Somatic Embryogenesis in Maize and Comparison of Genetic Variability Induced by Gamma Radiation and Tissue Culture Techniques

Sixteen inbred lines and one hybrid of manse were tested for their capability of somatic embryogenesis, and fully developed plants could be regenerated, from ten inbred, lines. The highest frequency of plant regeneration was expressed in the inbred line CHI 31, and when this line was crossed with a recalcitrant, non-regenerating line, the F₁ and BC hybrids were regenerable. The results of reciprocal crosses demonstrated that dominant nuclear genes and cytoplasmic factors are primarily responsible for the heritable determination of embryogenic callus proliferation and in vitro regeneration of maize plants. Somaclonal and radiation-induced variability was studied in maize to assess their nature and potential contribution to plant breeding., The inbred line CHI 31 possessing a high in vitro capacity of somatic embryo formation was used as experiments.] material. CHI 31 plants were selfed and twelve-day old zygotic embryos irradiated with ⁶⁰Co gamma radiation in situ. Mature caryopses were harvested and assigned as M₁ material. In another series, immature zygotic embryos (size 1.2—1.5 mm) were cultured in vitro on N-6 medium supplemented with 2,4-D (2.5 μM), and somatic embryos regenerated into plants; these were transplanted into soil and self-pollinated. Regenerants from non-irradiated cultures were grown as R₁ generation, while regenerants from irradiated explants were considered as M₁R₁ generation. The genetic variability was evaluated in the M₂, R₂ and M₂R₂ generations, respectively, and compared with a non-treated seed control. Irradiation induced a variety of chlorophyll and morphological variants segregating in the M; generation; however, the frequency of deviant types obtained in the R: generation (somaclonal variation) was significantly exceeding the one derived from the M₂ populations. The combination of expert irradiation and in vitro regeneration was most effective for the manifestation of chlorophyll and morphological o if types in the M₂R₂ generation, and increased drastically the frequency of early flowering variants. Differences in the segregation patterns of mutant phenotypes amonsister somaclones in the R₃ and M₃R₃ generations indicate a different genetic basis, of plants originating from the same explant. This phenomenon suggests a mutational sectoring of the callus during culture. Radiation induced and somaclonal variation exerted a similar spectrum of chlorophyll and morphological deviants.     

Induction and selection of mutations for resistance against bacterial leaf blight in rice

Summary Dry seed lots of a rice cultivar, Harebare, susceptible for bacterial leaf blight (BLB), were exposed to thermal neutrons with and without pre-treatment of seed for boron-enrichment, and to gamma-rays. M₁ plants of each of the treatments were grown and their seeds were separately harvested. M₂ populations were raised in rice fields of a farmer in a region where BLB is epidemic every year, and M₂ and control plants which expressed resistant reactions to BLB were selected. M₃ and control lines as plant progenies of the selected M₂ plants were raised in the same rice fields as in M₂ generation in order to investigate their BLB resistance and other agronomically important traits. Variances for disease severity of M₂ populations were significantly larger than those of the control population. whereas their means were not singificantly different from that of the control population, suggesting induction of mutations toward both resistance and susceptibility to BLB. Mean disease severity values of the M₃ lines from selected M₂ plants were significantly smaller than those of the control lines, indicating gains of selection in M₂ for quantitative resistance against BLB. Thermal neutrons, especially with boron-enrichment pre-treatment were effective to induce mutations for resistance against BLB. Some M₃ mutant lines with quantitatively enhanced resistance against BLB were not modified in other agronomic traits from those of the original cultivar. Significance of the induced quantitative resistance in breeding programmes for BLB resistance is discussed.     

Radiation and genetical studies to estimate the number of embryonic initials taking part in the formation of the shoot meristem of Vicia ervilia and Lathyrus sativus

Summary Dry seeds of V. ervilia and L. sativus were irradiated with 60Co gamma rays. The M₁ plants were grown to maturity. During harvest, ten randomly selected plants were arbitrarily numbered (symbols P₁ to P₁₀) and the branches and the pod positions along each branch of each plant were numbered (symbols for branches, B₁ to B₄ and pod positions, p₁ to p₁) from the lowest to the topmost one. The seeds of each pod position were collected, threshed, counted and, the next year, sown separately. Mutants were isolated from among the M₂ population and each mutant could be traced back to the particular topography of the M₁ plant. The seeds from the M₂ plants originating from different sectors of the M₁ plants were grown to M₃ generation to study the pattern of inheritance. The segregation data of L. sativus show that in some cases at least one and in other cases at least two embryonic initials were involved in the formation of the shoot meristem whereas in V. ervilia the number of embryonic initials involved was at least two or three.     

Induced mutations in winged bean (Psophocarpus tetragonolobus L. DC) with low tannin content

Four mutants of winged bean (Psophocarpus tetragonolobus (L.) DC) with altered tannin content were selected among the M₃ seeds present on M₂ plants following mutagenic treatment of seeds of cvs UPS 122 and Kade 6/16 using gamma radiation from a Co⁶⁰ gamma source. These mutants were selected from the most chimeric parts of the M₁ plant, which had earlier been identified to be the first mature pods on the M₁ plant and the earliest formed M₂ seeds in the M₁ pods. The indirect selection of tannin mutants was based on searching for seed coat colour changes in M₃ seeds. Cv UPS 122 yielded the mutants 3/1-10-12 and X22 from 1958 and 1883 M₂ plants respectively. Cv Kade 6/16 yielded the mutants 3/9-0-12 and 3/4-10-7 from 1442 and 1011 M₂ plants respectively. One of the mutants, 3/4-10-7, which was the only desirable one, had a level of tannin of about 25% of the wild type cv. Kade 6/16. The other three mutants had similar or increased tannin levels. This revised version was published online in July 2006 with corrections to the Cover Date.     

Screening of Chinese cabbage mutants produced by 60Co γ‐ray mutagenesis of isolated microspore cultures

Since the release of the Chinese cabbage genome sequence, increasing interest has focused on the functional analysis of unidentified genes in Chinese cabbage. Mutant analysis forms the basis of functional genomics research. To produce a variety of Chinese cabbage mutants in the same genetic background, buds containing late uninucleate spores from a doubled haploid line of the Chinese cabbage variety ‘Fukuda 50’ were irradiated with ⁶⁰Co γ‐rays at doses of 20, 40 and 60 Gy. Then, the treated microspores were isolated and cultured. A total of 492 putative M₀ mutants were isolated from 1483 regenerated plants. Of these, six M₁ mutants were verified; the mutant frequency was 0.41%. These mutants comprise a mutant library that includes one plant shape mutant, two flower mutants and three male sterile mutants. Pollen viability detection and DNA flow cytometry were used to determine the ploidy of the regenerated plants. Some of the mutants isolated in this study may be useful for Chinese cabbage breeding and functional genomics research.     

Effect of Mutagenic Treatments on Somaclonal Variation in Wheat (Triticum aestivum L.)

Different wheat genotypes were treated with gamma-rays, sodium azide (SA) and EMS before tissue culture and immature embryos from M₁ plants or plants shortly after exposure to gamma-rays were used to initiate callus culture. Thousands of plants were regenerated and used to investigate the effect of mutagenic treatments on the regenerated plants and somaclonal variation in the M₃R₂ and M₂R₂ generations. The results showed that mutagen-induced damage in terms of reduction in plant height, fertility and spike length were not outstanding in the regenerated plants as compared with the untreated control. In the M₃R₂ generation, only SA treatment had significantly higher frequencies of somaclonal variations than the control. Increases in the variation frequencies were observed when explant embryos were irradiated with 2.5 and 5 gy gamma-rays and the highest frequency appeared when embryos were exposed to 5 gy gamma-rays on the 5th day after anthesis. Increased variation spectra also resulted from mutagenic treatments and most of the variants recovered were unsuitable for plant improvement.     

The combined use of leaf irradiation and of the adventitious bud technique for inducing and detecting polyploidy,marker mutations and self-compatibility in clonal populations of Nicotiana alata Link and Otto

Summary The method of vegetative propagation by adventitious bud formation on detached leaves has been used, in combination with radiation treatment, for obtaining self-compatibility mutations and marker mutations in two clonal populations derived from single S ₂S₃plants of Nicotiana alata. Out of in all 345 individuals arisen from leaves exposed to various dosages of X-rays, 3 self-compatible and 3 marker mutants were selected, which maintain their phenotypes after several cycles of vegetative propagation. These results demonstrate that it is possible to recover, in a self-incompatible clone, mutant individuals which are characterized by the same genetic background as the mother plant and which are, consequently, free of the inbreeding effects usually resulting from the conventional methods of mutation induction and detection.In addition to these six mutants, a number of plants originated from both control and irradiated leaves were found to be tetraploid and self-compatible. Whereas the occurrence of tetraploidy is taken as an indication that some of the initial cells from which the plantlets originated were polyploid, the fact that no cytochimeras were detected, is considered to present a further demonstration of the one cell-one plant theory. Self-compatibility in the tetraploid plants is ascribed to a competition effect in heterogenic pollen.Attempts are now in progress to obtain more mutant individuals and to use the marker-mutations available for mapping the S-locus. A biochemical comparison of self-compatible and self-incompatible plants with almost identical genetic backgrounds will be initiated.This publication is contribution No. 702 of the EURATOM Biology Division     

Potential of gamma radiation enhancing the biosynthesis of tropane alkaloids in black henbane (Hyoscyamus niger L.)

Summary A productive yellow flowered mutant with high tropane alkaloid content was isolated in the M₂ generation of gamma irradiated (Co⁶⁰ at 40 kr) progenies ofHyoscyamus niger. Mutant progenies, on an average, were capable of synthetising more than twice crude alkaloid (0.147% to 0.221%) in M₃ through M₆ generations compared to the parental control (0.065 to 0.106%). In pilot scale trial (M₇), two elite lines, Y-15 and Y-17 were found to contain 0.390% and 0.318% crude drug in contrast to 0.211% in an improved inbred used as control. However, owing to conducive plant morphology, Y-17 registered high yield of dry biomass, hence gave more than double yield of crude drug than the improved control (inbred) (23 kg/ha against 11 kg/ha). Also being superior in hyoscyamine and hyoscine yields, it was redesignated as var.Aela, and released for commercial cultivation in India, thus signifying the efficiency of radiation breeding for crop improvement.     

EMS Induction of Early Flowering Mutants in Spring Rape (Brassica napus)

Studies were undertaken to induce early flowering mutants by ethyl methanesulphonate (EMS) treatments of Brassica napus seeds. EMS treatments for 12 h of a highly inbred B. napus line TBS had adverse effects on M¹ plant development and fertility only when concentrations were greater than 1%. However, an EMS concentration of 1.5% did not reduce M₁ plant fertility to an extent which significantly reduced production of M₂ seeds. Genetic changes induced by EMS treatment and affecting flowering time were of three main types: (1) Changes within a polygenic system reflected by increased variation in flowering time among M₂ families. As the increase in variation was due primarily to a higher frequency of later flowering plants, these polygenic changes would be of little value in developing better-adapted cultivars. (2) Induction of a recessive mutation at a major gene locus which caused M₃ plants homozygous for the mutant gene to flower at least 20 days earlier than the parental line TB8. (3) Induction of a dominant mutation at a major gene locus which affects flowering time by causing a substantial reduction in vernalization requirement. M₂ plants carrying the mutant gene flowered as early as 59 days before the parental line. These major gene mutations could be rapidly exploited in the development of agronomically superior cultivars for short-season, lower rainfall environments in Western Australia.     

All hermaphrodite progeny are derived by self-pollinating the sunrise papaya mutant

Self‐pollination of a hermaphroditic cultivar normally gives a ratio of 2 : 1 hermaphrodite to female papayas with genotypes M₂m and mm, respectively. Much effort has been dedicated to marking the sexual types of papaya at the seedling stage to distinguish hermaphroditic from female papayas. A hermaphroditic papaya mutant (SR*) has been obtained, derived from the ‘Sunrise’ papaya cultivar mutant. Self‐pollination of the mutant resulted in all progenies being hermaphroditic. The genotype of the female was lethal, as a result of a lethal gene being linked to the mm female gene complex in this case. However, a 3 : 1 segregation ratio was obtained from the progeny of the hermaphroditic cultivar ‘Thailand’ crossed with SR*, indicating that all genotypes survived. Homozygous genotypes (M₂M₂) would be lethal according to Storey's model. Randomly selected F1 plants of the ‘Thailand’ SR* combination were self‐pollinated to obtain an F₂ generation. The F₂ segregation ratio suggested that the SR* mutant had a different form of the M₂ allele, now designated as M_@, which allowed the dominant M_@M₂ to survive in cross combinations. Genetic study has proved that SR* has the M_@ml genotype, a new mutant. It is capable of producing all hermaphroditic papaya progenies.     

EMS-induced mutation of an endoplasmic reticulum oleate desaturase gene (<Emphasis Type="Italic">FAD2</Emphasis>-<Emphasis Type="Italic">2</Emphasis>) results in elevated oleic acid content in rapeseed (<Emphasis Type="Italic">Brassica napus</Emphasis> L.)

The development of rapeseed cultivars (Brassica napus L.) with high oleic acid and low linolenic acid is highly desirable for food and industrial applications. In this study, the Korean rapeseed cultivar Tamla was used for ethyl methanesulfonate (EMS)-induced mutagenesis and seed oils were screened up to generation M₇ for high oleate mutants. Two mutant populations (M₇) with an average of approximately 76% oleic acid content were isolated. Yield components between two mutant populations and untreated Tamla plants were not substantially different, although the mutants in the vegetative stage were slightly smaller in size than Tamla. Genomic analyses of six fatty acid desaturase (four FAD2 and two FAD6) genes revealed that the elevated oleic acid content in the mutants is the result of single gene mutations. Changes in DNA sequence were observed in two genes out of six fatty acid desaturase (four FAD2 and two FAD6). FAD2-2 exhibited a 2-bp deletion in the upstream region of the gene in the two mutants, resulting in a severely truncated polypeptide (57 aa instead of 469 aa), while six point mutations in the other gene did not result in changes in the amino acid sequence. Based on these results, FAD2-2, an endoplasmic reticulum (ER) oleic acid desaturase, is affected in the mutants, resulting in a ~ 7% increase in oleic acid content in comparison to untreated Tamla plants. The induced mutants could be utilized for the development of high oleic oil rapeseed varieties and for regulatory studies of lipid metabolism in seed oils.     

Inheritance of rol-genes from Agrobacterium rhizogenes through two generations in Kalancho?

Transformation of Kalancho? blossfeldiana ‘Molly’ using the naturally occurring bacterium Agrobacterium rhizogenes is a non-GMO strategy to breed compact plants. In the present study, crosses resembling a commercial breeding strategy were made to determine if the improved ornamental quality observed in the T1 generation was inherited to the offspring. F1 lines were produced by crossing the commercial Kalancho? cultivar ‘Sarah’ with a selected T1 line. Subsequently, F2 populations were produced by self-pollination of individual selected lines. The rol-genes were inherited to the progeny and the presence of rol-genes was confirmed in all F1 and many F2 plant lines exhibiting dwarfism. Screening of F1 and F2 plants showed that the rol-genes were inherited together. Besides decreased plant height, several F1 and F2 lines containing rol-genes exhibited changes in plant diameter, number of branches, flower diameter and time to first open flower and duration of flowering compared to the WT ‘Molly’. Furthermore, increased ethylene tolerance was observed in several lines containing rol-genes compared to the WT ‘Sarah’. Screening of three selected F2 populations derived from self-pollination of F1 lines containing rol-genes indicated a shift in distribution towards a lower mean plant height within the entire plant population compared to a control population of plants without rol-genes. Moreover, one of these F2 populations also exhibited earlier flowering compared to the control population. Compact potted plants and lines without delayed flowering and with improved ethylene tolerance were obtained and are valuable in commercial breeding programmes without using recombinant DNA technology.     

Induced mutations for reduced tuber glycoalkaloid content in potatoes

Mutation breeding was used to produce mutants of the potato breeding selection NDA1725-1 with reduced levels of tuber glycoalkaloids. Excised tuber eye-pieces were exposed to 35 Gy of gamma rays from a ⁶⁰Co source. In the M₁V₁, leaf tissue was screened using high-performance thin layer chromatography and each plant given a relative score based on a subjective rating of band intensity. Tubers were harvested from all plants that produced leaves with relatively low levels of glycoalkaloids. Tuber glycoalkaloids were quantified in the M₁V₂ and M₁V₃ using high-performance liquid chromatography, and 35% and 23% of clones with the lowest glycoalkaloid content were selected for further evaluation in each generation, respectively. In the M₁V₄, M₁V₅ and M₁V₆, selection was based on statistically significant differences between the mutant clones and the controls for glycoalkaloid content, as determined using a gravimetric quantification method, as well as on agronomic and quality characteristics. At the end of six clonal generations three selections exhibited acceptable levels of tuber glycoalkaloids and retained enough of the parent clone's exceptional traits to have potential in the commercial potato chip (crisp) industry.     

一个化学诱变的小麦斑点叶突变体的生理和遗传分析

通过EMS诱变普通小麦品系H261获得一个稳定遗传的斑点叶突变体LF2010。在自然条件下, 该突变体在三叶期叶片基部开始出现黄色斑点, 随后逐步扩散到全片叶、叶鞘、颖壳和麦芒。斑点部位不存在细胞死亡, 斑点性状的表达受光照和温度诱导, 突变体的色素含量、光合速率随着斑点的出现而显著下降。突变体的株高、有效穗数、单株产量、穗长、结实率和旗叶长等农艺性状显著下降, 但是千粒重和旗叶宽却与野生型无差异。将突变体与正常绿色品系杂交, 对其F₁、F₂和BC₁代的遗传分析表明, LF2010的突变性状由1对隐性核基因控制。     

Heavy-ion-induced mutants in sweet pepper isolated by M1 plant selection

We examined the effects of heavy-ion bombardment on mutagenesis in sweet pepper (Capsicum annuum L.). Dose–response studies indicated that 10 Gy irradiation of ¹²C or ²⁰Ne ions on dry seeds is suitable for inducing mutations in plants. From ²⁰Ne-irradiated M₁ plants, putative mutants included two dwarf plants and one plant whose pericarp was yellow were isolated. Phenotypes of their M₂ progeny were similar to those of the M₁ plants and did not segregate. F₁ plants resulting from reciprocal crosses between the mutants and wild-type plants showed the wild-type phenotype, but phenotypes of F₂ and BC₁F₁ segregated at 1:3 (mutant:wild) and 1:1, respectively. These crossing experiments indicate that the three mutants have monogenic recessive mutations in nuclear genes. In light of these data, we discuss the effectiveness of using heavy-ion bombardment to mutate sweet peppers.     

A dwarfing mutant caused by deactivation function of alpha subunit of the heterotrimeric G-protein in rice

Dwarf mutants in plants are crucial for elucidating regulatory mechanisms for plant growth and development. Previous studies suggested that the heterotrimeric G-protein alpha subunit known as D1/RGA1 in rice was involved in deactivation function of the G protein. However, so far no partner has been analyzed the spatial structure change acting with D1. In this study, a dwarf mutant designated Mu101 was obtained in M₂ population of rice indica cultivar M804 treated with ⁶⁰Co γ-ray. Genetic analysis of Mu101 indicated that the dwarf phenotype was controlled by a single dwarf gene, and the dwarf mutant was insensitive to gibberellin (GA), which was named dwarf 89 (d89). Using a large F₂ population derived from a cross between the d89 and a japonica rice variety, Taigeng16, the D89 gene was fine mapped into a 62.13 kb physical distance on chromosome 5, where eight open reading frames were predicted. Sequence analysis indicated that only one bp substitution (A-G) was found in LOC_Os05g26890 between M804 and the d89 mutant. The rice GA insensitive dwarf mutant DWARF1 gene was in this locus. The modeling analysis showed amino acid threonine to alanine mutation was likely to make the alpha helix short, and led to the G protein deactivation.     

Gamma Ray-Induced Field Tolerance to Phytophthora Blight in Sesame

Due to the non-availability of sources of resistance in Sri Lanka to Phytopbthora blight in sesame (Sesamum indicum) caused by Phytopbthora nicotianae var. parasitica, a mutation breeding programme was initiated. Seeds of three genotypes were subjected to six doses of gamma rays from 100 Gy to 750 Gy from a ⁶⁰Co source. Seeds of M₂ bulks, sampled by variety and treatment from the first five capsules formed on M₁ plants, were grown in a field with a history of repeated incidence of the disease. The best 21 lines, having the highest survival and seed production, identified in the screening of M₃ and M₄ progeny rows of selected M₂ single plants were then tested in a replicated field trial with the recommended cultivar ‘MI 3’ as a control. The plant survival in selected lines averaged 43.3 percent as against 7.2 percent in the recommended variety. Eight selections recorded significantly higher seed yield than ‘MI 3’ at P < 0.01 and another three at P < 0.05. Gamma ray treatments of 450 Gy and 600 Gy produced more lines tolerant to the disease than the other doses used.     

Mutation research in peas

Dry seeds of the pea variety Pauli were irradiated with 15,000 r X-rays, 18,000 r X-rays, 18,000 r rays and with neutrons; the latter treatment for 6 hours. In addition the chemical mutagen ethyl methane sulphonate (EMS) was used, in concentrations of 0.33% and 0.50% respectively.After raising the germlings in a glasshouse surviving plants were planted in the field. The germination capacity was only little disturbed after the irradiation; however, it was greatly reduced after the EMS treatment. A very large number of germlings did not grow further, their roots showing a deep brown discoloration, ultimately leading to death. Several other influences after planting in the field killed many other plants.The fertility of the M₁-generation was only little disturbed by the irradiation, but much by the EMS-treatment. In the M₂-generation, both after irradiation and after EMS-treatment, at higher dosages and concentrations lower percentages of mutated progenies were found than at lower dosages and concentrations. This was presumably due to the fact that the number of plants per M₂-line in the former case was on average twice as high. The percentage progenies with mutations and the percentage mutated plants was larger after EMS-treatment than after irradiation.Of normal looking plants with a good fertility, progenies were raised in non-replicated small plots. The average yield per plant and the distribution in the irradiations did not differ significantly from that of the untreated material. After EMS-treatment the average yield was significantly lower and the distribution significantly higher. The fertility disturbance caused by EMS, therefore, also became manifest in the M₃-generation.     

Selection of Semidwarf, Early Maturing and Blast Resistant Mutants after Mutagenic Seed Treatment in Two Locally Adapted Indian Rice Cultivars

The objective of this study was to induce semidwarf, early maturing and blast resistant mutants in two adapted Indian rice cultivars. Seeds of the rice cultivars ‘Madhu Malti’ and ‘Phul Patas 72’ were treated with gamma rays and ethyl methane sulfonate (EMS) as well as a combination of gamma rays and EMS. A total of 96 dwarf (< 75 cm) and semidwarf (< 100 cm) and 104 early flowering/maturing M₂ plants were identified among the 18,060 M₂ plants observed. Sixty-eight blast resistant M₂ plants were also identified. The true mutant status of these plants has yet to be determined in progeny tests. The induction of desired mutant types in locally adapted cultivars could prove to be superior over their introduction from non-adapted foreign materials via backcrossing in that the important agronomic characteristics of adaptation to the local environment are retained in the mutants.     

Increasing erucic acid content in Ethiopian mustard through mutation breeding

Ethylmethane sulphonate (EMS) was applied to seeds of the Ethiopian mustard (Brassica carinata A. Braun) line C-101. Bulk samples of M₃ seeds from 8331 M₂ plants were evaluated for the fatty acid composition of their oil by near-infrared reflectance spectroscopy (NIRS) and by further gas chromatography on selected samples. A putative mutant, N2-6230, showing very low oleic acid content (4.7% vs. average of 8.6% in C-101) and erucic acid content within the range of variation of the line C-101 (40-49.3%) was identified. The M₃ progeny of this mutant showed a wide segregation for erucic acid content (39.1-57.9% vs. 41.8-50.3% in C-101), and maintained levels of oleic acid lower than in line C-101. Selection for high erucic acid content in the M₃ and M₄ generations led to the fixation of this mutation in the M₅ generation (52.2-59.3% vs. 39.0-47.6% in C-101). This is the first high erucic acid line obtained in Brassica species through mutation breeding. Its utility in future programmes to develop very high erucic acid lines is discussed.