Selection of a mutant from adventitious shoots formed in X ray treated cherry leaves and differentiation of standard and mutant with RAPDs

Summary An obstacle when using scions orin vitro shoots for mutation induction is the occurrence of chimeras. When adventitious shoots are formed from irradiated material these usually are derived from single cells, this leading to homohistont mutants. SincePrunus avium regenerates adventitious shoots from leaves at a low rate only (Yang & Schmidt, 1992), leaves of the interspecific cherry rootstock ‘209/1’ (P. cerasus ×P. canescens) were irradiated. ‘209/1’ regenerates adventitious shoots readily. Dosages applied were 5, 10, 20 and 40 Gy. Shoot production following 5 Gy irradiation was similar to the control. The application of 40 Gy resulted in strong damage with only few leaves regenerating. Among the adventitious shoots from leaves irradiated with 20 Gy one shoot was evident alreadyin vitro with thicker and smaller leaves having a serrate margin. It was cloned as ‘209/1-20m’. The clone stayed stable since 1990in vitro, in the greenhouse and the field. Compared with standard ‘209/1’ the mutant is very dwarf. Research to differentiate between standard ‘209/1’ and ‘209/1-20m’ was done using RAPDs. Among the decamer primer kits D, J, and T from Operon Technologies, Calif. Only primer OPJ05 (5‘CTCCATGGGG3’) differentiated between ‘209/1’ and ‘209/1-20m’. Rootstock ‘209/1’ showed one band of 2 kb additionally. This band is missing in the mutant.     

Gamma-irradiation of cacao (Theobroma cacao L.) pollen: Effect on pollen grain viability,germination and mitosis and on fruit set

Summary Theobroma cacao pollen fertilization capability was studied after 0, 50, 100, 200, 500 and 1000 Gy gamma-irradiation. For all irradiation doses, no alteration of pollen grain viability and in vitro germination was observed. In situ, for all doses, pollen tubes penetrated into the styles and reached the ovules 20 hours after pollination. In vitro observations of the pollen grain nuclei after 20 hours incubation showed that pollen irradiation causes inhibition of the division of the generative nucleus. Fruit survival rate 30 days after pollination decreased as irradiation doses increased from 0 to 100 Gy, and over 100 Gy no fruit set was obtained. The possibility of using irradiated pollen as a method for obtaining haploid cacao plantlets is discussed.     

Induction of mutations for heat tolerance in potato by using in vitro culture and radiation

Heat tolerant mutants were obtained in two commercial potato cultivars, `Kufri Jyoti' and `Kufri Chandramukhi' through in vitro mutagenesis of in vitro propagated plantlets. Gamma-irradiated (20 and 40 Gy) shoots were micropropagated for three cycles (M₁V₃). A large number of the micropropagated shoots produced microtubers at 28 ^°C. Microtubers induced at high temperature had distorted shape but showed normal germination in field. Under stress conditions of high temperature, the frequency of chlorophyll variants increased in the gamma irradiation-derived material, however, nearly 40% of the plants had normal leaf tissue, whereas control plants showed completely damaged leaves. This revised version was published online in July 2006 with corrections to the Cover Date.     

High regeneration potential in vitro of sunflower (Helianthus annuus L.) lines derived from interspecific hybridization

Successful selection of interspecific hybrid progenies with superior ability to regenerate shoots from apical meristems was performed in sunflower which now allows for the development of lines for improved biotechnological applications. Early generations of interspecific hybrids originating from crosses between the two H. annuus CMS lines ‘HA89’ and ‘Baso’, and 9 wild species were screened for their ability to regenerate in vitro. Evaluation of 36 progenies allowed to identify seven progenies from crosses involving H. mollis, H. giganteus, H. strumosus, and H. decapetalus which showed a significantly higher regeneration potential than the commercial hybrid ‘Albena’ regarding the number of shoots per explant. Among these progenies, 47.2 to 62.4% of explants produced shoots with an average of 2.3 to 3.5 shoots per cultured explant. Regeneration in vitro was significantly determined by the genotype. More than half of the investigated interspecific hybrids performed better than the inbred ‘HA89’ demonstrating that the high regeneration potential available in the wild species can be efficiently transferred to cultivated sunflower. The seven progenies with high regeneration potential in vitro were characterised by agronomic performance in the field. Two of the interspecific hybrids derived from H. strumosus and H. decapetalus not only showed a superior regeneration potential but also proved to be competitive to commercial hybrids with regard to important agronomic traits, e.g. fat content and TGW. This revised version was published online in July 2006 with corrections to the Cover Date.     

Preliminary results of using in vitro axillary and adventitious buds in mutation breeding of Chinese gooseberry

Summary Aseptically cultured shoots of Chinese gooseberry exhibited growth disorder and morphological aberrances, and some died after being exposed to sufficient gamma-ray irradiation. The death rate was dose dependant and the LD₅₀ was 80–90 Gy and 50–60 Gy respectively for cv. Hayward and clone 4. All petiole explants irradiated with gamma-ray could form calli as the control, but the rate of differentiation of adventitious shoots of the petiole explants decreased and was dependant on dose. Sensitivity of the shoot or petiole explants to gamma-ray irradiation varied with species. Gamma-ray irradiation did not deter either the 2-node segments from producing axillary shoots M₁, M₂, and M₃ or the advantitious shoots originating in the petiole explants and the M₃ shoots from forming advantitious roots. Therefore, using aseptically cultured axillary or adventitious buds for mutation breeding of Chinese gooseberry is feasible. A bacterium surviving in the explants lessened the efficiency of these two in vitro techniques in mutation breeding of Chinese gooseberry.Abbreviations IAA 3-indole acetic acid - IBA -indole butyric acid - MS Murashige & Skoog (1962)     

Obtention of haploid embryos and plants through irradiated pollen technique in squash (Cucurbita pepo L.)

The influence of gamma ray doses (25, 50, 75, 100, 200, 300 and 400 Gy) and genotypes (Eskenderany F₁, Acceste F₁, Sakiz, Urfa Yerli) on the induction of haploid embryos obtained by irradiated pollen technique was studied in squash (Cucurbita pepo L.). Different shapes and stages of embryos were derived from seeds extracted from fruits harvested 4–5 weeks after pollination. As a result of the present study, haploid embryos and haploid plants were obtained, with haploid production strongly influenced by gamma ray doses, embryo stages and genotypes. Gamma ray doses of 25 and 50 Gy gave the highest parthenogenetical response. All of the point shape, globular shape, arrow tips and stick-shaped embryos developed into haploid plants. However, only 53.8% of torpedo and 23.1% of heart-shaped embryos gave haploid plants. In contrast, cotyledon-shaped and amorphous-shaped embryos produced only diploid plantlets. The number of embryos per 100 seeds was the highest in ‘Eskenderany F₁’ and ‘Sakiz’ genotypes. After in vitro culture, a total of 93 haploid plantlets were obtained. This revised version was published online in August 2006 with corrections to the Cover Date.     

Introgression of in vitro regeneration capability of Lycopersicon pimpinellifolium Mill. into recalcitrant tomato cultivars

The goal of this research was to study the introgression of the high regeneration capacity of Lycopersicon pimpinellifolium Mill line WV-700, in recalcitrant tomato cultivars (L. esculentum Mill cvs. Petomech, Santa Rita and VFN-8) using backcrossing. Hypocotyl explants of in vitro germinated seeds were cultivated in half strength MS medium supplemented with 5mg/l 6-BA to assess their shoot regeneration capacity. The apical shoot of the in vitro germinated seeds were cultured on a separate medium. Apical shoots from genotypes showing high regeneration rates were acclimated in a glasshouse and used as pollen donors for the next backcrossing. After four backcrossings, the material showed a similar mean fruit weight for the cultivated tomato and a high regeneration capacity similar to the wild species. It is shown that L. pimpinellifolium can be used with success as donor parent to introgress in vitro regeneration capacity to recalcitrant tomato cultivars. This revised version was published online in August 2006 with corrections to the Cover Date.     

An assessment of morphogenic and transformation efficiency in a range of varieties of potato (Solanum tuberosum L.)

Summary To provide a truly genotype-independent transformation system, it is necessary to be able to transform a wide range of potato genotypes. The ability to regenerate shoots in vitro was determined for 34 potato varieties using tuber disc explants. Following a culture regime used extensively in previous studies with the variety Desiree, half of the varieties could be regenerated from tuber discs and half could not. From a sample of varieties that could be regenerated from tuber discs, all but one variety gave transgenic plants. Twelve varieties were evaluated for the capacity to regenerate shoots from leaf and internode explants excised from in vitro grown plants. All of the varieties tested regenerated adventitious shoots. Leaf and internode explants from 5 varieties were subsequently used for transformation, and transgenic plants were produced from two potato varieties that did not give transgenic plants from tuber disc explants. Some varieties could not be transformed by either method, and will require modification of the in vitro regeneration and transformation system to be successful.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid     

Cytological Study of Callus and Regenerated Plants of Sunflower (Helianthus annuus L.)

In vitro culture of shoot apical meristems of Helianthus annuus was studied to determine the cytological condition of calli and regenerated shoots. A broad chromosome mosaicism (aneusomary) in bath callus and regenerated shoots was found, reflecting the cytological conditions of the explain. During in vitro plant development, a diplontic selection took place that was more rapid when very precocious in vitro flowering occurred.     

Oryzalin as an Efficient Agent for Chromosome Doubling of Haploid Apple Shoots in vitro

In an outbreeding species such as apple, haploid plants may be especially useful in breeding programmes for the production of homozygous material. However, methods must be available to induce chromosome doubling in the haploid plants. Two antimitotic agents, colchicine and oryzalin, were compared as regards their efficiency in inducing chromosome doubling of in vitro haploid apple shoots. Three colchicine levels (0.025, 0.25 and 1.25 mM) and three oryzalin levels (5, 15 and 30 μM) were evaluated. Three techniques were also used and compared. Survival rate and chromosome counts were determined. Differences were observed between the two antimitotic agents and between the three techniques. This study demonstrates that oryzalin could be a better choice than colchicine for chromosome doubling on haploid apple shoots in vitro.     

Overexpression of BpMADS4 from silver birch (Betula pendula Roth.) induces early-flowering in apple (Malus × domestica Borkh.)

To shorten the juvenile stage of apple (Malus × domestica Borkh.) the BpMADS4 gene from silver birch (Betula pendula Roth.) was constitutively overexpressed in 25 transgenic apple clones. All clones were characterized by PCR, RT‐PCR and Real Time PCR. Solitary flowers were produced on in vitro shoots of eight transgenic clones and most of them appeared to be morphologically normal. Twenty shoots of each clone were rooted and transferred to a glasshouse. Glasshouse plants of clones T1165, T1187 and T1190 developed flowers. Several plants of T1165 and T1187 started floral initiation within 3–4 months following transfer to the glasshouse. Primary flowers were solitary and in a terminal position on the main shoot. Lateral flower clusters, consisting of three to five individual flowers, were also found. Pollen vitality and tube germination of glasshouse‐grown flowers were investigated, and there were no significant differences compared to pollen of non‐transgenic control plants. Preliminary crosses using flowers of glasshouse plants resulted in small apple fruits. It would seem that this is the first report on in vitro flower induction in transgenic apple.     

Pear mutagenesis: In vitro treatment with gamma-rays and field selection for productivity and fruit traits

In vitro shoots of six pear (Pyrus communis L.) cultivars, ‘Conference’, ‘Doyennéd'Hiver’, ‘Passe Crassane’, ‘Bartlett’, ‘AbbéFetel’ and ‘Butirra Precoce Morettini’ were irradiated with gamma rays (3.5 Gy). After three subcultures, microcuttings from the irradiated shoots and from additional non-irradiated microcuttings were rooted to establish plants for survey orchards. All trees were individually observed for variation in fruit traits and for productivity. Trees were selected for improved characters related to production such as early bearing and consistent productivity. Variations observed in fruit appearance concerned degree of russeting, fruit shape and size. The frequencies of the observed variations in fruit traits depended on the cultivar, ranging from 0.81% in ‘Doyennéd'Hiver’ to 3.64% in ‘Passe Crassane’. Of the 97 variants selected, only two showed chimeral behavior. This revised version was published online in July 2006 with corrections to the Cover Date.     

Osmoregulation in birdseed millet under conditions of water stress II. Variation in F3 lines of Setaria italica and its relationship to plant morphology and yield

We report, for the first time, there generation of four homozygous lines in sweet cherry (Prunus avium L.) byin situ parthenogenesis followed by embryo and cotyledon culture. The sweet cherry cultivar ‘Altenburger’ was pollinated with marked pollen irradiated by γ-rays at doses ranging from 250 to1200 Gy. Pollination with such irradiated pollen affected fruit set and the quality of the embryos, and induced the formation of parthenogenic embryos. The immature embryos extracted from the stones, 35 or 75days after pollination, were cultivatedin vitro in an embryo or cotyledon culture. Although flow cytometrical analysis demonstrated the diploid level for all regenerants, four lines could be characterized as homozygous using isoenzyme analysis. This revised version was published online in July 2006 with corrections to the Cover Date.     

High frequency plantlet regeneration from nodal segment culture of female <Emphasis Type="Italic">Momordica dioica</Emphasis> (Roxb.)

An in vitro propagation method for female plants of Momordica dioica (Roxb.) has been established. The nodal segments were harvested and the cut ends of the explants were sealed with wax and then surface sterilized and cultured. Bud breaking occurred on Murashige and Skoog’s (MS) agar-gelled medium + 2.0 mg L⁻¹ 6-Benzylaminopurine (BAP) + 0.1 mg L⁻¹ Indole-3 acetic acid (IAA). The cultures were amplified by passages on MS medium supplemented with 1.0 mg L⁻¹ BAP + 0.1 mg L⁻¹ IAA. Further, shoot amplification (29.2 shoots per vessel) was achieved by subculturing of in vitro regenerated shoot clump on MS medium + 0.5 mg L⁻¹ BAP + 0.1 mg L⁻¹ IAA. The micropropagated shoots were subsequently transferred for root formation on half-strength MS medium + 2.0 mg L⁻¹ Indole-3 butyric acid (IBA) with 89% success rate. The in vitro-regenerated shoots were also rooted ex vitro with 34% success. These plantlets were hardened in the greenhouse and transferred to the field. The established protocol is suitable for true to type cloning of mature female plant of M. dioica.     

High-frequency transgenic plant regeneration and plumbagin production through methyl jasmonate elicitation from hairy roots of <Emphasis Type="Italic">Plumbago indica</Emphasis> L.

High-frequency transgenic plant regeneration and production of plumbagin were accomplished from hairy roots induced by Agrobacterium rhizogenes strain A₄M70GUS on Plumbago indica L. Of the two types of hairy roots developed on Murashige and Skoog (MS) basal medium, Type I was long and thick with lesser branches and root hairs, and Type II was highly-branched short and slender with tufts of root hairs. Of the different lines grown in half-strength MS liquid basal media, root line 5 (R5) of the Type I yielded the highest plumbagin (0.92% DW) and was significantly different to that of the in vitro control. R5 showed a stable production of plumbagin (1.09% DW) in subsequent cultures. Elicitation of R5 with 50 μM methyl jasmonate for 48 h increased the yield of plumbagin to 5.0% DW, and was superior to 100 μM acetylsalicylic acid (3.8% DW). Plumbagin yield was five times that of twoyear-old ex vitro roots. Histochemical assay and PCR analysis using the primers of uidA coding region confirmed the transformation. Hairy root segments cultured on MS medium containing 8.8 μM benzyladenine and 2.5 μM indole-3-butyric acid induced a mean of 9.1 shoots. Subsequent culture of the isolated shoots developed more than 50 normal shoots per culture. The root-free shoots were rooted on half-strength MS basal medium. The plantlets transferred in field conditions grew normally and exhibited 90% survival. Transgenic plant regeneration and hairy root induction in P. indica serves as reliable source of plumbagin which in turn cut off the mass destruction of the plant species.     

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.     

Variants of Digitalis obscura from irradiated shoot tips

Shoot tips excised from genotype T4 of Digitalis obscura were exposed to gamma rays (20–100 Gy). Radiosensitivity was assessed and LD₅₀ determined was about 60 Gy. The effect of the herbicide amitrole on shoot-tip culture was investigated, efficient bleaching was obtained with 10 mg/l amitrole. Shoot tips mutagenized (20–40 Gy) were cultured on several selective amitrole-containing media, but none of them permitted prolonged survival of the green shoots formed. Ploidy level of the plantlets developed was analysed by flow cytometry 8 and 18 months after culture establishment. Variations detected corresponded to aneuploid changes and increased in parallel with the gamma radiation dose. Plantlets developed from irradiated shoot tips presented a high variability in their cardenolide production (878 to 3291 μg/g d.w.), including variants with similar or even higher productivities than the native T4 plant. This revised version was published online in August 2006 with corrections to the Cover Date.     

In vitro chromosome doubling of Miscanthus sinensis

The aim was to develop an efficient chromosome doubling method for Miscanthus sinensis to enable the production of triploids and so avoid seed dispersal to the environment. Antimitotic treatments with colchicine or oryzalin were tested in M. sinensis cl. MS‐88‐110 on: (1) in vitro shoots and plants established in soil; (2) during propagation of embryogenic callus; and (c) during the initial stages of callus induction. All systems produced chromosome‐doubled plants. A higher percentage of tetraploids was found after antimitotic treatment at the explant or callus level compared with treatment of in vitro shoots or plants established in soil. In general, oryzalin was more toxic to plant material than colchicine. A higher frequency of chimeras was found among plants with altered ploidy level when the target was formed shoot buds compared with adventitious shoot formation from callus. Antimitotic treatment of embryogenic callus from shoot apices also resulted in a high degree of albinism.     

Morphogenic response of the alginate encapsulated nodal segment and antioxidative enzymes analysis during acclimatization of Ocimum basilicum L.

Synthetic seed technology is a potential tool for a more efficient and cost effective rapid clonal propagation system. In the present investigation, synthetic seeds were produced by encapsulating nodal segments of Ocimum basilicum in calcium alginate gel. For encapsulation of nodal segment, 3% (w/v) sodium alginate and 75 mM CaCl₂.2H₂O were found most suitable. The synthetic seeds when cultured on half-strength Murashige and Skoog (MS) medium supplemented with 5.0 μM benzyladenine (BA) and 0.5 μM Indole -3- acetic acid (IAA) produced maximum number of shoots (7.9 ± 0.54) after 8 weeks of culture exhibiting 80% in vitro conversion response. Further, synthetic seeds stored at 4 °C for 4 weeks resulted in maximum conversion response (90%) when placed back to regeneration medium. Both root and shoot formation took place in the same medium but the roots were thin and difficult to handle. Individual elongated shoots were rooted on MS medium supplemented with 1.0 μM Indole -3- butyric acid (IBA). Plants regenerated from the synseeds were hardened, acclimatized, and established in soil with 80% survival rate. Changes in antioxidative enzymes viz., Superoxide dismutase (SOD) and Catalase (CAT) in O. basilicum indicated the adaptation of micropropagated plants to ex vitro conditions.     

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.