Salt-tolerance in Brassica juncea L. I. In vitro selection,agronomic evaluation and genetic stability

Summary In vitro selection of salt tolerant plants of Brassica juncea L. (Indian mustard) cv. Prakash has been accomplished by screening highly morphogenic cotyledon explant cultures on high NaCl media. Out of a total of 2,620 cotyledons cultured on high salt medium, 3 survived, showed sustained growth and regenerated shoots. They were multiplied by axillary bud culture on NaCl free medium. The salt-selected shoots retained salt tolerance following 3 month of growth and multiplication on control medium. While two of these somaclones flowered and set seeds, third one grew slowly, had abnormal leaf morphology and was sterile. The seed of the two fertile plants were sown in the field to raise R₁ segregating generation. Data were recorded for field, other agronomic components and oil content. The somaclonal lines, both selected salt-tolerant and non-selected, showed tremendous amount of variation for all the characters studied. One of the two tolerant somaclones invariably showed reduced height, longer reproductive phase and higher 1000 seed weight. Based on the agronomic performance of R₁ plants of these somaclones, some plants were selected and their progeny were evaluated for agronomic performance under standard field conditions and salt-tolerance in the greenhouse using sand pot culture method. Most of the lines bred true for their specific characteristics. In the greenhouse, selected salt-tolerant somaclones (SR-2 and SR-3) performed better for plant growth, yield and other agronomic traits at higher salt treatments, indicating thereby that salt-tolerance trait selected in vitro was expressed in the whole plants and is genetically stable and transmitted onto the progeny. The two tolerant lines, however, differed in their salt-tolerance during vegetative and reproductive phases as indicated by their salt-tolerance and stress susceptibility indices. The mechanism of salt-tolerance is not clear and needs to be further investigated.     

In vitro induction and characterization of somaclonal variation for red rot and other agronomic traits in sugarcane

In the present investigation, optimal media compositions for enhancing the callus induction and subsequent plant regeneration were worked out in sugarcane (Saccharum spp. complex). The field grown somaclones were characterized for yield and quality contributing traits in comparison to donor variety CoJ 88. The MS + 2,4-D (4.0 mg/l) + kin (0.5 mg/l) + sucrose (30 g/l) was found to be the best for the callus induction among the nine media compositions used. The highest shoot regeneration was 34.65% on MS + BAP (0.5 mg/l) followed by 31.07% on MS + IAA (2.0 mg/l) + kin (0.5 mg/l) and the least 11.80% on MS + BAP (2.0 mg/l). The shoot elongation was optimum on MS + IBA (5.0 mg/l). The rooting was carried on MS + NAA (3.0 mg/l) + IBA (2.0 mg/l) + sucrose (70 g/l). The somaclones, on field evaluation, exhibited huge variability for characters viz., tiller number at 120 days of field transfer, number of millable canes, cane height, number of nodes and internodal length at time of harvest and also for HR Brix at 240 and 300 days after field transfer. In R₀ generation, the variations induced for the characters such as stalk height after 120 days, HR Brix after 240 and 300 days of field transfer were negatively skewed with most of somaclones having values higher than the population mean values thereby offering adequate scope for improvement cane and quality. Elite somaclones marked for each trait were reported. Out of 228 somaclones inoculated with Cf 08 pathotype of red rot in R₁, three were resistant, four were moderately resistant and 221 somaclones showed variable degree of susceptibility, whereas, to Cf 03 pathotype, fourteen were resistant, nineteen were moderately resistant and 195 showed variable degree of susceptibility. Three somaclones exhibited moderate resistance (MR) to both the pathotypes, whereas, donor variety CoJ 88 showed susceptible reaction. These somaclones will further enhance the germplasm for sugarcane breeding.     

Wheat somaclonal variants showing earliness, improved spot blotch resistance and higher yield

Somaclones (R₂, R₃ and R₄generations) were regenerated from immature embryos of two spring wheat varieties,HUW-206 and HUW-234. Many somaclones displayed improved earliness, enhanced resistance to spot blotch disease and increased yield over the parent. The superiority of variants for yield traits and disease resistance was established in R₄ generation, confirming the possibility of wheat improvement through somaclonal variation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Culture selected somaclonal variation in five Triticum aestivum L. genotypes

Somaclones (R₃ and R₄ generations) regenerated from five winter wheat (Triticum aestivum L.) genotypes were evaluated for variation in agronomic and morphological characters. Immature embryos were used as initial explant material. Comparisons for plant height, top internode length, spike length, number of seeds per spike and 100 seed weight were made between the somaclones and their parents. Some morphological variations of stem and spike characteristics were registered which demonstrate that plant height and spike length can be changed by using immature embryo culture. The results obtained may be considered a biotechnological contribution to wheat plant improvement. This revised version was published online in August 2006 with corrections to the Cover Date.     

Variation among progenies of diploid plants regenerated from haploid, microspore-derived cell-suspension protoplasts of rice (Oryza sativa L.)

Progenies of 110 diploid R₀ plants regenerated from haploid protoplasts derived from microspore callus cell suspension culture of rice (cv. Miara) were evaluated for 6 traits, along with control lines, in a replicated field trial. Complementary post-harvest observations were carried out for four panicle or grain characters. A wide range of variation was observed for all traits under study among the protoclonal lines. The mean of the protoclonal population was significantly different from that of the control for most traits and 64.5% of the lines differed from the control for at least one field-evaluated trait. A unidirectional shift towards later-maturing, taller, and lower-tillering lines was observed. Of all the lines, 39% were taller than the control by at least 10 cm, 31 % had flowering delayed by at least 5 days, and 13% exhibited significantly longer panicles (P = 0.05). As to the agronomical value of the lines, the variation resulted in a global negative drift although favourable variations were found in a few lines. All but two lines were homogeneous, suggesting that most variations existed in a homozygous form in the R₀ plants. R₂ lines grown from seeds of 17 R₁ protoclonal lines stably inherited the changes and did not segregate, thereby confirming that variations are fixed.     

High frequency regeneration and heritable somaclonal variation in Brassica juncea

Summary Multiple shoot formation in cotyledonary callus of Indian mustard (Brassica juncea cv. Prakash) was induced on modified MS media supplemented with high cytokinin (kinetin or zeatin) and low IAA concentrations. Complete plants were obtained on prolonged incubation of shoots on the same medium. 6-Benzyladenine alone or in combination with IAA or NAA did not support plantlet regeneration. A total of 71 plants were transferred to greenhouse. The seed, however, could be collected from 37 plants only. The seed was sown in the field to evaluate the material for somaclonal variation in R₁ generation. Data were recorded for yield, plant height, number of primary branches, siliqua number, 1,000 seed weight and oil content. Somaclonal lines showed tremendous amount of variation for all the characters studied. A number of plants in this generation showed significantly higher yield and/or other improved agriculturally important characteristics as compared to the control. A line with dwarf plant type was also identified. A number of plants were selected from this generation and carried forward to R₂ generation. Most of these lines bred true in R₂ generation. The material seems to be very promising for future breeding programmes.     

The influence of auxin type on the array of somaclonal variants generated from somatic embryogenesis of eggplant, Solanum melongena L.

Somaclonal variation is a possible source of variation in plant breeding. To apply this approach to eggplant breeding, somaclonal variations were observed among plants regenerated through somatic embryogenesis induced by 1-naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D). Variations including leaf shape, plant height, flower number per cluster, fruit shape, anther number per flower and pollen fertility were compared among ? 300 plants (R₀). Although these variations were confirmed among plants regenerated using both auxins, the frequencies of somaclonal variations in leaf shape, plant height, fruit shape and pollen fertility in the NAA experiment were higher than those in the 2,4-D experiment. Variations in flower number and anther number were also confirmed among plants from both experiments, although no significant differences in their frequency were observed. Subsequently, the inheritance of variations (leaf shape, fruit shape, and flower number) observed in R₀ was investigated from generation to generation (R₁). Variations in leaf shape and fruit shape were inherited while those in flower number were not. From these results we concluded that, although a high frequency of somaclonal variations were observed among plants from both methods, embryogenesis with NAA was more efficient than 2,4-D in eggplant.     

A comparison of somaclonal variation in rice plants derived and not derived from protoplasts

To clarify the effect of the type of in vitro culture on the generation of somaclonal variation, protoplast-derived rice plants were compared with rice plants derived from suspension culture or primary calli (not derived from protoplasts). Regenerated plants showing polyploid-like phenotypes appeared at a higher frequency (33–70%) in plants derived from protoplasts than in those not derived from protoplasts (3–6%). In the first progeny (R₁) generation of all regenerated plants, 120 of 368 lines (33%) segregated plants with mutated characters such as albino, dwarf and sterile. In quantitative traits, 62 (21%) and 144 (50%) of 290 Rj lines showed shorter culm and lower seed-fertility, respectively, compared with the control line derived from the selfed seeds of the original cultivar. The frequency of the mutant-possessing R₁ lines among lines derived from protoplasts was not significantly different from those not derived from protoplasts. These results indicate that isolation and culture of protoplasts does not enhance genetic changes other than cytogenetical changes.     

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.     

Detection of cross over genotype × environment interactions in pearl millet

Summary Somaclonal variation from a partially sterile intergeneric hybrid was obtained for sugarcane improvement and for understanding phylogeny. Over 1,000 somaclones were regenerated from callus cultures of intergeneric hybrid clone H83-9998 (2n = ca. 94) involving H62-4671 (Saccharum spp. hybrid, 2n = ca. 115) x 28NG7 (Erianthus arundinaceum, 2n = 60) as parents. One hundred twenty-six somaclones were selected on the basis of observed morphological variation and planted in large plots with the original hybrid and its parental clones for detailed studies. Data were collected for stalk (number, length, diameter), leaf (length, width) and sucrose characters. Obvious variations in dewlap shape, leaf and stalk colors, leaf angle, rhizome habit, and pollen fertility, as well as date of flowering, were recorded. Chromosome and isozyme analyses were conducted to confirm that the observed phenotypic variation among somaclones had a genetic basis. Chromosome numbers of somaclones selected on the basis of phenotypic variation ranged from 2n = 66 to 2n = 109. With isozyme analysis, 1 to 4 band variations in three systems (peroxidase, phosphoglucomutase, and phosphoglucoisomerase) were found. Significant genetic variance estimates (G²/P²) and genetic CVs (6.7–25.6%) indicate that the differences observed among somaclonal variants would likely remain stable in additional asexually propagated cycles. Yield was estimated from stalk volume x refractometer solids, and 9 out of 126 somaclones produced higher estimates yield than the hybrid. One somaclone showed estimated yield that was not significantly different from the Saccharum parent.Published as Paper No. 737 in the Journal Series of the Experiment Station, Hawaiian Sugar Planter's Association, Aiea, HI 96701.     

Plant Regeneration from Callus Cultures of Brassica carinata A. Br. and its Implications to Improvement of Oil Seed Brassicas

Protocols of plant regeneration have been developed for Brassica carinata for creating somaclonal variation for plant type and adaptability, so that this species can fit into cropping systems in Indian agriculture. The response of cotyledonary and stem explants was assessed for callus induction and shoot regeneration on MS and B₅ basal media containing different combinations of auxin and cytokinin concentrations. MS medium supplemented with BA and NAA favoured callus induction. Supplementing MS with combinations of BA and IAA, as also with BA alone, regenerated shoots from the ex pi ants with a high frequency. The frequency of shoot regeneration and the mean number of shoots per explant were higher in cotyledons than in stem explants on identical growth regulator combinations. On B₅ medium, supplemented with BA (2 mg/l) and IBA (0.4 mg/l), compact callus was produced which regenerated shoots on transfer to medium containing BA (0.8 mg/l). Genotypic differences among carinata accessions for regeneration were also observed.     

In vitro screening and field evaluation of tissue-culture-regenerated sorghum (Sorghum bicolor (L.) Moench) for soil stress tolerance

Summary Sorghum bicolor (L.) Moench is generally quite sensitive to salt and acid (high aluminium) soil stresses, but quite tolerant of drought stress. As with any stress phenomenon, intra-specific variability exists within the genus. In vitro cell selection and somaclonal variation offer an alternative to traditional breeding methodology for generating improved breeding lines for hybrid development. A field selection protocol was developed for the three soil stresses and inter-stress evaluations were conducted in an effort to find multiple, stress-tolerant genotypes. The acid soil-drought stress, super-tolerant selections were located by the R₇ generation when exposed to a combined aluminium-drought stress field environment and when the regeneration population (number of regenerated lines from one callus source) was maintained at 15,000 plants or higher. A variant frequency of 0.1 to 0.2% for stress tolerance and acceptable agronomic traits among the surviving somaclones, provided an adequate number of phenotypes with desirable agronomic characteristics and a high level of soil stress tolerance. Subsequent research verified that the stress-tolerant regenerants had superior acid soil and drought stress tolerance to that of the donor parents, that their yield capabilities under stress were superior to their parents, and that their stress tolerance attributes were transferred in hybrid combinations. In vitro selection was not effective in increasing the number of field stress survivors. In fact, superior germplasms were developed from non-stressed callus or salt-stressed callus. In vitro selection reduced regeneration frequency and subsequent survival of plants under field stress. In vitro-stressed regenerants should be subjected only to non-stressed environments to maintain population numbers for field selection and thereafter should be subjected to stress environments during later (R₅₊) generations. The optimal strategy for the exploitation of somaclonal variation may be through short-term cell culture (< 12 months) with no attempt at in vitro selection.     

Regeneration, establishment and evaluation of somaclones in Sorghum bicolor (L.) Moench

Novel in vitro techniques have the potential to aid conventional breeding programs. Somaclonal variation is considered to be a useful source of variation and has been demonstrated to be feasible in crop species like wheat, rice and maize. A study was taken up at Tissue Culture Laboratory, University of Agricultural Sciences Dharwad, India, for callus induction, maintenance and regeneration of two well established sorghum varieties, M35-1 and A-1 to study somaclonal variation for both qualitative and quantitative characters. The frequency of callus induction was 90 and 83.3 percent in M35-1 and A-1, respectively, on MS medium with 2 mg l-l of 2,4-D. On higher levels of sucrose (60 gl-l), M35-1 showed good response for both regeneration and rooting, while for variety A-1 BA(0.5 mg l-l) and NAA (0.2 mg l-l) had to be supplemented for satisfactory levels of regeneration (73.3%) and rooting respectively. The regenerated plants (SC₁) were selfed to obtain the seeds for the next generation. In M35-1, variations were observed for chlorophyll, phyllotaxy and midrib structure, while male sterile and branched phenotypes occurred in A-1 families. Combined analysis of variances showed that there was significant difference between and within families for most of the quantitative characters in both cultivars except for the character, fourth leaf breadth in the cultivar A-1. The variation, accompanied by a positive shift from the mean is an indication of the response to selection for any specific character. Occurrence of productive variants among the somaclones of established cultivars like M35-1 and A-1 indicates the possibility of their improvement through somaclonal variation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Towards development of Al-toxicity tolerant lines in indica rice by exploiting somaclonal variation

Somaclonal variations, induced in vitro, were used to enhance tolerance to aluminium (Al) toxicity in rice. Tolerant plants were developed through in vitro screening of embryogenic calli. The calli were derived from mature seed embryos and cultured on medium stressed with different concentrations of Al₂(SO₄)₃⋅18H₂O. Seed germination, callus induction, plantlet regeneration and callus health declined with increased concentration of Al. At higher Al concentrations, callus health deteriorated drastically with partial to total necrosis. Plantlet regeneration varied largely among varieties and treatments. The variety IR72 produced maximum plantlets among all genotypes tested. An amount of 60 ppm or more Al was highly toxic, which greatly reduced plantlet regeneration from callus. R₀ plantlets were grown under glasshouse. Based on the appearance of bronzing symptoms on leaves, the tolerant R₁ plants were selected. R₁ and R₂ lines derived from putative tolerant somaclones, were evaluated in fiberglass tanks filled with Al-toxic soil. R₃ population was evaluated in the field. A few lines derived from IR72 showed high yield and good plant type. The progenies at R₃ showed normal root growth under stressed environment in sand culture. The study revealed that in vitro screening would be an appropriate alternative to conventional breeding in evolving Al-tolerant lines as observed in case of other abiotic stresses. The technique was useful in creating de novo synthesized Al-tolerance character in rice.     

In vitro techniques for selecting wheat (Triticum aestivum L.) for Fusarium-resistance. II. Culture filtrate technique and inheritance of Fusarium-resistance in the somaclones

Summary Calli of resistant, intermediary and susceptible wheat (Triticum aestivum L.) varieties were selected using culture filtrates of Fusarium graminearum and F. culmorum and the regenerants were evaluated for resistance up to R₃. Czapek-Dox broth medium was inoculated with mycelia of Fusarium isolates and incubated for 2–6 weeks. Filtrates were added to MS callus growing medium, then 5 weeks-old calli were transferred onto this medium (MST) for 4–5 weeks. MST containing 30% filtrate was found to be suitable for selection. Resistant calli were transferred again to fresh MST for further two selection cycles. The surviving calli produced less fertile regenerated lines (R₀) than the non-selected ones. Among 18 R₁ lines tested for Fusarium-resistance in the seedling stage by artificial inoculation in the greenhouse, two (11.1%) were significantly more resistant, one (5.6%) was more susceptible than the original cultivar and the rest (83.3%) behaved similarly to the donor plants. Among unselected R₃ lines of three varieties, practically the same number of resistant plants were found as among the related selected ones. When the R₃ selfed generations obtained through double-layer and culture filtrate selection techniques were tested for Fusarium-resistance, 35.7% of the lines were found to be more resistant than the original cultivars, none was more susceptible and 64.3% had a reaction similar to that of the source materials. Thus, inheritance of the disease reaction was not stable in all cases. Success of in vitro selection for Fusarium-resistance depended also on the genotype, and toxin analysis showed that although being effective, the selective media contained deoxynivalenol only exceptionally. In selecting wheat for Fusarium-resistance in vitro, the culture filtrate technique proved better than the double-layer procedure.     

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.     

Somaclonal variation for some morphological traits, herb yield, essential oil content and essential oil composition in an Indian cultivar of rose-scented geranium

Somaclonal variation for plant height, plant spread, leaf shape, leaf size, leaf form, herb yield, essential oil content and 10 important constituents of the essential oil was studied in an Indian cultivar, ‘Bourbon’ of rose‐scented geranium (Pelargonium graveolens). Significantly larger variance was observed among in vitro‐regenerated plants of the SC1 generation (first generation following an in vitro phase) than among parental plants raised from stem cuttings for herb yield, plant height, leaf size, essential oil content, and for the contents of cis‐rose oxide, trans‐rose oxide, isomenthone and 10‐epi‐γ‐eudesmol in the essential oil. Plants of the SC₁ generation showing values beyond the higher or lower limit (depending on the trait) of the range observed among parental plants for different traits were developed into clones by their multiplication through stem cuttings, and evaluated again in the SC₂ generation (second generation following the in vitro phase). Somaclones selected for plant spread, and for the contents of linalool and trans‐rose oxide in their essential oils did not breed true in the SC₂ generation. However, for the remaining traits studied, 13–100% of the selected clones retained their selected traits in the SC₂ generation, suggesting that the changes in these somaclones were due to genetic causes.     

Genetic variability in plants regenerated from in vitro culture of sunflower (Helianthus annum L.)

In vitro regenerated sunflower (Helianthus annuus L.) plants (R₁) were self-fertilized and the R₂ generation was evaluated for qualitative traits. A broad range of phenotypic variation was observed and mutation frequencies were calculated. Some in vitro induced variant phenotypes were similar to known spontaneous or induced mutations in sunflower, while others were new. Chlorophyll and carotenoid deficiencies, chimaerical variegation, fasciated stem and capitulum, abnormal shoot development, and other morphological variations, were noted. Substitution of anthers with petaloid structures in a disk-floret mutant indicates a possible homeotic mutation induced by in vitro tissue culture.     

Analysis of Chinese Spring regenerants obtained from short- and long-term wheat somatic embryogenesis

Summary Somatic embryogenesis was initiated from immature embryos on Murashige-Skoog (MS) medium plus 2 mg.l^-1 2,4-dichlorophenoxyacetic acid, 2% sucrose and 0.6% agarose. Somatic embryos were isolated and regenerated into whole green plants on MS medium devoid of 2,4-D. These regenerants were previously demonstrated to differ in their mitochondrial DNA organization. In order to estimate their characteristics three progenies of short-term culture regenerants and three progenies of long-term culture regenerants were analyzed and compared to the parental line. These somaclones obtained from the wheat variety Chinese Spring were evaluated for variation of 13 agronomic and morphological quantitative characters in comparison to the parental line. Significant variation was observed for plant height, spike length, main tiller diameter, between the somaclones regenerated from long-term culture and their parent. Differences were observed to increase with the duration of culture, leading to a significant modification of the structure of the plants. Several changes occurred during the somatic tissue cultures, but to a lesser extent than has previously been described in the literature.     

Hybridization between pascal celery and parsley

Summary Pascal celery cv. Golden Spartan was hybridized with parsley which is immune to celery blight (Septoria apiicola). Three F₁ hybrids were obtained from over a thousand seedlings grown from the pascal celery parent. The F₁ plants were intermediate for most characters. Segregation for petiole color in the F₂ generation demonstrated that a hybrid actually occurred. Segregation for late blight resistance occurred independently from petiole color.Received for publication 6 December 1979. Michigan Agricultural Experiment Station Journal Article No. 9222.Department of Botany and Plant Pathology, Michigan State University.