the parameters for electric field treatment are more easily controlled.

Growth Rates of Tobacco Genotypes in Tissue Cultures and Their Relationship to Leaf Yields

Three experiments were conducted to determine the influence of tobacco, Nicotiana tabacum L., genotypes on in vitro relative growth rates of callus (RGR) and the relationship of this parameter to mature plant characters, cured leaf yields, days from transplanting to flowering, and number of leaves per plant. Ten anther-derived dihaploid lines from the Coker 139 cultivar and three anther-derived dihaploid lines from NC 95 differed greatly in their in vitro growth rates in the first experiment. The correlation of in vitro RGR with mature plants characteristics was negative for all three parameters, but significantly different from zero only for leaf yields. Significant genotypic effects on RGR were detected among nine dihaploids of NC 95 and conventional cultivars NC 95 and C 139 in a second study, but correlations with mature plant characters were not significant. Dihaploid line NC 95 DH4 was common to both experiments and exhibited the highest RGR in both sutdies. In a third experiment 15 F 8 breeding lines, previously classified as producing high, moderate, or low cured leaf yields, exhibited significant variability for in vitro RGR. Genotypic variability for RGR was primarily a function of differences among lines within groups, as group means did not differ. All mature plant parameters were negatively correlated with RGR but nonsignificantly. The studies demonstrate that plant genotype has a pronounced effect on callus growth rate and that this characteristic is probably negatively related to mature plant leaf yields for fluecured tobacco. This relationship, as measured in these studies, is not sufficiently high to be of value in predicting leaf yields of varying genotypes.

Effects of mutagens on androgenesis in some species of the genusNicotiana L.

Chosen species and cultivars(Nicotiana sanderae, N. glutinosa, N. otophora, N. paniculata, N. sylvestris, N. velutina, N. tabacum cv. Samsun, N. tabacum cv. Samsun — a dihaploid line, and an amphidiploid N.sylvestris x N tomentosiformis) were treated with adequate concentration of n-buthylmethane sulphonate. The anthers of M1 plants were cultured on the medium of NITSCH 1969 plus 0.1 mg IAA/1. Special attention was paid to quantitative characteristics of androgenesis. Androgenesis occurred more often in the treated variants. The conclusions have been confirmed of a heterosis effect of adequate chemomutagen concentration at the diploid level (an increase in the number of androgenic anthers in the treated variants, and a decrease in the mean number of haploids per androgenic anther) in the broad range of the material. We have recorded a high androgenic ability in the amphidiploidN. sylvestris x N. tomentosiformis, and androgenesis inN. velutina for the first time.