In vitro adventitious bud techniques for vegetative propagation and mutation breeding of potato (Solanum tuberosum L.). II. Significance for mutation breeding

Summary It was investigated what potentialities for mutation breeding of potato are offered by using adventitious sprouts that arise in vitro from leaf explants (rachis, petiole, leaflet-disc) after X-irradiation. Mutation frequency and chimerism were studied in subterranean and aerial parts in three vegetative generations (vM₁, vM₂, vM₃). Plants obtained from irradiated series produced a very high mutation frequency, a wide mutation spectrum and a very low rate of chimerism. Mutations were observed also in control series, especially in plants derived from the rachis and petiole explant group.     

Vegetative propagation of Beta vulgaris by leaf cuttings

Summary Leaf cuttings without axillary buds were made from young vegetative beet plants. Roots were formed at the petiole end. Adventitious bud formation occurred at a very low frequency and could not be stimulated by application of growth regulators. Leaf cuttings with axillary buds formed plants at a higher frequency but development of buds into shoots was slow and irregular. As no vascular connections were found between petiole and axillary bud, the slow growth of the buds was attributed to insufficient supply of assimilates from the leaves. Axillary buds rapidly developed into shoots when petiole explants with buds were placed on culture media. Nearly all cultures, however, were contaminated with bacteria that originated from the inner tissue of the explants.     

Mutation breeding of Chrysanthemum morifolium Ram. using in vivo and in vitro adventitious bud techniques

Summary During experiments, which are being carried out to study the factors which control the process of adventitious bud formation in vivo on detached leaves of Chrysanthemum morifolium Ram, adventitious shoots were produced from leaves, irradiated with 500 rad of X-rays. The most important but disadvantageous result was that the majority of the adventitious shoots proved to be of a chimeral nature and obviously developed from more than one cell.An in vitro adventitious bud technique was developed using different types of explants. Pedicel segments regenerated the highest number of adventitious shoots and, moreover, they developed faster as compared to explants of young flower heads or leaves. The mutants produced by irradiating the various explants were almost exclusively of a solid (non-chimeral) nature. In addition, histological observations suggest that single epidermal cells are involved in the initiation of the adventitious shoot apices.The optimum dose for mutant production is approximately 800 rad X-rays. Rather often, more than one phenotypically identical mutant was found, which was always derived from the same explant. They could for instance originate from a multi-apical meristem formed by a single mutated cell.     

Isolation of a pure thornless loganberry by meristem tip culture

Summary Thornless Loganberry (TL) is a periclinal chimeral blackberry in which a layer of mutant (thornless) epidermis surrounds a core of wild-type (thorny) tiusse. Due to its chimeral arrangement, TL produces thorny adventitious root cuttings and thorny offspring. To separate the chimera into its components parts, meristems of TL were grow in vitro on modified Murashige and Skoog medium to yield callus and adventitious shoots. One of these shoots has survived, flowered, and produced thornless offspring from seed. The importance of this non-chimeral TL is discussed.List of terms BA 6-benzylaminopurine - GA₃ gibberellic acid - NAA naphthalene acetic acid - hybridberry polyploid bramble interspecific hybrids - MS Murashige & Skoog (1962) high mineral salt medium - TL Thornless Loganberry - TL_tc tissue culture-derived (non-chimeral) Thornless Loganberry     

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     

The use of an in vitro adventitious bud technique for mutation breeding of Begonia x hiëmalis

Summary An in vitro propagation of two genotypes of Begonia x hiëmalis was achieved through adventitious shoot formation on (sub)cultured leaf-disc explants and subsequent transplantation to soil of explant-parts with adventitious shoots.After irradiation of detached leaves with different doses of X-rays and two cycles of adventitious shoot formation on in vitro (sub)cultured leaf-disc explants, plantlets were produced. About 30% of these plants was mutated with respect to e.g. the colour, size and form of the leaves and flowers. The great majority of the mutants (98.5%) proved to be solid (non-chimeric).     

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.     

Effect of gamma and ultraviolet irradiation on adventitious regeneration from in vitro cultured pear leaves

Summary Irradiation of in vitro explants and subsequent adventitious regeneration has been tested for 4 commercially important varieties of pear (Pyrus communis) with the aim to create mutants with a reduced susceptibility to fire-blight (Erwinia amylovora). The effect of gamma and ultraviolet irradiation of leaves on adventitious regeneration ability has been studied. The LD50 (50% decrease of regeneration) after gamma irradiation was genotype-dependent and was between 20 and 50 grays. The curves of regeneration showed a threshold dose underneath which none or a very slight decrease was registered. The decrease might result from cumulative events. After an ultraviolet irradiation as low as 62.5 J/m², the leaves became crumbly and rolled up, and their metabolism seemed to be altered. The LD50 was about 125 J/m² for all varieties and the decrease of regeneration was linear. Histological investigations showed leaves with flattened epidermal cells after ultraviolet irradiation and slack spongy parenchyma after gamma irradiation.Abbreviations Gy grays - J joules - LD lethal dose     

茶树不同组织体细胞胚、不定芽分化的研究

根据茶树子叶柄、叶柄断面离腋芽的距离,将外植体依次分为C1, C2, C3和L1, L2, L3。通过上述外植体和不同培养基的研究结果表明:子叶柄外植体在MT1和MT3培养基上培养,其不定芽及总分化率是C1>C2>C3;体细胞胚的分化率是C3>C2>C1。叶柄外植体只有L1在MT3培养基上培养分化了芽,MT1培养基的胚性愈伤组织诱导率比MT,培养基高,     

Chromosome doubling through adventitious shoot formation on in vitro cultivated leaf explants from diploid interspecific potato hybrids

Summary Nearly 450 plantlets were produced from 51 diploid Solanum etuberosum x S. pinnatisectum F₁ hybrids through adventitious shoot formation on in vitro cultivated rachis and petiole explants.On the basis of phenotypical assessment of the ploidy level of 425 plants, 84.7% of the plants were scored as doubled or doubled twice. A cytological analysis of ploidy in the three layers L₁.L₂ and L₃ of 112 plants revealed 83.9% complete doubling: periclinal ploidy chimeras were not found and only two sectorial ploidy chimeras were detected. Doubled plants were obtained from all 51 clones.Various flower colours and epinastic leaves (in 1 clone) may be indications of mutagenesis through the treatment.     

A tissue culture technique for vegetative propagation and low temperature preservation of Beta vulgaris

Summary A tissue culture technique for clonal propagation of Beta vulgaris is described. Flower buds or flower bud clusters formed adventitious shoots on half-strength Murashige & Skoog's medium supplemented with 10 mol/l benzyladenine (BA). The shoots were multiplied by axillary bud proliferation on a medium with 1 mol/l BA and rooted on a medium with 10 mol/l indolebutyric acid. The plants were vegetative. No mutations were observed. Genotypic variation was found in shoot formation, shoot multiplication and rooting. Some genotypes showed leaf malformations which were attributed to BA. Rooted plants in culture tubes could be stored for one year at 2 or 5°C and a low light intensity.     

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.     

Some methodological aspects of apple transformation by Agrobacterium

Summary Leaf explants of apple cvs Gala and Golden Delicious were infected with the Agrobacterium tumefaciens strain AGL0(pMOG410). The effects of a 2 d preculture of the explants before infection and the addition of 5-azacytidine to the selection medium were studied. The percentages of GUS-positive explants after 5 w did not significantly alter due to these treatments. One of the Gala shoots, which was removed from a leaf explant cultured for 8 w on selection medium, proved to be GUS-positive and will be analyzed further. In general, however, it should be concluded that regeneration of transgenic shoots directly from leaf tissue was not very effective.Abbreviations MS Murashige and Skoog medium - GD Golden Delicious     

Induced changes in growth and flowering of chrysanthemum after irradiation and in vitro culture of pedicels and petal epidermis

Summary To increase the genetic variation for yield in Chrysanthemum morifolium cv. White Spider, pedicel segments and petal epidermis were induced in vitro to regenerate adventitious buds either directly from the original tissue or indirectly via callus. Besides, pedicels were irradiated before in vitro culture with an X-ray dose of 8 Gy. All treatments yielded variation in flower morphology. Changes in flower colour were largely restricted to treatments involving irradiation.Treatments yielding many morphological variants also yielded most production variants. Plants regenerated from petals differed from those originating from pedicels. Clones were found that had retained the morphology of White Spider, but outperformed the controls in flowering time and flower number.     

“渝苏303”甘薯离体形态发生过程中生理生化特性的变化

甘薯茎段、叶片和叶柄在附加有1.0 mg/L NAA的MS培养基中培养5天可产生大量的不定根,培养20 d茎段和叶片分化出不定芽。本文以甘薯品种“渝苏303”为材料,研究了甘薯外植体在不定根和不定芽分化过程中可溶性蛋白质含量、过氧化物酶（POD）和超氧化物酶（SOD）活性的变化,以及可溶性蛋白质和SOD同工酶酶谱的变化。结果表明     

Mutation breeding for resistance to blackspot bruise and low temperature sweetening in the potato cultivar Lemhi Russet

A mutation breeding method was developed to select clones of Lemhi Russet that have resistance to blackspot bruise and low temperature sweetening. Following irradiation with gamma rays from a Cobalt⁶⁰ source, over 2000 tuber eye pieces were planted directly to the field and tubers from the resulting crop were individually evaluated for blackspot bruise potential. Selection for blackspot bruise resistance continued for five clonal cycles. Selection for low temperature sweetening began in the M₃ and continued for three cycles. Ten clones were selected, eight with significantly (p=0.05) better blackspot bruise resistance, and two with increased resistance to low temperature sweetening. The results confirm the possibility for the development of a system to improve single selectable quality characteristics in potatoes.     

In vitro production of male sterile Zinnia elegans

Summary Axillary bud proliferation of Zinnia elegans Jacq. was accomplished from vegetative bud explants of greenhouse-grown plants. Photoperiod extension via metalarc lamps in the greenhouse stock plant environment significantly increased both the quantity and quality of explants. Shoot cultures initiated from axillary buds of identified male sterile plants proliferated best on a medium with 1M BAP. Microshoots exhibited 80% rooting on media without growth regulators, and 100% acclimatization of rooted plantlets was accomplished in a lighted, high humidity chamber. In vitro- produced male sterile plants were more compact, more heavily branched, and demonstrated earlier flowering than seed-propagated plants. These male steriles were cross pollinated to male fertile lines to verify their ability to serve as a uniform female parent.     

Induction and inheritance of determinate growth habit in chickpea (Cicer arietinum L.)

Summary The character of determinate plant growth has not been reported for chickpea and has not been observed in the world germplasm collection at ICRISAT, Patancheru, India. A determinate growth habit would be desirable where growing conditions often lead to excessive vegetative growth. We attempted to generate this trait by mutation breeding. Seeds of the cultivar ICCV 6 were exposed to varying irradiation treatments, M₁ and M₂ populations were raised, and in the latter one plant was detected that showed the determinate growth habit and female sterility. The character of determinate growth segregated in a postulated digenic epistatic 3:13 fashion in the F₂ and confirmed its digenic mode of inheritance in the F₃ and F₄. The symbol cd is proposed for the allele conditioning for determinancy and Dt for the allele expressing the determinate trait. Continued mutation breeding with this and other material may result in identifying fully fertile, determinate plant types.Abbreviations DT - determinate - IDT - indeterminate ICRISAT Journal Article No. 1396.     

The Occurrence of Rust Resistance Gene Ur-13 in Common Bean Cultivars and Lines

Summary For the past 10 years, the Andean-type Phaseolus vulgaris cultivar Kranskop has played an important role in South African bean production and breeding. Kranskop shares an ancestor with the internationally important Andean-type rust differential cultivar Redlands Pioneer. The Ur-13 gene in Kranskop and Redlands Pioneer gives protection to numerous internationally reported races of Uromyces appendiculatus and it is imperative to retain this gene in local breeding programmes.In this study, three co-dominant SCAR markers (SE_AACM_ACC430/405, SE_ACAM_CTT310/288 and SE_AAGM_CGT436 Hha I_186/250) were used to trace the origin of Ur-13, and its presence in 71 germplasm accessions, including the international rust differential lines and additional sources of already characterized genes, as well as 78 breeding lines.Each marker was present in approximately 30% of the accessions tested. Only accessions belonging to the Kranskop or Redlands groups contained all three markers. Contrary to expectations, the first two markers, as well as Ur-13, appeared to have originated from a Middle-American-type parent of the Redlands group, California Small White 643, whereas the third marker probably came from the Andean-type cultivar Brown Beauty. This has important implications for the new set of differential lines, as Redlands Pioneer can no longer be regarded as a representative of the Andean gene pool. The markers, in particular SE_AACM_ACC430/405, will be useful in tracing Ur-13 in large seeded breeding material, except where lines such as Mexico 309, PI 181996 and A 286 are used as donors of additional rust resistance genes, as these have the positive alleles of both SE_AACM_ACC430/405 and SE_ACAM_CTT310/288.Part of a Ph.D thesis submitted by the first author to the Department of Plant Sciences, University of the Free State.     

Inheritance of resistance to turnip mosaic virus in Chinese cabbage

Summary Turnip mosaic virus (TuMV) is the most important virus of commercially grown cole crops in many Asian countries, affecting both yield and quality. TuMV-infected Chinese cabbage becomes unmarketable because of the presence of black spots and necrosis often induced by the virus. Resistance breeding is complicated by the existence of five strains of the virus, one of which was discovered in 1985 for the first time in Taiwan. Resistance to strains C1 to C3 is readily available among the Chinese cabbage germplasm at AVRDC, whereas resistance to strains C4 and C5 is rarely found. To elucidate the inheritance of resistance to TuMV, P₁, P₂, F₁, F₂ and BC₁ generations of crosses between the resistant line 0–2 and three susceptible lines, E-7, E-9 and FL-9, were inoculated with strains C4 and C5. Segregation ratios obtained by visual observation and enzyme-linked immunosorbent assay (ELISA) indicate that two recessive genes confer resistance to both TuMV-C4 and TuMV-C5.