Nitrogen addition has a stronger effect on stoichiometries of non-structural carbohydrates,nitrogen and phosphorus in <Emphasis Type="Italic">Bothriochloa ischaemum</Emphasis> than elevated CO<Subscript>2</Subscript>

The current study attempted to obtain candidate doubled haploid (DH) wheat lines by serially combining two approaches: conventional chemical mutagenesis and anther culture. Additionally, the salt tolerance levels were examined between stress-treated (100 mM NaCl) and non-treated DH groups. For the molecular analysis, IRAP markers were used to characterize retrotransposon insertion polymorphisms induced by haploidization, chromosome doubling, and/or mutagenesis in the DH lines. Various sodium azide (NaN₃) concentrations (from 0 to 5 mM) were applied to seeds of the Pehlivan wheat cultivar to obtain an M₁ generation mutant population. Anther culture was set up from the M₁ mutant population. Green plant regeneration, the frequency of selected candidate mutants within the DH form and the levels of salt tolerance between samples were screened. A total of eight thousand anthers were cultured, and sixteen candidate salt-tolerant DH mutant lines, twenty-seven candidate DH mutant lines with different characteristics and one hundred and two candidate DH lines with morphologically normal appearances were obtained from the NaN₃-mutagenized population. The IRAP patterns were quite similar between the control DH lines, and the genetic differences between the controls and DHs originating from possible mutants showed close relatedness. According to previous studies, chemical mutagenesis and anther culture were combined for the first time to detect candidate salt tolerant genotypes at the DH stage. This approach might also be useful for determining the threshold dose and efficiency of wheat mutagens.     

Generation of Peanut Drought Tolerant Plants by Pingyangmycin-Mediated In Vitro Mutagenesis and Hydroxyproline-Resistance Screening

In order to enlarge the potential resources of drought-tolerant peanuts, we conducted in vitro mutagenesis with Pingyangmycin (PYM) as the mutagen as well as directed screening on a medium supplemented with Hydroxyproline (HYP). After being extracted from mature seeds (cv. Huayu 20), the embryonic leaflets were cultured on somatic embryogenesis-induction medium with 4 mg/L PYM and the generated embryos were successively transferred to a germination medium with 4 and then 8 mmol/L HYP to screen HYP-tolerant plantlets. After that, these plantlets were grafted and transplanted to the experimental field. In the next generation, all seeds were sown in the field, and phenotype variation and trait segregation can be observed in most of the offspring (M₂ generation). The M₃ generation individuals were subjected to drought stress at the seedling stages. The activities of SOD and POD were substantially increased in eight offspring of 11 HYP-tolerant, regenerated plants than in their mutagenic parents. To determine the correlation between mutant phenotypes and genomic modification, we carried out a comparison of the DNA polymorphisms between the mutagenic parents and 13 M₃ generation individuals from different HYP-tolerant, regenerated plants with SSR primers. Results showed that most mutants and parent plants had signs of polymorphisms. Under drought stress, some M₃ generation individuals of 10 original HYP-tolerant, regenerated plants produced more pods than the mutagenic parent; twenty individuals among them produced >60 g pods/plant. M₄-generation seeds were tested for quality characteristics by Near Infrared Spectroscopy (NIS) and nine individuals with higher protein content (>30%) and 21 individuals with higher oil content (>58%) were screened. We concluded that the use of PYM-based in vitro mutagenesis in combination with directed screening with HYP is effective for the creation of potential drought-tolerant mutants of peanut.     

Rice <Emphasis Type="Italic">phot1a</Emphasis> mutation reduces plant growth by affecting photosynthetic responses to light during early seedling growth

The aim of this work was to characterize the phot1 mutant of rice during early seedling growth in various light conditions. We isolated the rice T-DNA insertion mutant phot1a-1 and compared it to the Tos17 insertion mutant phot1a-2. When phot1a mutants were grown under WL (100) and BL (40 μmol m⁻² s⁻¹), they demonstrated a considerable reduction in photosynthetic capacity, which included decreased leaf CO₂ uptake and plant growth. Pigment analysis showed no significant difference between wild-type and mutants in the Chl a:b ratios, whereas in the latter, total concentration was reduced (a 2-fold decrease). Carotenoid contents of the mutants were also decreased considerably, implying the involvement of phot1a in pigment degradation. Deletion of phot1a showed higher contents of H₂O₂ in leaves. Chloroplastic APX and SOD activities were lower in the mutants whereas the activities of cytosolic enzymes were increased. Immunoblotting indicated reduced accumulation of photosystem proteins (D1, D2, CP43, Lhca2, and PsaC) relative to the other light-harvesting complexes in the mutant. We conclude that the defect of Os Phot1a affects degradation of chlorophylls and carotenoids, and under photosynthetically active photon fluxes, mutation of phot1a results in loss of photosynthetic capacity owing to the damage of photosystems caused by elevated H₂O₂ accumulation, leading to a reduction in plant growth. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The induction of grain size and colour mutations in rice (Oryza sativa L.) by radioisotopes

Summary Seven mutants with changes in grain size and in pigmentation of various organs were isolated in the M ₂ and M ₃ generations of the Patnai variety of winter rice after treatment with ³² P and ³⁵ S. Some of these could be useful for breeding.The narrow grain mutant, isolated in the M ₃ generation after treatment with ³² P, was high-yielding and had fine grains and an increased number of long panicles.Two short grain mutants with changed husk colour were obtained in the M ₂ generation after treatment with ³² P. One was also early maturing.One purple apiculus and three purple husk mutants were isolated in the M ₂ generation after treatment with ³⁵ S. Some of these also had reduced plant height and short grains. The purple apiculus mutant bred true for stigma and apiculus colour, but segregated for leaf sheath colour. In purple husk mutants the segregation of mutant characteristics was unusual and there was manifestation of new characters.     

A revisit of mutation induction by gamma rays in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L<Emphasis Type="Italic">.</Emphasis>): implications of microsatellite markers for quality control

Mutation techniques have been used for generating genetic variation and breeding new varieties during the past decades. However, the skepticism has also persisted during the course on the sole mutational origin of genetic variation in mutated populations. We addressed this issue using three unique rice genetic lines in this study. First, we confirmed that gamma rays had significant effect on the growth of M₁ plants, leading to significant reduction of fertility, seed set and plant height at doses 200 Gy and above. Second, we proved that out-crossing derived genetic variants existed in M₂ population (0.8%) and among selected putative mutants (0–33.3%), in addition to induced mutants. Third, we demonstrated that true induced mutant lines had identical microsatellite haplotypes to their parents. We proposed microsatellite assay as a method to exclude any genetic contaminants from induced mutants, with appropriate numbers for different levels of power based on reported microsatellite mutation rate and microsatellite polymorphic index.     

Production of doubled haploids through anther culture of M<Subscript>1</Subscript> rice plants derived from mutagenized fertilized egg cells

To produce stable mutants from Mankeumbyeo, a japonica rice (Oryza sativa L.) variety, we estimated the mutation efficiency of ethyl methane sulfonate (EMS) and N-methyl-N-nitrosourea (MNU) on fertilized egg cells using doubled haploids (DHs) derived from anther culture of M₁ plants. M₁ seed production and germination were higher in 1 mM MNU than in 94.2 mM EMS. A total of 68 DHs (35.4%) were regenerated by anther culture of M₁ plants. Twenty-one DHs (30.9%) were stable mutants, 14 DHs (20.6%) were unstable mutants, and the remainder (48.5%) were normal. The frequencies of stable mutants following EMS and MNU treatments were 20.7% (three semidwarfs, one early maturation and one glabrous line) and 38.5% (three semidwarfs, two early maturation, four glabrous and one long grain line), respectively. In a field trial of seven stable mutants for yield potential, five mutants did not show a significant difference in yield as compared with the original variety. Among these five, three glabrous mutants (MK-MAC 1, MK-MAC 4 and MK-MAC 26) with a smooth leaf and hull may be considered to be improved mutant lines because of the health benefits (reduced skin damage and generation of less dust compared to the original variety) to farmers handling the plant materials. MK-MAC 26, a glabrous mutant, had also less shattering resistance than that of the original variety. These stable mutants could be used as new breeding materials.Communicated by P.P. Kumar     

Survival Chances of Mutants Starting With One Individual

A simple theoretical model of a Darwinian system (a periodic system with a multiplication phase and a selection phase) of entities (initial form of polymer strand, primary mutant and satellite mutants) is given. First case: one mutant is considered. One individual of the mutant appears in the multiplication phase of the first generation. The probabilities to find N mutants W_n^M(N) after the multiplication phase M of the n-th generation (with probability δ of an error in the replication, where all possible errors are fatal errors) and W_n^S(N) after the following selection phase S (with probability β that one individual survives) are given iteratively. The evolutionary tree is evaluated. Averages from the distributions and the probability of extinction W_∞^S(0) are obtained. Second case: two mutants are considered (primary mutant and new form). One individual of the primary mutant appears in the multiplication phase of the first generation. The probabilities to find N_p primary mutants and N_m of the new form W_n^M(N_p, N_m) after the multiplication phase M of the n-th generation (probability ε of an error in the replication of the primary mutant giving the new form) and W_n^S(N_p, N_m) after the following selection phase S (probabilities β_p and β_m that one individual each of the primary mutant and of the new form survives) are given iteratively. Again the evolutionary tree is evaluated. Averages from the distributions are obtained.     

Application of hydrotime model to predict early vigour of rapeseed (<Emphasis Type="Italic">Brassica napus</Emphasis> L.) under abiotic stresses

Rapeseed (Brassica napus L.) is important for edible oil production in semi-arid areas. Abiotic stresses are threatening rapeseed production in such areas. This study was conducted to find tolerant genotypes of rapeseed and to determine which traits of crop establishment is related to abiotic stress tolerance. Hydrotime model parameters were determined in a laboratory germination test, and seedling emergence and growth were evaluated in pot experiments under control, drought, salinity, deep sowing, low and high temperatures for 19 rapeseed genotypes. Results indicated that the predicted germination time courses at the various water potentials generally fitted well with the observed germination data. The estimated values of θ _H, ψ_b(50), and σψ_b differed significantly across genotypes. Seedling emergence and growth differed significantly under each environmental condition. PCA showed that genotypes of Hayola 401 and line 285 were the most tolerant to abiotic stresses during crop establishment and seedling growth. The first PC explained 40% of variations, and a correlation was observed between PC1 and ψ_b(50). Correlations among hydrotime model parameters and early seed vigour variables indicated that ψ_b(50) negatively correlated with seedling emergence percentage and rate (day^?1) under all abiotic stresses. It shows that genotypes with more negative values of ψ_b(50) have more seedling emergence percentage and a larger seedling emergence rate (days^?1) under a wide range of environmental conditions. Thus, it can be concluded that, to identify tolerant genotypes of rapesee to abiotic stresses, ψ_b(50) is a good trait and that breeders can focus on reducing ψ_b(50) to increase tolerance of abiotic stresses.     

OsCIPK31, a CBL-interacting protein kinase is involved in germination and seedling growth under abiotic stress conditions in rice plants

Calcineurin B-like protein-interacting protein kinases (CIPKs) are a group of typical Ser/Thr protein kinases that mediate calcium signals. Extensive studies using Arabidopsis plants have demonstrated that many calcium signatures that activate CIPKs originate from abiotic stresses. However, there are few reports on the functional demonstration of CIPKs in other plants, especially in grasses. In this study, we used a loss-of-function mutation to characterize the function of the rice CIPK gene OsCIPK31. Exposure to high concentrations of NaCl or mannitol effected a rapid and transient enhancement of OsCIPK31 expression. These findings were observed only in the light. However, longer exposure to most stresses resulted in downregulation of OsCIPK31 expression in both the presence and absence of light. To determine the physiological roles of OsCIPK31 in rice plants, the sensitivity of oscipk31::Ds, which is a transposon Ds insertion mutant, to abiotic stresses was examined during germination and seedling stages. oscipk31::Ds mutants exhibited hypersensitive phenotypes to ABA, salt, mannitol, and glucose. Compared with wild-type rice plants, mutants exhibited retarded germination and slow seedling growth. In addition, oscipk31::Ds seedlings exhibited enhanced expression of several stress-responsive genes after exposure to these abiotic stresses. However, the expression of ABA metabolic genes and the endogenous levels of ABA were not altered significantly in the oscipk31::Ds mutant. This study demonstrated that rice plants use OsCIPK31 to modulate responses to abiotic stresses during the seed germination and seedling stages and to modulate the expression of stress-responsive genes.     

Interaction of certain base-specific chemicals and diethylsulphate in the induction of chlorophyll mutations in Oryza sativa L.

The presoaked seeds of a rice cultivar, Tellakattera, were treated with three different concentrations of hydrazine (HZ) or hydroxylamine (HA) in combination with diethylsulphate (dES) (0.05%). In the M₁ generation more than additive effects were observed for increase in chlorophyll chimeras and decrease in seed fertility. A synergistic effect was also observed for both chlorophyll mutation and mutant frequencies, in the M₂ generation, in sequential treatments. However, the degree of synergism, based on M₂ chlorophyll mutant frequency, was greater in dES posttreatment combinations with HA or HZ, compared with dES pretreatments. These differences in reciprocal treatments may be due to more efficient fixation of premutational events by dES than HZ or HA.     

Pleiotropic morphological and abiotic stress resistance phenotypes of the hyper-abscisic acid producing Abo™ mutant in the periwinkleCatharanthus roseus

The pleiotropic properties of aabo abo (Abo^™) γ-ray induced mutant ofCatharanthus roseuscv. Nirmal, selected among the M₂ generation seeds for ability to germinate at 45°C, are described. The mutant produced seeds possessing tricotyledonous embryos, unlike the typically dicotyledonous embryos present in the wild type Abo⁺ seeds. In comparison to Abo⁺ adults, the mutant plants had short stature and lanceolate leaves. The vascular bundles in the leaves and stem were poorly developed. Leaf surfaces were highly trichomatous, epidermal, cortex and mesophyll cells were small sized and a large majority of stomata were closed. Besides high temperature, the mutant was salinity and water-stress tolerant. The abscisic acid (ABA) content in the leaves was about 500-fold higher. The genetic lesionabo responsible for the above pleiotropy was recessive and inherited in Mendelian fashion. The seedlings and adult plants of the mutant accumulated higher proline than Abo⁺ plants. The phenotypes ofabo abo mutants permitted the conclusions that (i) the mutant synthesizes ABA constitutively, (ii) both ABA-dependent and ABA independent pathways for proline and betaine accumulation are functional in the mutant, and (iii) cell division, elongation and differentiation processes in embryo and adult plant stages are affected in the mutant.     

Response of antioxidant systems in oxygen deprived suspension cultures of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

The effect of oxygen deprivation (anoxia) on the antioxidant system in suspension culture of anoxia-intolerant Malaysian rice mutants cells was examined. Abiotic stresses have been reported to adversely affect cell division, damage cellular and organelle membranes. The signaling defense mechanisms, such as molecular and biochemical aspects responding to stress have been proven to be very complex, and still largely untapped. The objective of this study was to determine the potential involvement of activated oxygen species, such as superoxide dismutase, catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase which occur in cells of rice plants exposed to anoxia stress in two Malaysian rice mutants, MR219-4 and MR219-9, and rice cultivar FR13A which is known to be tolerant to anoxia stress during 5–30 days of exposure. The antioxidative enzymes were decreased for MR219-4 and MR219-9 mutants for CAT and APX activities, and increased in FR13A cultivar starting at 20 days in suspension culture compared to that of control. CAT and APX activities were maintained higher in anoxia condition for all mutants and cultivar. These findings suggested that anoxia stress in suspension cultures induced the level of H₂O₂ to toxic levels.     

Screening of ethyl methane sulphonate mutagenized tef [Eragrostis tef (Zucc.) Trotter] population identifies Al-tolerant lines

About 15,000 M₂ seeds of ethyl-methane-sulphonate (EMS)-mutagenized population were screened along with Al-tolerant and sensitive checks and the M₀ variety. Strongly acidic soil with an external application of a toxic Al-solution and exposure to moisture stress was used to maximize selection pressure. Twenty-one M₂ plants with root lengths of greater than the mean of the tolerant check were selected and planted for seed production. Candidate M₃ plants were investigated for Al-tolerance and for morpho-agronomic traits under greenhouse and field conditions, respectively. Highly significant differences were observed for Al-tolerance between the candidate mutant lines and the M₀ (P?<?.001), and between mutant lines and the sensitive check (P?<?.001). Similarly, significant differences were observed between the mutant lines for 16 of the 20 quantitative traits measured. This study is the first to report successful induction of enhanced Al-tolerance in tef by using EMS mutagenized population.     

Molecular characterization of marker-free transgenic lines of <Emphasis Type="Italic">indica</Emphasis> rice that accumulate carotenoids in seed endosperm

A single Agrobacterium strain harbouring two binary plasmids was successfully used for the first time to develop a marker-free transgenic rice of improved nutritional value. Sixty-eight T₀ co-transformants were obtained in three indica rice cultivars—two popular high-yielding Bangladeshi varieties (BR28 and BR29), and one high-iron rice cultivar (IR68144). Marker-free lines were obtained from 14 out of 24 selected co-transformants screened in the T₁ generation. The accumulation of total carotenoids in polished T₂ rice seeds of the primary transgenic VPBR29-17-37 reached levels of up to 3.0 μg/g, with the level of β-carotene reaching 1.8 μg/g. In the cultivars BR28 and IR68144, total carotenoid levels in the transformants reached 2.0 μg/g of polished rice seeds. The levels of lutein and other carotenoids in the seeds were also significantly enhanced. T₁ plants obtained from primary transgenics with simple gene-integration patterns tended to have a lower carotenoid content than the original parental lines. This study describes the development of marker-free transgenic rice lines containing high levels of carotenoids, and addresses the relationship between the rearrangement of transgenes and the presence of metabolic end products in transgenic rice.     

Selection and initial characterization of partially nitrate tolerant nodulation mutants of soybean

Since NO₃⁻ availability in the rooting medium seriously limits symbiotic N₂ fixation by soybean (Glycine max [L.] Merr.), studies were initiated to select nodulation mutants which were more tolerant to NO₃⁻ and were adapted to the Midwest area of the United States. Three independent mutants were selected in the M₂ generation from ethyl methanesulfonate or N-nitroso-N-methylurea mutagenized Williams seed. All three mutants (designated NOD1-3, NOD2-4, and NOD3-7) were more extensively nodulated (427 to 770 nodules plant⁻¹) than the Williams parent (187 nodules plant⁻¹) under zero-N growth conditions. This provided evidence that the mutational event(s) affected autoregulatory control of nodulation. Moreover, all three mutants were partially tolerant to NO₃⁻; each retained greater acetylene reduction activity when grown hydroponically with 15 millimolar NO₃⁻ than did Williams at 1.5 millimolar NO₃⁻. The NO₃⁻ tolerance did not appear to be related to an altered ability to take up or metabolize NO₃⁻, based on solution NO₃⁻ depletion and on in vivo nitrate reductase assays. Enhanced nodulation appeared to be controlled by the host plant, being consistent across four Bradyrhizobium japonicum strains tested. In general, the mutant lines produced less dry weight than the control, with root dry weights being more affected than shoot dry weights. The nodulation trait has been stable through the M₅ generation in all three mutants.     

Nodulation and nitrogen fixation mutants of pea,Pisum sativum

Summary The 36 mutants which did not nodulate and 24 mutants which formed inefficient nodules with no or very low acetylene reduction activity were isolated among 86,000 M₂-seedlings of Finale pea, Pisum sativum L., after treatment with chemical mutagens. One mutant was found for approximately every 50 chlorophyll mutants. Most mutations were induced by ethyl methanesulfonate; some by diethyl sulfate, ethyl nitrosourea and acidified sodium azide. Putative mutants were selected as nitrogen deficient plants, yellowing from the bottom and up, when M₂ seedlings were grown in sand with a Rhizobium mixture and PK fertilizer. The mutants were verified in the M₃ generation by acetylene reduction assay on intact plants.     

Characterization and fine mapping of the glabrous leaf and hull mutants (gl1) in rice (Oryza sativa L.)

The glabrous leaf and hull (gl1) mutants were isolated from M₂ generation of indica cultivar 93-11. These mutants produced smooth leaves and hairless glumes under normal growth conditions. By analyzing through scanning electron microscope, it was revealed that the leaf trichomes, including macro and micro hairs, were deficient in these mutants. Genetic analysis indicated that the mutation was controlled by a single recessive gene. Using nine SSR markers and one InDel marker, the gl1 gene was mapped between RM1200 and RM2010 at the short arm of chromosome 5, which was consistent with the mapping of gl1 in previous studies. To facilitate the map-based cloning of the gl1 gene, 12 new InDel markers were developed. A high-resolution genetic and physical map was constructed by using 1,396 mutant individuals of F₂ mapping population. Finally, the gl1 was fine mapped in 54-kb region containing 10 annotated genes. Cloning and sequencing of the target region from four gl1 mutants (gl1-1, gl1-2, gl1-3 and gl1-4) and four glabrous rice varieties (Jackson, Jefferson, Katy and Lemont) all showed that the same single point mutation (A→T) occurred in the 5′-untranslated region (UTR) of the locus Os05g0118900 (corresponding to the 3′-UTR of STAR2). RT-PCR analysis of the locus Os05g0118900 revealed that its mRNA expression level was normal in gl1 mutant. RNA secondary structure prediction showed that the single point mutation resulted in a striking RNA conformational change. These results suggest that the single point mutation is most likely responsible for the glabrous leaf and hull phenotypes in rice.     

Efficient production of androgenic doubled-haploid mutants in barley by the application of sodium azide to anther and microspore cultures

 The aim of this study was to establish a protocol for an efficient production of agronomical and/or physiological mutants from model (cvs. Igri and Cobra) and low-androgenic-responding (cv. Volga) cultivars of barley through the application of a mutagenic agent, sodium azide, to anthers and isolated microspores cultured in vitro. This technology offers the possibilities of screening for recessive mutants in the first generation, selecting for novel genotypes from very large haploid populations, avoiding chimerism and rapidly fixing selected genotypes as fertile true breeding lines. The mutagenic treatment, 10^–3–10^–5 M sodium azide, was applied during the anther induction pre-treatment or immediately after the microspore isolation procedure. Out of 616 M₂ doubled-haploid lines characterised under field conditions, a total of 63 morphological and developmental independent mutant lines were identified. The percentage of M₂ doubled-haploid lines carrying mutations per line analysed was 3.8% when 10^–4 M sodium azide was applied to anthers from the low-responding cv. Volga; this increased to 8.6% and 15.6% when 10^–5 and 10^–4 M sodium azide were applied to freshly isolated microspores from model cultivars. Received: 18 April 2000 / Revision received: 28 September 2000 / Accepted: 28 September 2000     

Three <Emphasis Type="Italic">nth</Emphasis> homologs are all required for efficient repair of spontaneous DNA damage in <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis>

Deinococcus radiodurans is a bacterium that can survive extreme DNA damage. To understand the role of endonuclease III (Nth) in oxidative repair and mutagenesis, we constructed nth single, double and triple mutants. The nth mutants showed no significant difference with wild type in both IR resistance and H₂O₂ resistance. We characterized these strains with regard to mutation rates and mutation spectrum using the rpoB/Rif^r system. The Rif^r frequency of mutant MK1 (△dr0289) was twofold higher than that of wild type. The triple mutant of nth (ME3)generated a mutation frequency 34.4-fold, and a mutation rate 13.8-fold higher than the wild type. All strains demonstrated specific mutational hotspots. Each single mutant had higher spontaneous mutation frequency than wild type at base substitution (G:C → A:T). The mutational response was further increased in the double and triple mutants. The higher mutation rate and mutational response in ME3 suggested that the three nth homologs had non-overlapped and overlapped substrate spectrum in endogenous oxidative DNA repair.     

Frequency and spectrum of chlorophyll-deficient mutations in rice after treatment with radiation and alkylating agents

Three varieties of rice were treated with gamma rays and two alkylating agents EMS and DES, alone and in combinations, with a view to finding out the frequency and spectrum of chlorophyll mutations in relation to the genotype and the nature of the mutagen.Chlorophyll mutation frequency was enhanced with increasing dose but dropped at very high doses (doses that induced over 90% seedling lethality in M₁). The fall was attributed to either the increased mutated sector and diplontic selection after exposure to very high doses or relatively high resistance of some of the seeds.Among chlorophyll mutants in M₂ induced by radiations as well as alkylating agents, the albina type formed the majority class. EMS induced a significantly higher proportion of albinas than did gamma rays.